Analysis of pea mutants reveals the conserved role of FRUITFULL controlling the end of flowering and its potential to boost yield.

Monocarpic plants have a single reproductive phase in their life. Therefore, flower and fruit production are restricted to the length of this period. This reproductive strategy involves the regulation of flowering cessation by a coordinated arrest of the growth of the inflorescence meristems, optimizing resource allocation to ensure seed filling. Flowering cessation appears to be a regulated phenomenon in all monocarpic plants. Early studies in several species identified seed production as a major factor triggering inflorescence proliferative arrest. Recently, genetic factors controlling inflorescence arrest, in parallel to the putative signals elicited by seed production, have started to be uncovered in Arabidopsis, with the MADS-box gene FRUITFULL (FUL) playing a central role in the process. However, whether the genetic network regulating arrest is also at play in other species is completely unknown. Here, we show that this role of FUL is not restricted to Arabidopsis but is conserved in another monocarpic species with a different inflorescence structure, field pea, strongly suggesting that the network controlling the end of flowering is common to other plants. Moreover, field trials with lines carrying mutations in pea FUL genes show that they could be used to boost crop yield.

Arabidopsis thaliana SHOOT MERISTEMLESS Substitutes for Medicago truncatula SINGLE LEAFLET1 to Form Complex Leaves and Petals.

LEAFY plant-specific transcription factors, which are key regulators of flower meristem identity and floral patterning, also contribute to meristem activity. Notably, in some legumes, LFY orthologs such as Medicago truncatula SINGLE LEAFLET (SGL1) are essential in maintaining an undifferentiated and proliferating fate required for leaflet formation. This function contrasts with most other species, in which leaf dissection depends on the reactivation of KNOTTED-like class I homeobox genes (KNOXI). KNOXI and SGL1 genes appear to induce leaf complexity through conserved downstream genes such as the meristematic and boundary CUP-SHAPED COTYLEDON genes. Here, we compare in M. truncatula the function of SGL1 with that of the Arabidopsis thaliana KNOXI gene, SHOOT MERISTEMLESS (AtSTM). Our data show that AtSTM can substitute for SGL1 to form complex leaves when ectopically expressed in M. truncatula. The shared function between AtSTM and SGL1 extended to the major contribution of SGL1 during floral development as ectopic AtSTM expression could promote floral organ identity gene expression in sgl1 flowers and restore sepal shape and petal formation. Together, our work reveals a function for AtSTM in floral organ identity and a higher level of interchangeability between meristematic and floral identity functions for the AtSTM and SGL1 transcription factors than previously thought.

The SINGLE FLOWER (SFL) gene encodes a MYB transcription factor that regulates the number of flowers produced by the inflorescence of chickpea.

Legumes usually have compound inflorescences, where flowers/pods develop from secondary inflorescences (I2), formed laterally at the primary inflorescence (I1). Number of flowers per I2, characteristic of each legume species, has important ecological and evolutionary relevance as it determines diversity in inflorescence architecture; moreover, it is also agronomically important for its potential impact on yield. Nevertheless, the genetic network controlling the number of flowers per I2 is virtually unknown. Chickpea (Cicer arietinum) typically produces one flower per I2 but single flower (sfl) mutants produce two (double-pod phenotype). We isolated the SFL gene by mapping the sfl-d mutation and identifying and characterising a second mutant allele. We analysed the effect of sfl on chickpea inflorescence ontogeny with scanning electron microscopy and studied the expression of SFL and meristem identity genes by RNA in situ hybridisation. We show that SFL corresponds to CaRAX1/2a, which codes a MYB transcription factor specifically expressed in the I2 meristem. Our findings reveal SFL as a central factor controlling chickpea inflorescence architecture, acting in the I2 meristem to regulate the length of the period for which it remains active, and therefore determining the number of floral meristems that it can produce.

PYL8 mediates ABA perception in the root through non-cell-autonomous and ligand-stabilization-based mechanisms.

The phytohormone abscisic acid (ABA) plays a key role regulating root growth, root system architecture, and root adaptive responses, such as hydrotropism. The molecular and cellular mechanisms that regulate the action of core ABA signaling components in roots are not fully understood. ABA is perceived through receptors from the PYR/PYL/RCAR family and PP2C coreceptors. PYL8/RCAR3 plays a nonredundant role in regulating primary and lateral root growth. Here we demonstrate that ABA specifically stabilizes PYL8 compared with other ABA receptors and induces accumulation of PYL8 in root nuclei. This requires ABA perception by PYL8 and leads to diminished ubiquitination of PYL8 in roots. The ABA agonist quinabactin, which promotes root ABA signaling through dimeric receptors, fails to stabilize the monomeric receptor PYL8. Moreover, a PYL8 mutant unable to bind ABA and inhibit PP2C is not stabilized by the ligand, whereas a PYL85KR mutant is more stable than PYL8 at endogenous ABA concentrations. The PYL8 transcript was detected in the epidermis and stele of the root meristem; however, the PYL8 protein was also detected in adjacent tissues. Expression of PYL8 driven by tissue-specific promoters revealed movement to adjacent tissues. Hence both inter- and intracellular trafficking of PYL8 appears to occur in the root apical meristem. Our findings reveal a non-cell-autonomous mechanism for hormone receptors and help explain the nonredundant role of PYL8-mediated root ABA signaling.

AUXIN RESPONSE FACTOR3 Regulates Compound Leaf Patterning by Directly Repressing PALMATE-LIKE PENTAFOLIATA1 Expression in Medicago truncatula.

Diverse leaf forms can be seen in nature. In Medicago truncatula, PALM1 encoding a Cys(2)His(2) transcription factor is a key regulator of compound leaf patterning. PALM1 negatively regulates expression of SGL1, a key regulator of lateral leaflet initiation. However, how PALM1 itself is regulated is not yet known. To answer this question, we used promoter sequence analysis, yeast one-hybrid tests, quantitative transcription activity assays, ChIP-PCR analysis, and phenotypic analyses of overexpression lines and mutant plants. The results show that M. truncatula AUXIN RESPONSE FACTOR3 (MtARF3) functions as a direct transcriptional repressor of PALM1. MtARF3 physically binds to the PALM1 promoter sequence in yeast cells. MtARF3 selectively interacts with specific auxin response elements (AuxREs) in the PALM1 promoter to repress reporter gene expression in tobacco leaves and binds to specific sequences in the PALM1 promoter in vivo. Upregulation of MtARF3 or removal of both PHANTASTICA (PHAN) and ARGONAUTE7 (AGO7) pathways resulted in compound leaves with five narrow leaflets arranged in a palmate-like configuration. These results support that MtARF3, in addition as an adaxial-abaxial polarity regulator, functions to restrict spatiotemporal expression of PALM1, linking auxin signaling to compound leaf patterning in the legume plant M. truncatula.

Functional characterization of AGAMOUS-subfamily members from cotton during reproductive development and in response to plant hormones.

KEY MESSAGE: Expression analysis of the AG -subfamily members from G. hirsutum during flower and fruit development. Reproductive development in cotton, including the fruit and fiber formation, is a complex process; it involves the coordinated action of gene expression regulators, and it is highly influenced by plant hormones. Several studies have reported the identification and expression of the transcription factor family MADS-box members in cotton ovules and fibers; however, their roles are still elusive during the reproductive development in cotton. In this study, we evaluated the expression profiles of five MADS-box genes (GhMADS3, GhMADS4, GhMADS5, GhMADS6 and GhMADS7) belonging to the AGAMOUS-subfamily in Gossypium hirsutum. Phylogenetic and protein sequence analyses were performed using diploid (G. arboreum, G. raimondii) and tetraploid (G. barbadense, G. hirsutum) cotton genomes, as well as the AG-subfamily members from Arabidopsis thaliana, Petunia hybrida and Antirrhinum majus. qPCR analysis showed that the AG-subfamily genes had high expression during flower and fruit development in G. hirsutum. In situ hybridization analysis also substantiates the involvement of AG-subfamily members on reproductive tissues of G. hirsutum, including ovule and ovary. The effect of plant hormones on AG-subfamily genes expression was verified in cotton fruits treated with gibberellin, auxin and brassinosteroid. All the genes were significantly regulated in response to auxin, whereas only GhMADS3, GhMADS4 and GhMADS7 genes were also regulated by brassinosteroid treatment. In addition, we have investigated the GhMADS3 and GhMADS4 overexpression effects in Arabidopsis plants. Interestingly, the transgenic plants from both cotton AG-like genes in Arabidopsis significantly altered the fruit size compared to the control plants. This alteration suggests that cotton AG-like genes might act regulating fruit formation. Our results demonstrate that members of the AG-subfamily in G. hirsutum present a conserved expression profile during flower development, but also demonstrate their expression during fruit development and in response to phytohormones.

Dengue Virus Directly Stimulates Polyclonal B Cell Activation.

Dengue infection is associated to vigorous inflammatory response, to a high frequency of activated B cells, and to increased levels of circulating cross-reactive antibodies. We investigated whether direct infection of B cells would promote activation by culturing primary human B lymphocytes from healthy donors with DENV in vitro. B cells were susceptible, but poorly permissive to infection. Even though, primary B cells cultured with DENV induced substantial IgM secretion, which is a hallmark of polyclonal B cell activation. Notably, DENV induced the activation of B cells obtained from either DENV immune or DENV naïve donors, suggesting that it was not dependent on DENV-specific secondary/memory response. B cell stimulation was dependent on activation of MAPK and CD81. B cells cultured with DENV also secreted IL-6 and presented increased expression of CD86 and HLA-DR, which might contribute to B lymphocyte co-stimulatory function. Indeed, PBMCs, but not isolated B cells, secreted high amounts of IgG upon DENV culture, suggesting that interaction with other cell types in vivo might promote Ig isotype switching and IgG secretion from different B cell clones. These findings suggest that activation signaling pathways triggered by DENV interaction with non-specific receptors on B cells might contribute to the exacerbated response observed in dengue patients.

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.

Changing the spatial pattern of TFL1 expression reveals its key role in the shoot meristem in controlling Arabidopsis flowering architecture.

Models for the control of above-ground plant architectures show how meristems can be programmed to be either shoots or flowers. Molecular, genetic, transgenic, and mathematical studies have greatly refined these models, suggesting that the phase of the shoot reflects different genes contributing to its repression of flowering, its vegetativeness ('veg'), before activators promote flower development. Key elements of how the repressor of flowering and shoot meristem gene TFL1 acts have now been tested, by changing its spatiotemporal pattern. It is shown that TFL1 can act outside of its normal expression domain in leaf primordia or floral meristems to repress flower identity. These data show how the timing and spatial pattern of TFL1 expression affect overall plant architecture. This reveals that the underlying pattern of TFL1 interactors is complex and that they may be spatially more widespread than TFL1 itself, which is confined to shoots. However, the data show that while TFL1 and floral genes can both act and compete in the same meristem, it appears that the main shoot meristem is more sensitive to TFL1 rather than floral genes. This spatial analysis therefore reveals how a difference in response helps maintain the 'veg' state of the shoot meristem.

Pea VEGETATIVE2 Is an FD Homolog That Is Essential for Flowering and Compound Inflorescence Development.

As knowledge of the gene networks regulating inflorescence development in Arabidopsis thaliana improves, the current challenge is to characterize this system in different groups of crop species with different inflorescence architecture. Pea (Pisum sativum) has served as a model for development of the compound raceme, characteristic of many legume species, and in this study, we characterize the pea VEGETATIVE2 (VEG2) locus, showing that it is critical for regulation of flowering and inflorescence development and identifying it as a homolog of the bZIP transcription factor FD. Through detailed phenotypic characterizations of veg2 mutants, expression analyses, and the use of protein-protein interaction assays, we find that VEG2 has important roles during each stage of development of the pea compound inflorescence. Our results suggest that VEG2 acts in conjunction with multiple FLOWERING LOCUS T (FT) proteins to regulate expression of downstream target genes, including TERMINAL FLOWER1, LEAFY, and MADS box homologs, and to facilitate cross-regulation within the FT gene family. These findings further extend our understanding of the mechanisms underlying compound inflorescence development in pea and may have wider implications for future manipulation of inflorescence architecture in related legume crop species.

Regulation of compound leaf development by PHANTASTICA in Medicago truncatula.

Plant leaves, simple or compound, initiate as peg-like structures from the peripheral zone of the shoot apical meristem, which requires class I KNOTTED-LIKE HOMEOBOXI (KNOXI) transcription factors to maintain its activity. The MYB domain protein encoded by the ASYMMETRIC LEAVES1/ROUGH SHEATH2/PHANTASTICA (ARP) gene, together with other factors, excludes KNOXI gene expression from incipient leaf primordia to initiate leaves and specify leaf adaxial identity. However, the regulatory relationship between ARP and KNOXI is more complex in compound-leafed species. Here, we investigated the role of ARP and KNOXI genes in compound leaf development in Medicago truncatula. We show that the M. truncatula phantastica mutant exhibited severe compound leaf defects, including curling and deep serration of leaf margins, shortened petioles, increased rachises, petioles acquiring motor organ characteristics, and ectopic development of petiolules. On the other hand, the M. truncatula brevipedicellus mutant did not exhibit visible compound leaf defects. Our analyses show that the altered petiole development requires ectopic expression of ELONGATED PETIOLULE1, which encodes a lateral organ boundary domain protein, and that the distal margin serration requires the auxin efflux protein M. truncatula PIN-FORMED10 in the M. truncatula phantastica mutant.

Essential role of RIG-I in the activation of endothelial cells by dengue virus.

Dengue virus (DENV) infection is associated to exacerbated inflammatory response and structural and functional alterations in the vascular endothelium. However, the mechanisms underlying DENV-induced endothelial cell activation and their role in the inflammatory response were not investigated so far. We demonstrated that human brain microvascular endothelial cells (HBMECs) are susceptible to DENV infection, which induces the expression of the cytoplasmic pattern recognition receptor (PRR) RIG-I. Infection of HBMECs promoted an increase in the production of type I IFN and proinflammatory cytokines, which were abolished after RIG-I silencing. DENV-infected HBMECs also presented a higher ICAM-1 expression dependent on RIG-I activation as well. On the other hand, ablation of RIG-I did not interfere with virus replication. Our data suggest that RIG-I activation by DENV may participate in the disease pathogenesis through the modulation of cytokine release and expression of adhesion molecules, probably contributing to leukocyte recruitment and amplification of the inflammatory response.

VEGETATIVE1 is essential for development of the compound inflorescence in pea.

Unravelling the basis of variation in inflorescence architecture is important to understanding how the huge diversity in plant form has been generated. Inflorescences are divided between simple, as in Arabidopsis, with flowers directly formed at the main primary inflorescence axis, and compound, as in legumes, where they are formed at secondary or even higher order axes. The formation of secondary inflorescences predicts a novel genetic function in the development of the compound inflorescences. Here we show that in pea this function is controlled by VEGETATIVE1 (VEG1), whose mutation replaces secondary inflorescences by vegetative branches. We identify VEG1 as an AGL79-like MADS-box gene that specifies secondary inflorescence meristem identity. VEG1 misexpression in meristem identity mutants causes ectopic secondary inflorescence formation, suggesting a model for compound inflorescence development based on antagonistic interactions between VEG1 and genes conferring primary inflorescence and floral identity. Our study defines a novel mechanism to generate inflorescence complexity.

STENOFOLIA regulates blade outgrowth and leaf vascular patterning in Medicago truncatula and Nicotiana sylvestris.

Dicot leaf primordia initiate at the flanks of the shoot apical meristem and extend laterally by cell division and cell expansion to form the flat lamina, but the molecular mechanism of lamina outgrowth remains unclear. Here, we report the identification of STENOFOLIA (STF), a WUSCHEL-like homeobox transcriptional regulator, in Medicago truncatula, which is required for blade outgrowth and leaf vascular patterning. STF belongs to the MAEWEST clade and its inactivation by the transposable element of Nicotiana tabacum cell type1 (Tnt1) retrotransposon insertion leads to abortion of blade expansion in the mediolateral axis and disruption of vein patterning. We also show that the classical lam1 mutant of Nicotiana sylvestris, which is blocked in lamina formation and stem elongation, is caused by deletion of the STF ortholog. STF is expressed at the adaxial-abaxial boundary layer of leaf primordia and governs organization and outgrowth of lamina, conferring morphogenetic competence. STF does not affect formation of lateral leaflets but is critical to their ability to generate a leaf blade. Our data suggest that STF functions by modulating phytohormone homeostasis and crosstalk directly linked to sugar metabolism, highlighting the importance of coordinating metabolic and developmental signals for leaf elaboration.

Control of dissected leaf morphology by a Cys(2)His(2) zinc finger transcription factor in the model legume Medicago truncatula.

Plant leaves are diverse in their morphology, reflecting to a large degree the plant diversity in the natural environment. How different leaf morphology is determined is not yet understood. The leguminous plant Medicago truncatula exhibits dissected leaves with three leaflets at the tip. We show that development of the trifoliate leaves is determined by the Cys(2)His(2) zinc finger transcription factor PALM1. Loss-of-function mutants of PALM1 develop dissected leaves with five leaflets clustered at the tip. We demonstrate that PALM1 binds a specific promoter sequence and down-regulates the expression of the M. truncatula LEAFY/UNIFOLIATA orthologue SINGLE LEAFLET1 (SGL1), encoding an indeterminacy factor necessary for leaflet initiation. Our data indicate that SGL1 is required for leaflet proliferation in the palm1 mutant. Interestingly, ectopic expression of PALM1 effectively suppresses the lobed leaf phenotype from overexpression of a class 1 KNOTTED1-like homeobox protein in Arabidopsis plants. Taken together, our results show that PALM1 acts as a determinacy factor, regulates the spatial-temporal expression of SGL1 during leaf morphogenesis and together with the LEAFY/UNIFOLIATA orthologue plays an important role in orchestrating the compound leaf morphology in M. truncatula.

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.

Abscisic acid and desiccation-dependent expression of a novel putative SNF5-type chromatin-remodeling gene in Pisum sativum.

Snf5-like proteins are components of multiprotein chromatin remodeling complexes involved in the ATP-dependent alteration of DNA-histone contacts. Mostly described in yeast and animals, the only plant SNF5-like gene characterized so far has been BSH from Arabidopsis thaliana (L.) Heynh. We report the cloning and characterization of expression of a SNF5-like gene from pea (Pisum sativum L. cv. Lincoln), which has been designated PsSNF5. Southern analysis showed a single copy of the gene in the pea genome. The cDNA contained a 723bp open reading frame encoding a 240 amino acid protein of 27.4kDa with a potential nuclear localization signal. PsSNF5 protein sequence closely resembled BSH, with which it showed an overall amino acid identity of 78.5%. Two-hybrid experiments showed that PsSNF5 is functionally interchangeable with Arabidopsis BSH in the interactions with other components of the remodeling complex. Phylogenetic analysis demonstrated that PsSNF5 clustered with translated expressed sequence tags from other Leguminosae, hypothetically coding for new Snf5-like proteins. RT-PCR expression analysis demonstrated that the PsSNF5 gene is constitutively expressed in all the tissues examined, with minor differences in expression level in different tissues. Nevertheless, expression analysis revealed that PsSNF5 was up-regulated in the last stages of embryo development, when water content decreases. Moreover, abscisic acid and drought stress induced PsSNF5 accumulation in germinating embryos and vegetative tissues, suggesting that chromatin remodeling induced by PsSNF5-containing complexes might contribute to the response to that phytohormone.

Functional conservation of PISTILLATA activity in a pea homolog lacking the PI motif.

Current understanding of floral development is mainly based on what we know from Arabidopsis (Arabidopsis thaliana) and Antirrhinum majus. However, we can learn more by comparing developmental mechanisms that may explain morphological differences between species. A good example comes from the analysis of genes controlling flower development in pea (Pisum sativum), a plant with more complex leaves and inflorescences than Arabidopsis and Antirrhinum, and a different floral ontogeny. The analysis of UNIFOLIATA (UNI) and STAMINA PISTILLOIDA (STP), the pea orthologs of LEAFY and UNUSUAL FLORAL ORGANS, has revealed a common link in the regulation of flower and leaf development not apparent in Arabidopsis. While the Arabidopsis genes mainly behave as key regulators of flower development, where they control the expression of B-function genes, UNI and STP also contribute to the development of the pea compound leaf. Here, we describe the characterization of P. sativum PISTILLATA (PsPI), a pea MADS-box gene homologous to B-function genes like PI and GLOBOSA (GLO), from Arabidopsis and Antirrhinum, respectively. PsPI encodes for an atypical PI-type polypeptide that lacks the highly conserved C-terminal PI motif. Nevertheless, constitutive expression of PsPI in tobacco (Nicotiana tabacum) and Arabidopsis shows that it can specifically replace the function of PI, being able to complement the strong pi-1 mutant. Accordingly, PsPI expression in pea flowers, which is dependent on STP, is identical to PI and GLO. Interestingly, PsPI is also transiently expressed in young leaves, suggesting a role of PsPI in pea leaf development, a possibility that fits with the established role of UNI and STP in the control of this process.