Introduction Serine carboxypeptidases (SCPs) are members of the α / β hydrolase proteins, which contain the highly conserved catalytic triad, Ser‐Asp‐His (Fricker and Leiter ). The catalytic triad is able to cleave the carboxyl‐terminal peptide bonds of their protein or peptide substrates (Fraser et al. ). SCP proteins have been identified in a variety of organisms, including plants (Lehfeldt et al. ; Li et al. ; Shirley et al. ; Zhou and Li ), yeast (Hayashi et al. ; Endrizzi et al. ), and mammals (Naggert et al. ; Chen et al. ). The Arabidopsis genome encodes a family of 51 proteins that are homologous to known SCPs (Fraser et al. ). Although functions of most of the SCPs or SCP‐like proteins (SCPLs) remain unknown, studies have indicated a diversity of functions for the Arabidopsis SCPs and SCPLs. For example, BRS1 (BRI1 Suppressor 1), a member of SCP family, is a secreted and biochemically functional carboxypeptidase (Zhou and Li ) and has been shown to function genetically in the brassinosteroid (BR) signaling pathway (Li et al. ). Other reports, however, indicate that some SCPs or SCPLs may function as acyltransferases (Wajant et al. ; Lehfeldt et al. ; Li and Steffens ; Mugford and Osbourn ) or sinapoyltransferases (Shirley et al. ; Fraser et al. ; Stehle et al. ), although they are grouped into the carboxypeptidase family due to their sequence similarity and conserved catalytic triad. SCPs or SCPLs have been extensively investigated for their functions in protein turnover, biosynthesis of metabolites, and mobilization of storage proteins during seed germination and organ senescence (Schaller ; Stehle et al. ; Mugford and Osbourn ). Studies have also revealed that some SCPs or SCPLs may be involved in signal transduction (Li et al. ), programmed cell death (Dominguez and Cejudo ), seed development (Cercos et al. ), and production of secondary metabolites necessary for herbivory defense, UV protection, and disease resistance (Lehfeldt et al. ; Shirley et al. ; Mugford et al. ). Our previous study showed that overexpression of BRS1 suppresses multiple bri1 defects, suggesting that BRS1 might play an important role at the early stage of the BRI1 signaling pathway (Li et al. ). The protease activity of BRS1 is required for its function in suppressing the phenotypes of a weak BRI1 allele, bri1‐5 . The presence of an N‐terminal signal peptide in BRS1 predicts that the protein should enter into the secretory pathway. Sequence analysis failed to identify any obvious endoplasmic reticulum or Golgi apparatus retention sequences. BRS1 was subsequently shown to be a secreted protein (Zhou and Li ). These observations are consistent with the findings that BRS1 can suppress two extracellular domain weak bri1 mutants, bri1‐5 and bri1‐9 (Li et al. ), but failed to suppress a loss‐of‐function cytoplasmic domain mutant bri1‐1 . BRS1 is a member of the SCP family in Arabidopsis . The fact that a loss‐of‐function allele of BRS1 does not show any significant phenotypes suggests functional redundancy among the family members. The gene with the highest sequence similarity to BRS1 is ECS1 ( Extra Carpels and Seeds 1 ). Phylogenetic analysis indicates that ECS1 is a putative type II SCPL protein (Fig. A). In addition, like BRS1 , ECS1 contains a predicted N‐terminal signal peptide that should lead ECS1 to the secretory pathway. To test whether members of the BRS1 family play redundant roles in suppressing bri1‐5 , five close BRS1 homologs were overexpressed in bri1‐5 plants under the 35S promoter. Results showed that three of the five BRS1 ‐related genes were able to partially suppress the defective phenotypes of bri1‐5 . Interestingly, ECS1 overexpression produced extra phenotypes including an increase in the number of carpels and seeds per silique in Arabidopsis thaliana . Phylogenetic tree of ECS 1 and closely related family members in Arabidopsis . (A) The full amino acid sequences of BRS1 , ECS 1 , and four ECS 1 ‐like genes ( ECL 1–4) were aligned by Clustal V (Thompson et al. ), and the phylogenetic tree was generated by neighbor‐joining (N‐J) program ( http://clustalw.genome.ad.jp/ ) with website default parameters. (B) Partial amino acid sequence comparison between ECS 1 and At2g24000. The MIPS prediction of At2g24000 has a stretch of 10 amino acid residues that are truly not present in the experimentally confirmed ECS 1 . Materials and Methods Plant materials and binary vectors for plant transformation Full‐length ECS1 complementary DNA (cDNA) was amplified from total RNA isolated from 2‐week‐old wild‐type (Col‐0) seedlings by reverse transcription polymerase chain reaction (RT‐PCR) using primers ECS1‐F 5′‐CTT GAG CTC ATG GCA AGA ACC CAC TTA CTC TTT CT‐3′ and ECS1‐R 5′‐CTT GAG CTC CTA ATA AGA TCT TGA AAG CTC ATT TC‐3′ and cloned into the binary vector pBIBKAN at the SacI sites (Wen et al. ). The inactive form of ECS1 (S179R or H437Q) was generated by the QuickChange Site‐Directed Mutagenesis Kit (Stratagene, Cedar Creek, TX). The primers used for the mutagenesis are ECS1‐S179R‐F 5′‐CTA CAT TGT TGG TGA AAG GTA CGC CGG TCA TTA TG TCC‐3′ and ECS1‐S179R‐R 5′‐GGA ACA TAATGA CCG GCG TAC CTT TCA CCA ACA ATG TAG‐3′; ECS1‐H437Q‐F 5′‐GTA GAG GGG GCG GGG CAG GAG GTG CCA TTC TTC CA‐3′ and ECS1‐H437Q‐R 5′‐TGG AAG AAT GGC ACC TCC TGC CCC GCC CCT CTT AC‐3′. The ECS1 or mECS1 ( S179R or H437Q ) fragments were driven by a dual‐enhancer CaMV35S promoter. The transformation constructs used in these studies were designated as 35S::ECS1 , 35S::mECS1 ( S179R ), and 35S::mECS1 ( H437Q ). The transgenic plants were generated in either bri1‐5 or WS/Col backgrounds. In each case, the homozygous transgenic plants were selected from the T3 generation and used for the analyses described in this article except as otherwise specified. All plants were grown in a 16‐h light and 8‐h dark growth chamber at 22°C, whereas transgenic seedlings were screened on half‐strength Murashige and Skoog Salt Mix (Invitrogen, Carlsbad, CA) with the addition of 0.7% agar and 30 μ g/mL of kanamycin and grown under continuous light at 22°C. Total RNA isolation and RT‐PCR analyses Total RNA was isolated using the RNeasy Plant Midi Kit from Qiagen (Germantown, MD). For reverse transcription, SuperScript II RNase H reverse transcriptase from Invitrogen was used. Two micrograms of total RNA was reverse transcribed to the first strand of the cDNA in a 20 μ L of total volume. One microliter of the RT product was used as a subsequent PCR template. Thirty cycles were used for amplifying ECS1 cDNA, and 20 cycles were used for amplifying the quantity control, ACT7 . Northern blot analyses Total RNA samples were isolated from 4‐week‐old aboveground tissues with an RNeasy Midi Kit. Twenty micrograms of total RNA were separated by a formaldehyde‐containing agarose gel, blotted onto Biotrans nylon membranes (ICN, Aurora, OH), and probed with 32 P‐labeled ECS1 cDNA fragments. After hybridization, the blots were exposed to X‐ray film for 1–2 days to detect the ECS1 expression in wild‐type and transgenic plants. Generation of double mutants A homozygous ECS1 ‐overexpressing line in WS background was crossed with det2‐101 , clv1‐2 , clv2‐3, and clv3‐2 . The F1 plants showed ECS1 ‐overexpressing phenotypes but no det2 or clavata mutant phenotypes. The F1 plants were allowed to self‐fertilize to generate a segregating F2 population. In the F2 progeny, double mutants were identified by genotyping individuals by PCR and subsequent DNA sequencing. The plants containing the ECS1 transgene have a kanamycin resistance gene. Plants survived on 1/2 MS media with kanamycin were first confirmed for the presence of ECS1 transgene by PCR genotyping. All the plants with the presence of ECS1 transgene were then genotyped for the homozygous locus of det2‐101, clv1‐2, clv2‐3, or clv3‐2 . Results ECS 1 cloning ESC1 corresponds to At2g24000 in Arabidopsis genome. ECS1 cDNA was amplified by RT‐PCR from wild type (ecotype: Col‐0) using two primers covering the entire predicted open reading frame of At2g24000 . Interestingly, sequencing of the PCR products indicated that the gene prediction for At2g24000 was incorrect. A stretch of 30 bp that was included at the end of the first exon of At2g24000 was actually not present in the amplified and sequenced ECS1 cDNA. This incorrect prediction results in an extra 10 amino acids in At2g24000 that are not present in ECS1 (Fig. B). The corrected ECS1 cDNA sequence was deposited in GenBank under the accession number of DQ785742 . Overexpression of ECS 1 partially suppresses bri1‐5 phenotypes ECS1 is closely related to BRS1 , an SCP that suppresses the phenotypes of bri1‐5 when overexpressed (Li et al. ). ECS1 shows 71% amino acid identity with BRS1 . The sequence identity between ECS1 and ECL1, ECL2, ECL3, and ECL4 (ECS‐Like 1–4) is 75%, 60%, 55%, and 55%, respectively. The homology in the central region of these proteins is lower than that of the N‐terminal and C‐terminal regions. We tested whether overexpression of ECS1 driven by a dual‐enhancer‐containing 35S promoter (Fang et al. ) also suppresses bri1‐5 phenotypes as observed in the overexpression of BRS1 . Rosette leaves in bri1‐5 are curly, and thus, the rosette of bri1 ‐5 plants is small in appearance. When ECS1 is overexpressed in bri1‐5 mutant background, the transgenic plants have flattened and expanded leaves, though the leaves still have some dimples (Fig. A). bri1‐5 plants flower 7–10 days later than wild‐type plants (ecotype WS), whereas ECS1 ‐overexpressing bri1‐5 plants flower 5–7 days earlier than the bri1‐5 plants, but still 2–3 days later than wild‐type plants (Fig. A). The transgenic plants overexpressing ECS1 cDNA show partial bri1‐5 suppression phenotypes in rosette leaves and flowering time. ECS 1 ‐overexpressing phenotype observations in bri1‐5 background. (A) Overexpression of 35S:: ECS 1 suppresses bri1‐5 phenotypes. Rosette leaves in bri1‐5 are curled, whereas ECS 1 ‐overexpressing plants have expanded leaves (left panel). bri1‐5 plants flower 7–10 days later than wild‐type plants, whereas ECS 1 ‐overexpressing plants flower 5–7 days earlier than bri1‐5 plants. The mature ECS 1 ‐overexpressing plants are about 30% taller than mature bri1‐5 plants (right panel). Scale bar = 1 cm. (B) Mature green siliques in bri1‐5 (left) and 35S:: ECS 1 bri1‐5 (right). Siliques in ECS 1 ‐overexpressing plants look “fatter” than bri1‐5 siliques. Scale bar = 10 mm. (C) Dissected single silique of bri1‐5 (left) and 35S:: ECS 1 bri1‐5 (right). Four carpels in 35S:: ECS 1 bri1‐5 contrast two carpels in bri1‐5 . Scale bar = 1 mm. It is worth noting that ECL1 shares 75% identity to ECS1 but lacks a predicted N‐terminal signal peptide. Interestingly, overexpression of Arabidopsis ECL1 in bri1‐5 does not suppress the bri1‐5 defects and does not cause the ECS1 silique phenotype (see description below), possibly due to different subcellular localization (data not shown). Overexpression of ECS1 not only partially suppresses bri1‐5 phenotypes but also produces a new phenotype that is not observed in overexpressing lines of BRS1 and ECLs. Similar to the wild‐type WS, the majority of the siliques on bri1‐5 plants contain two carpels. However, siliques of the ECS1 ‐overexpressing plants in bri1‐5 have four carpels that make the siliques “fatter” than those of bri1‐5 plants (Fig. B and C). Carpels are the ovule (seed)‐bearing organ in the gynoecium and the increased carpel numbers result in an elevated seed number per silique. A two‐carpel silique from bri1‐5 plants contains an average of 43.2 ± 2.6 seeds, whereas the four‐carpel silique from the ECS1 ‐overexpressing bri1‐5 plants has a seed number of 58.3 ± 2.8 seeds per silique (Table ). Increased seed numbers in ECS 1 ‐overexpressing plants Genotype bri1‐5 ECS1 bri1‐5 WS 35S::ECS1 Seed numbers per silique ( n = 1) 43.2 ± 2.6 58.3 ± 2.8 66.2 ± 3.6 88.1 ± 4.1 Overexpression of ECS 1 results in strongly fasciated stems in a det2 mutant Initially, ECS1 was overexpressed in a BR‐insensitive mutant bri1‐5, which is defective in the BR signaling pathway. To investigate whether the ECS1 ‐induced multi‐carpel phenotype is dependent on BR, ECS1 was overexpressed in a det2 mutant by genetic crossing. det2‐101 is a BR biosynthetic mutant with significantly reduced BR biosynthesis. det2‐101 plants show dwarf phenotypes (Li et al. ). We crossed a homozygous ECS1 ‐overexpressing line (in WS) with det2‐101 . F1 plants showed the ECS1 ‐overexpressing phenotype. At the rosette stage of a segregating F2 population, we removed all the plants that showed no det2 phenotypes. The remaining plants in the population were all homozygous for the det2 mutation, regardless of presence of 35S::ECS1 . Twenty‐four det2 ‐like plants in the F2 population were genotyped by primers specific for the ECS1 transgene. Among them, six plants did not contain the ECS1 transgene, indicating that they were det2 single mutants. Other 18 plants showed PCR amplification of ECS1 transgene (data not shown), suggesting that they were either homozygous or heterozygous for the ECS1 transgene. All 24 plants were indistinguishable from the seedling stage to 1 week after bolting. However, from 2 weeks after bolting, 7 of the 18 plants with the ECS1 transgene began to develop strongly fasciated stems with clustered flowers (Fig. B). Based on the ratio of the plants with and without the ECS1 transgene, as well as the fasciated stem phenotypes, these seven plants are most likely homozygous for both ECS1 transgene and det2 . det2 single mutant plants are sterile (occasionally a few siliques set a very limited number of seeds) and the sterile siliques have only two carpels. However, the plants with fasciated stems were partially fertile and the fruits had three carpels (Fig. C). These results suggest that the extra‐carpel phenotype caused by the ECS1 overexpression is independent of BRs. Phenotypes of ECS 1 overexpression in clavata and det2 mutants. (A) Phenotype comparison between clv2‐3 and 35S:: ECS 1 clv2‐3 (left) and between clv3‐2 and 35S:: ECS 1 clv3‐2 (right). Enhanced club‐shaped siliques were observed in both 35S:: ECS 1 clv2‐3 and 35S:: ECS 1 clv3‐2 plants. Scale bar = 10 mm. (B) Inflorescence phenotype of det2‐101 and 35S:: ECS 1 det2‐101 plants. det2‐101 mutant plants have normal inflorescence but fruits are sterile, while 35S:: ECS 1 det2‐101 plants have strongly fasciated stems and thus clustered flowers with partially fertile fruits developed. Scale bar = 1 cm. (C) Comparison of fruits of det2‐101 and 35S:: ECS 1 det2‐101 plants. det2‐101 plants have sterile fruits with two carpels (middle panel) while 35S:: ECS 1 det2‐101 plants have some fruits normally developed and the developed fruits have three carpels (right panel). Scale bar = 5 mm. Wild‐type plants overexpressing ECS 1 show an increased carpel phenotype The extra‐carpel and seed phenotype caused by overexpression of ECS1 was observed in bri1‐5 mutant backgrounds. It is reasonable to raise the question whether this phenotype can also be observed in the wild‐type background. We introduced the ECS1 ‐overexpressing construct into wild‐type WS and Columbia (Col‐0) by both genetic crossing and transformation. When ECS1 is overexpressed in wild type, a similar phenotype of increased carpels was observed, although there were no phenotypic alterations in rosette leaves (Fig. A). Wild‐type Arabidopsis plants (both WS and Col) have two carpels in each silique. In contrast, siliques of ECS1 ‐overexpressing lines have three carpels or even have four carpels. Wild‐type plants have an average seed number of 66.2 ± 3.6 seeds per silique, whereas ECS1 ‐overexpressing lines have 88.1 ± 4.1 seeds per silique (Table ). The weight of 1000 seeds from ECS1‐ overexpressing plants is not significantly different from that of wild type (data not shown), indicating that the seeds from the ECS1‐ overexpressing plants are of normal size. Microscopic examination showed that the shape of the seeds is also normal (Fig. B). However, the total seed weight per silique is increased by about 33% in ECS1 ‐overexpressing plants due to the increased total number of seeds. ECS 1 overexpression in wild‐type ( WS ) background. (A) Two‐week‐old wild‐type ( WS ) and ECS 1 ‐overexpressing plants. No significant differences were observed in rosettes between wild‐type and ECS 1 ‐overexpressing plants. Scale bar = 1 cm. (B) Seeds from ECS 1 ‐overexpressing line (right) are normal in shape and size relative to seeds from wild type (left). Seeds shown here are from one silique of wild‐type or ECS 1 ‐overexpressing plant. (C) Overexpression of ECS 1 in wild‐type Arabidopsis . Both Columbia and WS ecotypes express a very low level of ECS 1 (below the detection limit of Northern blot analysis), while the ECS 1 ‐overexpressing line (in WS background) has an apparently elevated ECS 1 expression level (top panel). ACT 7 was used as a probe to show equal loading of total RNA (bottom panel). A population of 28 plants heterozygous or homozygous for the ECS1 transgene and a population of 29 wild‐type plants were grown to maturity in the same room of the greenhouse. Total seeds were collected from each individual plant and measured to determine the total seed weight per plant. ECS1 ‐overexpressing plants produce 0.82 ± 0.16 g of seeds per plant, whereas wild‐type (WS) plants produce 0.77 ± 0.13 g of seeds per plant. Statistical analysis suggests that the seed yield from two populations is not statistically different ( P value = 0.178). As ECS1 ‐overexpressing plants produce more seeds per silique, this result suggests that the ECS1 ‐overexpressing plants have fewer siliques per plant. This is consistent with our observation that the ECS1 plants used in this study are slightly smaller than wild‐type plants and appear to produce fewer flowers under the same growth conditions as wild‐type plants. Transgenic plants expressing 35S:: mECS1 (S179R) or 35S:: mECS1 (H437Q) transgenes were also generated in Col‐0 background. Serine 179 and histidine 437 are two amino acids of the catalytic triad in ECS1 . Mutation of either of the corresponding amino acids in BRS1 (S181F and H438A) inactivates the enzymatic activity of BRS1 and abolishes its function in bri1‐5 suppression (Li et al. ; Zhou and Li ). Among 50 transgenic plants overexpressing 35S:: mECS1 (S179R or H437Q), none of them showed the multi‐carpel ECS1 ‐overexpressing phenotype (data not shown). This result suggests that the ECS1 enzymatic activity is required for the multiple‐carpel phenotype. Overexpression of ECS 1 enhances the clavata multi‐carpel phenotype A club‐shaped, multi‐carpel silique is a typical phenotype of clavata pathway mutants (Dievart and Clark ). To determine if overexpression of ECS1 modifies the multi‐carpel phenotype of the clavata mutants, we crossed an ECS1 ‐overexpressing line (in WS) with clv1‐2 , clv2‐3, and clv3‐2 . All the F1 plants showed ECS1 ‐overexpressing phenotypes, indicating the dominant effect of ECS1 overexpression. From the segregated F2 populations, plants homozygous for clv1‐2 with 35S::ECS1 did not show an altered phenotype. However, homozygous clv2‐3 and clv3‐2 plants with 35S::ECS1 exhibited an enhanced clv2‐3 or clv3‐2 multi‐carpel silique phenotypes. For example, there were significantly more carpels in the siliques of the ECS1 clv2‐3 and ECS1 clv3‐2 plants compared with the siliques of clv2‐3 and clv3‐2 single mutants (Fig. A). Many of the increased carpels are partial valves. Other than the enhanced multi‐carpel silique phenotype, we did not observe any other additional phenotypes from overexpression of ECS1 in clavata mutants. Knock‐out allele of ECS 1 shows no visible phenotype A single T‐DNA insertion allele of ECS1 was isolated from the SALK T‐DNA insertion population. In the ecs1 mutant (SALK_114735), the T‐DNA is inserted in the last exon of ECS1 (Fig. ). RT‐PCR analysis verified that the ecs1 is a null allele (Fig. ). Homozygous ecs1 mutant shows no visible phenotypes under normal growth conditions, similar to what was found in the knock‐out allele of brs1 (Li et al. ). Furthermore, a double mutant of ecs1 brs1 was also generated by genetic crossing, and no visible morphological difference was observed between the double mutant and either of the single mutants, indicating strong functional redundancy among the family members. T‐ DNA insertion line of ECS 1 shows no visible phenotypes. (A) Schematic diagram of the genomic ECS 1 and the site of T‐ DNA insertion. Filled rectangular boxes represent exons of ECS 1 , whereas introns are shown in solid lines. (B) RT‐PCR confirms that the T‐ DNA insertion line ( ecs1 ) is RNA ‐null. (C) There are no visible phenotypes in ecs1 compared with wild type (Col‐0) under normal growth conditions. Scale bar = 1 cm. Discussion Overexpression of a putative SCP, ECS1 , results in an increased number of carpels and seeds in A. thaliana . The increased carpel number phenotype of ECS1 overexpression is independent of BR biosynthesis. Moreover, overexpression of ECS1 enhances the multi‐carpel phenotype of clavata mutants. Although ECS1 overexpression led to a 33% increase in seed production in single siliques, the total seed yield per plant in Arabidopsis was not significantly increased because the total number of siliques in ECS1 ‐overexpressing plants was less than that of wild‐type plants. One possible reason for this result is that under our growth condition, nutrition may become a limiting factor for ECS1 ‐overexpressing plants. It is postulated that at the reproductive development stage of an angiosperm plant, flower initiation and development is determined by the number of potential fruits and seeds (Dosio et al. ). Resource allocation is adjusted by ovary and fruit development. Under a given growth condition, the development and maturation of increased seeds in ECS1 ‐overexpressing plants may use up available resources and therefore restrain further flower development. In contrast, if no seeds or limited seeds develop in opened flowers, an Arabidopsis plant can initiate a few hundred flowers (Butenko et al. ). The high homology among ECS1 , BRS1 , and other type II SCPs suggests that ECS1 is a SCP II (carboxypeptidase D)‐like protein. In addition, like BRS1 , ECS1 is predicted to have an N‐terminal signal peptide and is likely secreted. Overexpression of ECS1 not only partially suppresses bri1‐5 phenotypes but produces an extra‐carpel phenotype which is typical of clavata pathway mutants. CLAVATA 2 (CLV2) is a leucine‐rich repeat (LRR) protein that is proposed to form a complex with CLAVATA 1 (CLV1), an LRR receptor‐like protein kinase (Dievart and Clark ). CLAVATA 3 (CLV3) is a small, predicted extracellular peptide that acts with CLV1 as a ligand–receptor pair in coordinating meristem proliferation and differentiation (Fletcher et al. ). Our observations showed that overexpression of ECS1 results in an enhanced multi‐carpel phenotype in both clv2 and clv3 mutants, suggesting that ECS1 modify the function of CVL2 and CLV3. More interestingly, many of the enhanced carpels are partial valves, suggesting that ECS1 activates cell division in the developing gynoecium (Durbak and Tax ). The regulatory roles of SCPs in plants have not yet been investigated. On the basis of an analogy with BRS1 , we hypothesize that ECS1 will either process an unidentified protein factor that is involved in the control of carpel development. The processing step by ECS1 may be rate‐limiting. Such a model would suggest that elevated expression of ECS1 can increase the amount of the active form of the factor, which subsequently activates the signal transduction pathway involved in carpel development. As a result, extra carpels are formed and the number of seeds increases. Alternatively, ECS1 could also be responsible for eliminating a protein/peptide that is negatively involved in carpel and seed development. Further investigations in identifying the target(s) of ECS1 will be valuable to prove the current hypothesis. To the best of our knowledge, there were no other reports showing that overexpression of ECS1 or related genes produce an increased carpel and seed‐number phenotype in any plants. Because this family of genes are conserved in all plants, it is possible that overexpression of ECS1 in closely related crop species will increase yield and seed productivity and provide a potential measure of food security. Acknowledgments We thank Shulan Zhang for assistance in taking care of Arabidopsis planting, seed harvesting, and sorting. Research support was provided by the University of Missouri Food for the 21st Century Program. The nucleotide sequence for the ECS1 gene reported in this article was submitted to GenBank under the accession number DQ785742 .
Food and Energy Security – Wiley
Published: Jul 1, 2012