Valérie Hecht

The Arabidopsis somatic embryogenesis receptor kinase 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture

We report here the isolation of the Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (AtSERK1) gene and we demonstrate its role during establishment of somatic embryogenesis in culture. The AtSERK1 gene is highly expressed during embryogenic cell formation in culture and during early embryogenesis. The AtSERK1 gene is first expressed in planta during megasporogenesis in the nucellus [corrected] of developing ovules, in the functional megaspore, and in all cells of the embryo sac up to fertilization. After fertilization, AtSERK1 expression is seen in all cells of the developing embryo until the heart stage. After this stage, AtSERK1 expression is no longer detectable in the embryo or in any part of the developing seed. Low expression is detected in adult vascular tissue. Ectopic expression of the full-length AtSERK1 cDNA under the control of the cauliflower mosaic virus 35S promoter did not result in any altered plant phenotype. However, seedlings that overexpressed the AtSERK1 mRNA exhibited a 3- to 4-fold increase in efficiency for initiation of somatic embryogenesis. Thus, an increased AtSERK1 level is sufficient to confer embryogenic competence in culture.

The Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES1 and 2 Control Male Sporogenesis

The Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family of plasma membrane receptors consists of five closely related members. The SERK1 and SERK2 genes show a complex expression pattern throughout development. Both are expressed in anther primordia up to the second parietal division. After this point, expression ceases in the sporocytes and is continued in the tapetum and middle layer precursors. Single knockout mutants of SERK1 and SERK2 show no obvious phenotypes. Double mutants of SERK1 and SERK2 are completely male sterile due to a failure in tapetum specification. Fertility can be restored by a single copy of either gene. The SERK1 and SERK2 proteins can form homodimers or heterodimers in vivo, suggesting they are interchangeable in the SERK1/SERK2 signaling complex.

Conservation of Arabidopsis Flowering Genes in Model Legumes

The model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have provided a wealth of information about genes and genetic pathways controlling the flowering process, but little is known about the corresponding pathways in legumes. The garden pea (Pisum sativum) has been used for several decades as a model system for physiological genetics of flowering, but the lack of molecular information about pea flowering genes has prevented direct comparison with other systems. To address this problem, we have searched expressed sequence tag and genome sequence databases to identify flowering-gene-related sequences from Medicago truncatula, soybean (Glycine max), and Lotus japonicus, and isolated corresponding sequences from pea by degenerate-primer polymerase chain reaction and library screening. We found that the majority of Arabidopsis flowering genes are represented in pea and in legume sequence databases, although several gene families, including the MADS-box, CONSTANS, and FLOWERING LOCUS T/TERMINAL FLOWER1 families, appear to have undergone differential expansion, and several important Arabidopsis genes, including FRIGIDA and members of the FLOWERING LOCUS C clade, are conspicuously absent. In several cases, pea and Medicago orthologs are shown to map to conserved map positions, emphasizing the closely syntenic relationship between these two species. These results demonstrate the potential benefit of parallel model systems for an understanding of flowering phenology in crop and model legume species.

Sexual and Apomictic Reproduction in Hieracium subgenus Pilosella Are Closely Interrelated Developmental Pathways

Seed formation in flowering plants requires meiosis of the megaspore mother cell (MMC) inside the ovule, selection of a megaspore that undergoes mitosis to form an embryo sac, and double fertilization to initiate embryo and endosperm formation. During apomixis, or asexual seed formation, in Hieracium ovules, a somatic aposporous initial (AI) cell divides to form a structurally variable aposporous embryo sac and embryo. This entire process, including endosperm development, is fertilization independent. Introduction of reproductive tissue marker genes into sexual and apomictic Hieracium showed that AI cells do not express a MMC marker. Spatial and temporal gene expression patterns of other introduced genes were conserved commencing with the first nuclear division of the AI cell in apomicts and the mitotic initiation of embryo sac formation in sexual plants. Conservation in expression patterns also occurred during embryo and endosperm development, indicating that sexuality and apomixis are interrelated pathways that share regulatory components. The induction of a modified sexual reproduction program in AI cells may enable the manifestation of apomixis in Hieracium.

Pea LATE BLOOMER1 is a GIGANTEA ortholog with roles in photoperiodic flowering, deetiolation, and transcriptional regulation of circadian clock gene homologs

Genes controlling the transition to flowering have been studied in several species, including Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), but have not yet received much attention in legumes. Here, we describe a new allelic series of late-flowering, photoperiod-insensitive mutants in the pea (Pisum sativum) LATE BLOOMER1 (LATE1) gene and show that LATE1 is an ortholog of Arabidopsis GIGANTEA. Mutants display defects in phytochrome B-dependent deetiolation under red light and in the diurnal regulation of pea homologs of several Arabidopsis circadian clock genes, including TIMING OF CAB1, EARLY FLOWERING4, and CIRCADIAN CLOCK ASSOCIATED1/LATE ELONGATED HYPOCOTYL. LATE1 itself shows strongly rhythmic expression with a small but distinct acute peak following dark-to-light transfer. Mutations in LATE1 prevent the induction of a FLOWERING LOCUS T (FT) homolog FTL in long days but cause only minor alteration to the rhythmic expression pattern of the only known group Ia CONSTANS homolog COLa. The late-flowering phenotype of late1 mutants can be completely rescued by grafting to the wild type, but this rescue is not associated with a significant increase in FTL transcript level in shoot apices. Genetic interactions of late1 with the photoperiod-insensitive, early-flowering sterile nodes (sn) mutant and impairment of the LATE1 diurnal expression rhythm in sn plants suggest that SN may also affect the circadian clock. These results show that several functions of Arabidopsis GIGANTEA are conserved in its pea ortholog and demonstrate that genetic pathways for photoperiodic flowering are likely to be conserved between these two species. They also suggest that in addition to its role in the floral transition, LATE1 also acts throughout reproductive development.

The Pea GIGAS Gene Is a FLOWERING LOCUS T Homolog Necessary for Graft-Transmissible Specification of Flowering but Not for Responsiveness to Photoperiod

The pea flowering gene GIGAS regulates a mobile flowering signal and is essential for flowering under long days but not for the ability to respond to photoperiod. This study characterizes the FLOWERING LOCUS T (FT) gene family in pea, identifies one gene (FTa1) as GIGAS, and associates another gene (FTb2) with a second mobile signal and a broader role in photoperiod responsiveness. Garden pea (Pisum sativum) was prominent in early studies investigating the genetic control of flowering and the role of mobile flowering signals. In view of recent evidence that genes in the FLOWERING LOCUS T (FT) family play an important role in generating mobile flowering signals, we isolated the FT gene family in pea and examined the regulation and function of its members. Comparison with Medicago truncatula and soybean (Glycine max) provides evidence of three ancient subclades (FTa, FTb, and FTc) likely to be common to most crop and model legumes. Pea FT genes show distinctly different expression patterns with respect to developmental timing, tissue specificity, and response to photoperiod and differ in their activity in transgenic Arabidopsis thaliana, suggesting they may have different functions. We show that the pea FTa1 gene corresponds to the GIGAS locus, which is essential for flowering under long-day conditions and promotes flowering under short-day conditions but is not required for photoperiod responsiveness. Grafting, expression, and double mutant analyses show that GIGAS/FTa1 regulates a mobile flowering stimulus but also provide clear evidence for a second mobile flowering stimulus that is correlated with expression of FTb2 in leaf tissue. These results suggest that induction of flowering by photoperiod in pea results from interactions among several members of a diversified FT family.

A conserved molecular basis for photoperiod adaptation in two temperate legumes

Legumes were among the first plant species to be domesticated, and accompanied cereals in expansion of agriculture from the Fertile Crescent into diverse environments across the Mediterranean basin, Europe, Central Asia, and the Indian subcontinent. Although several recent studies have outlined the molecular basis for domestication and eco-geographic adaptation in the two main cereals from this region, wheat and barley, similar questions remain largely unexplored in their legume counterparts. Here we identify two major loci controlling differences in photoperiod response between wild and domesticated pea, and show that one of these, HIGH RESPONSE TO PHOTOPERIOD (HR), is an ortholog of EARLY FLOWERING 3 (ELF3), a gene involved in circadian clock function. We found that a significant proportion of flowering time variation in global pea germplasm is controlled by HR, with a single, widespread functional variant conferring altered circadian rhythms and the reduced photoperiod response associated with the spring habit. We also present evidence that ELF3 has a similar role in lentil, another major legume crop, with a distinct functional variant contributing to reduced photoperiod response in cultivars widely deployed in short-season environments. Our results identify the factor likely to have permitted the successful prehistoric expansion of legume cultivation to Northern Europe, and define a conserved genetic basis for major adaptive changes in flowering phenology and growth habit in an important crop group.

Light regulation of gibberellin biosynthesis in pea is mediated through the COP1/HY5 pathway.

Light regulation of gibberellin (GA) biosynthesis occurs in several species, but the signaling pathway through which this occurs has not been clearly established. We have isolated a new pea (Pisum sativum) mutant, long1, with a light-dependent elongated phenotype that is particularly pronounced in the epicotyl and first internode. The long1 mutation impairs signaling from phytochrome and cryptochrome photoreceptors and interacts genetically with a mutation in LIP1, the pea ortholog of Arabidopsis thaliana COP1. Mutant long1 seedlings show a dramatic impairment in the light regulation of active GA levels and the expression of several GA biosynthetic genes, most notably the GA catabolism gene GA2ox2. The long1 mutant carries a nonsense mutation in a gene orthologous to the ASTRAY gene from Lotus japonicus, a divergent ortholog of the Arabidopsis bZIP transcription factor gene HY5. Our results show that LONG1 has a central role in mediating the effects of light on GA biosynthesis in pea and demonstrate the importance of this regulation for appropriate photomorphogenic development. By contrast, LONG1 has no effect on GA responsiveness, implying that interactions between LONG1 and GA signaling are not a significant component of the molecular framework for light–GA interactions in pea.

Update on the genetic control of flowering in garden pea

The garden pea has been a model for the genetics of flowering for several decades and numerous flowering loci have been identified, but until recently little was known about the molecular nature of these loci. This paper presents an update on recent work on the molecular genetics of flowering in pea, outlining progress in gene and mutant isolation, expression analyses, grafting and other physiological studies, and candidate gene assessment. Work so far has led to the identification of the LATE1 and DNE loci as orthologues of Arabidopsis GIGANTEA and ELF4, respectively, and candidate genes for several other loci are being evaluated. Expression analysis of an expanded FT-like gene family suggests a more complex role for this group of genes. These results provide the first insight into the circadian clock, photoperiod response mechanism, and mobile signals in pea, and identify both conserved and divergent features in comparison with Arabidopsis.

DIE NEUTRALIS and LATE BLOOMER 1 Contribute to Regulation of the Pea Circadian Clock

The DIE NEUTRALIS (DNE) locus in garden pea (Pisum sativum) was previously shown to inhibit flowering under noninductive short-day conditions and to affect a graft-transmissible flowering signal. In this study, we establish that DNE has a role in diurnal and/or circadian regulation of several clock genes, including the pea GIGANTEA (GI) ortholog LATE BLOOMER 1 (LATE1) and orthologs of the Arabidopsis thaliana genes LATE ELONGATED HYPOCOTYL and TIMING OF CHLOROPHYLL A/B BINDING PROTEIN EXPRESSION 1. We also confirm that LATE1 participates in the clock and provide evidence that DNE is the ortholog of Arabidopsis EARLY FLOWERING4 (ELF4). Circadian rhythms of clock gene expression in wild-type plants under constant light were weaker in pea than in Arabidopsis, and a number of differences were also seen in the effects of both DNE/ELF4 and LATE1/GI on clock gene expression. Grafting studies suggest that DNE controls flowering at least in part through a LATE1-dependent mobile stimulus, and dne mutants show elevated expression of a FLOWERING LOCUS T homolog under short-day conditions. However, the early flowering of the dne mutant is not associated with altered expression of a previously described CONSTANS-like gene. Collectively, our results characterize the clock system and reveal its importance for photoperiod responsiveness in a model legume.