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.

Transcription factors HB21/40/53 trigger inflorescence arrest through abscisic acid accumulation at the end of flowering.

Flowers, and hence, fruits and seeds, are produced by the activity of the inflorescence meristem after the floral transition. In plants with indeterminate inflorescences, the final number of flowers produced by the inflorescence meristem is determined by the length of the flowering period, which ends with inflorescence arrest. Inflorescence arrest depends on many different factors, such as the presence of seeds, the influence of the environment, or endogenous factors such as phytohormone levels and age, which modulate inflorescence meristem activity. The FRUITFULL-APETALA2 (FUL-AP2) pathway plays a major role in regulating the end of flowering, likely integrating both endogenous cues and those related to seed formation. Among AP2 targets, HOMEOBOX PROTEIN21 (HB21) has been identified as a putative mediator of AP2 function in the control of inflorescence arrest. HB21 is a homeodomain leucine zipper transcription factor involved in establishing axillary bud dormancy. Here, we characterized the role of HB21 in the control of the inflorescence arrest at the end of flowering in Arabidopsis (Arabidopsis thaliana). HB21, together with HB40 and HB53, are upregulated in the inflorescence apex at the end of flowering, promoting floral bud arrest. We also show that abscisic acid (ABA) accumulation occurs in the inflorescence apex in an HB-dependent manner. Our work suggests a physiological role of ABA in floral bud arrest at the end of flowering, pointing to ABA as a regulator of inflorescence arrest downstream of the HB21/40/53 genes.

A transcriptional complex of NGATHA and bHLH transcription factors directs stigma development in Arabidopsis.

The stigma is an angiosperm-specific tissue that is essential for pollination. In the last two decades, several transcription factors with key roles in stigma development in Arabidopsis thaliana have been identified. However, genetic analyses have thus far been unable to unravel the precise regulatory interactions among these transcription factors or the molecular basis for their selective roles in different spatial and temporal domains. Here, we show that the NGATHA (NGA) and HECATE (HEC) transcription factors, which are involved in different developmental processes but are both essential for stigma development, require each other to perform this function. This relationship is likely mediated by their physical interaction in the apical gynoecium. NGA/HEC transcription factors subsequently upregulate INDEHISCENT (IND) and SPATULA and are indispensable for the binding of IND to some of its targets to allow stigma differentiation. Our findings support a nonhierarchical regulatory scenario in which the combinatorial action of different transcription factors provides exquisite temporal and spatial specificity of their developmental outputs.

Seed production determines the entrance to dormancy of the inflorescence meristem of Pisum sativum and the end of the flowering period.

Flowering plants adjust their reproductive period to maximize the success of the offspring. Monocarpic plants, those with a single reproductive cycle that precedes plant senescence and death, tightly regulate both flowering initiation and flowering cessation. The end of the flowering period involves the arrest of the inflorescence meristem activity, known as proliferative arrest, in what has been interpreted as an evolutionary adaptation to maximize the allocation of resources to seed production and the viability of the progeny. Factors influencing proliferative arrest were described for several monocarpic plant species many decades ago, but only in the last few years studies performed in Arabidopsis have allowed to approach proliferative arrest regulation in a comprehensive manner by studying the physiology, hormone dynamics, and genetic factors involved in its regulation. However, these studies remain restricted to Arabidopsis and there is a need to expand our knowledge to other monocarpic species to propose general mechanisms controlling the process. In this work, we have characterized proliferative arrest in Pisum sativum, trying to parallel available studies in Arabidopsis to maximize this comparative framework. We have assessed quantitatively the role of fruits/seeds in the process, the influence of the positional effect of these fruits/seeds in the behavior of the inflorescence meristem, and the transcriptomic changes in the inflorescence associated with the arrested state of the meristem. Our results support a high conservation of the factors triggering arrest in pea and Arabidopsis, but also reveal differences reinforcing the need to perform similar studies in other species.

Flowering also has to end: knowns and unknowns of reproductive arrest in monocarpic plants.

All flowering plants adjust their reproductive period for successful reproduction. Flower initiation is controlled by a myriad of intensively studied factors, so it can occur in the most favorable conditions. However, the end of flowering is also a controlled process, required to optimize the size of the offspring and to maximize resource allocation. Reproductive arrest was described and mainly studied in the last century by physiological approaches, but it is much less understood at the genetic or molecular level. In this review, we present an overview of recent progress in this topic, fueled by highly complementary studies that are beginning to provide an integrated view of how the end of flowering is regulated. In this emerging picture, we also highlight key missing aspects that will guide future research and may provide new biotechnological avenues to improve crop yield in annual plants.

New roles of NO TRANSMITTING TRACT and SEEDSTICK during medial domain development in Arabidopsis fruits.

The gynoecium, the female reproductive part of the flower, is key for plant sexual reproduction. During its development, inner tissues such as the septum and the transmitting tract tissue, important for pollen germination and guidance, are formed. In Arabidopsis, several transcription factors are known to be involved in the development of these tissues. One of them is NO TRANSMITTING TRACT (NTT), essential for transmitting tract formation. We found that the NTT protein can interact with several gynoecium-related transcription factors, including several MADS-box proteins, such as SEEDSTICK (STK), known to specify ovule identity. Evidence suggests that NTT and STK control enzyme and transporter-encoding genes involved in cell wall polysaccharide and lipid distribution in gynoecial medial domain cells. The results indicate that the simultaneous loss of NTT and STK activity affects polysaccharide and lipid deposition and septum fusion, and delays entry of septum cells to their normal degradation program. Furthermore, we identified KAWAK, a direct target of NTT and STK, which is required for the correct formation of fruits in Arabidopsis These findings position NTT and STK as important factors in determining reproductive competence.

Study and QTL mapping of reproductive and morphological traits implicated in the autofertility of faba bean.

Autofertility describes the ability of faba bean flowers to self-fertilize thereby ensuring the productivity of this crop in the absence of pollinators or mechanical disturbance. In the legume crop faba bean (Vicia faba L.), lack of autofertility in a context of insufficient pollination can lead to a severe decrease in grain yield. Here we performed the first QTL analysis aimed at identifying the genomic regions controlling autofertility in this crop. We combined pod and seed setting scores from a recombinant inbred population (RIL) segregating for autofertility in different environments and years with measurements of morphological floral traits and pollen production and viability. This approach revealed 19 QTLs co-localizing in six genomic regions. Extensive co-localization was evident for various floral features whose QTLs clustered in chrs. I, II and V, while other QTLs in chrs. III, IV and VI revealed co-localization of flower characteristics and pod and seed set data. The percentage of phenotypic variation explained by the QTLs ranged from 8.9 for style length to 25.7 for stigma angle. In the three QTLs explaining the highest phenotypic variation (R 2 > 20), the marker alleles derived from the autofertile line Vf27. We further inspected positional candidates identified by these QTLs which represent a valuable resource for further validation. Our results advance the understanding of autofertility in faba bean and will aid the identification of responsible genes for genomic-assisted breeding in this crop.

Rice SEPALLATA genes OsMADS5 and OsMADS34 cooperate to limit inflorescence branching by repressing the TERMINAL FLOWER1-like gene RCN4.

The spatiotemporal control of meristem identity is critical for determining inflorescence architecture, and thus yield, of cereal plants. However, the precise mechanisms underlying inflorescence and spikelet meristem determinacy in cereals are still largely unclear. We have generated loss-of-function and overexpression mutants of the paralogous OsMADS5 and OsMADS34 genes in rice (Oryza sativa), and analysed their panicle phenotypes. Using chromatin immunoprecipitation, electrophoretic mobility-shift and dual-luciferase assays, we have also identified RICE CENTRORADIALIS 4 (RCN4), a TFL1-like gene, as a direct downstream target of both OsMADS proteins, and have analysed RCN4 mutants. The osmads5 osmads34 mutant lines had significantly enhanced panicle branching with increased secondary, and even tertiary and quaternary, branches, compared to wild-type (WT) and osmads34 plants. The osmads34 mutant phenotype could largely be rescued by also knocking out RCN4. Moreover, transgenic panicles overexpressing RCN4 had significantly increased branching, and initiated development of c. 7× more spikelets than WT. Our results reveal a role for OsMADS5 in panicle development, and show that OsMADS5 and OsMADS34 play similar functions in limiting branching and promoting the transition to spikelet meristem identity, in part by repressing RCN4 expression. These findings provide new insights to better understand the molecular regulation of rice inflorescence architecture.

Identification of Players Controlling Meristem Arrest Downstream of the FRUITFULL-APETALA2 Pathway.

The end of the reproductive phase in monocarpic plants is determined by a coordinated arrest of all active meristems, a process known as global proliferative arrest (GPA). GPA is linked to the correlative control exerted by developing seeds and, possibly, the establishment of strong source-sink relationships. It has been proposed that the meristems that undergo arrest at the end of the reproductive phase behave at the transcriptomic level as dormant meristems, with low mitotic activity and high expression of abscisic acid response genes. Meristem arrest is also controlled genetically. In Arabidopsis (Arabidopsis thaliana), the MADS-box transcription factor FRUITFULL induces GPA by directly repressing genes of the APETALA2 (AP2) clade. The AP2 genes maintain shoot apical meristem (SAM) activity in part by keeping WUSCHEL expression active, but the mechanisms downstream of this pathway remain elusive. To identify target genes, we performed a transcriptomic analysis, inducing AP2 activity in meristems close to arrest. Our results suggest that AP2 controls meristem arrest by repressing genes related to axillary bud dormancy in the SAM and negative regulators of cytokinin signaling. In addition, our analysis indicates that genes involved in the response to environmental signals also respond to AP2, suggesting that it could modulate the end of flowering by controlling responses to both endogenous and exogenous signals. Our results support the previous observation that at the end of the reproductive phase the arrested SAM behaves as a dormant meristem, and they strongly support AP2 as a master regulator of this process.

Comparative anatomy and genetic bases of fruit development in selected Rubiaceae (Gentianales).

PREMISE: The Rubiaceae are ideal for studying the diversity of fruits that develop from flowers with inferior ovary. We aimed to identify morpho-anatomical changes during fruit development that distinguish those derived from the carpel versus the extra-carpellary tissues. In addition, we present the fruit genetic core regulatory network in selected Rubiaceae species and compare it in terms of copy number and expression patterns to model core eudicots in the Brassicaceae and the Solanaceae. METHODS: We used light microscopy to follow morphoanatomical changes in four selected species with different fruit types. We generated reference transcriptomes for seven selected Rubiaceae species and isolated homologs of major transcription factors involved in fruit development histogenesis, assessed their homology, identified conserved and new protein motifs, and evaluated their expression in three species with different fruit types. RESULTS: Our studies revealed ovary-derived pericarp tissues versus floral-cup-derived epicarp tissues. Gene evolution analyses of FRUITFULL, SHATTERPROOF, ALCATRAZ, INDEHISCENT and REPLUMLESS homologs suggest that the gene complement in Rubiaceae is simpler compared to that in Brassicaceae or Solanaceae. Expression patterns of targeted genes vary in response to the fruit type and the developmental stage evaluated. CONCLUSIONS: Morphologically similar fruits can have different anatomies as a result of convergent tissues developed from the epicarps covering the anatomical changes from the pericarps. Expression analyses suggest that the fruit patterning regulatory network established in model core eudicots cannot be extrapolated to asterids with inferior ovaries.

Expression and function of the bHLH genes ALCATRAZ and SPATULA in selected Solanaceae species.

The genetic mechanisms underlying fruit development have been identified in Arabidopsis and have been comparatively studied in tomato as a representative of fleshy fruits. However, comparative expression and functional analyses on the bHLH genes downstream the genetic network, ALCATRAZ (ALC) and SPATULA (SPT), which are involved in the formation of the dehiscence zone in Arabidopsis, have not been functionally studied in the Solanaceae. Here, we perform detailed expression and functional studies of ALC/SPT homologs in Nicotiana obtusifolia with capsules, and in Capsicum annuum and Solanum lycopersicum with berries. In Solanaceae, ALC and SPT genes are expressed in leaves, and all floral organs, especially in petal margins, stamens and carpels; however, their expression changes during fruit maturation according to the fruit type. Functional analyses show that downregulation of ALC/SPT genes does not have an effect on gynoecium patterning; however, they have acquired opposite roles in petal expansion and have been co-opted in leaf pigmentation in Solanaceae. In addition, ALC/SPT genes repress lignification in time and space during fruit development in Solanaceae. Altogether, some roles of ALC and SPT genes are different between Brassicaceae and Solanaceae; while the paralogs have undergone some subfunctionalization in the former they are mostly redundant in the latter.

Evolution of Class II TCP genes in perianth bearing Piperales and their contribution to the bilateral calyx in Aristolochia.

Controlled spatiotemporal cell division and expansion are responsible for floral bilateral symmetry. Genetic studies have pointed to class II TCP genes as major regulators of cell division and floral patterning in model core eudicots. Here we study their evolution in perianth-bearing Piperales and their expression in Aristolochia, a rare occurrence of bilateral perianth outside eudicots and monocots. The evolution of class II TCP genes reveals single-copy CYCLOIDEA-like genes and three paralogs of CINCINNATA (CIN) in early diverging angiosperms. All class II TCP genes have independently duplicated in Aristolochia subgenus Siphisia. Also CIN2 genes duplicated before the diversification of Saruma and Asarum. Sequence analysis shows that CIN1 and CIN3 share motifs with Cyclin proteins and CIN2 genes have lost the miRNA319a binding site. Expression analyses of all paralogs of class II TCP genes in Aristolochia fimbriata point to a role of CYC and CIN genes in maintaining differential perianth expansion during mid- and late flower developmental stages by promoting cell division in the distal and ventral portion of the limb. It is likely that class II TCP genes also contribute to cell division in the leaf, the gynoecium and the ovules in A. fimbriata.

The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium.

Fruits and seeds are the major food source on earth. Both derive from the gynoecium and, therefore, it is crucial to understand the mechanisms that guide the development of this organ of angiosperm species. In Arabidopsis, the gynoecium is composed of two congenitally fused carpels, where two domains: medial and lateral, can be distinguished. The medial domain includes the carpel margin meristem (CMM) that is key for the production of the internal tissues involved in fertilization, such as septum, ovules, and transmitting tract. Interestingly, the medial domain shows a high cytokinin signaling output, in contrast to the lateral domain, where it is hardly detected. While it is known that cytokinin provides meristematic properties, understanding on the mechanisms that underlie the cytokinin signaling pattern in the young gynoecium is lacking. Moreover, in other tissues, the cytokinin pathway is often connected to the auxin pathway, but we also lack knowledge about these connections in the young gynoecium. Our results reveal that cytokinin signaling, that can provide meristematic properties required for CMM activity and growth, is enabled by the transcription factor SPATULA (SPT) in the medial domain. Meanwhile, cytokinin signaling is confined to the medial domain by the cytokinin response repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 6 (AHP6), and perhaps by ARR16 (a type-A ARR) as well, both present in the lateral domains (presumptive valves) of the developing gynoecia. Moreover, SPT and cytokinin, probably together, promote the expression of the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the gene encoding the auxin efflux transporter PIN-FORMED 3 (PIN3), likely creating auxin drainage important for gynoecium growth. This study provides novel insights in the spatiotemporal determination of the cytokinin signaling pattern and its connection to the auxin pathway in the young gynoecium.

Loss of LOFSEP Transcription Factor Function Converts Spikelet to Leaf-Like Structures in Rice.

SEPALLATA (SEP)-like genes, which encode a subfamily of MADS-box transcription factors, are essential for specifying floral organ and meristem identity in angiosperms. Rice (Oryza sativa) has five SEP-like genes with partial redundancy and overlapping expression domains, yet their functions and evolutionary conservation are only partially known. Here, we describe the biological role of one of the SEP genes of rice, OsMADS5, in redundantly controlling spikelet morphogenesis. OsMADS5 belongs to the conserved LOFSEP subgroup along with OsMADS1 and OsMADS34OsMADS5 was expressed strongly across a broad range of reproductive stages and tissues. No obvious phenotype was observed in the osmads5 single mutants when compared with the wild type, which was largely due to the functional redundancy among the three LOFSEP genes. Genetic and molecular analyses demonstrated that OsMADS1, OsMADS5, and OsMADS34 together regulate floral meristem determinacy and specify the identities of spikelet organs by positively regulating the other MADS-box floral homeotic genes. Experiments conducted in yeast also suggested that OsMADS1, OsMADS5, and OsMADS34 form protein-protein interactions with other MADS-box floral homeotic members, which seems to be a typical, conserved feature of plant SEP proteins.

Identification of Stipules reduced, a leaf morphology gene in pea (Pisum sativum).

Pea (Pisum sativum) is one of relatively few genetically amenable plant species with compound leaves. Pea leaves have a variety of specialized organs: leaflets, tendrils, pulvini and stipules, which enable the identification of mutations that transform or affect distinct parts of the leaf. Characterization of these mutations offers insights into the development and evolution of novel leaf traits. The previously characterized morphological gene Cochleata, conferring stipule identity, was known to interact with Stipules reduced (St), which conditions stipule size in pea, but the St gene remained unknown. Here we analysed Fast Neutron irradiated pea mutants by restriction site associated DNA sequencing. We identified St as a gene encoding a C2H2 zinc finger transcription factor that is regulated by Cochleata. St regulates both cell division and cell expansion in the stipule. Our approach shows how systematic genome-wide screens can be used successfully for the analysis of traits in species for which whole genome sequences are not available.

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.

Arabidopsis COGWHEEL1 links light perception and gibberellins with seed tolerance to deterioration.

Light is a major regulator of plant growth and development by antagonizing gibberellins (GA), and we provide evidence for a role of light perception and GA in seed coat formation and seed tolerance to deterioration. We have identified two activation-tagging mutants of Arabidopsis thaliana, cog1-2D and cdf4-1D, with improved seed tolerance to deterioration linked to increased expression of COG1/DOF1.5 and CDF4/DOF2.3, respectively. These encode two homologous DOF transcription factors, with COG1 most highly expressed in seeds. Improved tolerance to seed deterioration was reproduced in transgenic plants overexpressing these genes, and loss of function from RNA interference resulted in opposite phenotypes. Overexpressions of COG1 and CDF4 have been described to attenuate various light responses mediated by phytochromes. Accordingly, we found that phyA and phyB mutants exhibit increased seed tolerance to deterioration. The phenotype of tolerance to deterioration conferred by gain of function of COG1 and by loss of function of phytochromes is of maternal origin, is also observed under natural aging conditions and correlates with a seed coat with increased suberin and reduced permeability. In developing siliques of the cog1-2D mutant the expression of the GA biosynthetic gene GA3OX3 and levels of GA1 are higher than in the wild type. These results explain the antagonism between phytochromes and COG1 in terms of the inhibition and the activation, respectively, of GA action.

FRUITFULL controls SAUR10 expression and regulates Arabidopsis growth and architecture.

MADS-domain transcription factors are well known for their roles in plant development and regulate sets of downstream genes that have been uncovered by high-throughput analyses. A considerable number of these targets are predicted to function in hormone responses or responses to environmental stimuli, suggesting that there is a close link between developmental and environmental regulators of plant growth and development. Here, we show that the Arabidopsis MADS-domain factor FRUITFULL (FUL) executes several functions in addition to its noted role in fruit development. Among the direct targets of FUL, we identified SMALL AUXIN UPREGULATED RNA 10 (SAUR10), a growth regulator that is highly induced by a combination of auxin and brassinosteroids and in response to reduced R:FR light. Interestingly, we discovered that SAUR10 is repressed by FUL in stems and inflorescence branches. SAUR10 is specifically expressed at the abaxial side of these branches and this localized activity is influenced by hormones, light conditions and by FUL, which has an effect on branch angle. Furthermore, we identified a number of other genes involved in hormone pathways and light signalling as direct targets of FUL in the stem, demonstrating a connection between developmentally and environmentally regulated growth programs.

The NTT transcription factor promotes replum development in Arabidopsis fruits.

Fruits are complex plant structures that nurture seeds and facilitate their dispersal. The Arabidopsis fruit is termed silique. It develops from the gynoecium, which has a stigma, a style, an ovary containing the ovules, and a gynophore. Externally, the ovary consists of two valves, and their margins lay adjacent to the replum, which is connected to the septum that internally divides the ovary. In this work we describe the role for the zinc-finger transcription factor NO TRANSMITTING TRACT (NTT) in replum development. NTT loss of function leads to reduced replum width and cell number, whereas increased expression promotes replum enlargement. NTT activates the homeobox gene BP, which, together with RPL, is important for replum development. In addition, the NTT protein is able to bind the BP promoter in yeast, and when this binding region is not present, NTT fails to activate BP in the replum. Furthermore, NTT interacts with itself and different proteins involved in fruit development: RPL, STM, FUL, SHP1 and SHP2 in yeast and in planta. Moreover, its genetic interactions provide further evidence about its biological relevance in replum development.

Leaf expansion in Arabidopsis is controlled by a TCP-NGA regulatory module likely conserved in distantly related species.

The NGATHA (NGA) clade of transcription factors (TFs) forms a small subfamily of four members in Arabidopsis thaliana. NGA genes act redundantly to direct the development of apical tissues in the gynoecium, where they have been shown to be essential for style and stigma specification. In addition, NGA genes have a more general role in controlling lateral organ growth. The four NGA genes in Arabidopsis are expressed in very similar domains, although little is known about the nature of their putative regulators. Here, we have identified a conserved region within the four NGA promoters that we have used as a bait to screen a yeast library, aiming to identify such NGA regulators. Three members of the TCP family of TFs, named after the founding factors TEOSINTE BRANCHED 1, CYCLOIDEA and PROLIFERATING CELL FACTOR 1 AND 2), were recovered from this screening, of which two [TCP2 and TCP3, members of the CINCINNATA (CIN) family of TCP genes (CIN-TCP) subclade] were shown to activate the NGA3 promoter in planta. We provide evidence that support that CIN-TCP genes are true regulators of NGA gene expression, and that part of the CIN-TCP role in leaf development is mediated by NGA upregulation. Moreover, we have found that this TCP-NGA regulatory interaction is likely conserved in angiosperms, including important crop species, for which the regulation of leaf development is a target for biotechnological improvement.