Identification and characterization of putative targets of VEGETATIVE1/FULc, a key regulator of development of the compound inflorescence in pea and related legumes

Inflorescence architecture contributes to essential plant traits. It determines plant shape, contributing to morphological diversity, and also determines the position and number of flowers and fruits produced by the plant, thus influencing seed yield. Most legumes have compound inflorescences, where flowers are produced in secondary inflorescences (I2), formed at the flanks of the main primary inflorescence (I1), in contrast to simple inflorescences of plants like Arabidopsis, in which flowers are directly formed on the I1. The pea VEGETATIVE1/FULc (VEG1) gene, and its homologs in other legumes, specify the formation of the I2 meristem, a function apparently restricted to legumes. To understand the control of I2 development, it is important to identify the genes working downstream of VEG1. In this study, we adopted a novel strategy to identify genes expressed in the I2 meristem, as potential regulatory targets of VEG1. To identify pea I2-meristem genes, we compared the transcriptomes of inflorescence apices from wild-type and mutants affected in I2 development, such as proliferating inflorescence meristems (pim, with more I2 meristems), and veg1 and vegetative2 (both without I2 meristems). Analysis of the differentially expressed genes using Arabidopsis genome databases combined with RT-qPCR expression analysis in pea allowed the selection of genes expressed in the pea inflorescence apex. In situ hybridization of four of these genes showed that all four genes are expressed in the I2 meristem, proving our approach to identify I2-meristem genes was successful. Finally, analysis by VIGS (virus-induced gene silencing) in pea identified one gene, PsDAO1, whose silencing leads to small plants, and another gene, PsHUP54, whose silencing leads to plants with very large stubs, meaning that this gene controls the activity of the I2 meristem. PsHUP54-VIGS plants are also large and, more importantly, produce large pods with almost double the seeds as the control. Our study shows a new useful strategy to isolate I2-meristem genes and identifies a novel gene, PsHUP54, which seems to be a promising tool to improve yield in pea and in other legumes.Peer reviewe

Defining the components of the miRNA156-SPL-miR172 aging pathway in pea and their expression relative to changes in leaf morphology

International audienceThe timing of developmental phase transitions is crucial for plant reproductive success, and two microRNAs (miRNA), miR156 and miR172, are implicated in the control of these changes, together with their respective SQUAMOSA promoter binding-like (SPL) and APETALA2-like (AP2) targets. While their patterns of regulation have been studied in a growing range of species, to date they have not been examined in pea (Pisum sativum), an important legume crop and model species. We analysed the recently released pea genome and defined nine miR156, 21 SPL, four miR172, and five AP2-like genes. Phylogenetic analysis of the SPL genes in pea, Medicago and Arabidopsis confirmed the eight previously defined clades, and identified a ninth potentially legume-specific SPL clade in pea and Medicago. Among the PsSPL, 14 contain a miR156 binding site and all five AP2-like transcription factors in pea include a miR172 binding site. Phylogenetic relationships, expression levels and temporal expression changes identified PsSPL2a/3a/3c/6b/9a/9b/13b/21, PsmiR156d/j and PsmiR172a/d as the most likely of these genes to participate in phase change in pea. Comparisons with leaf morphology suggests that vegetative phase change is unlikely to be definitively marked by a change in leaflet number. In addition, the timing of FT gene induction suggests that the shift from the juvenile to the adult vegetative phase may occur within fourteen days in plants grown under inductive conditions, and calls into question the contribution of miR172/AP2 to the floral transition. This work provides the first insight into the nature of vegetative phase change in pea, and an important foundation for future functional studies

The Cryptochrome Gene Family in Pea Includes Two Differentially Expressed CRY2 Genes

The cryptochromes are a family of blue light photoreceptors that play important roles in the control of plant development. We have characterised the cryptochrome gene family in the model legume garden pea (Pisum sativum L.). Pea contains three expressed cryptochrome genes; a single CRY1 orthologue, and two distinct CRY2 genes that we have termed CRY2a and CRY2b. Genomic southern blots indicate that there are unlikely to be more CRY genes in pea. Each of the three genes encodes a full-length CRY protein that contains all the major domains characteristic of other higher plant cryptochromes. Database searches have identified Medicago truncatula expressed sequence tags (ESTs) corresponding to all three genes, whereas only a single CRY2 is represented in EST collections from the more distantly related legumes soybean and Lotus japonicus. The proteins encoded by the pea and Medicago CRY2b genes are distinguished from other CRY2 proteins by their shorter C-terminus. Expression analyses have identified marked differences in the regulation of the three genes, with CRY2b expression in particular distinguished by high-amplitude diurnal cycling and rapid repression in seedlings transferred from darkness to blue light

Cryptochrome 1 Contributes to Blue-Light Sensing in Pea

Cryptochromes are widespread in higher plants but their physiological roles as blue-light photoreceptors have been examined in relatively few species. Screening in a phyA null mutant background has identified several blue-light response mutants in pea (Pisum sativum), including one that carries a substitution of a highly conserved glycine residue in the N-terminal photolyase-homologous domain of the pea CRY1 gene. Analyses of cry1, phyA, and phyB mutants show that all three photoreceptors contribute to seedling photomorphogenesis under high-irradiance blue light, whereas phyA is the main photoreceptor active under low irradiances. Triple phyA phyB cry1 mutants grown under high-irradiance blue light are indistinguishable from dark-grown wild-type plants in length and leaf expansion but show a small residual response to higher-irradiance white light. Monogenic cry1 mutants have little discernable phenotype at the seedling stage, but later in development are more elongated than wild-type plants. In addition, the loss of cry1 moderates the short-internode phenotype of older phyA mutants, suggesting an antagonism between phyA and cry1 under some conditions. Pea cry1 has a small inhibitory effect on flowering under long and short days. However, the phyA cry1 double mutant retains a clear promotion of flowering in response to blue-light photoperiod extensions, indicating a role for one or more additional blue-light photoreceptors in the control of flowering in pea

Table_2_Identification and characterization of putative targets of VEGETATIVE1/FULc, a key regulator of development of the compound inflorescence in pea and related legumes.xlsx

Inflorescence architecture contributes to essential plant traits. It determines plant shape, contributing to morphological diversity, and also determines the position and number of flowers and fruits produced by the plant, thus influencing seed yield. Most legumes have compound inflorescences, where flowers are produced in secondary inflorescences (I2), formed at the flanks of the main primary inflorescence (I1), in contrast to simple inflorescences of plants like Arabidopsis, in which flowers are directly formed on the I1. The pea VEGETATIVE1/FULc (VEG1) gene, and its homologs in other legumes, specify the formation of the I2 meristem, a function apparently restricted to legumes. To understand the control of I2 development, it is important to identify the genes working downstream of VEG1. In this study, we adopted a novel strategy to identify genes expressed in the I2 meristem, as potential regulatory targets of VEG1. To identify pea I2-meristem genes, we compared the transcriptomes of inflorescence apices from wild-type and mutants affected in I2 development, such as proliferating inflorescence meristems (pim, with more I2 meristems), and veg1 and vegetative2 (both without I2 meristems). Analysis of the differentially expressed genes using Arabidopsis genome databases combined with RT-qPCR expression analysis in pea allowed the selection of genes expressed in the pea inflorescence apex. In situ hybridization of four of these genes showed that all four genes are expressed in the I2 meristem, proving our approach to identify I2-meristem genes was successful. Finally, analysis by VIGS (virus-induced gene silencing) in pea identified one gene, PsDAO1, whose silencing leads to small plants, and another gene, PsHUP54, whose silencing leads to plants with very large stubs, meaning that this gene controls the activity of the I2 meristem. PsHUP54-VIGS plants are also large and, more importantly, produce large pods with almost double the seeds as the control. Our study shows a new useful strategy to isolate I2-meristem genes and identifies a novel gene, PsHUP54, which seems to be a promising tool to improve yield in pea and in other legumes.</p

Table_3_Identification and characterization of putative targets of VEGETATIVE1/FULc, a key regulator of development of the compound inflorescence in pea and related legumes.xlsx

Inflorescence architecture contributes to essential plant traits. It determines plant shape, contributing to morphological diversity, and also determines the position and number of flowers and fruits produced by the plant, thus influencing seed yield. Most legumes have compound inflorescences, where flowers are produced in secondary inflorescences (I2), formed at the flanks of the main primary inflorescence (I1), in contrast to simple inflorescences of plants like Arabidopsis, in which flowers are directly formed on the I1. The pea VEGETATIVE1/FULc (VEG1) gene, and its homologs in other legumes, specify the formation of the I2 meristem, a function apparently restricted to legumes. To understand the control of I2 development, it is important to identify the genes working downstream of VEG1. In this study, we adopted a novel strategy to identify genes expressed in the I2 meristem, as potential regulatory targets of VEG1. To identify pea I2-meristem genes, we compared the transcriptomes of inflorescence apices from wild-type and mutants affected in I2 development, such as proliferating inflorescence meristems (pim, with more I2 meristems), and veg1 and vegetative2 (both without I2 meristems). Analysis of the differentially expressed genes using Arabidopsis genome databases combined with RT-qPCR expression analysis in pea allowed the selection of genes expressed in the pea inflorescence apex. In situ hybridization of four of these genes showed that all four genes are expressed in the I2 meristem, proving our approach to identify I2-meristem genes was successful. Finally, analysis by VIGS (virus-induced gene silencing) in pea identified one gene, PsDAO1, whose silencing leads to small plants, and another gene, PsHUP54, whose silencing leads to plants with very large stubs, meaning that this gene controls the activity of the I2 meristem. PsHUP54-VIGS plants are also large and, more importantly, produce large pods with almost double the seeds as the control. Our study shows a new useful strategy to isolate I2-meristem genes and identifies a novel gene, PsHUP54, which seems to be a promising tool to improve yield in pea and in other legumes.</p