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.

Assessing Flowering Time Under Different Photoperiods.

Flowering time is one of the most important developmental transitions in plants, especially in annuals such as Arabidopsis thaliana. However, flowering is also a critical agronomic trait, as it impacts the level of vegetative biomass produced (e.g., leaves) or the amount of seed (grain) generated. Therefore, uncovering flowering phenotypes would help understand the impact of any regulatory network on the overall plant life cycle, since flowering integrates multiple cues, both environmental (e.g., photoperiod, temperature) and internal (e.g., induction/repression of specific genes, phytohormone accumulation, plant age). Although the photoperiod flowering pathway has been extensively studied, and its gene circuitry characterized in great detail, specific flowering time protocols are mostly accessible to specialized laboratories in this field. In this report, we address this knowledge gap by generating a reproducible, non-expensive, and step-by-step protocol to assess flowering time under different photoperiods. We provide a comprehensive description and highlight the major pitfalls in the process. Moreover, this protocol could be expanded to include temperature changes and thus contribute to assess the impact of both environmental conditions in the plant's decision to flower.

LEAFY activity is post-transcriptionally regulated by BLADE ON PETIOLE2 and CULLIN3 in Arabidopsis.

The Arabidopsis LEAFY (LFY) transcription factor is a key regulator of floral meristem emergence and identity. LFY interacts genetically and physically with UNUSUAL FLORAL ORGANS, a substrate adaptor of CULLIN1-RING ubiquitin ligase complexes (CRL1). The functionally redundant genes BLADE ON PETIOLE1 (BOP1) and -2 (BOP2) are potential candidates to regulate LFY activity and have recently been shown to be substrate adaptors of CULLIN3 (CUL3)-RING ubiquitin ligases (CRL3). We tested the hypothesis that LFY activity is controlled by BOPs and CUL3s in plants and that LFY is a substrate for ubiquitination by BOP-containing CRL3 complexes. When constitutively expressed, LFY activity is fully dependent on BOP2 as well as on CUL3A and B to regulate target genes such as APETALA1 and to induce ectopic flower formation. We also show that LFY and BOP2 proteins interact physically and that LFY-dependent ubiquitinated species are produced in vitro in a reconstituted cell-free CRL3 system in the presence of LFY, BOP2 and CUL3. This new post-translational regulation of LFY activity by CRL3 complexes makes it a unique transcription factor subjected to a positive dual regulation by both CRL1 and CRL3 complexes and suggests a novel mechanism for promoting flower development.

Sumoylation in plants: mechanistic insights and its role in drought stress.

Post-translational modification by SUMO is an essential process that has a major role in the regulation of plant development and stress responses. Such diverse biological functions are accompanied by functional diversification among the SUMO conjugation machinery components and regulatory mechanisms that has just started to be identified in plants. In this review, we focus on the current knowledge of the SUMO conjugation system in plants in terms of components, substrate specificity, cognate interactions, enzyme activity, and subcellular localization. In addition, we analyze existing data on the role of SUMOylation in plant drought tolerance in model plants and crop species, paying attention to the genetic approaches used to stimulate or inhibit endogenous SUMO conjugation. The role in drought tolerance of potential SUMO targets identified in proteomic analyses is also discussed. Overall, the complexity of SUMOylation and the multiple genetic and environmental factors that are integrated to confer drought tolerance highlight the need for significant efforts to understand the interplay between SUMO and drought.

Genetic control of inflorescence architecture in legumes.

The architecture of the inflorescence, the shoot system that bears the flowers, is a main component of the huge diversity of forms found in flowering plants. Inflorescence architecture has also a strong impact on the production of fruits and seeds, and on crop management, two highly relevant agronomical traits. Elucidating the genetic networks that control inflorescence development, and how they vary between different species, is essential to understanding the evolution of plant form and to being able to breed key architectural traits in crop species. Inflorescence architecture depends on the identity and activity of the meristems in the inflorescence apex, which determines when flowers are formed, how many are produced and their relative position in the inflorescence axis. Arabidopsis thaliana, where the genetic control of inflorescence development is best known, has a simple inflorescence, where the primary inflorescence meristem directly produces the flowers, which are thus borne in the main inflorescence axis. In contrast, legumes represent a more complex inflorescence type, the compound inflorescence, where flowers are not directly borne in the main inflorescence axis but, instead, they are formed by secondary or higher order inflorescence meristems. Studies in model legumes such as pea (Pisum sativum) or Medicago truncatula have led to a rather good knowledge of the genetic control of the development of the legume compound inflorescence. In addition, the increasing availability of genetic and genomic tools for legumes is allowing to rapidly extending this knowledge to other grain legume crops. This review aims to describe the current knowledge of the genetic network controlling inflorescence development in legumes. It also discusses how the combination of this knowledge with the use of emerging genomic tools and resources may allow rapid advances in the breeding of grain legume crops.

Crystal structure and functional characterization of photosystem II-associated carbonic anhydrase CAH3 in Chlamydomonas reinhardtii.

In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO2 and water into carbohydrates. The light-driven oxidation of water that provides the electrons and protons for the subsequent CO2 fixation takes place in photosystem II (PSII). Recent studies show that in higher plants, HCO3 (-) increases PSII activity by acting as a mobile acceptor of the protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested to improve proton removal from PSII, possibly by rapid reformation of HCO3 (-) from CO2. In this study, we investigated the interplay between PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometry measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and 2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in α-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 function with dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO2/HCO3 (-) on PSII activity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSII preparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at low pH and CO2 concentration.

Analysis of conifer FLOWERING LOCUS T/TERMINAL FLOWER1-like genes provides evidence for dramatic biochemical evolution in the angiosperm FT lineage.

In flowering plants, homologs of the Arabidopsis phosphatidylethanolamine-binding protein (PEBP) FLOWERING LOCUS T (FT) are key components in controlling flowering time. We show here that, although FT homologs are found in all angiosperms with completed genome sequences, there is no evidence to date that FT-like genes exist in other groups of plants. Through phylogeny reconstructions and heterologous expression, we examined the biochemical function of the Picea (spruces) and Pinus (pines) PEBP families - two gymnosperm taxa phylogenetically distant from the angiosperms. We have defined a lineage of gymnosperm PEBP genes, termed the FT/TERMINAL FLOWER1 (TFL1)-like genes, that share sequence characteristics with both the angiosperm FT- and TFL1-like clades. When expressed in Arabidopsis, FT/TFL1-like genes repressed flowering, indicating that the proteins are biochemically more similar to the angiosperm TFL1-like proteins than to the FT-like proteins. This suggests that the regulation of the vegetative-to-reproductive switch might differ in gymnosperms compared with angiosperms. Molecular evolution studies suggest that plasticity at exon 4 contributes to the divergence of FT-like function in floral promotion. In addition, the presence of FT-like genes in basal angiosperms indicates that the FT-like function emerged at an early stage during the evolution of flowering plants as a means to regulate flowering time.

Analysis of the developmental roles of the Arabidopsis gibberellin 20-oxidases demonstrates that GA20ox1, -2, and -3 are the dominant paralogs.

Gibberellin (GA) biosynthesis is necessary for normal plant development, with later GA biosynthetic stages being governed by multigene families. Arabidopsis thaliana contains five GA 20-oxidase (GA20ox) genes, and past work has demonstrated the importance of GA20ox1 and -2 for growth and fertility. Here, we show through systematic mutant analysis that GA20ox1, -2, and -3 are the dominant paralogs; their absence results in severe dwarfism and almost complete loss of fertility. In vitro analysis revealed that GA20ox4 has full GA20ox activity, but GA20ox5 catalyzes only the first two reactions of the sequence by which GA(12) is converted to GA(9). GA20ox3 functions almost entirely redundantly with GA20ox1 and -2 at most developmental stages, including the floral transition, while GA20ox4 and -5 have very minor roles. These results are supported by analysis of the gene expression patterns in promoter:ß-glucuronidase reporter lines. We demonstrate that fertility is highly sensitive to GA concentration, that GA20ox1, -2, and -3 have significant effects on floral organ growth and anther development, and that both GA deficiency and overdose impact on fertility. Loss of GA20ox activity causes anther developmental arrest, with the tapetum failing to degrade. Some phenotypic recovery of late flowers in GA-deficient mutants, including ga1-3, indicated the involvement of non-GA pathways in floral development.

Integrating long-day flowering signals: a LEAFY binding site is essential for proper photoperiodic activation of APETALA1.

The transition to flowering in Arabidopsis is characterized by the sharp and localized upregulation of APETALA1 (AP1) transcription in the newly formed floral primordia. Both the flower meristem-identity gene LEAFY (LFY) and the photoperiod pathway involving the FLOWERING LOCUS T (FT) and FD genes contribute to this upregulation. These pathways have been proposed to act independently but their respective contributions and mode of interaction have remained elusive. To address these questions, we studied the AP1 regulatory region. Combining in vitro and in vivo approaches, we identified which of the three putative LFY binding sites present in the AP1 promoter is essential for its activation by LFY. Interestingly, we found that this site is also important for the correct photoperiodic-dependent upregulation of AP1. In contrast, a previously proposed putative FD-binding site appears dispensable and unable to bind FD and we found no evidence for FD binding to other sites in the AP1 promoter, suggesting that the FT/FD-dependent activation of AP1 might be indirect. Altogether, our data give new insight into the interaction between the FT and LFY pathways in the upregulation of AP1 transcription under long-day conditions.

The Arabidopsis thaliana Med25 mediator subunit integrates environmental cues to control plant development.

Development in plants is controlled by abiotic environmental cues such as day length, light quality, temperature, drought, and salinity. These signals are sensed by a variety of systems and transmitted by different signal transduction pathways. Ultimately, these pathways are integrated to control expression of specific target genes, which encode proteins that regulate development and differentiation. The molecular mechanisms for such integration have remained elusive. We here show that a linear 130-amino-acids-long sequence in the Med25 subunit of the Arabidopsis thaliana Mediator is a common target for the drought response element binding protein 2A, zinc finger homeodomain 1, and Myb-like transcription factors which are involved in different stress response pathways. In addition, our results show that Med25 together with drought response element binding protein 2A also function in repression of PhyB-mediated light signaling and thus integrate signals from different regulatory pathways.

An antagonistic pair of FT homologs mediates the control of flowering time in sugar beet.

Cultivated beets (Beta vulgaris ssp. vulgaris) are unable to form reproductive shoots during the first year of their life cycle. Flowering only occurs if plants get vernalized, that is, pass through the winter, and are subsequently exposed to an increasing day length (photoperiod) in spring. Here, we show that the regulation of flowering time in beets is controlled by the interplay of two paralogs of the FLOWERING LOCUS T (FT) gene in Arabidopsis that have evolved antagonistic functions. BvFT2 is functionally conserved with FT and essential for flowering. In contrast, BvFT1 represses flowering and its down-regulation is crucial for the vernalization response in beets. These data suggest that the beet has evolved a different strategy relative to Arabidopsis and cereals to regulate vernalization.

Analysis of B function in legumes: PISTILLATA proteins do not require the PI motif for floral organ development in Medicago truncatula.

The B-class gene PISTILLATA (PI) codes for a MADS-box transcription factor required for floral organ identity in angiosperms. Unlike Arabidopsis, it has been suggested that legume PI genes contribute to a variety of processes, such as the development of floral organs, floral common petal-stamen primordia, complex leaves and N-fixing root nodules. Another interesting feature of legume PI homologues is that some of them lack the highly conserved C-terminal PI motif suggested to be crucial for function. Therefore, legume PI genes are useful for addressing controversial questions on the evolution of B-class gene function, including how they may have diverged in both function and structure to affect different developmental processes. However, functional analysis of legume PI genes has been hampered because no mutation in any B-class gene has been identified in legumes. Here we fill this gap by studying the PI function in the model legume species Medicago truncatula using mutant and RNAi approaches. Like other legume species, M. truncatula has two PI homologues. The expression of the two genes, MtPI and MtNGL9, has strongly diverged, suggesting differences in function. Our analyses show that these genes are required for petal and stamen identity, where MtPI appears to play a predominant role. However, they appear not to be required for development of the nodule, the common primordia or the complex leaf. Moreover, both M. truncatula PI homologues lack the PI motif, which indicates that the C-terminal motif is not essential for PI activity.

Structural basis for LEAFY floral switch function and similarity with helix-turn-helix proteins.

The LEAFY (LFY) protein is a key regulator of flower development in angiosperms. Its gradually increased expression governs the sharp floral transition, and LFY subsequently controls the patterning of flower meristems by inducing the expression of floral homeotic genes. Despite a wealth of genetic data, how LFY functions at the molecular level is poorly understood. Here, we report crystal structures for the DNA-binding domain of Arabidopsis thaliana LFY bound to two target promoter elements. LFY adopts a novel seven-helix fold that binds DNA as a cooperative dimer, forming base-specific contacts in both the major and minor grooves. Cooperativity is mediated by two basic residues and plausibly accounts for LFY's effectiveness in triggering sharp developmental transitions. Our structure reveals an unexpected similarity between LFY and helix-turn-helix proteins, including homeodomain proteins known to regulate morphogenesis in higher eukaryotes. The appearance of flowering plants has been linked to the molecular evolution of LFY. Our study provides a unique framework to elucidate the molecular mechanisms underlying floral development and the evolutionary history of flowering plants.

Genetic analysis reveals that C19-GA 2-oxidation is a major gibberellin inactivation pathway in Arabidopsis.

Bioactive hormone concentrations are regulated both at the level of hormone synthesis and through controlled inactivation. Based on the ubiquitous presence of 2beta-hydroxylated gibberellins (GAs), a major inactivating pathway for the plant hormone GA seems to be via GA 2-oxidation. In this study, we used various approaches to determine the role of C(19)-GA 2-oxidation in regulating GA concentration and GA-responsive plant growth and development. We show that Arabidopsis thaliana has five C(19)-GA 2-oxidases, transcripts for one or more of which are present in all organs and at all stages of development examined. Expression of four of the five genes is subject to feed-forward regulation. By knocking out all five Arabidopsis C(19)-GA 2-oxidases, we show that C(19)-GA 2-oxidation limits bioactive GA content and regulates plant development at various stages during the plant life cycle: C(19)-GA 2-oxidases prevent seed germination in the absence of light and cold stimuli, delay the vegetative and floral phase transitions, limit the number of flowers produced per inflorescence, and suppress elongation of the pistil prior to fertilization. Under GA-limited conditions, further roles are revealed, such as limiting elongation of the main stem and side shoots. We conclude that C(19)-GA 2-oxidation is a major GA inactivation pathway regulating development in Arabidopsis.

Floral initiation and inflorescence architecture: a comparative view.

BACKGROUND: A huge variety of plant forms can be found in nature. This is particularly noticeable for inflorescences, the region of the plant that contains the flowers. The architecture of the inflorescence depends on its branching pattern and on the relative position where flowers are formed. In model species such as Arabidopsis thaliana or Antirrhinum majus the key genes that regulate the initiation of flowers have been studied in detail and much is known about how they work. Studies being carried out in other species of higher plants indicate that the homologues of these genes are also key regulators of the development of their reproductive structures. Further, changes in these gene expression patterns and/or function play a crucial role in the generation of different plant architectures. SCOPE: In this review we aim to present a summarized view on what is known about floral initiation genes in different plants, particularly dicotyledonous species, and aim to emphasize their contribution to plant architecture.

Isolation of mtpim proves Tnt1 a useful reverse genetics tool in Medicago truncatula and uncovers new aspects of AP1-like functions in legumes.

Comparative studies help shed light on how the huge diversity in plant forms found in nature has been produced. We use legume species to study developmental differences in inflorescence architecture and flower ontogeny with classical models such as Arabidopsis thaliana or Antirrhinum majus. Whereas genetic control of these processes has been analyzed mostly in pea (Pisum sativum), Medicago truncatula is emerging as a promising alternative system for these studies due to the availability of a range of genetic tools. To assess the use of the retrotransposon Tnt1 for reverse genetics in M. truncatula, we screened a small Tnt1-mutagenized population using degenerate primers for MADS-box genes, known controllers of plant development. We describe here the characterization of mtpim, a new mutant caused by the insertion of Tnt1 in a homolog to the PROLIFERATING INFLORESCENCE MERISTEM (PIM)/APETALA1 (AP1)/SQUAMOSA genes. mtpim shows flower-to-inflorescence conversion and altered flowers with sepals transformed into leaves, indicating that MtPIM controls floral meristem identity and flower development. Although more extreme, this phenotype resembles the pea pim mutants, supporting the idea that M. truncatula could be used to complement analysis of reproductive development already initiated in pea. In fact, our study reveals aspects not shown by analysis of pea mutants: that the mutation in the AP1 homolog interferes with the specification of floral organs from common primordia and causes conversion of sepals into leaves, in addition to true conversion of flowers into inflorescences. The isolation of mtpim represents a proof of concept demonstrating that Tnt1 populations can be efficiently used in reverse genetics screenings in M. truncatula.