Plant Physiology

Minorsky, Peter V.

doi: 10.1104/pp.104.900142pmid: N/A

RNAi Silencing of Isoflavone Synthase The isoflavones play diverse roles in the plant-microbe interactions of the Papilionoideae. Isoflavones function as antibiotics as well as precursors for defense-related phytoalexins. They also serve as signal molecules in the induction of microbial genes involved in soybean (Glycine max) nodulation. Although isoflavones are normally present at relatively low levels in mature soybean tissues, their accumulation is strongly induced in response to pathogen attack or defense elicitors. Isoflavone synthase (IFS) is a key enzyme for the formation of the isoflavones. Subramanian et al. (pp. 1345–1353) show that the silencing of both copies of IFS genes in roots initiated from soybean cotyledons leads to the effective spread of silencing throughout the nontransformed cotyledon tissues. Distal silencing was established within 5 d of transformation and was highly efficient for a 3- to 4-d period, after which it faded. Distal silencing effected a nearly complete suppression of mRNA accumulation for both the IFS1 and IFS2 genes and of isoflavone production induced by wounding or treatment with the cell wall glucan elicitor from Phytophthora sojae. Preformed isoflavone conjugates were not reduced in distal tissues, suggesting little turnover of these stored isoflavone pools. Silencing of IFS led to enhanced susceptibility to P. sojae. The soybean cotyledon system may prove to be a convenient and effective system for the functional analysis of other plant genes through gene silencing. Oxalate Oxidase Confers Resistance to Sclerotinia Blight in Peanut Sclerotinia minor is the causal agent of Sclerotinia blight, a highly destructive disease of peanut (Arachis hypogaea). Much evidence indicates that oxalic acid serves as a pathogenicity factor during Sclerotinia infection. Direct application of oxalic acid to stem or leaf tissue causes tissue injury and wilting, similar to plant responses to fungal infection by S. sclerotiorum. Mutant isolates of S. sclerotiorum, deficient in oxalic acid production, are not pathogenic on bean (Phaseolus vulgaris), but revertants became pathogenic once they regain the ability to produce oxalic acid. Oxalic acid may aid in fungal infection through a number of proposed routes, including the facilitation of cell wall-degrading enzyme activity, through pH-mediated tissue damage, or via sequestration of Ca2+ ions. There is also evidence that oxalic acid can directly suppress the oxidative burst associated with the detection of pathogens by plants and disturb guard cell function during infection by S. sclerotiorum by inducing stomatal opening and inhibiting abscisic acid (ABA)-induced stomatal closure. Oxalate oxidase (OO) has received considerable attention for its possible utility in plant defense. OO catalyzes the degradation of oxalic acid to produce carbon dioxide and hydrogen peroxide. It has been proposed that through the production of hydrogen peroxide, OO may cause cross-linking of plant cell wall proteins at the site of infection or play a role in the plant hypersensitive response. Livingstone et al. (pp. 1354–1362) report that the size of S. minor-induced lesions were reduced by 75% to 97% in OO-transformed peanut plants, providing evidence that OO confers enhanced resistance to Sclerotinia blight in peanut. Kinematic Analysis of an Aphid Infestation The pea aphid (Acyrthosiphon pisum) is a phloem-sap feeder known to reduce growth and dry-mass production under field conditions (Fig. 1 Figure 1. Open in new tabDownload slide Pea aphids, seen here infesting a pea (Pisum sativum) fruit, infest mostly the stems of alfalfa, causing them to become such strong nutrient sinks that the shoot apices actually become source tissues (photograph by Bob Lamb). Figure 1. Open in new tabDownload slide Pea aphids, seen here infesting a pea (Pisum sativum) fruit, infest mostly the stems of alfalfa, causing them to become such strong nutrient sinks that the shoot apices actually become source tissues (photograph by Bob Lamb). ). Pea aphids preferentially settle on the elongating internodes of alfalfa (Medicago sativa) and only rarely colonize leaves. Thus, their impact on stem elongation is particularly pronounced. Because pea aphids neither transmit viruses nor deliver toxic substances by salivary secretions, it is generally assumed that their effect on growth is mainly due to removal of phloem sap from their host plants. Kinematic studies offer a powerful tool for assessing the fluxes and deposition rates that sustain the growth of plant organs. Although kinematic studies have previously been used to study the effects of a wide variety of abiotic stresses on growth, Girousse et al. (pp. 1474–1484) provide the first kinematic study of a biotic stress, namely pea aphid infestation of alfalfa. They report that severe short-term aphid infestation induces a strong and synchronized reduction in elongation and in water and carbon deposition. Reduced nitrogen contents were observed in some parts of the infested stems, especially in the apex. This suggests that aphid infestation converts the shoot apex, normally a sink tissue, into a source tissue. Because aphid infestation reduced longitudinal elongation 1.4 times more than radial expansion, and in a manner similar to mechanical stimulation, the authors speculate that aphid infestation stress may also involve a thigmomorphogenic component. Novel Anion Transporter in the Symbiosome Membrane During development of a legume-Rhizobium symbiosis, the bacterium ultimately becomes enclosed in a specialized, plant-derived organelle known as the symbiosome. It is across the symbiosome that reduced carbon compounds from the plant cytosol are exchanged for reduced nitrogen from the bacteroid. Vincill et al. (pp. 1435–1444) have isolated a cDNA from soybean nodules that encodes a putative transporter (GmN70) of the Major Facilitator Superfamily. GmN70 is expressed predominantly in the symbiosome membrane of mature nitrogen-fixing root nodules. Outward currents, carried by anions and with a selectivity of nitrate > nitrite ≫ chloride, were observed in Xenopus laevis oocytes expressing GmN70. No apparent transport of organic anions was observed. Half maximal currents were induced by nitrate concentrations between 1 to 3 mm. These currents showed little sensitivity to pH or the nature of the counter cation in the oocyte bath solution. Voltage clamp records of an ortholog of GmN70 from Lotus japonicus (LjN70) also showed anion currents with a similar selectivity profile. These findings suggest that GmN70 and LjN70 are inorganic anion transporters of the symbiosome membrane with enhanced preference for nitrate. Previously, it has been shown that the membrane potential generated by the H+-ATPase in isolated intact soybean symbiosomes can be collapsed by the addition of anion salts, suggesting the existence of an anion transporter in the symbiosome membrane. Interestingly, the effectiveness of the various anion salts to dissipate the soybean symbiosome membrane potential is similar to the permeability profile of GmN70. Proteomics of Seed Filling During a 4- to 5-week period of seed filling, most of the storage reserves in soybeans are synthesized. At maturation, approximately 41% of soybean seed dry weight is storage protein. The two most prevalent seed storage proteins are glycinin and β-conglycinin. Despite the importance of seed filling, systematic proteomic analyses of this phase of seed development are only beginning to be initiated in legumes. Hajduch et al. (pp. 1397–1419) employed a high-throughput proteomic approach to determine the expression profiles and identity of hundreds of proteins during seed filling in soybean. Soybean seed proteins were analyzed at 2, 3, 4, 5, and 6 weeks after flowering using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). This led to the establishment of high-resolution proteome reference maps, expression profiles of 679 spots, and corresponding MALDI-TOF-MS spectra for each spot during five key stages of seed development in soybean. From these 679 protein spot groups, the authors were able to identify 422 proteins representing 216 nonredundant proteins. These proteins were classified into 14 major functional categories. Proteins involved in metabolism, protein destination and storage, metabolite transport, and disease/defense were the most abundant. An overall decrease in metabolism-related proteins versus an increase in proteins associated with destination and storage was observed during seed filling. Sucrose transport and cleavage enzymes, cysteine and methionine biosynthesis enzymes, 14-3-3 like proteins, lipoxygenases, storage proteins, and allergenic proteins also accumulate during seed filling. A database has been developed to allow access to these data for soybean and related data for other oilseeds. Aphid Resistance in Medicago truncatula Phloem feeding insects such as aphids harm plants by direct feeding damage and by serving as vectors for the spread of microbial pathogens. Bluegreen aphid or blue alfalfa aphid (Acyrthosiphon kondoi) is an important pest of pasture legumes, particularly Medicago spp. Klingler et al. (pp. 1445–1455) provide insights into aphid resistance by means of a side-by-side comparison of two closely related M. truncatula cultivars, Jemalong/A17 and Jester. They report that alatae (the winged, migratory morphs) prefer susceptible line A17 over the resistant line Jester, suggesting that antixenotic (deterrent) factors are present in aphid-resistant plants. The proportion of time that aphids spent ingesting phloem sap was dramatically reduced for aphids on previously infested Jester plants. Antibiosis against A. kondoi is enhanced by prior infestation, indicating induction of this phloem-specific defense. The finding that shoot excision eliminates A. kondoi resistance in M. truncatula raises the possibility that a resistance factor(s) is imported to the feeding site and that resistance may not be tissue autonomous. Aphid resistance segregates as a single dominant gene, AKR (Acyrthosiphon kondoi resistance), in two mapping populations, which have been used to map the locus to a region flanked by resistance gene analogs predicted to encode the CC-NBS-LRR subfamily of resistance proteins. These results suggest that AKR may reside within a cluster of defense-related genes. © 2005 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Stacey, Gary; VandenBosch, Kate

doi: 10.1104/pp.104.900141pmid: 15824278

The last twenty years have been a period of rapid advance in our understanding of plant biology, metabolism, and genomics. In large part, these advances were facilitated by adoption of plant models, the first and most important being Arabidopsis thaliana. This paradigm was quickly adopted by other communities, resulting in, for example, the sequencing of the rice (Oryza sativa) genome. The legume community has not been immune and, for a wide variety of reasons, adopted two models, Medicago truncatula and Lotus japonicus. The genome sequences of these two plants are expected by the end of 2006 (Young et al., 2005). With this as a backdrop, the legume community came together recently to access progress and to set future goals for legume comparative biology (Cross-Legume Advances through Genomics [CATG] Conference, Santa Fe, NM, December 14–15, 2004). A major topic was how to harness the information available in both legume and non-legume models to address needs across a wide variety of legume species. Comparative biology and, more specifically, comparative genomics (Zhu et al., 2005) were the major topics of conversation. Details and specific recommendations of this meeting can be found in this issue (Gepts et al., 2005). One notable outcome of this meeting was a community consensus to select soybean (Glycine max L. Merr.) as the representative species (model) for the phaseolid legumes, which comprise many of the major legume crops, including common bean (Phaseolus vulgaris). As shown by the research articles in this special issue, as well as those in the preceding legume issue (Vol. 131[3], 2003), it is clear that our knowledge of legumes is accelerating in step with other advances in plant biology. However, are there areas in which progress is moving slower than desired? Although molecular biology and genomics have clearly had a major impact on our general understanding of plant mechanisms, they have had significantly less impact on our understanding of crop plants or development of new bioproducts. The actions, such as outlined in the CATG conference report, seek to utilize the basic information gathered from models to investigate crop plants. These are indeed important steps. However, what concerted efforts are being made to translate this information into real benefit for farmers, the agricultural industry (including biotechnology), and consumers (you and me)? Webster's dictionary states that “translation implies the rendering from one language into another,” whereas “genomics” is generally considered the use of high-throughput methods to study both form and function of genomes. Therefore, plant translational genomics implies going from the language of genomics to that of practical application. In the future, metabolic engineering of legume products in cultures of transformed plants or microbes may convert current findings into advances for the pharmaceutical or food science industries. Currently, though, in most cases translation to applications uses the language of plant breeding. Even biotechnology applications require plant breeding in order to move target traits into suitable germplasm. In other words, today translational genomics implies the direct application of genomic resources to make plant breeding programs easier, effective, and more efficient. An examination of legume plant breeding shows a mixed record with a few notable successes in applying legume genomic information through marker-assisted selection. In other cases, little progress is being made in translating information, obtained at considerable public expense, into real utility. However, we note with some optimism the new emphasis by the U.S. Department of Agriculture National Research Initiative to establish Coordinated Agricultural Projects with a specific focus on translational genomics. This and meetings such as the CATG conference should hopefully focus more effort and resources on the “language” gap that exists between basic discovery and practical application in legumes, as well as other important plant groups. We hope that the information found in this special issue will continue to convince young scientists of the wonderful careers that exist in both basic and applied legume biology. LITERATURE CITED Gepts P, Beavis WD, Brummer EC, Shoemaker RC, Stalker HT, Weeden NF, Young ND ( 2005 ) Legumes as a model plant family. Genomics for Food and Feed Report of the Cross-Legume Advances through Genomics Conference. Plant Physiol 137 : 1228 –1235 Young ND, Cannon SB, Sato S, Kim D, Cook DR, Town CD, Roe BA, Tabata S ( 2005 ) Sequencing the genespaces of Medicago truncatula and Lotus japonicus. Plant Physiol 137 : 1174 –1181 Zhu H, Choi H-K, Cook DR, Shoemaker RC ( 2005 ) Bridging model and crop legumes through comparative genomics. Plant Physiol 137 : 1189 –1196 Author notes [email protected] [email protected] © 2005 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Young, Nevin D.; Cannon, Steven B.; Sato, Shusei; Kim, Dongjin; Cook, Douglas R.; Town, Chris D.; Roe, Bruce A.; Tabata, Satoshi

doi: 10.1104/pp.104.057034pmid: 15824279

Two model legumes, Medicago truncatula (Mt) and Lotus japonicus (Lj), are currently targets of large-scale genome sequencing projects. As a result, legumes are one of few plant families with extensive genome sequence in multiple species. The prospect of integrating genome information from Mt and Lj together into a reference for legume genomics will provide exciting opportunities for plant biologists. Because the Mt and Lj sequencing efforts are both clone by clone (as opposed to shotgun or filtered genome sequencing strategies), syntenic comparisons between these two genomes and with other plant taxa will be straightforward and highly informative. Already, the Mt and Lj genome sequences offer novel insights into the organization and evolution of legumes, as well as the similarities and differences with genomes of other plant families, such as Arabidopsis (Arabidopsis thaliana; Zhu et al., 2003) and Populus trichocarpa (G. Tuskan, personal communication). A growing number of researchers are using the Mt and Lj genomes to positionally clone genes of biological importance, especially those involved in symbiosis (Schauser et al., 1999; Endre et al., 2002; Krusell et al., 2002; Nishimura et al., 2002; Stracke et al., 2002; Madsen et al., 2003; Ane et al., 2004; Levy et al., 2004). Increasingly, researchers working in broader aspects of plant biology will find the genome sequences of Mt and Lj essential to their research. In this review, we briefly describe basic features of the Mt and Lj genomes, gleaned from the growing body of genome sequence data. We compare the two genomes through direct sequence comparisons, based on a total of 122 Mb of finished (phase 3) sequence available between the two genomes. These comparisons lay a foundation for integrating knowledge about these two systems and increasing their utility as reference legumes. WHY M. TRUNCATULA AND L. JAPONICUS? Since the 1990s, Mt and Lj have played central roles in symbiosis research (Pichon et al., 1992; Crespi et al., 1994; Kapranov et al., 1997; Schauser et al., 1998). Like Arabidopsis before them, Mt and Lj evolved from systems for studying biological questions into models for genomics and genome sequencing. Both exhibit diploid genetics and modest genome sizes (around 500 Mb each), both are tractable to genetic manipulation, and both have strong international research communities. Recent progress in comparative genomics confirms that genomic discoveries made in these two model species can frequently be extended to other legumes, including most members of the large and agriculturally important Papilionoid subfamily. The decision to focus genome sequencing on reference species played a prominent role in the recent U.S. National Plant Genome Initiative report, published by the U.S. National Academy of Science in 2002 (www.nap.edu/openbook/0309085217/html/). This highly influential report recommended that the plant genomics community concentrate “on a small number of key species for in-depth development of genome-sequence data” (p. 3), and legumes were highlighted for the substantial investment required. The Kazusa DNA Research Institute in Japan had already chosen Lj as its target for legume sequencing with the backing of a large international research community and financial support from the local government of Chiba, Japan. Soon afterward, the University of Oklahoma, with support from the Samuel Roberts Noble Foundation, initiated similar efforts in Mt. This was followed by large-scale support for Mt sequencing by the U.S. National Science Foundation and the European Union 6th Framework Program. At first, sequencing in two model legumes was viewed as a wasteful duplication of effort. Now, it is clear that having two substantially sequenced legume genomes will lead to valuable new discoveries. Both projects decided to pursue similar strategies in their sequencing efforts. Previous research had indicated that most Mt and Lj genes would be found in euchromatic regions throughout chromosome arms and would be largely absent from the heterochromatin of centromeres and pericentromeres (Kulikova et al., 2001; Pedrosa et al., 2002). Thus, a judicious choice of bacterial artificial chromosome (BAC) and P1 transformation-competent artificial chromosome (TAC) clones (Liu et al., 1999) rich in genes would be expected to uncover the majority of genes in these two genomes. These large insert clones could be anchored to extensive map resources available in Mt (mtgenome.ucdavis.edu) and Lj (www.kazusa.or.jp/lotus/), efficiently creating contiguous or near-contiguous assemblies of genome sequence that comprise most of the genes, the so-called “genespace.” The decision to adopt this anchored, clone-by-clone approach to genome sequencing, rather than a whole-genome shotgun (WGS; Venter et al., 1998) or genome filtering method (Palmer et al., 2003; Whitelaw et al., 2003), was considered essential for making the Mt and Lj genomes as useful as possible to the broader community of legume and plant biologists. As of January 2005, approximately 134 Mb of the genome sequence in Mt (77 Mb finished, 57 Mb draft) and 165 Mb of the genome sequence in Lj (45 Mb finished, 120 Mb draft) were publicly available. Analysis of these genome sequences demonstrates the wisdom in adopting a clone-by-clone strategy. Gene density is reasonably high in sequenced clones: 149 genes/Mb (1 gene every 6.7 kb) in Mt and 158 genes/Mb (1 gene per 6.3 kb) in Lj. (These estimates are based on Fgenesh predictions [Salamov and Solovyev, 2000] using a Mt-trained matrix, retaining peptides with a BLASTP match at 10e−4 to the UniProt NREF100 database of peptides [Apweiler et al., 2004]. This estimate for Lj differs from the published value of 1 gene per 10.1 kb [Asamizu et al., 2003a] due to the use here of the Fgenesh gene-calling algorithm so Mt and Lj could be compared directly.) Based on these estimates, gene densities in Mt and Lj are lower than in Arabidopsis by a factor of approximately 1.5 (Arabidopsis Genome Initiative, 2000) but still quite high. While there are repetitive elements on most sequenced clones, there are no cases of BACs or TACs from euchromatic regions without genes or consisting entirely of repeats. There also is no indication that Mt or Lj genes are found in islands separated by extensive stretches of retroelements, as observed in maize (Zea mays; SanMiguel et al., 1996), although the longest contiguous stretches examined so far are just 1 Mb. In the case of Mt, for example, 77 Mb of phase 3 (finished) BACs contains approximately 11,500 genes. Assuming there are 35,000 to 40,000 genes overall, then sequencing a total of 230 to 270 Mb will discover essentially all. A growing body of data suggests that other legume species, even those with a much larger genome, such as soybean (Glycine max), may actually comprise genespaces on par with Mt and Lj (Mudge et al., 2004). GENOME SEQUENCING IN M. TRUNCATULA Mt (2n = 16) is an annual diploid in the tribe Trifolieae, cultivated as a forage crop and closely related to tetraploid alfalfa (Medicago sativa). In the past few years, more than 190,000 Mt expressed sequence tags (ESTs) have been produced (www.medicago.org/MtDB2/ and www.tigr.org/tdb/tgi/plant.shtml), with corresponding microarray and DNA chips now available. There are also 155,000 sequenced BAC ends (ftp.tigr.org/pub/data/m_truncatula) plus detailed physical and genetic maps (mtgenome.ucdavis.edu). Gene knockout systems involving T-DNA and Tnt1 (Scholte et al., 2002; d'Erfurth et al., 2003), RNA interference (Limpens et al., 2004), and gene TILLING (D. Cook, personal communication) are under development. Combined with the rapidly emerging sequence of its genespace, Mt provides an impressive array of genomic tools to legume biologists. Fluorescent in situ hybridization (FISH) has been especially influential in guiding the Mt sequencing effort (Kulikova et al., 2001; Choi et al., 2004a; Kulikova et al., 2004). The pachytene chromosomes of Mt are relatively easy to visualize, and all eight chromosomes can be identified by appearance. Significantly, they all show distinct euchromatic arms and heterochromatic centromeres/pericentromeres, although chromosome 6 is substantially more heterochromatic than the rest. Kulikova et al. (2004) found that each of 20 gene-rich BAC clones hybridized exclusively to euchromatic arms, and subsequent FISH analysis confirmed and extended this initial observation, with 80 gene-rich BACs all localizing to euchromatin (Choi et al., 2004a; R. Guerts, personal communication). This analysis also uncovered a translocation involving chromosomes 4 and 8 between the parents of a widely studied Mt mapping population: A20 and Jemalong, the genotype now being sequenced. In summary, cytogenetic and FISH results provided critical support for the BAC-by-BAC strategy adopted for sequencing. It demonstrated that if BAC clones could be efficiently identified as gene rich (initially through the use of EST-based overgoes), then most of the Mt genespace would be uncovered in the course of BAC-by-BAC sequencing. Genome sequencing began in earnest in 2002 through a collaboration between Bruce Roe at the University of Oklahoma and Doug Cook and Dongjin Kim at the University of California, Davis. This was expanded significantly in 2003 with grants from the National Science Foundation and the European Union (see www.medicago.org/genome/people.php for a complete list of participants). Sequencing is coordinated by an international steering committee, with most of the genespace sequencing scheduled for completion by the end of 2006. Altogether, slightly more than 2,000 BAC clones will be sequenced in the course of the project by the four centers performing the work (Bruce Roe et al., Oklahoma; Chris Town et al., The Institute for Genomic Research [TIGR]; Jane Rogers et al., Sanger Centre; Francis Quétier et al., Genoscope). The most important product of this initiative will be 16 chromosome arm-length sequences, called pseudomolecules after the model of Arabidopsis and rice (Oryza sativa), comprising the complete sequence of each chromosome arm. Realistically, every one of these molecules will still contain gaps, but the gaps will be sized through FISH. The pseudomolecules will extend approximately from telomeres to pericentromeres, and annotation in the form of computer-based predictions of genes and other genomic features will be performed. An international committee known as the International Medicago Genome Annotation Group is coordinating the annotation process and utilizing training sets of Mt gene models fully supported by EST sequence data to train gene prediction algorithms. As of January 2005, sequencing of 1,165 BAC clones, constituting approximately 133 Mb of the Mt genome, was complete or in progress. After accounting for overlap, this represents about 118 Mb of nonredundant sequence. As noted earlier, approximately 11,500 genes have been predicted among finished BAC clones so far. Most Mt BAC clones are anchored to chromosomal locations through the use of microsatellite and other BAC-based markers or by BAC sequence overlap. In this way, 820 of the sequenced BAC clones have been assigned to a specific chromosome and genetic map location. Information about the Mt genome sequence can be accessed through a variety of Web sites. Because of the project's international and collaborative nature, data production, storage, and visualization tools are broadly distributed. These resources include the primary Mt genome sequence portal, www.medicago.org/genome at the University of Minnesota, as well as related sites at the University of Oklahoma (www.genome.ou.edu), TIGR (www.tigr.org/tdb/e2k1/mta1/), and the Munich Information Center for Protein Sequences (mips.gsf.de/proj/plant/jsf/medi/index.jsp). Along with the finger-print-contig-based physical and genetic map Web site (mtgenome.ucdavis.edu), the Mt genome sites provide query and visualization tools for BAC-based sequence assemblies, marker-BAC associations, BAC-sequence browsers showing tentative gene calls, and FTP downloads of large genome sequence datasets. GENOME SEQUENCING IN L. JAPONICUS Lj (2n = 12) is a diploid self-fertile perennial pasture legume. Several mutants in symbiosis and nitrogen fixation have previously been isolated and the underlying genes identified. Insertional mutagenesis and TILLING systems are available (Schauser et al., 1999; Webb et al., 2000; Perry et al., 2003), as are 110,000 ESTs derived from a variety of different organs (Szczyglowski et al., 1997; Endo et al., 2000; Asamizu et al., 2003b). High-density molecular marker maps plus TAC and BAC genomic libraries facilitate gene identification, map-based cloning, and genome sequencing (Hayashi et al., 2001; Sato et al., 2001). Cytogenetic analysis of Lj distinguished all six chromosomes based on patterns of heterochromatic regions (Ito et al., 2000; Hayashi et al., 2001; Pedrosa et al., 2002). FISH analysis integrated the genetic and cytogenetic maps of Lj with BAC and plasmid clones from 32 genome regions (Pedrosa et al., 2002), a process that continues with new seed clones from the genome sequencing project (N. Ohmido, personal communication). Like Mt, a difference in chromosome morphology between the two accessions used for genetic mapping (Gifu B-129 and Miyakojima MG-20) revealed a reciprocal translocation between chromosomes 1 and 2. FISH analysis pinpointed the borders and map location of this translocation using sequenced seed clones as probes. Large-scale genome sequencing of Lj began in 2000 using genotype Miyakojima MG-20. Seed points were chosen along the entire genome based on sequences of ESTs, cDNAs, and gene segments from Lj and other legumes, and corresponding TAC clones were selected for sequencing by PCR. TAC clones were sequenced by shotgun and standard finishing methods, and then gene annotation was performed by a combination of semiautomatic and manual methods. Microsatellite and single nucleotide polymorphism markers generated from genome sequence localized TACs onto the genetic linkage map. As of October 2004, a total of 1,659 clones had been selected for sequencing in Lj and a total of 162 Mb had been sequenced, including clones still in draft (phase 1) stage. In the 44.9 Mb of finished sequence, 4,089 potential protein-encoding genes are predicted (Sato et al., 2001; Nakamura et al., 2002; Asamizu et al., 2003a; Kaneko et al., 2003; Kato et al., 2003). (This estimate increases to 6,500 when Lj genes are predicted by the Mt-trained Fgenesh algorithm described earlier.) Altogether, 1,310 TACs have been placed on the genetic map using 691 microsatellite and 80 cleaved amplified polymorphic sequence markers and by overlaps among sequenced clones. These TAC-based markers and associated sequence information provide enormous value in gene mapping and map-based cloning in Lj and other legumes. A Web-based database (www.kazusa.or.jp/lotus/) supports easy access to Lotus genome information generated through the sequencing project. One can retrieve information on DNA markers, genetic linkage maps, recombinant inbred lines, nucleotide sequences of the chloroplast and TAC clones, annotation of predicted genes, and results of similarity searches. Legume Base (www.shigen.nig.ac.jp/legume/legumebase/) is a materials resource database for Lj and soybean, supported by the Japan National Bioresource Project. Resources such as seeds, recombinant inbred lines, and TAC genomic libraries can be obtained through this Web site. REPEAT SEQUENCES OF M. TRUNCATULA AND L. JAPONICUS An important feature of the Mt and Lj genomes that can be examined with existing sequence data is the diversity and organization of repeat elements. Of course, both sequencing projects have sought to avoid the highly repetitive sequences found in centromeres and pericentromeres, as this is the rationale for the underlying gene-rich BAC/TAC sequencing strategy. Still, a combination of random and clone-by-clone sequencing plus FISH analysis reveals a great deal about the repeat space of these two legume genomes. To survey the Mt genome for repeat sequences, Roe and colleagues carried out a pilot WGS of 25,000 reads early in the genome sequencing effort (Roe and Kupfer, 2004). In addition to assembling the entire Mt chloroplast genome sequence, the WGS displayed several novel Mt-specific repeat families. Altogether, 23% of nonchloroplast reads were repetitive and 25% of these clustered into groups of 50 or more, strongly suggesting they were high copy. Four centromere-associated, short-tandem repeat families were examined in detailed. Altogether, these repeats were found to comprise nearly 10% of the Mt genome. Three of these high copy repeats, MtR1, MtR2, and MtR3, were subsequently characterized by FISH (Kulikova et al., 2004). MtR3 is found in stretches 450 kb to 1 Mb in length within centromeres, whereas MtR1 and MtR2 occupy distinct and diagnostic regions within pericentromeric heterochromatin. In a similar fashion, 37,000 random TAC-end sequences from Lj were characterized and clustered by sequence similarity. Approximately 47% of the TAC ends could be clustered, with 25% of this fraction clustering into high copy repeats. Analyzing consensus sequences for each of these groups revealed five different short tandem repeats, two retroelements, and nine unclassified repeats, including a previously characterized centromere-associated repeat, Ljcen1 (GenBank accession no. AF390569; Pedrosa et al., 2002). Many of these repeats demonstrated characteristic patterns of distribution when examined by FISH. For example, LjRE2 was present only in pericentromeric heterochromatin, LjTR1 was found in chromosome knobs, and LjRE1 was found along the entire lengths of chromosome arms (N. Ohmido, personal communication). While the WGS and random TAC-end approaches enable comparisons of high copy tandem repeats, full-length BAC and TAC sequences provide opportunities to compare intergenic retrotransposons and DNA transposons of Mt and Lj. With this in mind, we carried out a preliminary RepeatMasker (www.repeatmasker.org) analysis of sequenced BACs and TACs to investigate the interspersed repeats in the available genomic sequence. In contrast with the WGS and random TAC-end results described earlier, just 4.7% of the BAC-by-BAC Mt sequence and 5.7% of the TAC-by-TAC Lj sequence could be classified as repetitive in this analysis (though some repeat classes that had previously been observed in Mt and Lj, including SIRE [Laten and Morris, 1993] and MIRE1 [GenBank accession no. AY196987], were not yet represented in the underlying RepBase database [www.girinst.org/]). Since the BAC and TAC clones sequenced so far were chosen because they were gene rich, this low percentage is not surprising. Indeed, just four sequenced Mt BACs and none of the Lj TACs contain centromeric repeats, and one of these Mt BACs had been chosen expressly so that the centromeric element MtR1 could be examined in detail. Based on this preliminary analysis, the Mt and Lj genespaces appear to be quite similar in their retrotransposon and DNA transposon composition. Both contain large numbers of the same family of LINEs (L1/CIN4), the same families of retrotransposons (Ty1/Copia and Gypsy), as well as similar families of DNA transposons. It is also interesting that the distribution of different repeat families is nonuniform among sequenced clones in both Mt and Lj. For example, LjRE1, a Ty1/Copia element, is found on 27% of sequenced Lj TACs, whereas LjRE2, a Gypsy element, is found on just 0.5% of clones, even though LjRE1 is only 4 times more abundant than LjRE2. COMPARATIVE GENOMICS WITH OTHER LEGUMES The most compelling rationale for sequencing genomes of model plant species is the opportunity to extend this information to important crops. A growing number of studies demonstrate macro- and microsynteny among legumes (Menancio-Hautea et al., 1993; Boutin et al., 1995; Simon and Muehlbauer, 1997; Lee et al., 2001; Brauner et al., 2002; Gualtieri et al., 2002; Cannon et al., 2003; Yan et al., 2003, 2004; Choi et al., 2004a, 2004b; Kalo et al., 2004). In a recent study that spanned multiple legume species, macrosynteny between Mt, Lj, and four other legume species was examined in detail (Choi et al., 2004b). The results indicate extensive genome-wide synteny between Mt and Galegoid legumes (such as alfalfa, pea [Pisum sativum], chickpea [Cicer arietinum], and Lj). The high level of macrosynteny between Mt and pea is notable, as the pea genome is roughly 10 times larger than Mt. Long tracts of macrosynteny were also observed between Mt and the Phaseoleae species soybean and mungbean (Vigna radiata), though at levels lower than with Galegoid legumes. These comparisons enabled the construction of a genome-wide picture of legume synteny in the form of concentric circles of corresponding chromosomes anchored by Mt (Choi et al., 2004b), similar to the model previously developed for rice and other grasses (Gale and Devos, 1998). These macrosynteny results complement a growing number of microsynteny studies that describe similarities at the scale of individual BAC clones or clone contigs between legume genomes. Of course, microsynteny between Mt or Lj and crop species like alfalfa and pea has already enabled the positional cloning of symbiosis genes (Endre et al., 2002; Stracke et al., 2002). However, microsynteny with Mt also extends to the more evolutionarily distant soybean. Specifically, two sequenced regions of soybean totaling 1 Mb in length have been compared to the Mt genome sequence, and the extent of colinearity is impressive (Choi et al., 2004b; J. Mudge, personal communication). Altogether, the syntenic regions comprised nearly 500 predicted genes, with 75% of soybean genes colinear with their Mt homologs, including one segment where 33 of 35 soybean genes with hits to the GenBank nonredundant database were colinear with Mt. In fact, microsynteny between Mt and soybean appears to be widespread throughout the genomes. One study compared 50 pairs of Mt and soybean BAC contigs, comprising nearly 10 Mb in each species, and found that 35% of contigs compared through cross-hybridization and physical mapping exhibited substantial microsynteny, with another 20% exhibiting more limited levels of microsynteny (Yan et al., 2003). INTEGRATING THE M. TRUNCATULA AND L. JAPONICUS GENOMES With the growing body of genome sequence for both Mt and Lj, it is clear that detailed comparisons between these two genomes (and also with Arabidopsis and poplar) will reveal exciting new aspects of plant genome organization and evolution. More importantly, detailed comparisons between Mt and Lj will provide a foundation for researchers in other systems to mine these model genomes in a systematic and integrated fashion. Marker-based comparisons between Mt and Lj have already demonstrated substantial macrosynteny (Choi et al., 2004b). This macrosynteny is punctuated by multiple rearrangements involving translocation of chromosomes arms, as the two species have different chromosome numbers (Mt with eight; Lj with six). Individual BAC- and TAC-based comparisons between Mt and Lj also revealed substantial levels of microsynteny (Cannon et al., 2003; Choi et al., 2004b). For example, 10 BAC/TAC clone pairs broadly spaced throughout the two genomes were compared at the sequence level (Choi et al., 2004b). Within these regions, 91 and 84 genes were identified in Mt and Lj, respectively, and 82% of the Mt genes were conserved between the genomes. With just four exceptions, homologs were present in conserved order and orientation. When all currently available phase 2 and 3 Mt and Lj genome sequences are compared, striking large-scale similarities become apparent (Fig. 1 Figure 1. Open in new tabDownload slide Sequence-based synteny between Mt and Lj. Provisional assemblies for all eight of Mt's chromosomes are displayed along side regions of sequence homology in Lj. Mt chromosome assemblies were constructed from sequenced phase 2 and 3 BACs, with order inferred from a combination of genetic map anchors, finger print contigs, and overlapping BAC sequence data. For each BAC along the assembly, the top reciprocal BLAST hits to predicted Mt genes are shown as colored bars for each of the six Lj chromosomes. Broader bars indicate regions of predicted synteny, corresponding to four or more genes with conserved order and orientation in both genomes. The heights of broader bars also indicate the length along Mt chromosome assemblies where synteny is observed. Figure 1. Open in new tabDownload slide Sequence-based synteny between Mt and Lj. Provisional assemblies for all eight of Mt's chromosomes are displayed along side regions of sequence homology in Lj. Mt chromosome assemblies were constructed from sequenced phase 2 and 3 BACs, with order inferred from a combination of genetic map anchors, finger print contigs, and overlapping BAC sequence data. For each BAC along the assembly, the top reciprocal BLAST hits to predicted Mt genes are shown as colored bars for each of the six Lj chromosomes. Broader bars indicate regions of predicted synteny, corresponding to four or more genes with conserved order and orientation in both genomes. The heights of broader bars also indicate the length along Mt chromosome assemblies where synteny is observed. ). These results significantly expand the scope of earlier comparative mapping studies, where macrosynteny was based on segregation analysis of conserved DNA markers and microsynteny was examined one BAC or TAC clone at a time. Since long stretches of anchored genome sequences can now be compared directly, microsynteny can be integrated into the larger picture of macrosynteny, and commonalities in genome organization can be inferred genome wide. This is illustrated in Figure 1, where 71 Mb of anchored Mt sequence is compared to 34 Mb of anchored Lj sequence. In the figure, all top hits of the Lj genome to Mt are shown. When four or more Lj top hits are colinear on the same Mt BAC, a wide colored block running the length of the Mt BAC is shown, with each Lj chromosome assigned a different color. For example, the bottom of Mt chromosome 3 has 11 clustered BACs, each with blocks of 4 or more colinear homologs on Lj chromosome 1. Altogether, 101 Mt BACs spanning approximately 10 Mb were microsyntenic with a comparable portion of the Lj genome in this analysis. The many genome segments that fail to exhibit conservation in this study might represent nonsyntenic regions. However, segments that appear to lack synteny are more likely to be cases where corresponding genome regions have not yet been sequenced in one or the other genome sequencing project. Even with all the sequencing that has been accomplished so far, finished and anchored BACs cover just 28% of the Mt genespace, whereas finished and anchored TACs cover just 13% of Lj. For this reason, sequence-based comparisons would be expected to discover just 4% of all potential overlap at this level of genome coverage, assuming unbiased distribution of sequences across the two genespaces. Even with the relatively limited genome sequence available, it is clear that Mt chromosome 1 shows modest synteny with Lj chromosome 5 (gold); Mt-2 is largely syntenic with Lj chromosomes 3 and 6 (green and purple, respectively); Mt-3 with Lj-1; Mt-4 with Lj-3 and Lj-4; Mt-5 with Lj-2; Mt-6 with Lj-2; Mt-7 with Lj-1; and Mt-8 with Lj-4. Mt chromosome 6, which exhibits the lowest synteny with Lj, is unusual in its high proportion of heterochromatin (Kulikova et al., 2004), lack of marker-based synteny with pea (Choi et al., 2004b), and abundance of nucleotide-binding site-Leu-rich repeat genes (Zhu et al., 2002). If all putative cases of synteny are considered (observed syntenic blocks plus nonsyntenic regions flanked on both sides by syntenic blocks), then more than 75% of the Mt and Lj genespaces are conserved. Not surprisingly, these genome sequence-based relationships mirror and extend marker-based synteny predictions made previously (Choi et al., 2004b). PERSPECTIVES In the future, genome-scale comparisons will become increasingly informative as genome sequencing of Mt and Lj nears completion. These comparisons will reveal the detailed processes that shaped the evolution of these two legume genomes and provide increasingly detailed insights into plant genome evolution and organization. Moreover, by viewing genome information from Mt and Lj in an integrated manner, researchers working in other species will find a much richer resource than would have been available with just one. 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Funding for Lotus japonicus sequencing comes from the Kazusa DNA Research Institute Foundation. * Corresponding author; e-mail [email protected]; fax 612–625–9728. www.plantphysiol.org/cgi/doi/10.1104/pp.104.057034. © 2005 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Davis, Eric L.; Mitchum, Melissa G.

doi: 10.1104/pp.104.054973pmid: 15824280

From an agronomic perspective, the interactions of phytoparasitic nematodes with leguminous crops can be devastating (Barker, 1998). On a cellular and molecular level, the complexities of the interactions of these microscopic worms with legumes are comparable to those of well-known symbionts (Mathesius, 2003). Because of their agricultural importance, the soybean cyst nematode Heterodera glycines and species of root-knot nematodes (Meloidogyne spp.) that infect soybean (Glycine max) and other legumes have emerged as primary research models to understand the signaling, perception, and response events during plant-nematode interactions (Davis et al., 2004). The recent advances in our understanding of the cellular, physiological, and molecular basis of legume-nematode interactions are merging at the crossroads of plant-microbe interactions and plant developmental biology. All plant-parasitic nematodes have evolved a hollow, protrusible mouth spear called a stylet that is used to inject secretions into host tissues and pierce plant cell walls to withdraw nutrients (Hussey and Grundler, 1998). Some phytonematode species feed upon and quickly destroy plant cells as migratory parasites, whereas other nematode species become sedentary in later life stages and must modify plant cells to provide a sustained local source of nutrition for parasitic success. Although species within a few nematode genera, such as Ditylenchus and Aphelenchoides, plague the shoot tissues of cultivated legumes such as alfalfa (Medicago sativa), the majority of phytonematode species are parasites of plant roots (Hussey and Grundler, 1998). The root-knot nematodes and cyst nematodes (Heterodera and Globodera spp.) are sedentary endoparasites of plant roots and the primary nematode pathogens of most crop species worldwide, including many cultivated legumes (Barker, 1998). The major species of Meloidogyne have a wide host range that includes at least 1,700 plant species (Barker, 1998). By contrast, individual species of cyst nematodes are relatively host specific, such as the soybean cyst nematode H. glycines that infects relatively few plant species beyond soybean (Hussey and Grundler, 1998). As members of the same family (Heteroderidae), however, both root-knot and cyst nematodes share similar parasitic habits, most notably the induction of elaborate modifications of selected plant root cells into complex feeding sites (Fig. 1 Figure 1. Open in new tabDownload slide Cross sections of feeding cells induced in plant roots by sedentary endoparasitic nematodes. A, Multinucleate giant cells (GC) induced by the root-knot nematode Meloidogyne incognita, derived from karyokinesis uncoupled from cytokinesis of plant cells adjacent to the nematode (N) head. B, A multinucleate syncytium (S) induced adjacent to the head of the cyst nematode (N) H. glycines, formed by dissolution of cell walls to incorporate neighboring plant cells into the feeding site (photo courtesy of Burton Y. Endo, U.S. Department of Agriculture Agricultural Research Service, Beltsville, MD). Reprinted from Davis et al. (2004) with permission from Elsevier. Figure 1. Open in new tabDownload slide Cross sections of feeding cells induced in plant roots by sedentary endoparasitic nematodes. A, Multinucleate giant cells (GC) induced by the root-knot nematode Meloidogyne incognita, derived from karyokinesis uncoupled from cytokinesis of plant cells adjacent to the nematode (N) head. B, A multinucleate syncytium (S) induced adjacent to the head of the cyst nematode (N) H. glycines, formed by dissolution of cell walls to incorporate neighboring plant cells into the feeding site (photo courtesy of Burton Y. Endo, U.S. Department of Agriculture Agricultural Research Service, Beltsville, MD). Reprinted from Davis et al. (2004) with permission from Elsevier. ). MUTUALISM AND PARASITISM The conspicuous galls (knots) formed on plant roots by species of Meloidogyne draw immediate comparisons to root nodules formed on legumes by rhizobia (Mathesius, 2003; Bird, 2004). Mycorrhizae and cyst nematodes (Hussey and Grundler, 1998; Parniske, 2004) also form complex cellular relationships within plant root tissues, but neither biotroph forms galls on roots. What are the molecular mechanisms and developmental pathways in the plant that ultimately determine the different outcomes of these interactions, and do they share any common features? Is there a fine line between mutualism and parasitism, or are they distinct phenomena? These questions provide a central theme for this treatise as we explore the interaction of nematodes with legumes. An overview of the parasitic relationship of root-knot and cyst nematodes with their plant hosts allows us to put these interactions into perspective. The infective (preparasitic) stage of root-knot and cyst nematodes is the motile second-stage juvenile (J2) that hatches from the egg in soil and penetrates the root directly behind the tip at the zone of elongation (Hussey and Grundler, 1998). The parasitic J2 of cyst nematodes migrate through root cortical cells (intracellularly) using thrusts of their stylet to breach cell walls, while the J2 of root-knot nematodes migrate between root cortical cells (intercellularly), to reach the root vascular cylinder. The esophageal gland cells of both nematodes actively synthesize and mobilize secretions from the stylet during migration within tissues and subsequent formation of feeding cells (Hussey, 1989). Parasitic J2 of root-knot nematodes induce the formation of three to six multinucleate giant cells (Fig. 1A) from individual root xylem parenchyma cells surrounding the nematode head that serve as the feeding site for the progression of swollen nematode sedentary stages to reproductive adult stages. Giant cells form from repeated nuclear division without cellular division, giving rise to multinucleate, hypertrophied feeding cells up to 100 times the size of normal root vascular parenchyma cells. In many but not all hosts, pericycle and cortical root cells immediately surrounding the giant cells are stimulated to divide (hyperplasia), giving rise to the gall characteristic of root-knot nematode infection. Parasitic J2 of cyst nematodes select a single root vascular parenchyma or pericycle cell to induce an initial syncytial cell. Enzymes of plant origin (Goellner et al., 2001) promote coordinated dissolution of cell walls neighboring the initial syncytial cell and subsequently give rise to a multinucleate feeding site for the cyst nematode called a syncytium (Fig. 1B). The syncytium continues to incorporate up to several hundred cells along the root vasculature as the nematode begins to feed and swells to a rounded, sedentary state through a series of molts to become a reproductive adult. Although the ontogeny of syncytia and giant cells differ fundamentally, these nematode-induced feeding cells do share many common features similar to natural plant transfer cells (Offler et al., 2003), including multiple lobed nuclei, dense cytoplasm with increased metabolic activity, and thickening and numerous ingrowths of the peripheral cell walls to increase solute transport and structural integrity. Similar modifications of plant cell walls and plasma membranes to accommodate solute transport to either colonizing rhizobia or vesicular arbuscular mycorrhizae are common features of symbioses (Parniske, 2004). Both phytoparasitic nematodes and rhizobia induce the expression of host plant cell wall-modifying enzymes at the infection site, but, interestingly, induction of this endogenous mechanism of plant cell wall modification has not been detected during mycorrhizal infection of roots (Goellner et al., 2001; Gheysen and Fenoll, 2002; Parniske, 2004). The localized hypertrophy and hyperplasia of plant cells induced during infection by rhizobia, Agrobacterium, and even the clubroot root fungus Plasmodiophora produce tissue swellings similar to the galls of root-knot nematodes (Agrios, 1997), but the giant cells induced within galls by Meloidogyne are distinctly multinucleate. Studies may need to distinguish between the formation of essential feeding cells versus associated gall tissues when drawing comparisons to Rhizobium nodules since giant cells can support normal root-knot nematode growth and development in the absence of surrounding galls (Webster, 1969). In total, the giant cells and syncytia formed by root-knot and cyst nematodes, respectively, are unique plant host modifications induced by these parasitic nematodes. The parasitic J2 of both cyst and root-knot nematodes are absolutely dependent on feeding site formation to progress through subsequent parasitic stages, and, conversely, the integrity of the feeding site is dependent on the presence of the nematode. The feeding sites serve as a nutrient sink for the parasitic needs of the nematodes resulting in disease of the host. This interaction is in stark contrast with the mutual benefits derived by both the microbe and plant during mycorrhizal colonization of plant roots or nodulation of legume roots. NEMATODE PARASITISM GENES Proteinaceous stylet secretions from nematodes that are synthesized in the esophageal gland cells are considered as primary signaling molecules at the plant-nematode interface because the morphology, contents, and activity of the gland cells change in relation to nematode migration within plant tissues, feeding cell formation, and nematode feeding activity (Hussey, 1989; Davis et al., 2004). The genes encoding these secretions have been termed parasitism genes (Davis et al., 2000), and a number of different research strategies have identified multiple parasitism genes encoding secreted products in several nematode species (for review, see Jasmer et al., 2003; Davis et al., 2004). The first phytonematode parasitism genes identified encoded cellulases (endoglucanases) synthesized in the esophageal gland cells of cyst nematodes (Smant et al., 1998) that were expressed and secreted only during nematode migration within roots (Davis et al., 2004). These were the first endogenous endoglucanase genes cloned from an animal, and phylogenetic analyses (Yan et al., 1998) indicated strong similarity to cellulase genes of soil bacteria, suggesting the potential for ancient horizontal gene transfer as a mechanism of gene acquisition in nematodes (Smant et al., 1998; Davis et al., 2000). A number of nematode parasitism genes encoding other cell wall-modifying proteins, including the first non-plant expansin (Qin et al., 2004), have since been identified that are expressed in the esophageal gland cells during nematode migration in plant tissues (Jasmer et al., 2003). Beyond cell wall modifications, phytoparasitic nematodes appear to be armed with a suite of stylet secretions (Fig. 2 Figure 2. Open in new tabDownload slide A model of potential interactions of secreted products of phytonematode parasitism genes with host plant cells. Nematode esophageal gland cell secretions are released through valves within ampulla for transport out of the stylet (feeding spear) into host tissues. Cell wall (CW)-modifying proteins (endoglucanases, pectolytic enzymes, xylanases, and expansins) may be secreted to aid the migration of infective juveniles through host plant tissues. Other nematode gland cell secretions might have multiple roles in the formation of specialized feeding cells by the nematode, including effects on host cell metabolism by secreted CM; signaling by secreted nematode peptides, such as homologs to plant CLE peptides; selective degradation of host proteins through the ubiquitin (UBQ)-proteosome pathway by ubiquitin, Skp-1, and RING-H2 secreted from the nematode; and potential effects of secreted nematode proteins that contain NLS within the host cell nucleus. Figure designed by Bill Baverstock (North Carolina State University Creative Services). Reprinted from Davis et al. (2004) with permission from Elsevier. Figure 2. Open in new tabDownload slide A model of potential interactions of secreted products of phytonematode parasitism genes with host plant cells. Nematode esophageal gland cell secretions are released through valves within ampulla for transport out of the stylet (feeding spear) into host tissues. Cell wall (CW)-modifying proteins (endoglucanases, pectolytic enzymes, xylanases, and expansins) may be secreted to aid the migration of infective juveniles through host plant tissues. Other nematode gland cell secretions might have multiple roles in the formation of specialized feeding cells by the nematode, including effects on host cell metabolism by secreted CM; signaling by secreted nematode peptides, such as homologs to plant CLE peptides; selective degradation of host proteins through the ubiquitin (UBQ)-proteosome pathway by ubiquitin, Skp-1, and RING-H2 secreted from the nematode; and potential effects of secreted nematode proteins that contain NLS within the host cell nucleus. Figure designed by Bill Baverstock (North Carolina State University Creative Services). Reprinted from Davis et al. (2004) with permission from Elsevier. ) to modulate many of the features observed in nematode feeding cells (Davis et al., 2004). Genes encoding secreted chorismate mutase (CM) that are most similar to bacterial CM have been isolated from root-knot and soybean cyst nematodes (Doyle and Lambert, 2003). CM is a pivotal enzyme in the shikimic acid pathway that modulates synthesis of Phe and Tyr, having pleiotropic effects on cellular metabolism, auxin synthesis, and as precursors of plant defense compounds. Expression of nematode CM in soybean tissues affected vascular tissue differentiation and was indirectly related to local indole-3-acetic acid concentrations and cellular partitioning of chorismate (Doyle and Lambert, 2003). Another group of candidate secreted nematode parasitism gene products that may also augment host cellular metabolism includes members of the proteasome (Skp-1, RING-H2, and ubiquitin extension protein) with significant similarity to plant genes involved in selective host cell protein degradation (Gao et al., 2003; Tytgat et al., 2004). If secreted into plant cells by nematodes, mimics of the plant proteasome may regulate host cell phenotype at the protein level to promote a compatible plant-nematode interaction. Two other intriguing observations have been made among putative parasitism genes isolated from H. glycines. More than 25% of the predicted nematode parasitism proteins encode putative nuclear localization signals (NLS; Gao et al., 2003), some of which contain DNA-binding motifs, suggesting that the secreted nematode products could be targeted to interact directly within the recipient host cell nucleus. Transient expression of some of the H. glycines NLS parasitism proteins as green fluorescent protein/β-glucuronidase fusion proteins in onion (Allium cepa) epidermal cells has localized the nematode gene products within the plant cell nucleus (A. Elling and T.J. Baum, personal communication). The most abundantly expressed candidate parasitism gene in H. glycines (Gao et al., 2003) was first isolated as clone HG-SYV46 (Wang et al., 2001) through secretion signal-peptide selection of an esophageal gland cell cDNA library. Computational analyses predicted that the C-terminal domain of HG-SYV46 is related to members of the CLAVATA3-ESR-like (CLE) family of signaling proteins in Arabidopsis (Arabidopsis thaliana; Olsen and Skriver, 2003) involved in controlling the balance between shoot meristem cell proliferation and differentiation by interacting with the CLAVATA1 (CLV1)/CLAVATA2 (CLV2) receptor complex (Brand et al., 2000). WUSCHEL is a homeodomain transcription factor (Mayer et al., 1998) that acts antagonistically to the CLV pathway to promote stem cell formation and maintenance (Brand et al., 2000). Remarkably, a WUSCHEL phenotype that includes premature termination of the shoot apical meristem and development of flowers lacking the central gynoecium (Laux et al., 1996) is observed when HG-SYV46 is expressed at high levels in wild-type Arabidopsis (Wang et al., 2005), similar to overexpression of CLV3 (Brand et al., 2000). In addition, expression of HG-SYV46 in a clv3 mutant in which the mutant plants have enlarged shoot and floral meristems with extra floral organs in each whorl (Clark et al., 1995) restored the wild-type phenotype (Wang et al., 2005). Although it is unclear if secreted nematode CLEs promote parasitism in roots via similar CLV1-like receptor-mediated cell differentiation as proposed for plant root CLEs (Casamitjana-Martinez et al., 2003), several models have been proposed (Wang et al., 2005). One possibility is that the secreted HG-SYV46 from the nematode is a CLE mimic that functions as one component of a pathway to redirect and maintain the differentiation of root vascular cells into elaborate feeding cells, potentially via a CLV1-like receptor complex. Alternatively, HG-SYV46 may function through competitive inhibition of a similar endogenous root-expressed CLE ligand for a host CLV1-like receptor in the roots to augment normal root vascular cell redifferentiation into feeding cells (Wang et al., 2005). Interestingly, the Lotus japonicus HAR1 gene encodes a Leu-rich repeat (LRR) receptor-like kinase with the highest level of similarity to the Arabidopsis CLAVATA1 gene (Krusell et al., 2002; Nishimura et al., 2002). The har-1 mutant displays a hypernodulating phenotype and was recently reported to be hyperinfected by root-knot nematodes (Lohar and Bird, 2003), implicating the involvement of the CLAVATA pathway in both rhizobia and nematode-plant interactions. In addition to potential nematode-secreted CLE ligand mimics, a NodL ortholog was recently identified among expressed sequence tags of the root-knot nematode (McCarter et al., 2003), and initial evidence suggests that signals from infective juveniles of root-knot nematodes can have similar effects as Nod factor on developing root hairs of the legume model L. japonicus (Weerasinghe et al., 2005). The combined evidence suggests that phytoparasitic nematodes may have evolved an extraordinary ability to modulate cellular processes of their host plants to promote parasitism. PLANT RESPONSE DURING NEMATODE PARASITISM The morphology of syncytia and giant cells is similar among plant hosts, including legumes, suggesting that fundamental mechanisms of plant cell development are manipulated during feeding cell ontogeny across diverse plant species. The limited host range of most cyst nematode species contrasts with the observed similarity in syncytial morphology among hosts, suggesting coevolutionary selection for an intrinsic level of specificity in cyst nematode-plant signaling events. Do phytoparasitic nematodes tap, directly or indirectly, into host developmental pathways similar to those recruited by other microbial symbionts of legumes? Evidence above suggests that the CLE/CLV1 developmental pathway may be a common mechanism exploited by both cyst nematodes and rhizobia during infection of roots. Orthologs of the PHAN and KNOX genes involved in transcript regulation and meristem maintenance in tomato (Lycopersicon esculentum), respectively, are expressed in giant cells and developing nodules in roots of the model legume Medicago truncatula (Koltai et al., 2001). Likewise, the early nodulation mitogen ENOD40 and the cell cycle regulator ccs52 active in nodules are also stimulated in giant cells and galls induced in M. truncatula by root-knot nematodes (Koltai et al., 2001; Favery et al., 2002). ENOD40 is involved in both the initiation and stimulation of cortical cell division for nodule formation and may play a similar role in stimulating the proliferation of cells around giant cells for gall formation. In nodules, CCS52 functions as a cell cycle regulator that promotes endoreduplication and cell enlargement in the nondividing submeristematic cell layers of zone II (Cebolla et al., 1999), two major characteristics of nematode-induced giant cells. By contrast, expression of the auxin-regulated cell cycle A2-type cyclin gene (cycA2;2) of M. truncatula, shown to play a role exclusively in the mitotic cycles and not expressed during cell differentiation coupled to endoreduplication, was suppressed in the endoreduplicating, nondividing cells of developing nodules and nematode-induced giant cells (Roudier et al., 2003). Further analysis of 192 nodule-expressed genes of M. truncatula revealed only two additional genes induced in both nodules and galls (Favery et al., 2002), suggesting that the morphological divergence in symbiotic phenotype is reflected at the molecular level. Like M. truncatula, L. japonicus has potential as a model legume host for root-knot nematodes (Lohar and Bird, 2003), and, most recently, induction of a cytokinin-responsive ARR5 gene promoter::β-glucuronidase transgene has been observed in the early stages of both nodule and giant-cell formation in L. japonicus (Lohar et al., 2004). Although the direct or indirect effects of observed phytohormone accumulation associated with nematode feeding sites is still unclear, perturbation of local concentrations of cytokinin (Lohar et al., 2004) and auxin (Goverse et al., 2000) suppress successful root-knot and cyst nematode infection, respectively. Auxin accumulation around giant cells was suggested to trigger gall formation during root-knot infection of white clover (Trifolium repens; Hutangura et al., 1999) and, as with H. glycines infection of soybean roots (Kennedy et al., 1999), was associated with an increase in isoflavonoid expression. A similar accumulation of auxin and induction of the flavonoid pathway has been observed during the formation of Rhizobium nodules (Mathesius et al., 1998). RESISTANCE VERSUS SUSCEPTIBILITY How does a symbiont induce such dramatic modifications in plant cells and get away with it (without invoking host defense)? This is especially true for intimate parasites such as nematodes, where there is sustained irritation with no “return on investment” for the host. As intimated above and by others (Parniske, 2004), active suppression of host defense may be required of the invader to maintain the interaction. Although unclear at present, the potential manipulation of the host proteasome via nematode secretions may be targeted, in part, specifically toward suppression of the host defense response (Davis et al., 2004). Similar manipulation of host defense may occur at the level of synthesis of defense compounds, perhaps via augmentation of subcellular chorismate levels (Doyle and Lambert, 2003). Conversely, the potential ability of nematodes to mimic signals in natural plant pathways may provide a measure of stealth that does not invoke host defense unless in the presence of an appropriate receptor. Evidence from many plant-microbe systems supports the model that (avirulence) variants of effector molecules from a microbe function as pathogenicity factors when not in the presence of an appropriate resistance gene (van't Slot and Knogge, 2002). Molecular comparisons of near-isogenic lines of the root-knot nematode that vary in virulence on tomato containing the Mi resistance gene have identified a number of secreted gene products that are differentially expressed between the avirulent and virulent strains (Neveu et al., 2003). Genes encoding peptides that contain conserved motifs of other pathogen avirulence products (Bos et al., 2003), including conserved Cys residues for potential three-dimensional architecture, are represented among the candidate parasitism genes of H. glycines (Gao et al., 2003). Most cloned plant resistance genes against nematode pathogens encode proteins containing nucleotide-binding site and LRR domains similar to those against other microbes (Williamson, 1999). Recently, candidate soybean genes at two loci, Rhg1 and Rhg4, which may condition resistance to H. glycines, were cloned (Lightfoot and Meksem, 2002). Both genes encode Xa21-like receptor kinases with an extracellular LRR domain, a transmembrane domain, and a protein kinase domain (Lightfoot and Meksem, 2002). The considerable variability in response to biotypes of H. glycines and the fact that genomic regions surrounding these loci in soybean are rich with nucleotide-binding site-LRR genes and other genes involved in plant-microbe interactions (Ghassemi and Gresshoff, 1998) suggests a mechanism for receptor variability among plant genotypes. It is tempting to consider the potential specificity of variants of effectors, such as a secreted CLE of H. glycines binding to variants of a CLV1-like or a Xa21-like receptor complex in the host either to promote parasitism or activate defense when in the appropriate combination. THE FUTURE IS NOW Will genomics play a major role in unraveling the differences and similarities among symbioses (mutualism and parasitism) in legumes? The host specificity observed in global plant gene expression to successful infection by Heterodera schachtii versus nonhost response to H. glycines has demonstrated the utility of Arabidopsis microarrays (Puthoff et al., 2003) to dissect plant-nematode interactions. Several microarray platforms are now available to the legume research community and may be combined with laser capture microdissection of nematode feeding sites (Ramsay et al., 2004) to greatly refine and expand analyses of nematode parasitism of legumes. Information on the organization and structure of legume genomes is keeping pace with expressed sequence discovery, and observed syntenic relationships among legume genomes (Young et al., 2003) should allow extrapolation to species of agronomic importance, such as soybean (until their genome sequences are generated). Development of robust functional genomic analyses of legume-nematode interactions will be of primary importance in the post-genomics era as potential gene targets are discovered. As described above, the utility of mutants in model plants such as Arabidopsis, and the legumes Medicago and Lotus, is beginning to be realized for this purpose. Natural variation in Medicago and Lotus is also being explored to map resistance to root-knot nematodes and to identify a potential model legume-cyst nematode pathosystem (M. Dhandayham and C. Opperman, personal communication). 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Zhu, Hongyan; Choi, Hong-Kyu; Cook, Douglas R.; Shoemaker, Randy C.

doi: 10.1104/pp.104.058891pmid: 15824281

The Fabaceae, or legumes, constitute the third largest family of flowering plants, comprising more than 650 genera and 18,000 species (Polhill and Raven, 1981). Economically, legumes represent the second most important family of crop plants after Poaceae (grass family), accounting for approximately 27% of the world's crop production (Graham and Vance, 2003). On a worldwide basis, legumes contribute about one-third of humankind's protein intake, while also serving as an important source of fodder and forage for animals and of edible and industrial oils. One of the most important attributes of legumes is their unique capacity for symbiotic nitrogen fixation, underlying their importance as a source of nitrogen in both natural and agricultural ecosystems. Legumes also accumulate natural products (secondary metabolites) such as isoflavonoids that are beneficial to human health through anticancer and other health-promoting activities (Dixon and Sumner, 2003). The legumes are highly diverse and can be divided into three subfamilies: Mimosoideae, Caesalpinioideae, and Papilionoideae (Doyle and Luckow, 2003). Of these, the Papilionoideae subfamily contains nearly all economically important crop legumes, including soybean (Glycine max), peanut (Arachis hypogaea), mungbean (Vigna radiata), chickpea (Cicer arietinum), lentil (Lens culinaris), common bean (Phaseolus vulgaris), pea (Pisum sativum), and alfalfa (Medicago sativa). With the notable exception of peanut, all these important crop legumes fall into two Papilionoid clades, namely, Galegoid and Phaseoloid, which are often referred to as cool season and tropical season legumes, respectively (Fig. 1 Figure 1. Open in new tabDownload slide Dendrogram depicting phylogenetic relationships of Papilionoideae legumes. (Figure reprinted from Choi et al. [2004b], based on figure 5 of Doyle and Luckow [2003].) Figure 1. Open in new tabDownload slide Dendrogram depicting phylogenetic relationships of Papilionoideae legumes. (Figure reprinted from Choi et al. [2004b], based on figure 5 of Doyle and Luckow [2003].) ). Despite their close phylogenetic relationships, crop legumes differ greatly in their genome size, base chromosome number, ploidy level, and self-compatibility (Table I Table I. Chromosome number and genome size of major model and crop legumesa Tribe . Genus . Species . Chromosome No. . Genome Size . Self-Compatibility . Mb/1C Trifolieae Medicago M. truncatula (barrel medic) 2n = 2x = 16 466 Selfing Alfalfa 2n = 4x = 32 1,715 Outcrossing Trifolium Trifolium pretense (red clover) 2n = 2x = 14 637 Outcrossing Trifolium repens (white clover) 2n = 4x = 32 956 Outcrossing Melilotus Melilotus officinalis (sweet clover) 2n = 2x = 16 1,103 Outcrossing Viceae Pisum Garden pea 2n = 2x = 14 4,337 Selfing Vicia Vicia faba (faba bean) 2n = 2x = 12 13,059 Selfing Lens Lentil 2n = 2x = 14 4,116 Selfing Cicereae Cicer Chickpea 2n = 2x = 16 931 Selfing Loteae Lotus L. japonicus 2n = 2x = 16 466 Selfing Phaseoleae Phaseolus Common bean 2n = 2x = 22 588 Selfing Vigna Mungbean 2n = 2x = 22 515 Selfing Glycine Soybean 2n = 4x = 40 1103 Selfing Cajanus Cajanus cajan (pigeon pea) 2n = 2x = 22 858 Selfing Tribe . Genus . Species . Chromosome No. . Genome Size . Self-Compatibility . Mb/1C Trifolieae Medicago M. truncatula (barrel medic) 2n = 2x = 16 466 Selfing Alfalfa 2n = 4x = 32 1,715 Outcrossing Trifolium Trifolium pretense (red clover) 2n = 2x = 14 637 Outcrossing Trifolium repens (white clover) 2n = 4x = 32 956 Outcrossing Melilotus Melilotus officinalis (sweet clover) 2n = 2x = 16 1,103 Outcrossing Viceae Pisum Garden pea 2n = 2x = 14 4,337 Selfing Vicia Vicia faba (faba bean) 2n = 2x = 12 13,059 Selfing Lens Lentil 2n = 2x = 14 4,116 Selfing Cicereae Cicer Chickpea 2n = 2x = 16 931 Selfing Loteae Lotus L. japonicus 2n = 2x = 16 466 Selfing Phaseoleae Phaseolus Common bean 2n = 2x = 22 588 Selfing Vigna Mungbean 2n = 2x = 22 515 Selfing Glycine Soybean 2n = 4x = 40 1103 Selfing Cajanus Cajanus cajan (pigeon pea) 2n = 2x = 22 858 Selfing a Data were from the Plant DNA C-values Database (http://www.rbgkew.org.uk/cval/homepage.html). Open in new tab Table I. Chromosome number and genome size of major model and crop legumesa Tribe . Genus . Species . Chromosome No. . Genome Size . Self-Compatibility . Mb/1C Trifolieae Medicago M. truncatula (barrel medic) 2n = 2x = 16 466 Selfing Alfalfa 2n = 4x = 32 1,715 Outcrossing Trifolium Trifolium pretense (red clover) 2n = 2x = 14 637 Outcrossing Trifolium repens (white clover) 2n = 4x = 32 956 Outcrossing Melilotus Melilotus officinalis (sweet clover) 2n = 2x = 16 1,103 Outcrossing Viceae Pisum Garden pea 2n = 2x = 14 4,337 Selfing Vicia Vicia faba (faba bean) 2n = 2x = 12 13,059 Selfing Lens Lentil 2n = 2x = 14 4,116 Selfing Cicereae Cicer Chickpea 2n = 2x = 16 931 Selfing Loteae Lotus L. japonicus 2n = 2x = 16 466 Selfing Phaseoleae Phaseolus Common bean 2n = 2x = 22 588 Selfing Vigna Mungbean 2n = 2x = 22 515 Selfing Glycine Soybean 2n = 4x = 40 1103 Selfing Cajanus Cajanus cajan (pigeon pea) 2n = 2x = 22 858 Selfing Tribe . Genus . Species . Chromosome No. . Genome Size . Self-Compatibility . Mb/1C Trifolieae Medicago M. truncatula (barrel medic) 2n = 2x = 16 466 Selfing Alfalfa 2n = 4x = 32 1,715 Outcrossing Trifolium Trifolium pretense (red clover) 2n = 2x = 14 637 Outcrossing Trifolium repens (white clover) 2n = 4x = 32 956 Outcrossing Melilotus Melilotus officinalis (sweet clover) 2n = 2x = 16 1,103 Outcrossing Viceae Pisum Garden pea 2n = 2x = 14 4,337 Selfing Vicia Vicia faba (faba bean) 2n = 2x = 12 13,059 Selfing Lens Lentil 2n = 2x = 14 4,116 Selfing Cicereae Cicer Chickpea 2n = 2x = 16 931 Selfing Loteae Lotus L. japonicus 2n = 2x = 16 466 Selfing Phaseoleae Phaseolus Common bean 2n = 2x = 22 588 Selfing Vigna Mungbean 2n = 2x = 22 515 Selfing Glycine Soybean 2n = 4x = 40 1103 Selfing Cajanus Cajanus cajan (pigeon pea) 2n = 2x = 22 858 Selfing a Data were from the Plant DNA C-values Database (http://www.rbgkew.org.uk/cval/homepage.html). Open in new tab ). Nevertheless, earlier studies indicated that members of the Papilionoideae subfamily exhibited extensive genome conservation based on comparative genetic mapping (Weeden et al., 1992; Menancio-Hautea et al., 1993). To establish a unified genetic system for legumes, two legume species in the Galegoid clade, Medicago truncatula and Lotus japonicus, which belong to the tribes Trifolieae and Loteae, respectively, were selected as model systems for studying legume genomics and biology (Cook, 1999; Stougaard, 2001). Unlike many of the major crop legumes, M. truncatula and L. japonicus are of small genome size, amenable to forward and reverse genetic analyses, and well suited for studying biological issues important to the related crop legume species. An immediate goal of legume genomics is to transfer knowledge between model and crop legumes. Accordingly, an in-depth understanding of conservation of genome structure among legume species is a prerequisite to achieving this goal. The idea that conserved genome structure can facilitate transfer of knowledge among related plant species is best addressed in grasses in which genome macrosynteny and microsynteny have been extensively maintained (Bennetzen, 2000; Devos and Gale, 2000). These studies, however, also revealed many exceptions to the conserved synteny, with frequent local genic rearrangements including gene inversion, duplication, translocation, and insertion/deletion. Although the degeneracy of local genome microstructure has been widely documented, it is less clear the extent to which such alterations to genome microstructure contribute to the divergence of genome function. In this review, we focus on recent results of comparative genome analysis between model and crop legumes, and also highlight the recent successes of using comparative genomics tools for cross-species gene isolation. DUPLICATIONS THAT SHAPE THE LEGUME GENOMES It has been estimated that upwards of 80% of all angiosperms are likely to have a polyploid origin (Masterson, 1994). It is unlikely that legumes are an exception to this. Soybean, for example, has long been known to be an ancient polyploid with putative homoeologous chromosomal regions readily identified by genetic mapping (Shoemaker et al., 1996; Lee et al., 1999, 2001) and by characterization of homoeologous bacterial artificial chromosome (BAC) clones (Foster-Hartnett et al., 2002; Yan et al., 2003). And recently, segmental duplications within the soybean genome were visualized by fluorescence in situ hybridization of BACs (Pagel et al., 2004). Segmental duplications also were identified in the M. truncatula and L. japonicus genomes through high-throughput genome sequencing (Zhu et al., 2003; N. Young, personal communication). Extensive expressed sequence tag (EST) collections exist for two legumes representing distinct phylogenetic clades, soybean (tropical legumes) and M. truncatula (cool season legumes). Much of a genome's evolutionary history can be read in these transcripts. Schlueter et al. (2004) identified duplicate transcripts from the EST collections of both of these legumes and estimated genetic distances of the pairs using synonymous substitution measurements. It was estimated that soybean probably underwent two major genome duplications events: one at 15 million years ago (MYA) and another at 44 MYA. A genome duplication event also was estimated to have occurred in M. truncatula at approximately 58 MYA. A subsequent analysis using a multigene approach concluded that the more ancient duplication events probably represent a single event that occurred before soybean and Medicago diverged (Pfeil et al., 2005). If this is true, then approximately 7,000 other legumes share the same genome duplication event (Pfeil et al., 2005). Genome duplications often are followed by gene loss, rearrangements, tandem gene or segmental duplications, and divergence of duplicated gene sequences. All of these events are involved in the process of diploidization (Ohno, 1970) and complicate the interpretation of comparative genomic data. MACROSYNTENY AMONG PAPILIONOID LEGUMES Macrosynteny generally refers to conserved gene order between species revealed by comparative genetic mapping of common DNA markers or in silico mapping of homologous sequences. Early comparative studies of legume genomes were focused on closely related species of the same genus or tribe, based primarily on comparative mapping of common RFLP markers. Weeden et al. (1992) first reported conserved gene order between pea and lentil, accounting for approximately 40% of the lentil genome. Later, Menancio-Hautea et al. (1993) demonstrated that mungbean and cowpea (Vigna unguiculata) also exhibited a high degree of linkage conservation, whereas chromosomal rearrangements have occurred since the divergence of the two species. Comparative mapping among mungbean, common bean, and soybean in the Phaseoleae tribe indicated that mungbean and common bean linkage groups were highly conserved, but synteny with soybean was limited only to the short linkage blocks (Boutin et al., 1995). A more recent study, however, using Arabidopsis (Arabidopsis thaliana) as a bridging species revealed that homoeologous segments of soybean chromosomes showed a higher degree of synteny with chromosomes of common bean and mungbean than previously thought (Lee et al., 2001). The most in-depth analysis of legume macrosynteny recently was reported by Choi et al. (2004a, 2004b) using M. truncatula as a central point of comparison. This research took advantage of abundant EST sequence information from the model legume M. truncatula to develop cross-species genetic markers where locus orthology was tested through phylogenetic analysis. Gene-specific PCR primers were designed to anneal to highly conserved exon sequences that span predicted introns, which allowed for efficient PCR amplification across species and for developing single nucleotide polymorphism markers for linkage mapping in multiple taxa. These putatively orthologous markers were mapped in M. truncatula, alfalfa, pea, mungbean (Choi et al., 2004a, 2004b), and chickpea (H. Zhu and D. Cook, unpublished data). In addition, 60 markers developed based on homology to mapped genetic markers of soybean were mapped in M. truncatula. Furthermore, the macrosyntenic relationship between M. truncatula and L. japonicus was evaluated based on 63 pairs of sequenced BAC clones, representing putatively orthologous loci with known genetic position in both species. A simplified consensus comparative map of eight legume species is shown in Figure 2 Figure 2. Open in new tabDownload slide A simplified consensus map for eight legume species. The figure is based on figure 5 of Choi et al. (2004b) with modification. Mt, M. truncatula; Ms, alfalfa; Lj, L. japonicus; Ps, pea; Ca, chickpea; Vr, mungbean; Pv, common bean; Gm, soybean. S and L denote the short and long arms of each chromosome in M. truncatula. Syntenic blocks are drawn to scale based on genetic distance. Figure 2. Open in new tabDownload slide A simplified consensus map for eight legume species. The figure is based on figure 5 of Choi et al. (2004b) with modification. Mt, M. truncatula; Ms, alfalfa; Lj, L. japonicus; Ps, pea; Ca, chickpea; Vr, mungbean; Pv, common bean; Gm, soybean. S and L denote the short and long arms of each chromosome in M. truncatula. Syntenic blocks are drawn to scale based on genetic distance. . As expected, the degree of synteny is correlated with the phylogenetic distance of these legume species. M. truncatula and alfalfa share highly conserved nucleotide sequences and exhibit nearly perfect synteny between the two genomes (Choi et al., 2004a). Although the pea genome is approximately 10 times larger than that of M. truncatula and has one less chromosome, the colinearity of genes is also remarkably conserved between the two genomes, with major evident differences being inferred interchromosomal rearrangements (Choi et al., 2004b). It was suggested that chromosomal rearrangements involving Medicago (alfalfa and M. truncatula) chromosome 6 might be responsible for the difference in chromosome number between Medicago and pea (Choi et al., 2004b; Kalo et al., 2004). Interestingly, the same chromosome seems to have also been associated with the interchromosomal rearrangements between M. truncatula and chickpea (H. Zhu and D. Cook, unpublished data). Even though M. truncatula and chickpea share the same base chromosome number of 8, one-to-one relationships do not hold true for M. truncatula linkage groups 5 and 6 and chickpea linkage groups 2 and 8. In particular, M. truncatula LG5 can be aligned with chickpea LG2 and LG8. Similarly, the genomes of M. truncatula and L. japonicus also are highly syntenic, but the synteny often is punctuated by chromosomal rearrangements, reflecting the difference of chromosome numbers between the two genomes. By contrast, macrosyntenic relationships between M. truncatula and Phaseoloid legumes were more complicated and less informative. Twenty-nine of the 38 (approximately 76%) markers mapped between M. truncatula and mungbean revealed evidence of conserved gene order, whereas the remaining markers mapped to nonsyntenic positions. Similarly, 23 of the 60 mapped markers identified 11 syntenic blocks between M. truncatula and soybean. The finding that synteny was limited only to small genetic intervals between more distantly related legumes suggests correlation between the frequency of chromosomal rearrangement and divergence time, which also is reflected by the differences in chromosome number between Galegoid and Phaseoloid legumes. In the case of soybean, duplication (polyploidization) followed by gene loss and segmental reshuffling (diploidization) may make it difficult to identify lengthy stretches of syntenic chromosome segments between soybean and related legumes. MICROSYNTENY AMONG M. TRUNCATULA, L. JAPONICUS, AND SOYBEAN In contrast with macrosynteny, microsynteny often refers to conserved gene content and order at sequence level over a short, physically defined DNA contig. Nearly all the interspecies analyses of microsynteny reported so far have been based on comparisons of a limited number of specific regions, and the conclusions drawn therein may not be extended to the global level because genome microstructure is highly dynamic and the level of conservation varies with different parts of a genome. Yan et al. (2003) estimated the level of microsynteny between M. truncatula and soybean using a hybridization strategy involving BAC contigs. Twenty-seven of 50 soybean contigs (54%) were shown to possess some level of microsynteny with M. truncatula. Sequence analysis of regions around the putatively orthologous apyrase genes between M. truncatula and soybean also revealed conserved gene order, with at least 6 genes in common over 70 kb (Cannon et al., 2003). Similar comparison was conducted between the rgh1 locus of soybean and the putatively orthologous region of M. truncatula (Choi et al., 2004b). From a total of 29 distinct genes identified in M. truncatula and soybean within the syntenic interval, 14 (approximately 48%) were conserved between the two genomes. More extensive analysis of microsynteny between M. truncatula and L. japonicus was facilitated by the ongoing genome sequencing efforts in both species. Sixty-three pairs of the sequenced BAC clones analyzed shared an average of nine microsyntenic gene pairs (Choi et al., 2004b). Results from detailed analysis of 10 of the 63 clone pairs with broadly spaced genetic positions in the two genomes showed that approximately 82% of identified genes were syntenic between M. truncatula and L. japonicus. Tandem duplication accounts for a 12% and a 17% increase in the number of predicted genes in L. japonicus and M. truncatula, respectively, with only one case that the same homolog duplicated in both species. This observation suggests that the majority of tandem duplication events occurred independently after the divergence of the two species. Intriguingly, in many cases, the regions that were conserved between legumes were also conserved with one or more regions of the Arabidopsis genome, despite at a lower level. CROSS-SPECIES GENE PREDICTION AND ISOLATION The conserved genome structure between M. truncatula and crop legumes has allowed for map-based cloning of genes required for nodulation in crop legumes, using M. truncatula as a surrogate genome (Endre et al., 2002; Limpens et al., 2003). One example is a nodulation receptor kinase (NORK) gene that is required for both bacterial and fungal symbiosis (Endre et al., 2002). Three loci with similar nonnodulation mutant phenotypes were mapped to syntenic locations of M. truncatula, pea, and alfalfa. The closely linked flanking markers in alfalfa were used as probes to pull out M. truncatula BACs that cover the orthologous locus in M. truncatula. Map-based cloning and a complementation test were performed in M. truncatula and eventually led to the simultaneous cloning of three orthologous genes (i.e. DOES NOT MAKE INFECTION2 [DMI2] in M. truncatula, NORK in alfalfa, and SYM19 in pea). At the same time, the ortholog of L. japonicus (called symbiosis receptor-like kinase, or SYMRK), which is located in a syntenic region of M. truncatula, alfalfa, and pea, also was isolated (Stracke et al., 2002, 2004). A similar strategy also was successful for cloning the pea SYM2 orthologous genes of M. truncatula (Limpens et al., 2003). Pea SYM2 is a putative Nod-factor entry receptor involved in the rhizobial infection process (Geurts et al., 1997). Map-based cloning of SYM2 in pea was difficult due to its large genome and the lack of efficient transformation methods. However, the pea SYM2 region is highly syntenic with M. truncatula (Gualtieri et al., 2002). The tightly linked markers flanking the SYM2 in pea were used to identify M. truncatula BACs, and a physical contig (approximately 300 kb) covering the SYM2 orthologous region of M. truncatula was sequenced to identify candidate genes. Using the RNA interference reverse genetic tool, Limpens et al. (2003) showed that two LysM-domain receptor kinases were specifically involved in infection thread formation, and, therefore, are potential orthologs of the SYM2 in pea. LEGUME-ARABIDOPSIS COMPARISON: IMPLICATION OF CORRELATED DIVERGENCE OF GENOME STRUCTURE AND FUNCTION? The degree of conservation of genome structure between legumes and Arabidopsis is less straightforward. Grant et al. (2000) reported substantial macrosynteny between soybean and Arabidopsis, while comparison between M. truncatula and Arabidopsis revealed a lack of extended macrosynteny between the two genomes (Zhu et al., 2003). Nevertheless, it is obvious that synteny is frequently maintained over small chromosomal segments. In cases of localized synteny, genetically linked loci in M. truncatula often are collinear with several segments of Arabidopsis, consistent with the fact that the Arabidopsis genome has experienced extensive segmental duplication and reshuffling accompanied by selective gene loss (Vision et al., 2000; Bowers et al., 2003). Sequence analyses also revealed networks of microsynteny that are often highly degenerate, similar to that reported by Ku et al. (2000). The erosion of microsynteny could be ascribed to either the selective gene loss from duplicated loci or the absence of close homologs of legume genes in Arabidopsis. The divergence of genome microstructure has been widely documented, but it is unknown whether such divergence contributes to the divergence of genome function. Comparisons of regions comprising genes responsible for species-specific or family specific phenotypes provide a unique opportunity to answer this question. As described above, the NORK orthologs required for nodulation are located in the syntenic regions of the four legume species (i.e. M. truncatula, alfalfa, L. japonicus, and pea). Comparative sequence analysis of the M. truncatula and L. japonicus regions revealed highly conserved microsynteny, with 9 of the 11 predicted genes being conserved over an approximately 130-kb interval (Fig. 3 Figure 3. Open in new tabDownload slide Microsynteny of the MtDMI2/LjSYMRK regions and comparison with four segments of Arabidopsis. Lines indicate significant homology matches between predicted genes. The orientations of predicted genes are indicated by arrows. The maps are drawn to scale. Figure 3. Open in new tabDownload slide Microsynteny of the MtDMI2/LjSYMRK regions and comparison with four segments of Arabidopsis. Lines indicate significant homology matches between predicted genes. The orientations of predicted genes are indicated by arrows. The maps are drawn to scale. ). Seven of the 11 distinct genes from the MtDMI2/LjSYMRK regions also exhibited microsynteny with four segments of the Arabidopsis genome. The individual Arabidopsis syntenic regions have experienced significant genic rearrangement, with less than four genes in a block being conserved with M. truncatula and L. japonicus. Nevertheless, a combination of 10 distinct Arabidopsis genes from the four duplicated segments is maintained from a total of 14 distinct genes predicted from the syntenic segments of all three species, identical to the numbers observed in M. truncatula and L. japonicus. Interestingly, the ortholog of MtDMI2/LjSYMRK is missing in the syntenic segments of Arabidopsis. In particular, a legume lectin gene was amplified in syntenic regions of both M. truncatula and L. japonicus, but not in the Arabidopsis regions. In fact, the syntenic counterparts of the legume lectin genes in Arabidopsis are lectin-like protein kinases comprising both a lectin domain and a Ser/Thr kinase domain, suggesting that domain shuffling might have occurred during the divergence of gene structure among plant genomes. Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis (Hirsch, 1999). It is unknown whether such gene amplification in legumes has any particular role in nodulation and nitrogen fixation. The fact that NORK orthologs are extremely conserved in terms of sequence similarity (87%–97%), function, and genomic location among multiple legumes suggests that such divergence of genome microstructure has occurred before the divergence of legume family. The two closest homologs of MtDMI2/LjSYMRK in Arabidopsis are At1g67720 and At2g37050 with a sequence identity of approximately 33%. A TBLASTn search of the M. truncatula Gene Index using the sequences of At1g67720 and At2g37050 identified highly conserved genes from M. truncatula, suggesting that At1g67720 and At2g37050 likely are not the ancient orthologs of MtDMI2/LjSYMRK. Therefore, MtDMI2/LjSYMRK likely is absent in the lineages giving rise to Arabidopsis. Similar divergence of genome microstructure also was observed in comparisons of maize (Zea mays), sorghum (Sorghum bicolor), and rice (Oryza sativa), where a cluster of maize zein storage protein genes are conserved with those of sorghum (kafirin), while the homologs of the storage protein genes are completely missing from the rice orthologous segment and elsewhere of the rice genome (Song et al., 2002). These observations indicate that selective gene loss/retention have played an important role in the divergence of species-specific or family specific phenotypes (e.g. nodulation in legumes). Another legume receptor-like kinase gene required for regulation of the root nodule number recently was cloned from soybean (GmNARK for G. max nodule autoregulation receptor kinase; Searle et al., 2003) and L. japonicus (LjHAR1 for hypernodulation aberrant roots; Krusell et al., 2002; Nishimura et al., 2002). The putative M. truncatula ortholog MtSUNN (supernumerary nodules) also was identified and mapped to the syntenic regions of GmNARK/LjHAR1 (Penmetsa et al., 2003; Schnabel et al., 2003; Choi et al., 2004a). Similar to that observed in the MtDMI2/LjSYMRK regions, the GmNARK/LjHAR1 regions can be aligned with at least seven duplicated segments of the Arabidopsis genome, but none of them contain the GmNARK/LjHAR1 ortholog. The ortholog also was missing in a paralogous region of L. japonicus. In this case, however, the closest homolog of GmNARK/LjHAR1, AtCLAVATA1, is located in a nonsyntenic region of Arabidopsis. Despite the uncertainty of their orthology, AtCLAVATA1 and GmNARK/LjHAR1 share both highly conserved sequence similarity and gene structure, indicating that they are descendants of a common ancestor (Krusell et al., 2002; Nishimura et al., 2002; Searle et al., 2003). Apparently, these homologous genes have experienced functional divergence between plant families, with AtCLAVATA1 controlling stem cell proliferation in Arabidopsis, whereas GmNARK/LjHAR1 functions in the leaf and exerts long-distance control of nodulation (Searle et al., 2003). Such functional divergence appears to have been associated with its relocation in either or both of the lineages leading to Arabidopsis or legumes, and this event should have occurred before the divergence of legume family. As there are two copies of AtCLAVATA1-like genes in soybean (GmCLAVATA1-A and GmCLAVATA1-B; Yamamoto et al., 2000), it was suggested that GmNARK (GmCLAVATA1-B) is likely a duplicated version of AtCLAVATA1 and has been recruited into nodulation function in legumes (Searle et al., 2003). The recruitment of the GmNARK into its current roles is associated with changes in gene expression, leading to differential tissue-specific expression to AtCLAVATA1 and function in controlling the nodule number (Searle et al., 2003). Alternatively, the ancestor of CLAVATA1 might be bifunctional, and the descendent duplicated genes evolve to partition the ancestral function by showing organ- and/or time-specific expression (Adams et al., 2003). Thus, losing one copy of the duplicated and subfunctionalized gene during speciation eventually may lead to species-specific phenotypes. WHAT MAKES A LEGUME? The divergence of plant phenotypes that distinguish one species from another is a big puzzle for plant biologists. In the case of legumes, an interesting question we often ask is as follows: Are genes required for nodulation and nitrogen fixation legume specific? Recent studies have shown that some of the genes required for nodulation (e.g. DMI1 and DMI3 in M. truncatula) are highly conserved between legumes and non-legumes, and their orthologs can be unambiguously defined in non-legumes such as rice and/or Arabidopsis through microsyntenic analysis (Ane et al., 2004; Levy et al., 2004). These results suggested that at least some of the genes involved in nodulation are evolved from broadly conserved pathways of plant development (Parniske and Downie, 2003; Szczyglowski and Amyot, 2003). This view was further supported by Fedorova et al. (2002), who identified 340 tentative consensus sequences with nodule-specific expression patterns, approximately 40% of which shared sequence homology to sequences from non-legumes. It will be a challenging task to undertake the discovery and dissection of the function of the genes that are required for nodulation but are conserved across plant families (e.g. DMI1 and DMI3) in non-legumes. In addition to the recruitment of broadly conserved genes for novel legume functions, legumes also may have evolved novel genes that are involved in legume-specific functions. By comparing unigene sets from M. truncatula, L. japonicus, and soybean to non-legume unigene sets and to the genomic sequences of rice and Arabidopsis, Graham et al. (2004) were able to identify more than 2,500 legume-specific EST contigs, accounting for approximately 6% of genes in legume unigene sets. Motif analysis identified three major gene families from this putative legume-specific gene set, including F-box-related proteins, Pro-rich proteins, and Cys cluster proteins. In particular, the more than 300 Cys cluster proteins, with predicted similarity to defensins, represent approximately 1% of expressed genes in Medicago, primarily from nodules (Fedorova et al., 2002; Mergaert et al., 2003; Graham et al., 2004). Work is under way to characterize these putative legume-specific genes using both forward and reverse genetic tools (K. VandenBosch, personal communication). CONCLUSION The past several years have seen tremendous progress in the study of legume genomics, thanks to the development of abundant genetic and genomic resources for two model legumes, M. truncatula and L. japonicus, and the important crop legume soybean. Undoubtedly, model systems will continue to play a critical role in contributing to our understanding of the mechanisms underlying nodulation and symbiotic nitrogen fixation, the most conserved phenotype in legumes. There is still much to learn about the genomic organization of crop legumes such as soybean, groundnut, and common bean. A major challenge for comparative legume genomics is to translate information gained from model species into improvements in crop legumes. The complexity of that challenge may well be defined by the structural and functional similarities and dissimilarities among these very fascinating genomes. ACKNOWLEDGMENTS We thank Dr. Steve Cannon and Dr. Kathryn VandenBosch for their helpful comments on the manuscript. This article (05–06–009) is published with the approval of the Director of the Kentucky Agricultural Experiment Station. 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Kondorosi, Eva; Redondo-Nieto, Miguel; Kondorosi, Adam

doi: 10.1104/pp.105.060004pmid: 15824282

The cell cycle plays a crucial role in plant development. Organogenesis takes place throughout the lifetime and most cells maintain their ability to reenter and to regulate the cell cycle in response to a wide range of endogenous and external signals. In planta, phytohormones, particularly auxin and cytokinin, are essential for cell proliferation. New organs arise from local foci of proliferating cells, called meristems, which either persist or are de novo formed. In the organ primordium (initial stage of an organ before reaching maturity), cell differentiation starts with cell division arrest. These nondividing cells can exit the mitotic cycle with either complete loss or partial activity of the cell cycle. In the latter case, plant cells frequently enter an altered version of the cell cycle, known as endoreduplication cycles or endocycles, where the genome is duplicated without mitosis. Single or multiple rounds of endoreduplication cycles result in the formation of polyploid cells. The physiological role of ploidy is poorly understood. Cell cycle progression is controlled by ordered action of cyclin-dependent kinases (CDKs), activated by defined cyclins, appearing for given periods in the cycle. When the function of a CDK-cyclin complex is accomplished, the associated cyclin partner becomes polyubiquitinated and destroyed by the ubiquitin-26S proteasome system (UPS). The vital importance of the UPS became evident during the last few years and its discovery was awarded by the Nobel Prize in Chemistry 2004 to Aaron Ciechanover, Avram Hershko, and Irwin Rose. The UPS is essential for many cellular processes including cell cycle, signal transduction and regulation of gene expression, circadian clocks, or phytohormone signaling pathways (Vierstra, 2003). This Update focuses on the possible implications of the ubiquitin-mediated proteolysis in differential regulation of the cell cycle in plant development using nitrogen-fixing root nodules of Medicago truncatula as a model organ. Nodules develop on the roots of legume plants in symbiosis with Rhizobium soil bacteria. One can ask whether studies on this legume-specific symbiotic organ can provide general information on cell cycle and differentiation that is also valid for other plant organs. The answer is yes. The plant encodes nodule development, which resembles, in many respects, lateral root development. Moreover, nodules have several advantages over other plant organs. First, their development can be programmed by application of Rhizobium signal molecules, the Nod factors, which allows studying the mechanisms of cell cycle reactivation and meristem formation from the instant of addition of the morphogen signal. Second, Medicago nodules are indeterminate, which means that the meristem remains active and generates cells that constantly enter differentiation. Thus, different stages of development can be monitored even in a mature nitrogen-fixing nodule. Third, in the submeristematic cell layers, endoreduplication cycles occur permanently. Such a local concentration of endocycling cells is rare and ideal to elucidate the mechanisms that generate polyploid cells in plants. In the following, we concentrate on two critical steps of nodule development: (1) how cell cycle is activated; and (2) how proliferating cells exit the mitotic cycle and enter differentiation via endoreduplication cycles. NITROGEN-FIXING NODULES: A PLANT ORGAN INDUCED BY BACTERIAL SIGNAL MOLECULES WITH RESEMBLANCE TO LATERAL ROOTS Nodule development requires active photosynthesis and limited nitrogen supply. There are two major nodule types: the indeterminate and determinate nodules with permanently or transiently active meristem that originate from the inner and outer cortex, respectively. Indeterminate nodule development (Fig. 1 Figure 1. Open in new tabDownload slide Developmental stages of indeterminate-type nodule (N) and lateral root (LR) formation. The root zones competent for N or LR development are indicated on the longitudinal root section. Transverse section of the root (R) shows epidermis (Ep), cortex (C), endodermis (En), pericycle (P), phloem (Ph), and xylem (X). Colors indicate expression of cycA2 (red), 1-aminocyclopropane-1-carboxylate synthase (yellow), and ccs52A, the overlapping expression of cycA2 and ccs52A (green), and the position of protoxylem poles (brown). R + aux, auxin-treated root section. Figure 1. Open in new tabDownload slide Developmental stages of indeterminate-type nodule (N) and lateral root (LR) formation. The root zones competent for N or LR development are indicated on the longitudinal root section. Transverse section of the root (R) shows epidermis (Ep), cortex (C), endodermis (En), pericycle (P), phloem (Ph), and xylem (X). Colors indicate expression of cycA2 (red), 1-aminocyclopropane-1-carboxylate synthase (yellow), and ccs52A, the overlapping expression of cycA2 and ccs52A (green), and the position of protoxylem poles (brown). R + aux, auxin-treated root section. ) has been studied mainly in the symbiosis of Medicago sativa/M. truncatula with Sinorhizobium meliloti and Pisum sativum/Trifolium repens with Rhizobium leguminosarum. In these plants, Nod factors elicit dedifferentiation and cell cycle reentry of the cortical cells in front of the protoxylem poles in the emerging root hair zone. Cell division in the inner cortex and in the pericycle below the activated cortical cells results in the formation of the nodule primordium and the vasculature, respectively (Yang et al., 1994). When the nodule primordium is formed and emerged from the root, it differentiates, generating a complex structure composed of different peripheral and central tissues (Vasse et al., 1990). The central region of a nitrogen-fixing nodule contains the persistent apical meristem zone I, the infection zone II, the nitrogen fixation zone III and, in old nodules, the proximal senescent zone IV (Fig. 1, N4). Infection of plant cells and differentiation of symbiotic cells take place in zone II. In this zone, the bacteria still produce Nod factors and, although the cells do not divide, they are able to undergo successive rounds of endoreduplication cycles. As a consequence, the nuclear DNA content increases from 2C up to 64C and, proportional to the genome size, the cells enlarge as they become older and more distant from the meristem (Cebolla et al., 1999). Zone III contains terminally differentiated giant plant cells hosting thousands of nitrogen-fixing bacteria, called bacteroids. The bacteroids stop Nod factor production and the expression of cell cycle genes is switched off. In contrast to zones I and II, the size of zone III increases during the lifetime of the nodule by continuous production of nitrogen-fixing cells. Development of nodules and lateral roots displays common but also distinct features (Fig. 1). Both organs originate from de novo formed meristems initiated in front of the protoxylem/xylem poles. However, lateral roots develop from a more distal root zone than nodules and arise from division of pericycle cells. The lateral root primordium is smaller than the nodule primordium and it starts differentiation before its outgrowth from the root. Endoreduplication cycles also occur during lateral root development; however, only in a few cells and not exceeding the 8C ploidy level (Cebolla et al., 1999). In lateral roots, the vasculature is central, while in nodules the vascular bundles are branched and localized at the nodule periphery. Certain aberrant nodules display properties of both organs, such as the outgrowth of lateral roots from nodule-like structures where the nodule-specific meristem is overtaken by a lateral root meristem (Ferraioli et al., 2004). CONCERTED ACTION OF NOD FACTORS AND PHYTOHORMONES IN CELL CYCLE ACTIVATION AND PRIMORDIUM FORMATION Auxin is a key signal in plant development. The asymmetric distribution of auxin (termed auxin maxima) affects polarity and pattern formation and is required for embryonic, root, and shoot organogenic processes. Auxin is mobilized by auxin influx and efflux carriers, encoded by the AUX/LAX and PIN genes, respectively (Kramer, 2004). During lateral root development, division of pericycle founder cells and cell proliferation in the young lateral root primordium are auxin-dependent (Casimiro et al., 2003). At a later stage, the lateral root primordium becomes independent of externally applied auxin, indicating the existence of an internal auxin source. Several studies suggest that Nod factors affect local distribution and concentration of auxin. The use of the auxin-sensitive GH3 promoter-reporter gene fusion indicated transient inhibition of auxin transport by rhizobia and Nod factors, leading to transient accumulation of auxin at the site where indeterminate root nodules initiate (Mathesius et al., 1998). In Lotus japonicus, which develops determinate nodules, a local up-regulation in auxin transport was detected in the root after inoculation with Nod factors and a strong GH3 promoter-β-glucuronidase expression was present in the dividing outer cortical cells leading to nodule primordium formation (Pacios-Bras et al., 2003). These results indicate that indeterminate and determinate type legumes might have different auxin distribution patterns that could lead to cell division either in the inner or outer cortex. In M. truncatula, expression studies on the AUX1-like genes suggest that auxin is required at two common stages of lateral root and nodule development, for the formation of primordia and differentiation of the vasculature (de Billy et al., 2001). Moreover, application of a polar auxin transport inhibitor resulted in the formation of pseudonodules (Hirsch et al., 1989). Cytokinin treatment of Medicago roots increased amyloplast accumulation and the number of cell division foci in the inner cortex recapitulating the responses to Nod factors (Bauer et al., 1996). Cytokinin has effects on the G1/S and G2/M transitions as well as on progression through S-phase (Dewitte and Murray, 2003). In L. japonicus, a legume forming determinate nodules, expression of the cytokinin-responsive ARR5 gene was absent in pericycle founder cells of lateral roots and at the initial divisions of cortical cells but was present in the nodule primordium (Lohar et al., 2004). This suggests that cytokinin is not needed for cell cycle reactivation, while it is necessary for maintaining cell proliferation. Or, local changes in cytokinin levels at the site of nodule initiation may alter auxin redistribution, thereby stimulating nodule organogenesis. In the indeterminate legumes, ethylene provides positional information on cortical cell division. Expression of 1-aminocyclopropane-1-carboxylate synthase, encoding the last enzyme in ethylene biosynthesis, is localized to pericycle cells opposite to the phloem poles (Fig. 1, R; Heidstra et al., 1997). As ethylene inhibits division of cortical cells and nodule primordium formation, the absence of ethylene production in front of the xylem poles may explain why nodules or even lateral roots develop at the xylem but not at the phloem poles. CDK-CENTRIC VIEW OF THE CELL CYCLE In eukaryotes, regulation of cell cycle has been attributed to the sequential activation of CDKs by cyclins. In Arabidopsis, 30 to 43 cyclins are predicted and the CDK family is composed of 12 proteins grouped in 6 types, from A to F (Vandepoele et al., 2002; Wang et al., 2004). The genome sequence of M. truncatula has not been completed yet; therefore, the exact numbers of cyclins and CDKs are not known but based on the identification of the six CDK types in Medicago (Magyar et al., 1997), similar complexity of the CDK-cyclin network is expected. The CDKs with the hallmark of PSTAIRE motif in the cyclin binding site are conserved in all eukaryotes. In plants, these are the A-type CDKs that express throughout the cell cycle and control both the G1/S and G2/M transitions, while the B-type CDKs are mitotic and plant specific (Fig. 2A Figure 2. Open in new tabDownload slide A, Plant CDKs and cyclins control different phases of the mitotic cycle. B, Inhibition of mitotic CDKs converts the mitotic cycle to endocycle. Figure 2. Open in new tabDownload slide A, Plant CDKs and cyclins control different phases of the mitotic cycle. B, Inhibition of mitotic CDKs converts the mitotic cycle to endocycle. ). The C-type CDKs are involved in the regulation of transcription, whereas the D- and F-type CDKs are CDK-activating kinases. In the cell cycle, specific cyclins are associated with G1 (cyclin D), S-phase (cyclin E and cyclin A), and mitosis (cyclin A and cyclin B). Cyclin E is missing from plants, while other cyclin types are present and represented by multiple members. In Arabidopsis (Arabidopsis thaliana), there are 9 or 10 D-type cyclins, 10 A-type, and 9 B-type cyclins (Vandepoele et al., 2002; Wang et al., 2004). With the exception of a few plant cyclins, it is unknown when and where they are expressed and what their functions are. By responding to nutrient and other signals, D-type cyclins are believed to have primary roles during G1 and G1-S transition. In Arabidopsis, cycD2 and cycD4 respond to sugar availability, while D3-type cyclins to cytokinin and brassinosteroid (Riou-Khamlichi et al., 1999, 2000). In Medicago roots, cycD3;1 was transiently induced in the reactivated cortical cells in response to Nod factors, while cycD3;2 expression was linked to endocycles in nodule zone II (Foucher and Kondorosi, 2000), indicating that different sets of CycDs may operate in mitotic and endocycles. Unlike in animals, specific CycDs in association with CDKBs act in G2-M (Kono et al., 2003). The diversity of A-type cyclins is plant specific. In contrast to a single cyclin A in animal cells, plants have three groups of A-type cyclins with multiple members in each. A-type cyclins function from S- to M-phase, but some of them may control S-phase entry (Roudier et al., 2000; Menges et al., 2005). Expression of B-type cyclins is confined to G2-M and cyclin-B associated CDKs are required for mitosis. In the absence of mitotic CDK activities, cells stop to divide and either exit the mitotic cycle and become quiescent or enter endoreduplication cycle(s), which operates with G1-S-G2 activities of the cell cycle (Fig. 2B). If cell cycle activity is maintained for DNA replication and mitotic CDKs are inhibited, endoreduplication cycles can be repeated in multiple rounds leading to the formation of polyploid cells. The presented CDK-cyclin centric view on cell cycle control is an extreme oversimplification. Many important components such as the Rb-E2F pathway or CDK inhibitors have not been discussed here, as this minimal information is sufficient to discuss nodule primordium formation and differentiation. CELL CYCLE CONTROL AND PHYTOHORMONE SIGNALING PATHWAYS DEPEND ON TARGETED DEGRADATION OF PROTEINS BY THE UBIQUITIN-PROTEASOME SYSTEM UPS is the primary mechanism in eukaryotic cells for degrading unwanted and misfolded proteins (Fig. 3A Figure 3. Open in new tabDownload slide A, The ubiquitin proteasome system. The ubiquitin (Ub) is covalently attached to substrate proteins (Sub) through sequential action of the E1, E2, and E3 enzymes. The polyubiquitinated proteins are recognized by the 26S proteasome, which degrades the substrate proteins and recycles the ubiquitin. The E3 enzymes achieve the specificity of ubiquitin-dependent proteolysis. B, The SCF and the APC E3 enzymes are dedicated to basic cell-cycle control. Rbx/APC11 and Cullin/APC2 are related subunits. SCF is constitutively active, while the WD40-repeat proteins Cdc20 and Cdh1/Ccs52A,B activate APC from M-phase to S-phase. The cullin/APC2 and the Rbx/APC11 subunits are related. The substrate-specificity determinants (Ssd) are the F-box proteins in the SCF and Cdc20 and Cdh1/Ccs52A,B in the APC. In addition to polyubiquitination of cell cycle proteins, the SCF and the APC are also active in nonproliferating cells mediating protein degradation in various cellular processes. The SCF via different F-box proteins is involved in phytohormone signaling pathways. Figure 3. Open in new tabDownload slide A, The ubiquitin proteasome system. The ubiquitin (Ub) is covalently attached to substrate proteins (Sub) through sequential action of the E1, E2, and E3 enzymes. The polyubiquitinated proteins are recognized by the 26S proteasome, which degrades the substrate proteins and recycles the ubiquitin. The E3 enzymes achieve the specificity of ubiquitin-dependent proteolysis. B, The SCF and the APC E3 enzymes are dedicated to basic cell-cycle control. Rbx/APC11 and Cullin/APC2 are related subunits. SCF is constitutively active, while the WD40-repeat proteins Cdc20 and Cdh1/Ccs52A,B activate APC from M-phase to S-phase. The cullin/APC2 and the Rbx/APC11 subunits are related. The substrate-specificity determinants (Ssd) are the F-box proteins in the SCF and Cdc20 and Cdh1/Ccs52A,B in the APC. In addition to polyubiquitination of cell cycle proteins, the SCF and the APC are also active in nonproliferating cells mediating protein degradation in various cellular processes. The SCF via different F-box proteins is involved in phytohormone signaling pathways. ; Ciechanover et al., 2000). Through the cascade of the E1 ubiquitin activating, E2 ubiquitin conjugating, and E3 ubiquitin ligase enzymes, ubiquitin monomers are attached sequentially to the target proteins. The polyubiquitinated proteins are then recognized by the 26S proteasome, a large ATP-dependent multicatalytic protease, which removes the ubiquitin chain and degrades the proteins to short peptides. The UPS appears to be the most elaborate regulatory mechanism in plants as 5% of their genome encodes core components of UPS and more than 1,000 E3 ubiquitin ligases are predicted (for review, see Vierstra, 2003; Schwechheimer and Villalobos, 2004). The selection and specific timing of polyubiquitination of the target proteins are conferred by different E3 ubiquitin ligases. In the cell cycle, two structurally related multicomponent ubiquitin ligases, the anaphase-promoting complex (APC) and the Skp1/Cul1/F-box protein (SCF) complexes (Fig. 3B) have essential and complementary functions by temporally controlled degradation of various cell cycle proteins (Peters, 2002; Vodermaier, 2004). Different F-box proteins provide the substrate-specificity of SCF. In Arabidopsis, the presence of almost 700 F-box proteins indicates the involvement of SCF in a wide range of cellular processes including various hormone responses. Auxin signaling is mediated by auxin-induced degradation of the Aux/IAA proteins by SCFTIR1, where TIR1 is an F-box protein (Gray et al., 1999, 2001). Elimination of the Aux/IAA proteins leads to the release of the interacting ARF transcription factors that regulate the expression of auxin responsive genes. In a similar way, in response to GA3, the SCFSLY1/2 mediates degradation of the putative transcription factors RGA and GAI (Dill et al., 2004; Fu et al., 2004), while SCFEBF1/2 is involved in ethylene signaling by degrading EIN3 in the absence of ethylene (Potuschak et al., 2003). The APC is composed of 11 to 13 subunits in human and yeast (Saccharomyces cerevisiae) and homologous APC subunits have also been found in plants (Capron et al., 2003). Except for APC2 and APC11, relatively little is known about the role of the other APC subunits or the assembly of the complex. APC functions both in mitotic and nondividing postmitotic cells. Binding of the APC substrates and activation of the APC are controlled by 2 WD40-repeat activator proteins, Cdc20 and Cdh1. They determine stage-specific activation of the APC from metaphase until S-phase and degradation of various cell cycle proteins during the cell cycle (Harper et al., 2002; Peters, 2002). Cdc20 appears to be active only in proliferating cells. In contrast, Cdh1 functions in both mitotic and differentiating cells. In plants, there are multiple cdc20 genes and Cdh1-type activators, identified as cell cycle switch Ccs52 proteins (Cebolla et al., 1999). The latter form 2 classes: Ccs52A, representing a plant ortholog of the yeast and animal Cdh1 proteins; and Ccs52B, which is plant specific (Tarayre et al., 2004). These proteins differ in their expression pattern during the cell cycle and plant development (Tarayre et al., 2004) and interact with distinct sets of APC substrate proteins (Z. Kelemen, G. Horvath, and E. Kondorosi, unpublished data). Mitotic cyclins that contain a destruction or D-box in their N terminus were the first identified substrates of the APC. Both the Cdc20 and the Cdh1/Ccs52 proteins can mediate the degradation of mitotic cyclins; however, at different phases of the cell cycle. A-type cyclins are not only substrates but also regulators of the APC as phosphorylation of Cdh1 by cyclin A-associated CDK inactivates Cdh1 and abolishes its binding to the core APC. Similarly, phosphomimetic amino acid replacements in the Medicago Ccs52A protein inhibit the interaction of Ccs52A with the APC (Tarayre et al., 2004). The Cdh1/Ccs52 proteins interact with many different proteins containing D-, KEN, A-, or GxEN boxes or other, yet unidentified degradation motifs and activate the APC both in and outside the cell cycle with essential roles in the differentiation of specific cell types. In plants, the number of APC substrates can be estimated from a few hundred up to a few thousand. CELL CYCLE ACTIVATION AND NODULE MERISTEM FORMATION REQUIRE AN AUXIN-REGULATED A2-TYPE CYCLIN Previous studies showed that Nod factors trigger reactivation of G0-arrested cells (Savouré et al., 1994; Yang et al., 1994). Surprisingly, one of the earliest Nod factor induced cell cycle genes was a cyclin that, based on its structure, was classified as mitotic A2-type cyclin, cycA2 (Foucher and Kondorosi, 2000; Roudier et al., 2003). In the nodulation competent root zone, activation of cycA2 coincided with the induction of G1-S regulators (such as cycD3;1). Unlike other mitotic cyclins, the level of cycA2 mRNA and the protein did not display marked oscillation from late G1 until prometaphase where the CycA2 protein was abruptly degraded (Roudier et al., 2000). Expression of cycA2 in late G1 as well as its activation by the Nod factors suggested that CycA2 might be involved in the cell cycle reentry. This was studied in roots, lateral roots and nodules, and in galls, abnormal swellings of roots, where endoparasitic root-knot nematodes trigger division of cortical cells and formation of polyploid feeding cells (Roudier et al., 2003). In the primary root, cycA2 expression was observed in the root apical meristem and faintly in the phloem cells (Fig. 1, R). cycA2 was induced at the onset of lateral root development, in the dividing cells and the lateral root primordium (Fig. 1, LR1 and 2). By differentiation of the primordium, cycA2 expression becomes restricted to the meristem (Fig. 1, LR3). During nodule organogenesis, cycA2 was induced 5 h after Nod factor treatment and the expression was maintained in the dividing cortical cells and in the nodule primordium (Fig. 1, N1–3). In nitrogen-fixing nodules, cycA2 was expressed only in the nodule meristem (Fig. 1, N4). In galls, expression of cycA2 was undetectable. Therefore, it is possible that the cycA2 function is linked to mitotic cycles, which lead to the formation of secondary meristems, but it is dispensable or even incompatible with endoreduplication cycles (Roudier et al., 2003). If cycA2 is involved in cell cycle reentry during lateral root and nodule initiation, it is expected that its expression is regulated by auxin. The cycA2 promoter contains two auxin-response-like elements. Treatment of M. truncatula roots with auxin or with a polar auxin transport inhibitor demonstrated that cycA2 is indeed auxin regulated (Roudier et al., 2003). Auxin-treatment resulted not only in the up-regulation of cycA2 but affected also the spatial expression pattern. Instead of phloem-associated expression, auxin induced de novo transcription of cycA2 in front of the xylem poles, where both lateral roots and nodules initiate (Fig. 1, R and R + aux). This was also consistent with the Nod factor-triggered expression of the auxin-responsive GH3 promoter at the inner cortex prior to nodule initiation (Mathesius et al., 1998). It is still unknown, however, how this auxin response emerges on cycA2, which IAA protein(s) are degraded, and whether only the SCFTIR1 system or other E3 ubiquitin ligases are involved in auxin signaling in Medicago roots. ENDOREDUPLICATION CYCLES MEDIATED BY THE APC ACTIVATOR CCS52A ARE INDISPENSABLE FOR SYMBIOTIC CELL DIFFERENTIATION After the formation of the nodule primordium, the next critical step is nodule differentiation that involves cell cycle arrest in the various nodule cell types but modified regulation of the cell cycle in the symbiotic cells. This raises the questions of how cell proliferation is arrested, how endocycles are triggered, and whether genome amplification has any biological meaning. Endoreduplication cycles result in periodic replication of the genome. This is achieved by the loss of M-phase and oscillations in the activity of S-phase cyclin-dependent kinase. In nodule zone II, expression of CDKA, G1-, and S-phase specific marker genes indicates that cell cycle activities for DNA replication and endoreduplication cycles are present (Foucher and Kondorosi, 2000). On the other hand, mitotic B-type cyclins are also expressed in the infected cells, albeit formation of mitotic cyclin-CDK complexes should be avoided during endocycles (Cebolla et al., 1999). How are the mitotic CDKs inactivated? Amongst many possible mechanisms, such as inhibitory phosphorylation of CDKs or binding of CDK inhibitors, mitotic CDKs can also be inactivated by destruction of the cyclin partner. This latter mechanism operates in nodules and involves the cell cycle switch gene ccs52A. Ccs52A binds and targets mitotic cyclins to the APC, resulting in their polyubiquitination and degradation (S. Tarayre, Z. Kelemen, and E. Kondorosi, unpublished data; Cebolla et al., 1999). In the absence of mitotic cyclins, the mitotic CDKs are inactive and M-phase progression as well as cell division is inhibited. If the cell cycle is otherwise active, the cells switch to endocycles entering directly G1-phase instead of M and synthesize the DNA in S-phase. If M-phase is blocked again by degradation of mitotic cyclins by APCCcs52A, the cells can enter a second or on a similar way repeated endoreduplication cycles (Fig. 2B). The ccs52A gene is not expressed in the dividing cortical cells and in the growing nodule primordium. Ccs52A becomes activated in the fully grown primordium before differentiation and expresses in zones I and II of nitrogen-fixing nodules (Fig. 1, N3 and N4; Vinardell et al., 2003). The ccs52A mRNA and the Ccs52A protein are present in all endoreduplicating cells in zone II but absent in zone III (Cebolla et al., 1999; Vinardell et al., 2003). Inversely, ccs52B expression is present in the root and declines with the formation of nodule primordium. As it was discussed before, phosphorylation by cyclin A-CDK inactivates the Cdh1-type APC activators. In human cells, depletion of cyclin A provoked a nonperiodic APC activity and endoreduplication cycles (Sorensen et al., 2000). It is unknown which cyclin A associated CDK regulates the activity of CCS52A in Medicago. It is tempting to speculate that it is CycA2. If this cyclin were a negative regulator of Ccs52A, its absence in nodule zone II, could lead to permanently unphosphorylated state of Ccs52A, providing constitutive APCCCS52A activity. Therefore, mitotic cyclins produced in zone II could be degraded at the instant of their production, generating multiple rounds of endocycles. This would also mean that CycA2 is dispensable for DNA replication and S-phase functions in the endoreduplicating cells, which are ensured by other A-type Medicago cyclins. But why are the nodule cells polyploid? Will a nodule be functional without endocycles? This was tested in ccs52A antisense plants where reduction of the ccs52A transcript level did not affect the formation of nodule primordia but aborted nodule development (Vinardell et al., 2003). These nodules displayed significantly a lower degree of ploidy, and the nodule cells were smaller and poorly or not infected and contained no nitrogen-fixing cells. These results demonstrated that repeated endoreduplication cycles controlled by Ccs52A are indispensable for nitrogen-fixing nodule development (Vinardell et al., 2003). In the determinate nodules, the symbiotic cells are also big and polyploid. Ccs52A is highly conserved in legumes and present in nodules; therefore, Ccs52A likely mediates nodule ploidy in all nodule types. CONCLUSIONS AND FUTURE DIRECTIONS During the past few years, enormous progress on the UPS highlighted the vital importance of this regulatory mechanism that turns off protein functions in the right place and at the right moment. Most knowledge on UPS arises from cell cycle studies where ordered destruction of proteins by the APC and SCF ensures unidirectional progression of the cycle. In plants, the discovery of the APC- and SCF-controlled processes is still at the elementary stage and out of hundreds or much more potential candidates, only a few are known as APC or SCF substrates. Future studies on the identification of novel targets and the APC- and SCF-regulated pathways will likely result in significant breakthroughs in understanding plant development. Data on degradation of plant cell cycle proteins are rather limited and it is unknown how hormone-signaling pathways communicate with the cell cycle. In M. truncatula, lateral root and nodule initiation depends on auxin maxima, formed de novo in front of the xylem/protoxylem poles and associated with the induction of an auxin-responsive cell cycle gene. Studies on nodule organogenesis have led to the identification of the plant APC activators, Ccs52A and Ccs52B, as well as to the discovery of APCCcs52A-mediated degradation of mitotic cyclins as a key regulatory mechanism inducing and driving endoreduplication cycles. Though endoreduplication is widespread in plants, until recently its mechanism and biological significance of polyploidy were poorly understood. Are the endocycles the cause or the consequence of differentiation? Several studies suggest that increased genome size may control cell size and may be required for faster cell growth and for increasing the storing capacity of cells. 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Plant Cell 6 : 1415 –1426 Author notes 1 This work was supported by the Spanish Ministerio de Educación y Ciencia Progam Becas Postdoctorales en España y en el extranjero 2003 (to M.R.-N.). * Corresponding author; e-mail [email protected]; fax 33–1–69–82–36–95. www.plantphysiol.org/cgi/doi/10.1104/pp.105.060004. © 2005 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Oldroyd, Giles E.D.; Harrison, Maria J.; Udvardi, Michael

doi: 10.1104/pp.104.057661pmid: 15824283

Terrestrial autotrophs such as higher plants are confronted with challenges largely unknown to their counterparts in the seas, including extreme patchiness of inorganic nutrients. Evolution of higher plants has yielded interesting and important solutions to the problem of nutrient acquisition on land. Some of the most intriguing of these involve mutually beneficial symbioses. In fact, evolution of a fungal-plant symbiosis around 450 million years ago may have been the key innovation that enabled plants to colonize the land (Pirozynski and Malloch, 1975; Remy et al., 1994). Related mycorrhizal associations continue to exist for more than 90% of land plants, a reflection of their ancient origin and importance. Mycorrhizal associations enhance the ability of plants to scavenge nutrients such as phosphate from the soil, by virtue of the greater volume of soil exploited by the filamentous fungal partner (Smith and Read, 1997; Smith et al., 2003). In return for its services, the fungus is provided with a carbon source, derived from plant photosynthesis, for biosynthesis and energy metabolism. To avoid exploitation, however, plants have evolved a series of checkpoints that help to discern friend from foe, which presumably switch off defense responses against robbers when a friend comes to visit. A different kind of beneficial plant-microbe interaction that provides a more restricted range of plants with the often-limiting macronutrient nitrogen is symbiotic nitrogen fixation (SNF). This type of symbiosis evolved more recently, some 60 million years ago, and is confined to legumes and a few nonlegumes (Doyle, 1998), which form intracellular symbioses with rhizobia or other nitrogen-fixing bacteria, respectively (Pawlowski and Bisseling, 1996). Once again, the plant provides its beneficial endosymbiont with photosynthate, together with other nutrients, in exchange for valuable fixed nitrogen, in the form of ammonium and amino acids (Udvardi and Day, 1997). Interestingly, some of the signaling pathways that mediate peace between legumes and mycorrhizal fungi also function in the rhizobial symbiosis, and stunning progress has been made recently in identifying a few of the genes involved. Significant progress has also been made in identifying changes in transport and metabolism during symbiosis development, which will contribute to a better understanding of the nature of trade between legumes and their microsymbionts. These breakthroughs largely stem from focused research on two model legumes, Lotus japonicus and Medicago truncatula, and the conjunction of genetics, genomics, and functional genomics. This Update focuses on recent discoveries in the areas of legume-microbe communication and trade, two essential aspects of stable mutualism. UNDERGROUND PEACE TALKS: RECENT ADVANCES IN LEGUME MICROBE SIGNALING Symbiotic interactions involve molecular communication between the host plant and its microbial symbiont in the rhizosphere. The legume symbioses, SNF and arbuscular mycorrhizae (AM), share common features in early signaling. Discoveries in the signaling pathways that underpin these symbioses are among the most exciting recent advances in legume research. It is becoming clear that calcium plays a crucial role in the symbiotic signaling and may be a common feature of legume/symbiont peace talks. NOD FACTOR SIGNALING The invasion of the bacteria into the plant root occurs through an invagination of the plant cell (termed the infection thread) that initiates at the primary site of the interaction, the root hair cell, but spans the entire root cortex, allowing bacterial invasion into the dividing cells of the nodule primordium. Bacteria are released from the infection thread into membrane bound compartments, where they differentiate into bacteroids. Nod factors, or lipo-chito-oligosaccharide signaling molecules, are central to the initial establishment of the legume-rhizobial symbiosis (Dénarié et al., 1996; Long, 1996; Oldroyd, 2001). Production of this signaling molecule is activated by the release of plant phenolic signals, predominantly flavonoids, into the rhizosphere, where they activate Nod factor production through induction of a set of nod genes in the appropriate rhizobial strain. The nature of both the flavonoid signal and the structure of Nod factor are central to the maintenance of specificity in this interaction, ensuring that the plant only accommodates a friendly rhizobial strain. Nod factors are critical both at the early stages of the interaction and during infection thread development, and may play a role during bacterial release (Ardourel et al., 1994; Downie and Walker, 1999). It is clear that understanding the plant's perception of this signaling molecule is key to understanding this symbiosis. Since the work of Ehrhardt nearly a decade ago (Ehrhardt et al., 1996), it is becoming more apparent that calcium is an important component of Nod factor signaling, and such a calcium-centric viewpoint has been validated by more recent work. A number of studies using calcium dyes and ion-selective electrodes have indicated significant Nod factor-induced calcium changes in root hair cells (Cardenas et al., 2000). This work can be summarized into two main calcium events: an initial calcium flux that occurs at the tip of the root hair and repetitive cytosolic oscillations of calcium, termed calcium spiking, in the region surrounding the nucleus (Oldroyd and Downie, 2004). These two calcium responses are separated both spatially and temporally, but can also be separated by Nod factor concentrations: 10−12 to 10−9 m Nod factor activates spiking without inducing the flux (Shaw and Long, 2003). Calcium is a ubiquitous secondary messenger in diverse organisms and can affect a wide variety of cellular events. The Nod factor-induced calcium changes are simply observations and provide little insight into the actual roles that calcium plays during this symbiosis. In animal systems, calcium spiking has been shown to regulate gene expression in response to a signaling molecule (Dolmetsch et al., 1998; Li et al., 1998). A similar signaling role for calcium spiking in Nod factor signal transduction is supported by the fact that a number of genes essential for Nod factor signaling are also required for activation of calcium spiking (Wais et al., 2000; Walker et al., 2000b; Oldroyd and Downie, 2004; Fig. 1 Figure 1. Open in new tabDownload slide The Nod factor and mycorrhizal signaling pathways. This signaling pathway has been defined through genetics in the model legumes M. truncatula and L. japonicus, and Medicago sativa. The genes identified are defined in boxes. Mutations in all these genes have been characterized for calcium spiking except SYMRK. In addition, it has been shown that mutations in NFR5 and NFR1 lack the calcium flux, whereas mutations in DMI1 and DMI2 show the first phase of the flux response. Components of the Nod factor (NF) signaling pathway are conserved with mycorrhizal signaling. We presume that genes specific to mycorrhizae must exist at equivalent positions to NFR1 and NFR5 and possibly NSP1 and NSP2. Figure 1. Open in new tabDownload slide The Nod factor and mycorrhizal signaling pathways. This signaling pathway has been defined through genetics in the model legumes M. truncatula and L. japonicus, and Medicago sativa. The genes identified are defined in boxes. Mutations in all these genes have been characterized for calcium spiking except SYMRK. In addition, it has been shown that mutations in NFR5 and NFR1 lack the calcium flux, whereas mutations in DMI1 and DMI2 show the first phase of the flux response. Components of the Nod factor (NF) signaling pathway are conserved with mycorrhizal signaling. We presume that genes specific to mycorrhizae must exist at equivalent positions to NFR1 and NFR5 and possibly NSP1 and NSP2. ). The molecular identity of the gene products that link Nod factor perception with induction of calcium spiking (Fig. 1) is highly informative of the regulation of calcium signaling in plants. The Nod factor receptor is most likely a heterodimer of two classes of receptor-like kinases that contain LysM domains in the extracellular region (Limpens et al., 2003; Madsen et al., 2003; Radutoiu et al., 2003). LysM domains are present in a number of proteins and have been shown to bind polysaccharides, particularly glucosamine chains, which form the backbone of the Nod factor molecule. Mutations in these LysM receptor-like kinases abolish all Nod factor-induced responses, including calcium spiking and the calcium flux, which supports a role for these proteins at a very early stage of the Nod factor signal transduction pathway (Amor et al., 2003; Limpens et al., 2003; Madsen et al., 2003; Radutoiu et al., 2003). Functioning downstream of these LysM receptor-like kinases are DMI1, a putative cation channel, and NORK/SYMRK/DMI2, another receptor-like kinase with Leu-rich repeat domains in the extracellular portion (Endre et al., 2002; Stracke et al., 2002; Ane et al., 2004). Mutations in both of these proteins abolish Nod factor-induced calcium spiking and restrict the calcium flux response to a single rapid calcium increase, rather than a calcium increase that is maintained for a number of minutes as is seen in wild-type plants (Wais et al., 2000; Shaw and Long, 2003). The presence of two receptor-like kinases that most likely make up the Nod factor receptor and a second receptor-like kinase functioning downstream implicates a phosphorylation cascade early in Nod factor signaling, and defining the targets of these kinases is crucial for linking Nod factor perception with the activation of the downstream calcium responses. The putative cation channel DMI1 may have a direct role in calcium spiking, in that it could function as a calcium channel during both the calcium flux and calcium spiking. However, two proteins homologous to DMI1, CASTOR and POLLUX, have recently been identified in L. japonicus that are required for Nod factor signaling and show plastid localization (Imaizumi-Anraku et al., 2005). The plastid is an unlikely internal calcium store for calcium spiking, and, therefore, this localization suggests that this class of putative cation channels is not the calcium channel involved in calcium spiking. Defining the ions transported by these channels is crucial for assessing their role in the Nod factor signaling pathway. DMI3 of M. truncatula is essential for Nod factor signaling, and mutations in dmi3 are phenotypically identical to dmi1and dmi2 except for the fact that dmi3 mutants can activate calcium spiking (Catoira et al., 2000; Wais et al., 2000). This indicates that DMI3 functions downstream of calcium spiking and is a strong candidate for a protein able to perceive and transduce the calcium spiking signal. This hypothesis is validated by the identity of DMI3: a chimeric calcium/calmodulin-dependent protein kinase (CCaMK; Levy et al., 2004; Mitra et al., 2004a). Clearly, such a calcium activatable kinase has the hallmarks of a protein able to decode the calcium spiking signal. If this is indeed the function of DMI3, the absolute requirement for this activity in Nod factor signaling highlights the central role that calcium spiking plays in transducing the Nod factor signal. EARLY SIGNALING IN THE AM SYMBIOSIS The symbiotic organelle of the AM symbiosis, the arbuscule, forms within the root cortex. Entry into the root is achieved through fungal appressoria that develop on the plant epidermal cell surface. While this is the first visible stage of the interaction, there is evidence that the plant and fungus communicate prior to physical contact. AM fungi respond to signals in plant root exudates by altering their respiratory activity and hyphal morphology, while a diffusible signal from the fungus elicits alterations in plant gene expression (Nagahashi and Douds, 1997; Buee et al., 2000; Kosuta et al., 2003; Tamasloukht et al., 2003). The nature of the signaling molecules is not known. The signaling pathways involved in triggering the cellular events required for development of the association are beginning to be revealed. The DMI genes of M. truncatula and their reciprocal proteins in L. japonicus and pea are not only required for nodulation, but also the early development of the mycorrhizal association (Fig. 1). Mutations in all these genes fail to allow entry of the fungus into the cortex (Sagan et al., 1995; Wegel et al., 1998). This implicates the NORK/SYMRK/DMI2/SYM19 Leu-rich repeat receptor kinase, the DMI1 channel protein, and the DMI3 CCaMK in early mycorrhizal signaling (Endre et al., 2002; Stracke et al., 2002; Ane et al., 2004; Levy et al., 2004; Mitra et al., 2004a). Since the AM symbiosis is the older of the two associations, the legume/rhizobial symbiosis probably co-opted part of a signaling pathway that had been established initially for development of the AM symbiosis. The fact that proteins involved in the induction and perception of calcium spiking in Nod factor signaling are also involved in mycorrhizal signaling implicates calcium as a secondary messenger in the mycorrhizal pathway. Nod factor signaling contains nodulation-specific proteins both upstream and downstream of the conserved pathway, and, similarly, one would expect mycorrhiza-specific signaling proteins. Genetic screens for these mycorrhiza-specific mutants are currently under way in a number of laboratories. Recently, transcriptional profiling has identified genes whose expression is differentially regulated in M. truncatula in response to appressoria formation and the early stages of development of the symbiosis (Liu et al., 2003; Brechenmacher et al., 2004). Defense gene transcripts are among those that show a transient increase in the early stages of the AM symbiosis, followed by a decrease coincident with proliferation of the fungus within the roots. While similar defense gene expression patterns had been noted earlier, the genome-scale analyses have identified coregulated signal transduction proteins that may be involved in regulating defense responses in the symbiosis (Liu et al., 2003). It would appear that the plant initially reacts in a defensive manner, but, following communications with the fungus, peace ensues and the plant reduces its defenses. SPECIALIZATION AND TRADE: TWO KEYS TO MUTUALISM Effective and sustained communication is necessary for any long-term relationship, but it is not sufficient. The evolutionary success of legume-rhizobia and mycorrhizal associations derives from the trade of goods (metabolites) of value to each. The division of labor that underpins such exchanges is achieved by metabolic specialization/differentiation in each symbiont during development of the symbiosis. The following sections describe recent advances in our understanding of these processes. DIFFERENTIATION OF LEGUMES AND RHIZOBIA DURING SNF Development of functional nodules requires differentiation of both plant and bacterial cells, the latter being converted to a distinct nitrogen-fixing form called the bacteroid. Transcriptomics and proteomics are beginning to reveal the true extent of plant and bacterial differentiation during nodule development. Hundreds of novel plant genes that are either induced or repressed during nodule development have now been identified using cDNA arrays (Colebatch et al., 2002, 2004; El Yahyaoui et al., 2004; Kouchi et al., 2004; Kuster et al., 2004; Lee et al., 2004), oligonucleotide microarrays (Mitra et al., 2004b), and bioinformatic approaches (Fedorova et al., 2002; Journet et al., 2002). Many of these are involved in metabolism and transport (Colebatch et al., 2004; El Yahyaoui et al., 2004; Kouchi et al., 2004). Coordinate up-regulation of plant genes involved in glycolysis, carbon fixation, and amino acid biosynthesis highlight the importance of these processes in carbon supply for bacteroid nitrogen fixation and plant ammonium assimilation. While compatible solutes may not be a currency of trade between legumes and rhizobia, induction of plant genes involved in polyamine, polyol, and Pro synthesis indicate that nodule cells may have to work overtime for osmotic homeostasis (Colebatch et al., 2004). Of more interest from the point of view of trade between host and microsymbionts is the growing list of nodule-induced plant genes encoding transporters (Colebatch et al., 2004; El Yahyaoui et al., 2004; Kouchi et al., 2004), some of which appear to be located on the symbiosome membrane, based on proteomics data (Saalbach et al., 2002; Wienkoop and Saalbach, 2003; Catalano et al., 2004). The symbiosome membrane is the specialized plant membrane that separates bacteroids from the host cell cytoplasm and controls the traffic of nutrients between the two. Among the most interesting of the nodule-induced transporters identified by transcriptomics are homologs of AgDCAT1, a dicarboxylate transporter of the nonlegume Alnus, which probably delivers carbon substrates to nitrogen-fixing Frankia in Alnus nodules (Jeong et al., 2004). The legume counterparts of AgDCAT1 may play a crucial role in SNF, as dicarboxylic acids are believed to be the primary source of carbon for bacteroid metabolism. Other nodule-induced transporters include putative amino acid transporters, which may provide a missing link in intriguing models of amino acid cycling between legumes and rhizobia (Lodwig and Poole, 2003). Transcriptome analysis is also making important in-roads in rhizobium biology. DNA arrays containing essentially all the genes of Mesorhizobium loti (Uchiumi et al., 2004) and Sinorhizobium meliloti (Becker et al., 2004) have now been produced and used to obtain the first global view of gene expression in symbiotic rhizobia. Nodule development is accompanied by declining levels of free oxygen, which is a prerequisite for activity of oxygen-labile nitrogenase in rhizobia. Oxygen levels control the expression of many genes in rhizobia (Batut and Boistard, 1994), and low oxygen is believed to be an important trigger for differentiation of rhizobia into bacteroids in nodules. Transcript profiling of oxygen-limited free-living bacteria indicated that up to 5% of S. meliloti genes may be oxygen regulated (Becker et al., 2004). However, microoxic and bacteroid transcriptomes overlapped only partially, indicating that low oxygen in nodules can account for only a fraction of the changes in gene expression observed during symbiotic development. Clearly, other physiological or biochemical factors in nodules are important for bacteroid differentiation, and it will be interesting to learn what these are in the future. Proteomic analysis has also contributed to knowledge about bacterial and plant cell differentiation during nodule development. The most comprehensive work has been done on free-living and symbiotic forms of S. meliloti isolated from Melilotus alba or M. truncatula (Natera et al., 2000; Djordjevic et al., 2003, 2004). Of 170 bacteroid proteins identified, 27 appear to be symbiosis specific, including nif and fix gene products involved directly or indirectly in nitrogen fixation. Also in this list are a raft of transporters that are presumably involved in nutrient transfer between host and microsymbiont (Djordjevic et al., 2003, 2004). Among the proteins that were reduced or absent in bacteroids compared to cultured cells were several involved in nitrogen regulation and nitrogen assimilation, which is consistent with past observations that ammonium assimilation is repressed in bacteroids. Not all of the differences in the proteomes of free-living and bacteroid forms of S. meliloti are mirrored by corresponding changes in gene transcript levels (Becker et al., 2004). Such discrepancies may reflect different levels of regulation (i.e. transcriptional versus posttranscriptional regulation), which is an interesting area for future research. DIFFERENTIATION DURING ARBUSCULE FORMATION Unlike the rhizobium-legume symbiosis, the AM symbiosis does not culminate in the formation of a new plant organ. Instead, there are major rearrangements within the root cortex where terminally differentiated hyphae, termed arbuscules, form extensive dichotomous branching within cortical cells, enveloped within the plant derived periarbuscular membrane (Bonfante-Fasolo, 1984). Transcriptional profiling has allowed the identification of several novel plant genes whose expression is activated coincident with arbuscule development (Liu et al., 2003; Wulf et al., 2003). Further spatial expression analyses revealed at least two distinct gene expression patterns: genes whose expression occurs only in cells with arbuscules and genes whose expression is activated more broadly throughout the cortex in both colonized and noncolonized cells. In addition to identifying candidate genes implicated in development of the biotrophic interface, these analyses point to the existence of both cell autonomous and systemic signaling pathways operating in the AM symbiosis. Current information about the genomes of AM fungi is limited, but analyses of one species, Glomus intraradices, suggest a genome size of 15 Mb (Hijiri and Sanders, 2004). Genome sequencing is in progress and promises to provide the first insights into the genome of a broad-host-range, obligate symbiont. Recent physiological and molecular data suggest that when plants form an AM symbiosis, they alter their phosphate acquisition pathways significantly. Phosphate transporters operating at the root-soil interface are down-regulated, and the plant relies largely on phosphate delivered by the fungal symbiont (Liu et al., 1998; Chiou et al., 2001; Smith et al., 2003). In mycorrhizal roots, phosphate is acquired by the extraradical fungal hyphae and is then transferred to the arbuscules, where it is released from the fungus and transported across the periarbuscular membrane into the cortical cell. In the past few years, there has been progress in understanding the molecular basis of phosphate transport in the symbiosis, and, most recently, plant phosphate transporters implicated in the uptake of phosphate released from the arbuscule have been reported (Rausch et al., 2001; Harrison et al., 2002; Paszkowski et al., 2002). In potato (Solanum tuberosum), expression of a high-affinity phosphate transporter, StPT3, is induced in mycorrhizal roots, particularly in cells containing arbuscules, making it a strong candidate for involvement in symbiotic phosphate transport (Rausch et al., 2001). Bioinformatic analyses of M. truncatula expressed sequence tag collections and the complete rice (Oryza sativa) genome enabled the identification of the mycorrhiza-specific phosphate transporters MtPT4 and OsPT11. These two transporters share a high level of sequence identity but are less closely related to StPT3 (Harrison et al., 2002; Paszkowski et al., 2002). They also differ from StPT3 in that they are expressed exclusively in cells containing arbuscules, with the protein located on the periarbuscular membrane (Harrison et al., 2002). Finally, in contrast to StPT3, MtPT4 mediates low-affinity phosphate transport in yeast (Harrison et al., 2002). The relative contribution of these transporters to phosphate transport across the periarbuscular membrane remains to be determined. In the meantime, the differences between the phosphate transporters identified in these different plant species are intriguing, particularly because the phosphate transporters operating in nonmycorrhizal roots are relatively well conserved across species. Have these plant species evolved different styles of phosphate transporters to mediate symbiotic phosphate transport, or are there additional transporters, as yet unidentified, in each species? SUMMARY The development of model legume systems has revolutionized our understanding of plant symbioses. These models have provided the genetic and genomic platforms essential for dissecting the biological components that underpin these fascinating interactions. The recent isolation of a number of genes essential for both rhizobial and mycorrhizal signal perception provides significant insights into symbiotic signaling pathways and the communication that is required to establish peaceful relationships between these organisms. However, gene isolation is just the beginning, and defining the function of these proteins, in particular how they link Nod factor perception with activation and recognition of calcium signals, is a major challenge for the future. Although impressive in terms of the amount of data that has been generated, the output of the various “omics” technologies has so far been largely descriptive. Ultimately, we must work toward a better understanding of the genes/proteins and processes that are central to both SNF and AM and the integration of data from molecular, cellular, and physiological levels into seamless models of the whole system. 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Plant Physiol 131 : 1080 –1090 Wulf A, Manthey K, Doll J, Perlick AM, Linke B, Bekel T, Meyer F, Franken P, Kuster H, Krajinski F ( 2003 ) Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula. Mol Plant Microbe Interact 16 : 306 –314 Author notes * Corresponding author; e-mail [email protected]; fax 44–1603–450045. www.plantphysiol.org/cgi/doi/10.1104/pp.104.057661. © 2005 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Ramírez, Mario; Graham, Michelle A.; Blanco-López, Lourdes; Silvente, Sonia; Medrano-Soto, Arturo; Blair, Matthew W.; Hernández, Georgina; Vance, Carroll P.; Lara, Miguel

doi: 10.1104/pp.104.054999pmid: 15824284

Gepts, Paul; Beavis, William D.; Brummer, E. Charles; Shoemaker, Randy C.; Stalker, H. Thomas; Weeden, Norman F.; Young, Nevin D.

doi: 10.1104/pp.105.060871pmid: 15824285

On December 14 to 15, 2004, some 50 legume researchers and funding agency representatives (the latter as observers) met in Santa Fe, New Mexico, to develop a plan for cross-legume genomics research. This conference was one of the outcomes of the Legume Crops Genome Initiative (LCGI), an organization bringing together the major U.S. legume commodity associations and their respective research communities. The commodities include alfalfa (Medicago sativa), common bean (Phaseolus vulgaris), the cool-season food legumes (pea [Pisum sativum], lentil [Lens culinaris Med.], and chickpea [Cicer arietinum]), peanut (Arachis hypogaea), and soybean (Glycine max L. Merr.). In recent years, legume genomics has been focused primarily on the development of resources and information of two species considered to be model legumes (Medicago truncatula Gaertner and Lotus japonicus [Regel] K. Larsen) and soybean, the legume of principal economic importance in the United States (VandenBosch and Stacey, 2003). The development of similar genomics research in other legumes has lagged, which has limited the overall impact of genomics of the three reference species. Conversely, these three reference species could benefit from the sharing of biological information that already exists in other legume crops. Therefore, the main goal of this conference was to forge a common plan with specific objectives for cross-legume genomics research. The specific objectives of the conference were to: (1) identify a unifying goal for a cross-legume genome project; (2) identify cross-cutting themes to help integrate the different legume crop genomics programs, including a unified legume genomics information system, nutritional and health-related aspects of legumes, and detailed synteny and comparative genomics of legumes; and (3) outline specific components and milestones for this initiative. The conference was funded by the National Science Foundation (NSF) Plant Genome Research and the U.S. Department of Agriculture (USDA)/Cooperative State Research, Education, and Extension Service/National Research Initiative Plant Genome programs and organized by the authors of this report. A more detailed white paper resulting from this meeting will be posted on the meeting Web site (http://catg.ucdavis.edu). THE MULTIPLE ROLES OF LEGUMES An overview of the societal importance of legumes (Leguminosae or Fabaceae) and some of their most salient biological features provides ample justification for a significant investment in genomics of this botanical family. It also helps orient and prioritize this investment according to goals, species, and tools. With some 20,000 species, the legumes are the third largest family of higher plants. In comparison with other families with model species, the Gramineae have only some 10,000 species and the Brassicaceae some 3,500 species. This situation represents a challenge for comparative genomics, the identification of model species, and the determination of synteny. The Leguminosae are second to cereal crops in agricultural importance based on area harvested and total production. In 2004, more than 300 million metric tons of grain legumes were produced on 190 million ha (or about 13% of total land under cultivation, including arable land and land under permanent crops; http://faostat.fao.org/faostat/collections?subset=agriculture). The diverse roles of legume plants are often overlooked. Grain legumes provide about one-third of all dietary protein nitrogen and one-third of processed vegetable oil for human consumption (Graham and Vance, 2003). Seeds of grain legumes contain at least 20% to 40% of protein. In many places of the world, legumes complement cereals or root crops, the primary source of carbohydrates, in terms of amino acid composition. Whereas cereal seed proteins are deficient in Lys, legume seed proteins are deficient in sulfur-containing amino acids and Trp (Wang et al., 2003). This situation may explain why in most centers of crop domestication, legumes and cereals have been domesticated together (Gepts, 2004). Legumes are also important forages in temperate (e.g. alfalfa, clover [Trifolium spp.]) and tropical (Stylosanthes sp., Desmodium sp.) regions. Of note are tropical legume trees, which play a particularly important role as forage in arid areas (National Academy of Sciences, 1979) and as abundant timber in humid areas (Doyle and Luckow, 2003). Legumes also provide essential minerals required by humans (Grusak, 2002a) and produce health-promoting secondary compounds that can protect against human cancers (Grusak, 2002b; Madar and Stark, 2002) and protect the plant against the onslaught of pathogens and pests (Dixon et al., 2002; Ndakidemi and Dakora, 2003). In addition to their blood cholesterol-reducing effect (e.g. Andersen et al., 1984), grain legumes generally also have a hypoglycemic effect, reducing the increase in blood Glc after a meal and, hence, blood insulin. Legumes are, therefore, included in the diet of insulin-dependent diabetics (Jenkins et al., 2003). Certain legumes, however, produce antinutritional factors, such as trypsin inhibitors and phytohemagglutinins (Gupta, 1987) and allergens, the latter being a severe problem in peanut (Spergel and Fiedler, 2001). Genomics approaches, including metabolomics and proteomics, are essential to understanding the metabolic pathways that produce these antinutritional compounds and to eliminating these factors from the plant. The molecular signaling taking place between legumes and rhizobial symbionts, pollinators, and herbivores and carnivores suggests that legumes are an excellent model for the study of molecular signaling among organisms. Legume crops are of great significance because they produce substantial amounts of organic nitrogen fertilizer resulting from a symbiosis between the plant and bacterial symbionts (e.g. Jensen and Hauggaard-Nielsen, 2003; Hirsch, 2004). Rapid progress is being made in unraveling the molecular basis of this nitrogen-fixing symbiotic relationship, principally in the two model legumes M. truncatula and L. japonicus (Oldroyd et al., 2005). Translating this success to other legume species can be realized with genomic approaches (Stougaard, 2001; Broughton et al., 2003). In contrast with other botanical families, wind-pollinated species are extremely rare in the legumes, which are largely insect-pollinated or self-fertilized. Although not unique to the legumes, insect pollination is accompanied by adaptations in the plant host such as the development of specific morphological traits and the production of volatile attractants. Morphological traits include specific inflorescence types such as racemes and pseudoracemes and a zygomorphic (bilateral) flower symmetry (Tucker, 2003). Floral volatiles have been studied in several legumes, including, and not limited to, alfalfa and clover (Henning et al., 1992; Tava and Pecetti, 1997). Interestingly, leaf volatiles also play an important role in communications with insects, particularly as a defense mechanism to attract predators of herbivores (Pichersky and Gershenzon, 2002) in lima bean (Arimura et al., 2000) and L. japonicus (Ozawa et al., 2000). Traditionally, the legume family has been divided into three subfamilies: Caesalpinieae, Mimosoideae, and Papilionoideae. The grain legumes are included in the latter subfamily. Within the Papilionoideae, there are four important clades, which group most of the economically important food and feed legumes (Fig. 1 Figure 1. Open in new tabDownload slide Simplified schematic tree of legume family (modified from Doyle and Luckow, 2003). The three subfamilies (Caesalpinioideae, Mimosoideae, and Papilionoideae) and major subclades identified by recent molecular phylogenetic studies are shown in boldface (Kajita et al., 2001; Wojciechowski et al., 2004) and their positions are indicated by black circles (estimated number of taxa from Lewis et al., 2005, and ages [in millions of years] from Lavin et al., 2005). Figure 1. Open in new tabDownload slide Simplified schematic tree of legume family (modified from Doyle and Luckow, 2003). The three subfamilies (Caesalpinioideae, Mimosoideae, and Papilionoideae) and major subclades identified by recent molecular phylogenetic studies are shown in boldface (Kajita et al., 2001; Wojciechowski et al., 2004) and their positions are indicated by black circles (estimated number of taxa from Lewis et al., 2005, and ages [in millions of years] from Lavin et al., 2005). ; Doyle and Luckow, 2003). The genistoid clade includes the genus Lupinus and the aeschynomenoid/dalbergioid clade, the peanut. The third and fourth clades have a common ancestor. The Hologalegina clade is split into two subclades, one that includes the Loteae (L. japonicus) and the other that includes species with a chloroplast DNA characterized by the loss of one copy of the inverted repeat found in most angiosperm plants (hence called inverted repeat loss clade). The inverted repeat loss clade includes many species with temperate adaptation (cool-season legumes), such as alfalfa (and M. truncatula), chickpea, faba bean (Vicia faba), lentil, and pea. The fourth clade, the phaseoloid/millettioid clade, includes several legumes that are better adapted to more tropical climates (warm-season legumes), such as common bean, cowpea (Vigna unguiculata L. Walp.), pigeon pea (Cajanus cajan L. Millsp.), and soybean (Doyle and Luckow, 2003). Phylogenetic relationships within the legume family (Wojciechowski et al., 2004) are reflected in relatively high similarity or synteny at the genome level among the cool-season legumes, including Medicago sp. and pea (Kalo et al., 2004) or between the warm-season legumes common bean and soybean (Lee et al., 2001), but limited synteny is present among other legumes (for example, between cool-season and warm-season legumes; Choi et al., 2004; Zhu et al., 2005). Through a comprehensive assessment of synteny, comparative genomics to assess synteny can facilitate back-and-forth use of genomics resources between different legume species, making the research cost-effective and efficient. In addition, it can speed up gene identification in species that are less tractable because they have a large genome or are less easily transformed. Despite these studies assessing overall levels of synteny, no systematic determination of (micro- and macro-) syntenic relationships among legume species has been attempted as has been accomplished in cereals. Thus, a detailed determination of these relationships is a critical need to allow translation of genomic information among the legume reference and crop species. Bringing the genomic and biological knowledge in reference legumes to bear on other food and feed legumes of major economic importance, including cool-season pulses (e.g. pea, lentil, and chickpea), warm-season food legumes (e.g. peanut and common bean), and forage legumes (e.g. alfalfa and clover) represents a major scientific opportunity. Each legume presents unique features of economic and scientific interest. Examples include the geotropic peg and pod development of peanut (Pattee et al., 1998), drought tolerance and vernalization in chickpea (Abbo et al., 2002), forage nutritive value in alfalfa (Riday et al., 2002), biologically interesting mutants in pea (e.g. DeMason and Villani, 2001; Novak, 2003), evolution of domestication (Koinange et al., 1996), coevolution of host and pathogen/pests in common bean (e.g. Geffroy et al., 2000; Aguilar et al., 2004), and differential resistance of legume species to closely related groups of pathogens such as fusarium and rust (Gray et al., 1999; Tenuta, 2004). Examination of these specific features will require a combination of genomic resources from the reference species together with those developed in the crops themselves. A VISION: LEGUMES AS A MODEL PLANT FAMILY: GENOMICS FOR FOOD AND FEED The CATG conference participants agreed for the first time on the development of a 10-year prioritized plan for cross-legume genomics focused on the single theme of legume genomics for food and feed. Cross-legume genomics seeks to advance: (1) knowledge about the legume family as a whole; (2) understanding about the evolutionary origin of legume-characteristic features such as rhizobial symbiosis, flower and fruit development, and its nitrogen economy; and (3) pooling of genomic resources across legume species to address issues of scientific, agronomic, environmental, and societal importance. Thus, the CATG initiative seeks to develop the study of the organization and function of a unified legume genome in all its diversity. This implies translation of genomic information and tools developed for the reference legumes to other legumes and, conversely, utilization of the extensive biological and agronomic knowledge accumulated in crop legumes to improve our understanding of the biology of reference legumes. To be fully effective, a genome project across a botanical family like the Leguminosae needs to allow researchers to go back and forth among species and not just in one direction, i.e. from reference or model species to crop species. However, because resources are limited, the development of genomic tools needs to be carefully prioritized. The general theme of improvement of food and feed represents a clear vision for the future for legume genomics, as well as an emphatic statement directed primarily toward the public, who will be the ultimate beneficiary of genomic activities. This unified theme combines several areas of research (Fig. 2 Figure 2. Open in new tabDownload slide Major research areas supporting improved food and feed as a major goal of cross-legume genomics. Figure 2. Open in new tabDownload slide Major research areas supporting improved food and feed as a major goal of cross-legume genomics. ). First and foremost, it recognizes the importance of grain legumes (also known as pulses) as essential sources of dietary protein for humans and animals, as well as health-related phytochemicals such as dietary fiber, hormone analogs, and antioxidants. Genomics provides essential tools to fully understand the molecular and metabolic basis of the synthesis of these compounds, to increase their content in seeds and pods, and to better manipulate interactions between the plant's genetic makeup and its environment. A focus on seeds also underscores the importance of the genomics of reproductive biology in the development of higher-yielding, more nutritious legume cultivars. One of the signature features of legumes is the association between plants and rhizobial and mycorrhizal symbionts. The application of genomics has led to substantial and rapid advances in our understanding of the molecular basis of the two types of symbioses in M. truncatula and L. japonicus (Oldroyd et al., 2005). Studies of the rhizosphere in legumes are among the most developed of all botanical families and can lead to significant advances in plant health and growth. The common currency underlying protein-rich seed/forage and rhizobial symbiosis is nitrogen. The nitrogen-rich life style can explain, in part, the success and diversity of the legume family (McKey, 1994) and represents a significant contribution to agricultural and natural ecosystems. Comparative and functional genomics will extend the knowledge gained in the model legumes to crop legumes and should lead to more efficient nitrogen-fixing cultivars. Finally, the contributions of legumes could not be fully realized without low incidence of diseases and pests that affect the family. Recent advances in our understanding of disease resistance genes will be complemented by the application of genomics to fully understand the mechanisms by which legumes resist or tolerate pathogens and pests. Toward this end, molecular markers developed during genomics projects will assist breeders in developing new, resistant cultivars. A STRATEGY TOWARD UNIFIED LEGUME GENOMICS The path to better food and forage legumes requires a detailed knowledge of the different genes involved in the biochemical pathways leading up to key nutritional compounds, including the expression patterns and levels of these genes and their interactions. The tools of genomics, including bioinformatics and synteny analysis, provide the opportunity not only to obtain this comprehensive type of metabolical information, but also to integrate research efforts across different species. At the CATG conference, participants considered information on legume phylogeny and economic importance, among other factors, to establish a cross-legume genomics strategy and priorities for the development of legume genomic resources over the period of the coming 10 years. Specifically, four tiers of investment of genomic tools were recognized in the legumes. These tiers are described below (in decreasing order of priority and investment). The Reference Legumes M. truncatula, L. japonicus, and Soybean Cross-legume genomics research will be organized around two major clades that include most of the economically important legumes. These two clades correspond to the Hologalegina (cool-season legumes) and phaseoloid/millettioid (warm-season legumes) clades (Fig. 1; Doyle and Luckow, 2003). Jointly, they represent a large part of the economic legumes, especially for food and feed, and capture approximately 40% of the phenotypic variation among all legumes (J. Doyle, personal communication). The relatively broad taxonomic distance separating the two clades (Doyle and Luckow, 2003; Wojciechowski et al., 2004; Lavin et al., 2005) warrants the development of one or two reference systems within each one. M. truncatula and L. japonicus represent models for the cool-season legumes and soybean for the warm-season legumes. For each of these reference systems, considerable investments have already been made and should be continued in order to develop the full range of genomics resources, including sequencing of the entire genome or at the very least the gene-rich regions and extensive transcriptomics, proteomics, and metabolomics resources. Extended Genomic Tools for Targeted Species Phaseolus and Arachis Phaseolus and Arachis will be targeted for the development of a range of extended genomic resources. These include development of a physical map accompanied by bacterial artificial chromosome (BAC)-end sequencing, molecular markers based on BAC-end sequencing, anchoring of physical and genetic maps, expressed sequence tags (ESTs) of the major organs, especially those involved in reproductive development, microarray and DNA chip resources, and ultimately sequencing of the Phaseolus genome and gene-rich regions for Arachis. In addition to their economic importance on a worldwide basis, special arguments support more extensive development of genomic tools in these two crops. The small, diploid genome of Phaseolus is a key to understanding genome structure and expression in the phaseoloid/millettioid group (Fig. 1), which includes soybean. Arachis occupies a phylogenetically more distant position from the two foci proposed here (Fig. 1), providing perspective on the evolution of the foci. Translational Genomic Tools for Other Major Legume Crops and Targeted Experimental Systems All legumes belonging to the two foci will benefit from the development of translational tools that enable the sharing of genetic and genomics information among the various species. The translation tools consist primarily of cross-legume markers (extending the preliminary work of Brauner et al., 2002; Choi et al., 2004; and Kalo et al., 2004 by expanding the number of markers and species across the two major foci and peanut), EST libraries from several critical tissues, and BAC libraries with extensive coverage of the genome. In addition, for those species still lacking genetic maps and recombinant inbred populations, development of these resources should be assigned a high priority. The crops recommended for targeting include pea, lentil, chickpea, faba bean, alfalfa, clover, cowpea, pigeon pea, and lupin (Lupinus spp.). With progress in sequencing, some genomes in this group may be sequenced at least in part (gene-rich regions). They include the genomes of pea (to permit more productive use of the extensive biochemical and physiological literature for this species) and chickpea, one of the most drought-tolerant species among legumes of major economic importance. Develop Additional, Selected Legume Experimental Systems Some of the fundamental biological questions, such as the origin of legume-characteristic traits, require an evolutionary approach that encompasses the entire legume family. Such traits include reproductive development (especially floral and pod development), the origin of nodulation, and the evolution and importance of polyploidy. The two foci, while providing coverage for most economic legumes involved in food and feed, do not come close to covering the biodiversity included in the Fabaceae. Hence, to address the issue of legume-characteristic traits, other species may have to be considered, including species in the basal clades of the Papilionoideae, the Caesalpinieae (e.g. Chamaecrista sp.), and the Mimosoideae. In these species, ad hoc genomic resources targeted toward evolutionary genomics questions of interest will have to be developed to allow comparisons with reference and other legume species. TOOLS AND TIMELINE TO ADDRESS STRATEGIC GOALS The timeline is divided into short-term (years 1–3), medium-term (years 4–6), and long-term (years 7–10) periods. Structural Genomics, Genome Sequencing, and Synteny Mapping Genomics resources need to be developed or expanded within the reference legumes as well as for the major crop legumes to enhance the translation of genomics information across species. The CATG consensus, benchmarked over the next decade, envisions the following. Within 3 Years A legume synteny project should be completed, in which 500 gene-based markers developed primarily from important biochemical genes will be mapped across major crop legumes in at least one mapping population per species. These markers should be explicitly tied to whole genome sequences of reference species. Additional segregating populations useful for mapping important traits should be developed. Physical mapping should be initiated by end-sequencing BAC libraries currently available in crop species and fingerprinting BAC libraries that have been constructed from parents of mapping populations. A legume-wide and plant-wide workshop specifically addressing potential solutions to the long-term maintenance and conservation of genomic resources is called for. Within 6 Years A genome-wide survey of genetic diversity among germplasm accessions of each major legume species should be undertaken, using common markers to facilitate translation of the results. Evaluation of the extent of linkage disequilibrium in exotic and domesticated germplasm should be started. Phenotypic evaluation of multiple populations per species should be conducted so that the locations of quantitative trait loci for important agronomic traits, such as seed composition and yield, can be identified by genetic and association mapping. The accumulation of mapping information will facilitate the exploration of syntenic region across legumes. Physical maps should be constructed for common bean and diploid Arachis. The soybean genome, at least its gene-rich regions, should be sequenced by the fourth or fifth year. Within 10 Years The genome sequences of Phaseolus and Arachis and selected BAC sequencing in Pisum and Cicer should be completed. Further genetic mapping in all species will continue. Bioinformatics Scientists at the CATG conference made the following recommendations. Within 3 Years Develop a virtual, easy-to-navigate one-stop legume information network. By one-stop, we refer by analogy to Google and how it can be seen as a single, yet nonexclusive, information resource. Such a system should be able to semantically respond to queries for both information and services. Current and future legume information resources can be viewed as components to this network. Attribution to the sources of the data will help assure a willingness to register with the network and provide a peer-review mechanism for quality assurance. While it was beyond the scope of this meeting to recommend a particular technological approach to assure these attributes, there was considerable enthusiasm expressed for the utilization of the BioMOBY technology platforms (Schiltz et al., 2004) as a possible solution. Species-level biologist/curator of data and information and cross-species working groups composed of biologist/curators will be put in place. Within 6 Years Legume information resources will accommodate proteomic and metabolomic data and information as well as analysis and visualization tools for these emerging sources of information. Within 10 Years The legume community should become a leader in the development of new bioinformatics tools. There is an opportunity to do so with development of a one-stop legume information network. There are also opportunities to develop novel analysis and visualization tools for both integrative and comparative research questions. For example, there are needs for: (1) visualization of comparative maps at the level of linkage maps, physical maps (BAC-level), and homologous (orthologous) sequences; and (2) the development of researcher-centric (breeders, geneticists, biochemists, and molecular biologists) interfaces. These tools can be extended to develop common biological and bioinformatic frameworks for all plants and animals to maximize the benefits of comparative biology. Throughout the 10-Year Period As new resources become available, workshops, education, and ongoing feedback from user community will be needed for all bioinformatics efforts. The Increasingly Prominent Role of Functional Genomics (ESTs, Transcriptomics, Proteomics, and Metabolomics) Breakthroughs in understanding the relationship between genotype and phenotype will come about through proteomics and metabolomics. Legumes are especially appropriate for research in the areas of proteomics and metabolomics. As information on DNA sequences becomes more common in legumes, especially the reference legumes, the next steps will naturally focus increasingly on the expression of DNA sequence. Transcript profiling, proteomics, and metabolomics are essential tools to fully understand the synthesis of compounds that are at the basis of this research community's focus on food and feed. In addition to protein, carbohydrates, and lipids, an understanding of the secondary metabolism and mineral nutrition of legumes is an important goal. The secondary metabolism involves interactions between legumes and pathogens, symbiotic organisms, predators, and pollinators. It is also the basis for many of the nutritional benefits ascribed to legumes. Extensive transcriptomics (e.g. Colebatch et al., 2004), proteomics (Watson et al., 2004), and metabolomics (Sumner et al., 2003) programs are already under way in the model legumes M. truncatula and L. japonicus. Within 3 Years These resources need to be further developed in the two model legume species in parallel with the ongoing whole-genome sequencing efforts. The modest EST resources in major legume crops (other than soybean) must be expanded to include a broader range of tissues and environmental conditions. Within 6 Years Transcriptomic, proteomic, and metabolomic resources should be extended to soybean. Additional resources to be developed include full-length cDNA libraries from seed, mature leaf, nodule, flower, and pod for common bean and peanut from which DNA microarray and oligonucleotide gene chips can be developed for these two species. Within 10 Years Selected resources should be extended to major crop legumes. Protein fragment databases, gel fingerprints for the important tissues of the major legumes, chips, including arrays of legume transcription factors and proteins (or antibodies to the proteins) of the developing flower, seed, and root, should be developed. Education, Outreach, and Recruitment of Young Scientists Because of their important role in human nutrition and the environment, legumes offer many opportunities in education and outreach. Ever since Mendel first discovered the fundamental laws of genetics through his studies of garden peas, legumes have helped people to understand biology. Now classroom and laboratory activities can introduce important concepts like genetic inheritance, symbiosis, and seed and flower development to young people. Already, new legume-based projects for K-12 students are being developed, like the “Bacteria and Plants Team Up!” packet created at Indiana University with NSF Plant Genome support. In addition, the role of legumes on a worldwide basis as a source of food, forage, and timber can be highlighted, as well as their role in ecosystem health and the nitrogen cycle. In the process, the benefits of good nutrition can be introduced to young people by making them familiar with the central role of legumes and health-promoting compounds in their diet (e.g. the place of legumes in the USDA food pyramid). Furthermore, research experience in legume genomics and genetics for K-12 teachers can, for example, also be provided through the Research Experience for Teachers (RET) program of NSF. Workshops describing legume genomics and bioinformatics for applied plant scientists and crop breeders need to be developed and expanded. Bringing in the International Legume Community, Including Researchers Working on Legume Crops of the Developing World A mechanism will be established to ensure international coordination of legume genomics efforts and involving researchers, funding agencies, farmers, and other stakeholders. A large proportion of worldwide legume production occurs in developing countries, where legumes fulfill a vital role in human nutrition, as tropical forages and as timber species. For several species, such as common bean and chickpea, strong collaborative links among scientists throughout the world, including those of developing countries, will provide additional opportunities for funding and research and will significantly increase the impact of legume genomics. The Phaseolus genomics initiative (Phaseomics; Broughton et al., 2003), with representatives of over 20 countries, provides an example of current collaboration. The scope of some projects, such as genome sequencing is beyond the capacity of single countries (e.g. the International Medicago truncatula Genome Sequencing Initiative). In addition, legume genomics projects and research centers in other countries provide opportunities for collaborations. These include the European Grain Legumes Integrated Project, which is part of the Framework 6 Food Quality and Safety research program of the European Union (http://www.eugrainlegumes.org), the Australian Centre of Excellence for Integrative Legume Research at the University of Queensland (http://www.cilr.uq.edu.au), and the Generation Challenge Program of the Consultative Group for International Agricultural Research (http://www.generationcp.org/vw/index.php). The introduction of Asian soybean rust (Phakopsora pachyrhizi Sydow) into the United States highlights the potential not only for cross-legume genomics research (e.g. between common bean and soybean) but also for international collaborations given the widespread distribution of the pathogen in Asia, Africa, and the Americas. CONCLUSION This white paper marks a major departure from previous legume genomic research, which was focused primarily on the development of genomic tools and biological investigations for individual species. The approach endorsed by CATG participants and described in this paper represents a coordinated effort for the development and research involving genomics across the legume family. To paraphrase Bennetzen and Freeling (1997) when they spoke about cereals, the legume genomics community is now pursuing a unified legume genome in the hope of achieving synergy in synteny. ACKNOWLEDGMENTS We are grateful to our fellow legume researchers for their enthusiasm and positive spirits, which made the CATG meeting a success. We thank D. Zamir, J. Doyle, A. Hirsch, M. Grusak, and E. Pichersky for exciting and stimulating plenary lectures, and J. Doyle and M. Wojciechowsky for their assistance with Figure 1. LITERATURE CITED Abbo S, Lev-Yadun S, Galwey N ( 2002 ) Vernalization response of wild chickpea. New Phytol 154 : 695 –701 Aguilar OM, Riva O, Peltzer E ( 2004 ) Analysis of Rhizobium etli and of its symbiosis with wild Phaseolus vulgaris supports coevolution in centers of host diversification. Proc Natl Acad Sci USA 101 : 13548 –13553 Andersen JW, Story L, Sieling B, Chen W-JL, Petro MS, Story J ( 1984 ) Hypocholesterolemic effects of oat-bran or bean intake for hypercholesterolemic men. 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Plant Physiol 137 : 1189 –1196 Author notes 1 This work was supported by the National Science Foundation Plant Genome Research Program and by the U.S. Department of Agriculture/Cooperative State Research, Education, and Extension Service/National Research Initiative Plant Genome Program. * Corresponding author; e-mail [email protected]; fax 530–752–4361. www.plantphysiol.org/cgi/doi/10.1104/pp.105.060871. © 2005 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Rolletschek, Hardy; Hosein, Felicia; Miranda, Manoela; Heim, Ute; Götz, Klaus-Peter; Schlereth, Armin; Borisjuk, Ljudmilla; Saalbach, Isolde; Wobus, Ulrich; Weber, Hans

doi: 10.1104/pp.104.056523pmid: 15793070