The Plant Journal

O'Malley, David M.; Whetten, Ross; Bao, Wuli; Chen, Chen‐Loung; Sederoff, Ronald R.

doi: 10.1046/j.1365-313X.1993.04050751.xpmid: N/A

The enzymatic mechanism of monolignol polymerization in lignin biosynthesis is not known, although it has been the subject of significant interest for more than 60 years. Peroxidase had been considered to be the exclusive plant enzyme involved in the oxidative polymerization of lignin precursors. Recently, laccase and laccase‐like oxidase activities have been associated with lignification. Laccase is bound to lignifying plant cell walls and can polymerize lignin precursors in vitro. Strong circumstantial evidence from different species implicates this enzyme in the polymerization of lignin precursors. Lignin has a complex structure and it has been difficult to analyze the heterogeneity of lignin by chemical and physical techniques. If lignin precursors are polymerized by enzymes that differ in their catalytic properties, then lignin heterogeneity could be produced by differential expression of multiple enzymes during plant development. When laccase genes are correctly identified, these ideas can be tested in genetic experiments where gain or loss of function can be predicted by the presence or absence of the functional gene.

Youssefian, Shohab; Nakamura, Michimi; Sano, Hiroshi

doi: 10.1046/j.1365-313X.1993.04050759.xpmid: 8275097

O‐acetylserine (thiol) lyase (EC4.2.99.8) is the key enzyme in the cysteine biosynthetic pathway of plants and prokaryotes. The gene, cys1, encoding this enzyme was isolated from a wheat (Triticum aestivum L.) cDNA library, and its deduced amino acid sequence found to show 53% sequence identity with the O‐acetyl‐serine (thiol) lyase of Escherichia coli and Salmonella typhimurium. The deduced peptide consists of 325 amino acids (34.1 kDa), contains a conserved motif for the binding of pyridoxal phosphate, a co‐factor required for enzymatic activity, and an N‐terminal region of 37 amino acid residues resembling chloroplast transit peptides. The identity of cys1 was further demonstrated through complementation of an E. coli cysteine auxotroph, which lacks O‐acetylserine (thiol) lyase, by expression of the wheat gene. Northern blot analysis showed that cys1 is highly expressed in green vegetative and reproductive tissues and in the roots of wheat, as well as in the leaves of several plant species. Southern blot analysis demonstrated that the gene exists as a single copy in the wheat genome. Tobacco plants transformed with cys1 in the sense orientation (sense plants) or antisense orientation (antisense plants), under the control of the CaMV 35S promoter, showed high levels of transcripts. The O‐acetylserine (thiol) lyase activity in transgenic plants was determined, and found to be three‐ to fivefold higher in sense plants than in control plants, but unaffected in antisense transformants. Fumigation experiments with toxic levels of hydrogen sulphide (H2S) gas showed that while sense transformants were highly resistant, control and antisense plants were severely damaged by the treatment.

Garcia‐Maroto, Federico; Salamini, Francesco; Rohde, Wolfgang

doi: 10.1046/j.1365-313X.1993.04050771.xpmid: 7903890

Three alleles of the Deficiens‐homologous potato gene St‐Deficiens (St‐Def) present in the genome of a tetraploid Solanum tuberosum variety were isolated and characterized. For one allele (St‐Def pD13) the complete molecular structure was determined by sequence analysis and comparison with its cDNA, while for the other two alleles (pD10, pD12) only partial sequences of regulatory and coding regions were obtained. All three alleles showed (except for one amino acid exchange in pD10) identical sequences in the coding region. While sequence variation was observed within the respective promoters starting some 300 nt upstream of the putative transcriptional start site, the 3′ terminal promoter sequences were highly conserved. Within this region, a sequence of 81 nt was identified which showed 73% sequence identity to a corresponding sequence in the Deficiens promoter. This region which contains a putative serum response element was previously shown to regulate the expression of the Deficiens gene in Antirrhinum majus. Expression patterns for the three alleles in transgenic potato lines expressing St‐Def promoter/Gus constructs were identical. GUS activity was predominantly located in petals and stamens as expected for the activity of a Def‐homologous gene, but a significant level of expression was also detected in the ovary wall and in the vascular bundles supporting anthers and petals. The promoters were also active in abscission structures at the junction of flower stem and pedicel, as well as in anther stomium.

Dubald, Manuel; Barakate, Abdellah; Mandaron, Paul; Mache, Régis

doi: 10.1046/j.1365-313X.1993.04050781.xpmid: 8275098

Exopolygalacturonase (exoPG) is a pectin‐degrading enzyme abundant in maize pollen. Using immunochemistry and in situ hybridization it is shown that in addition to its presence in pollen, exoPG is also present in sporophytic tissues, such as the tapetum and mesophyll cells. The enzyme is located in the cytoplasm of pollen and of some mesophyll cells. In other mesophyll cells, the tapetum and the pollen tube, exoPG is located in the cell wall. The measurement of enzyme activity shows that exoPG is ubiquitous in the vegetative organs. These results suggest a general function for exoPG in cell wall edification or degradation. ExoPG is encoded by a closely related multigene family. The regulation of the expression of one of the exoPG genes was analyzed in transgenic tobacco. Reporter GUS activity was detected in anthers, seeds and stems but not in leaves or roots of transgenic plants. This strongly suggests that the ubiquitous presence of exoPG in maize is the result of the expression of different exoPG genes.

Hara‐Hishimura, Ikuko; Takeuchi, Yuka; Inoue, Kaori; Nishimura, Mikio

doi: 10.1046/j.1365-313X.1993.04050793.xpmid: 8275099

Cell fractionation of pulse‐chase‐labeled developing pumpkin cotyledons demonstrated that proprotein precursor to 2S albumin is transported from the endoplasmic reticulum to dense vesicles and then to the vacuoles, in which pro2S albumin is processed to the mature 2S albumin. Immunocytochemical analysis showed that dense vesicles of about 300 nm in diameter mediate the transport of pro2S albumin to the vacuoles.

Araki, Takashi; Komeda, Yoshibumi

doi: 10.1046/j.1365-313X.1993.04050801.xpmid: N/A

A modified method for studying the initiation of flowering in darkness (dark flowering, DF) in Arabidopsis thaliana has been developed, and the DF process has been examined with the aid of late‐flowering mutants. A majority of plants developed floral buds by the use of liquid‐shaken cultures in darkness. The late‐flowering phenotype in gi and co mutants and early‐flowering phenotype in a hy2 mutant disappeared in DF. It was found that wild‐type plants grown under DF conditions express light‐regulated genes and develop appropriate leaf architecture, as do the light‐grown plants, without the apparent differentiation of chloroplasts. The shift experiments from darkness to light revealed the critical duration of growth in darkness for the initiation of DF. These results indicate that the DF process to the initiation of flowering is a mode of development distinct from that in light in Arabidopsis.

Innes, Roger W.; Bisgrove, Sherryl R.; Smith, Nadine M.; Bent, Andrew F.; Staskawicz, Brian J.; Liu, Yi‐Chun

doi: 10.1046/j.1365-313X.1993.04050813.xpmid: 8275100

A new disease resistance locus in Arabidopsis, RPS3, was identified using a previously cloned avirulence gene from a non‐Arabidopsis pathogen. The avrB avirulence gene from the soybean pathogen Pseudomonas syringae pv. glycinea was transferred into a P. syringae pv. tomato strain that is virulent on Arabidopsis, and conversion to avirulence was assayed on Arabidopsis plants. The avrB gene had avirulence activity on most, but not all, Arabidopsis ecotypes. Of 53 ecotypes examined, 45 were resistant to a P. syringae pv. tomato strain carrying avrB, and eight were susceptible. The inheritance of this resistance was examined using crosses between the resistant ecotype Col‐0 and the susceptible ecotype Bla‐2. In F2 plants from this cross, the ratio of resistant:susceptible plants was approximately 3:1, indicating that resistance to P. syringae expressing avrB is determined by a single dominant locus in ecotype Col‐0, which we have designated RPS3. Using RFLP analysis, RPS3 was mapped to chromosome 3, adjacent to markers M583 and G4523, and ≤ 1 cM from another disease resistance locus, RPM1. In soybean, resistance to P. syringae strains that carry avrB is controlled by the locus RPG1. Thus, RPG1 and RPS3 both confer avrB‐specific disease resistance, suggesting that these genes may be homologs.

Parker, Jane E.; Szabó, Véronique; Staskawicz, Brian J.; Lister, Clare; Dean, Caroline; Daniels, Michael J.; Jones, Jonathan D.G.

doi: 10.1046/j.1365-313X.1993.04050821.xpmid: N/A

Peronospora parasitica causes downy mildew on crucifers. An isolate of P. parasitica (denoted NoCO2) was identified that infected Arabidopsis plants of the land race Columbia (Col‐0) but not plants of land race Landsberg erecta (La‐er). Segregation analysis of F2 plants derived from a La‐er x Col‐0 cross established that the resistance was inherited as a single locus, denoted RPP5. Macroscopic and microscopic examinations of inoculated La‐er and Col‐0 cotyledons showed that restriction of fungal growth in La‐er was accompanied by massive callose accumulation and death of plant cells in direct contact with points of attempted fungal penetration. La‐er x Col‐0 F1 plants exhibited an intermediate resistance response in all aspects of fungal development, indicating that RPP5 is semi‐dominant in its action. F8 recombinant inbred lines generated between La‐er and Col‐0 were used to map RPP5 to a narrow interval (<1.1 cM) on chromosome 4, utilizing existing restriction fragment length polymorphic (RFLP) markers and newly generated random amplified polymorphic DNA (RAPD) markers. The data provide a basis for the isolation of the RPP5 locus by positional cloning as a first step towards understanding recognitional specificity in plant‐pathogen interactions at a molecular level.

Misawa, Norihiko; Yamano, Shigeyuki; Linden, Hartmut; Felipe, Maria R.; Lucas, Mercedes; Ikenaga, Hiroshi; Sandmann, Gerhard

doi: 10.1046/j.1365-313X.1993.04050833.xpmid: 8275101

Among the enzymes involved in carotenoid biosynthesis, phytoene desaturase is considered to be a rate‐limiting enzyme in this pathway and is also the target of many bleaching herbicides. This enzyme shows diversity concerning its function and amino acid homology among various organisms. The phytoene desaturase gene crtl of Erwinia uredovora was expressed, the 5′‐region of which was fused to the sequence for the transit peptide of a pea Rubisco small subunit, in tobacco plants under the control of the CaMV 35S promoter. This chimeric gene product was targeted into chloroplasts and processed in the transgenic plants. The production and processing of the corresponding protein could be demonstrated by Western blotting. Immunogold localization showed that the location of the gene product Crtl was preferentially in the thylakoids. A radioactive labeling study using the leaves demonstrated enhanced activity for the synthesis of β‐carotene. In addition, the transgenic tobacco acquired elevated resistance to the bleaching herbicide norflurazon.

Rechinger, K.B.; Simpson, D.J.; Svendsen, I.; Cameron‐Mills, V.

doi: 10.1046/j.1365-313X.1993.04050841.xpmid: 7506098

Hordein synthesis, transport and deposition was analysed by immunocytochemistry in developing endosperm cells of wild‐type (Carlsberg II) and mutant varieties deficient in B hordein (hor2ca), γ1 hordein (Donetsky), γ2 hordein and minor B hordein polypeptides (Haisa), or γ3 hordein (Nevsky). In all varieties, hordein polypeptides were detected both in the cytoplasm as globules, ranging in diameter from 50 nm to 1.24 µm, and in the vacuole as protein bodies. In the cytoplasmic globules B and C hordein polypeptides are assembled as a core and are surrounded by an outer layer of γ1 and γ2 hordein. The globules apparently fuse several times in the cytoplasm before entering the vacuole. Absence of γ3 hordein in the mutant Nevsky leads to a dramatic change in hordein polypeptide targeting, the hordein storage proteins being largely deposited in the lumen of the rough endoplasmic reticulum. γ3 Hordein is unique among the sulphur‐rich hordein polypeptides, being monomeric and forming only intramolecular disulphide bridges, while the other B and γ hordein polypeptides are aggregated by intermolecular disulphide bridges. Retention of hordein in the rough endoplasmatic reticulum in the absence of γ3 hordein suggests that γ3 hordein may maintain the prolamin storage polypeptides in a transport competent state. The sequence of the mature γ3 hordein polypeptide was deduced from a cDNA clone, and compared with γ2 hordein. The epitope recognized by the γ1 + γ2 hordein‐specific BX monoclonal antibody used for immunocytochemistry was mapped to include E190 and K193, by synthesizing overlapping oligopeptides.