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Tea (Camellia sinensis) is concocted from tea plant shoot tips that produce catechins, caffeine, theanine, and terpenoids, which collectively determine the rich flavors and health benefits of the infusion. However, little is known about the integrated regulation of shoot tip development and characteristic secondary metabolite biosynthesis in tea plants. Here, we demonstrate that MYB transcription factors (TFs) play key and yet diverse roles in regulating leaf and stem development, secondary metabolite biosynthesis, and environmental stress responses in tea plants. By integrating transcriptomic and metabolic profiling data in different tissues at a series of developmental stages or under various stress conditions, alongside biochemical and genetic analyses, we predicted the MYB TFs involved in regulating shoot development (CsMYB2, 98, 107, and 221), epidermal cell initiation (CsMYB184, 41, 139, and 219), stomatal initiation (CsMYB113 and 153), and the biosynthesis of flavonoids (including catechins, anthocyanins, and flavonols; CsMYB8 and 99), caffeine (CsMYB85 and 86), theanine (CsMYB9 and 49), carotenoids (CsMYB110), mono-/sesquiterpenoid volatiles (CsMYB68, 147, 148, and 193), lignin (CsMYB164 and 192), and indolic compounds (CsMYB139, 162, and 198), as well as the MYB TFs that are likely involved in hormone signaling-mediated environmental stress and defense responses. We characterized the functions of some key MYBs in regulating flavonoid and carotenoid biosynthesis for tea quality and flavor. This study provides a cross-family analysis of MYBs in tea alongside new insights into the coordinated regulation of tea plant shoot development and secondary metabolism, paving the way towards understanding of tea quality trait formation and genetic improvement of quality tea plants.

In most legumes, two typical features found in leaves are diverse compound forms and the pulvinus-driven nyctinastic movement. Many genes have been identified for leaf-shape determination, but the underlying nature of leaf movement as well as its association with the compound form remains largely unknown. Using forward-genetic screening and whole-genome resequencing, we found that two allelic mutants of Medicago truncatula with unclosed leaflets at night were impaired in MtDWFARF4A (MtDWF4A), a gene encoding a cytochrome P450 protein orthologous to Arabidopsis DWARF4. The mtdwf4a mutant also had a mild brassinosteroid (BR)-deficient phenotype bearing pulvini without significant deficiency in organ identity. Both mtdwf4a and dwf4 could be fully rescued by MtDWF4A, and mtdwf4a could close their leaflets at night after the application of exogenous 24-epi-BL. Surgical experiments and genetic analysis of double mutants revealed that the failure to exhibit leaf movement in mtdwf4a is a consequence of the physical obstruction of the overlapping leaflet laminae, suggesting a proper geometry of leaflets is important for their movement in M. truncatula. These observations provide a novel insight into the nyctinastic movement of compound leaves, shedding light on the importance of open space for organ movements in plants.

The LAM1 transcription factor regulates expansion of primary leaflets in the compound leaves of tomato, and also affects floral organ development, fruit size, and initiation of secondary leaflets., Plant-specific WOX family transcription factors play important roles ranging from embryogenesis to lateral organ development. The WOX1 transcription factors, which belong to the modern clade of the WOX family, are known to regulate outgrowth of the leaf blade specifically in the mediolateral axis; however, the role of WOX1 in compound leaf development remains unknown. Phylogenetic analysis of the whole WOX family in tomato (Solanum lycopersicum) indicates that there are 10 members that represent the modern, intermediate, and ancient clades. Using phylogenetic analysis and a reverse genetic approach, in this study we identified SlLAM1 in the modern clade and examined its function and tissue-specific expression pattern. We found that knocking out SlLAM1 via CRISPR/Cas9-mediated genome editing led to narrow leaves and a reduced number of secondary leaflets. Overexpression of tomato SlLAM1 could rescue the defects of the tobacco lam1 mutant. Anatomical and transcriptomic analyses demonstrated that floral organ development, fruit size, secondary leaflet initiation, and leaf complexity were altered due to loss-of-function of SlLAM1. These findings demonstrate that tomato SlLAM1 plays an important role in the regulation of secondary leaflet initiation, in addition to its conserved function in blade expansion.

WOX family transcription factors regulate multiple developmental programs. The intermediate clade transcriptional activator WOX9 functions together with the modern clade transcriptional repressor WOX genes in embryogenesis and meristems maintenance, but the mechanism of this interaction is unclear. STF and LAM1 are WOX1 orthologs required for leaf blade outgrowth in Medicago truncatula and Nicotiana sylvestris, respectively. Using biochemical methods and genome editing technology, here we show that WOX9 is an abaxial factor and functions antagonistically to STF and LAM1 to regulate leaf blade development. While NsWOX9 ectopic expression enhances the lam1 mutant phenotype, and antisense expression partially rescues the lam1 mutant, both overexpression and knockout of NsWOX9 in N. sylvestris resulted in a range of severe leaf blade distortions, indicating important role in blade development. Our results indicate that direct repression of WOX9 by WUS clade repressor STF/LAM1 is required for correct blade architecture and patterning in M. truncatula and N. sylvestris. These findings suggest that controlling transcriptional activation and repression mechanisms by direct interaction of activator and repressor WOX genes may be required for cell proliferation and differentiation homeostasis, and could be an evolutionarily conserved mechanism for the development of complex and diverse morphology in flowering plants.

A 3-ketoacyl-CoA synthase involved in biosynthesis of very long chain fatty acids and cuticular wax plays a vital role in aerial organ development in M. truncatula. Cuticular wax is composed of very long chain fatty acids and their derivatives. Defects in cuticular wax often result in organ fusion, but little is known about the role of cuticular wax in compound leaf and flower development in Medicago truncatula. In this study, through an extensive screen of a Tnt1 retrotransposon insertion population in M. truncatula, we identified four mutant lines, named wrinkled flower and leaf (wfl) for their phenotype. The phenotype of the wfl mutants is caused by a Tnt1 insertion in Medtr3g105550, encoding 3-ketoacyl-CoA synthase (KCS), which functions as a rate-limiting enzyme in very long chain fatty acid elongation. Reverse transcription-quantitative PCR showed that WFL was broadly expressed in aerial organs of the wild type, such as leaves, floral organs, and the shoot apical meristem, but was expressed at lower levels in roots. In situ hybridization showed a similar expression pattern, mainly detecting the WFL transcript in epidermal cells of the shoot apical meristem, leaf primordia, and floral organs. The wfl mutant leaves showed sparser epicuticular wax crystals on the surface and increased water permeability compared with wild type. Further analysis showed that in wfl leaves, the percentage of C20:0, C22:0, and C24:0 fatty acids was significantly increased, the amount of cuticular wax was markedly reduced, and wax constituents were altered compared to the wild type. The reduced formation of cuticular wax and wax composition changes on the leaf surface might lead to the developmental defects observed in the wfl mutants. These findings suggest that WFL plays a key role in cuticular wax formation and in the late stage of leaf and flower development in M. truncatula.

Summary DELAY OF GERMINATION1 (DOG1) is a primary regulator of seed dormancy. Accumulation of DOG1 in seeds leads to deep dormancy and delayed germination in Arabidopsis. B3 domain‐containing transcriptional repressors HSI2/VAL1 and HSL1/VAL2 silence seed dormancy and enable the subsequent germination and seedling growth. However, the roles of HSI2 and HSL1 in regulation of DOG1 expression and seed dormancy remain elusive.Seed dormancy was analysed by measurement of maximum germination percentage of freshly harvested Arabidopsis seeds. In vivo protein–protein interaction analysis, ChIP‐qPCR and EMSA were performed and suggested that HSI2 and HSL1 can form dimers to directly regulate DOG1.HSI2 and HSL1 dimers interact with RY elements at DOG1 promoter. Both B3 and PHD‐like domains are required for enrichment of HSI2 and HSL1 at the DOG1 promoter. HSI2 and HSL1 recruit components of polycomb‐group proteins, including CURLY LEAF (CLF) and LIKE HETERCHROMATIN PROTEIN 1 (LHP1), for consequent deposition of H3K27me3 marks, leading to repression of DOG1 expression.Our findings suggest that HSI2‐ and HSL1‐dependent histone methylation plays critical roles in regulation of seed dormancy during seed germination and early seedling growth.

Nyctinastic leaf movement of Fabaceae is driven by the tiny motor organ pulvinus located at the base of the leaf or leaflet. Despite the increased understanding of the essential role of ELONGATED PETIOLULE1 (ELP1)/PETIOLE LIKE PULVINUS (PLP) orthologs in determining pulvinus identity in legumes, key regulatory components and molecular mechanisms underlying this movement remain largely unclear. Here, we used WT pulvinus and the equivalent tissue in the elp1 mutant to carry out transcriptome and proteome experiments. The omics data indicated that there are multiple cell biological processes altered at the gene expression and protein abundance level during the pulvinus development. In addition, comparative analysis of different leaf tissues provided clues to illuminate the possible common primordium between pulvinus and petiole, as well as the function of ELP1. Furthermore, the auxin pathway, cell wall composition and chloroplast distribution were altered in elp1 mutants, verifying their important roles in pulvinus development. This study provides a comprehensive insight into the motor organ of the model legume Medicago truncatula and further supplies a rich dataset to facilitate the identification of novel players involved in nyctinastic movement.

Carotenoids are a group of natural tetraterpenoid pigments with indispensable roles in the plant life cycle and the human diet. Although the carotenoid biosynthetic pathway has been well characterized, the regulatory mechanisms that control carotenoid metabolism, especially in floral organs, remain poorly understood. In this study, we identified an anthocyanin-related R2R3-MYB protein, WHITE PETAL1 (WP1), that plays a critical role in regulating floral carotenoid pigmentation in Medicago truncatula. Carotenoid analyses showed that the yellow petals of the wild-type M. truncatula contained high concentrations of carotenoids that largely consisted of esterified lutein and that disruption of WP1 function via Tnt1 insertion led to substantially reduced lutein accumulation. WP1 mainly functions as a transcriptional activator and directly regulates the expression of carotenoid biosynthetic genes including MtLYCe and MtLYCb through its C-terminal acidic activation motif. Further molecular and genetic analyses revealed that WP1 physically interacts with MtTT8 and MtWD40-1 proteins and that this interaction facilitates WP1’s function in the transcriptional activation of both carotenoid and anthocyanin biosynthetic genes. Our findings demonstrate the molecular mechanism of WP1-mediated regulation of floral carotenoid pigmentation and suggest that the conserved MYB-basic-helix-loop-helix-WD40 regulatory module functions in carotenoid biosynthesis in M. truncatula, with specificity imposed by the MYB partner.

Plant lateral organ development is a complex process involving both transcriptional activation and repression mechanisms. The WOX transcriptional repressor WOX1/STF, the LEUNIG (LUG) transcriptional corepressor and the ANGUSTIFOLIA3 (AN3) transcriptional coactivator play important roles in leaf blade outgrowth and flower development, but how these factors coordinate their activities remains unclear. Here we report physical and genetic interactions among these key regulators of leaf and flower development. We developed a novel in planta transcriptional activation/repression assay and suggest that LUG could function as a transcriptional coactivator during leaf blade development. MtLUG physically interacts with MtAN3, and this interaction appears to be required for leaf and flower development. A single amino acid substitution at position 61 in the SNH domain of MtAN3 protein abolishes its interaction with MtLUG, and its transactivation activity and biological function. Mutations in lug and an3 enhanced each other's mutant phenotypes. Both the lug and the an3 mutations enhanced the wox1 prs leaf and flower phenotypes in Arabidopsis. Our findings together suggest that transcriptional repression and activation mediated by the WOX, LUG and AN3 regulators function in concert to promote leaf and flower development, providing novel mechanistic insights into the complex regulation of plant lateral organ development.

Somatic embryogenesis is widely used in plant biotechnology, but regulatory networks underlying this process are not fully understood. This process is regulated by many protein and hormonal factors, among which WUSCHEL-related homeobox proteins are believed to play key roles. In this study, we aimed to identify the role of Medicago truncatula WOX9-1 (MtWOX9-1), an Arabidopsis thaliana WOX9 (STIMPY) homolog, in somatic embryogenesis. We demonstrated that the MtWOX9-1 promoter is active in somatic embryos, and overexpression of MtWOX9-1 leads to the increase in embryogenic capacity, as well as to the changes in expression levels of two MADS-box genes (AGL15 and AGL8 homologs), associated with embryogenesis. The data obtained suggest that stimulation of MtWOX9-1 expression may have positive impact on plant biotechnology in improving the transformation and regeneration capacity of recalcitrant plants. MtWOX9-1 participates in somatic embryogenesis and can stimulate this process in tissue culture changing the expression level of different embryogenesis-associated genes.

The growth of leaves and biosynthesis of characteristic secondary metabolites are critically important for tea production and quality control. However, little is known about the coordinated regulation of leaf development and catechin biosynthesis in tea plants. Here, we reported that TCP TFs are involved in both catechin biosynthesis and leaf development. An integrated analysis of catechin profiling and CsTCP expression in different tissues of plants under various environmental conditions at different developmental stages indicated significant correlations between the transcript levels of CIN-type TCPs and catechin production. CIN-type CsTCP3 and CsTCP4 and PCF-type CsTCP14 interacted with the MYB-bHLH-WD40 repeat (MBW) complex by forming a CsTCP3-CsTT8 heterodimer and modulating the transactivation activity of the promoters of anthocyanin synthase (CsANS1) and anthocyanidin reductase (CsANR1). Four types of microRNA/target modules, miR319b/CsTCP3-4, miR164b/CsCUC, miR396/CsGRF-GIF, and miR165b/HD-ZIPIII ones, were also identified and characterized for their functions in the regulation of the development of tea plant shoot tips and leaf shape. The results of these modules were reflected by their different expression patterns in developing buds and leaves that had distinctly different morphologies in three different tea plant varieties. Their roles in the regulation of catechin biosynthesis were also further verified by manipulation of microRNA319b (miR319b), which targets the transcripts of CsTCP3 and CsTCP4. Thus, CsTCPs represent at least one of these important groups of TFs that can integrate tea plant leaf development together with secondary metabolite biosynthesis. Our study provides new insight into shoot tip development and catechin production in tea plants and lays a foundation for further mechanistic understanding of the regulation of tea plant leaf development and secondary metabolism.

The STENOFOLIA homeodomain binds promoter DNA as a tetramer and the α3 helix binds DNA in both the major and minor grooves., Homeobox transcription factors are key regulators of morphogenesis and development in both animals and plants. In plants, the WUSCHEL-related homeobox (WOX) family of transcription factors function as central organizers of several developmental programs ranging from embryo patterning to meristematic stem-cell maintenance through transcriptional activation and repression mechanisms. The Medicago truncatula STENOFOLIA (STF) gene is a master regulator of leaf-blade lateral development. Here, the crystal structure of the homeodomain (HD) of STF (STF-HD) in complex with its promoter DNA is reported at 2.1 Å resolution. STF-HD binds DNA as a tetramer, enclosing nearly the entire bound DNA surface. The STF-HD tetramer is partially stabilized by docking of the C-terminal tail of one protomer onto a conserved hydrophobic surface on the head of another protomer in a head-to-tail manner. STF-HD specifically binds TGA motifs, although the promoter sequence also contains TAAT motifs. Helix α3 not only serves a canonical role as a base reader in the major groove, but also provides DNA binding in the minor groove through basic residues located at its C-terminus. The structural and functional data in planta reported here provide new insights into the DNA-binding mechanisms of plant-specific HDs from the WOX family of transcription factors.

PvArf regulate proline biosynthesis by physically interacting with PvP5CS1 to improve salt tolerance in switchgrass. The genetic factors that contribute to stress resiliency are yet to be determined. Here, we identified three ADP-ribosylation factors, PvArf1, PvArf-B1C, and PvArf-related, which contribute to salinity tolerance in transgenic switchgrass (Panicum virgatum L.). Switchgrass overexpressing each of these genes produced approximately twofold more biomass than wild type (WT) under normal growth conditions. Transgenic plants accumulated modestly higher levels of proline under normal conditions, but this level was significantly increased under salt stress providing better protection to transgenic plants compared to WT. We found that PvArf genes induce proline biosynthesis genes under salt stress to positively regulate proline accumulation, and further demonstrated that PvArf physically interact with PvP5CS1. Moreover, the transcript levels of two key ROS-scavenging enzyme genes were significantly increased in the transgenic plants compared to WT, leading to reduced H2O2 accumulation under salt stress conditions. PvArf genes also protect cells against stress-induced changes in Na+ and K+ ion concentrations. Our findings uncover that ADP-ribosylation factors are key determinants of biomass yield in switchgrass, and play pivotal roles in salinity tolerance by regulating genes involved in proline biosynthesis.

Homeobox transcription factors are key regulators of morphogenesis and development in both animals and plants1. In plants, the WUSCHEL-related homeobox (WOX) family transcription factors function as central organizers of several developmental programs from embryo patterning to meristematic stem cell maintenance through transcriptional activation and repression2-4. The structure of WOX Homeodomain (HD) and the molecular mechanism of its interaction with DNA are unknown. Here, we report the 2.1 Å crystal structure of the STENOFOLIA (STF) HD fromMedicago truncatulain complex with DNA. STF binds DNA as a novel cooperative tetramer, enclosing nearly entire bound DNA surface. The STF tetramer is partially stabilized by docking of the C-terminal tail from one protomer onto a conserved hydrophobic surface on the head of another in a head-to-tail manner. Helix α3 not only serves a canonical role as a base reader in the major groove, but also provides extensive binding to DNA in the minor groove. Our structural and functional data reveal that STF specifically targets ‘TGA’ sequence and the cooperative tetrameric binding with DNA is key to transcriptional repression in plants. Our data reveal an unprecedented HD:DNA recognition mechanism, representing the first plant HD structure from WOX family of transcription factors.

The F-box protein MIO1/SLB1 regulates lateral organ size and pulvinus development by forming part of an SCF E3 ubiquitin ligase to modulate stability of BIG SEEDS1 in Medicago truncatula., The size of leaf and seed organs, determined by the interplay of cell proliferation and expansion, is closely related to the final yield and quality of forage and crops. Yet the cellular and molecular mechanisms underlying organ size modulation remain poorly understood, especially in legumes. Here, MINI ORGAN1 (MIO1), which encodes an F-box protein SMALL LEAF AND BUSHY1 (SLB1) recently reported to control lateral branching in Medicago truncatula, was identified as a key regulator of organ size. We show that loss-of-function of MIO1/SLB1 severely reduced organ size. Conversely, plants overexpressing MIO1/SLB1 had enlarged organs. Cellular analysis revealed that MIO1/SLB1 controlled organ size mainly by modulating primary cell proliferation during the early stages of leaf development. Biochemical analysis revealed that MIO1/SLB1 could form part of SKP1/Cullin/F-box (SCF) E3 ubiquitin ligase complex, to target BIG SEEDS1 (BS1), a repressor of primary cell division, for degradation. Interestingly, we found that MIO1/SLB1 also played a key role in pulvinus development and leaf movement by modulating cell proliferation of the pulvinus as leaves developed. Our study not only demonstrates a conserved role of MIO1/SLB1 in the control of organ size in legumes, but also sheds light on the novel function of MIO1/SLB1 in leaf movement.

International audience; The formation of nitrogen-fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen-fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin-motif receptor-like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild-type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild-type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan-defective strains was achieved by in trans rescue with a Nod factor-deficient S. meliloti mutant. While the Nod factor-deficient strain was always more abundant inside nodules, the succinoglycan-deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.

Plant specific WOX family transcription factors are known to regulate embryogenesis, meristem maintenance and lateral organ development. Modern clade WOX genes function through a transcriptional repression mechanism, and the intermediate clade transcriptional activator WOX9 functions with the repressor WOX genes in embryogenesis and meristems maintenance, but the mechanism of this interaction is unclear.WOX1homologuesSTFandLAM1are required for leaf blade outgrowth inMedicago truncatulaandNicotiana Sylvestris, respectively. Here we show thatWOX9negatively regulates leaf blade outgrowth and functions antagonistically toSTFandLAM1. WhileNsWOX9ectopic expression enhances thelam1mutant phenotype, and antisense expression partially rescues thelam1mutant, both overexpression ofNsWOX9and knockout by CRISPR/Cas9 genome editing inN. sylvestrisresulted in a range of severe leaf blade distortions, indicating that controlled negative regulation by NsWOX9 is required for proper blade development. Our results indicate that direct repression of WOX9 transcriptional activation activity by the transcriptional repressor STF/LAM1 is required for correct blade architecture and patterning inM. truncatulaandN. sylvestris. These findings suggest that a balance between transcriptional activation and repression mechanisms by direct interaction of activator and repressor WOX genes may be required for cell proliferation and differentiation homeostasis, and could be an evolutionarily conserved mechanism for the development of complex and diverse morphology in higher plants.One sentence summaryWOX9 negatively regulates blade outgrowth antagonizing STF function but directly repressed by STF indicating WOX-mediated homeostasis in cell proliferation and differentiation during leaf morphogenesis.

Male sterility is an important tool for plant breeding and hybrid seed production. Male-sterile mutants are largely due to an abnormal development of either the sporophytic or gametophytic anther tissues. Tapetum, a key sporophytic tissue, provides nutrients for pollen development, and its delayed degeneration induces pollen abortion. Numerous bHLH proteins have been documented to participate in the degeneration of the tapetum in angiosperms, but relatively little attention has been given to the evolution of the involved developmental pathways across the phylogeny of land plants. A combination of cellular, molecular, biochemical and evolutionary analyses was used to investigate the male fertility control in Medicago truncatula. We characterized the male-sterile mutant empty anther1 (ean1) and identified EAN1 as a tapetum-specific bHLH transcription factor necessary for tapetum degeneration. Our study uncovered an evolutionarily conserved recruitment of bHLH subfamily II and III(a + c)1 in the regulation of tapetum degeneration. EAN1 belongs to the subfamily II and specifically forms heterodimers with the subfamily III(a + c)1 members, which suggests a heterodimerization mechanism conserved in angiosperms. Our work suggested that the pathway of two tapetal-bHLH subfamilies is conserved in all land plants, and likely was established before the divergence of the spore-producing land plants.

The WOX family transcription factor HEADLESS exhibits a repressive activity and performs conserved and novel functions in the regulation of shoot meristems and leaf shape in Medicago truncatula., The formation and maintenance of the shoot apical meristem (SAM) are critical for plant development. However, the underlying molecular mechanism of regulating meristematic cell activity is poorly understood in the model legume Medicago truncatula. Using forward genetic approaches, we identified HEADLESS (HDL), a homolog of Arabidopsis WUSCHEL, required for SAM maintenance and leaf development in M. truncatula. Disruption of HDL led to disorganized specification and arrest of the SAM and axillary meristems, resulting in the hdl mutant being locked in the vegetative phase without apparent stem elongation. hdl mutant leaves are shorter in the proximal–distal axis due to reduced leaf length elongation, which resulted in a higher blade width/length ratio and altered leaf shape, uncovering novel phenotypes undescribed in the Arabidopsis wus mutant. HDL functions as a transcriptional repressor by recruiting MtTPL through its conserved WUS-box and EAR-like motif. Further genetic analysis revealed that HDL and STENOFOLIA (STF), a key regulator of M. truncatula lamina outgrowth, act independently in leaf development although HDL could recruit MtTPL in the same manner as STF does. Our results indicate that HDL has conserved and novel functions in regulating shoot meristems and leaf shape in M. truncatula, providing new avenues for understanding meristem biology and plant development.

DELAY OF GERMINATION1(DOG1) represents a major quantitative locus for the genetic regulation of seed dormancy in Arabidopsis. Accumulation of DOG1 in seeds leads to deep dormancy and delayed germination. Here, we report that the conserved B3 DNA binding domains of the transcriptional repressors HIGH-LEVEL EXPRESSION OF SUGAR INDICIBLE GENE2/ VIVIPAROUS-1/ABSCISIC ACID INSENSITIVE 3-LIKE1 (HSI2/VAL1) and HSI2-LIKE1/ VIVIPAROUS-1/ABSCISIC ACID INSENSITIVE 3-LIKE2 (HSL1/VAL2), which play critical roles in the developmental transition from seed maturation to seedling growth, interact with RY elements in theDOG1proximal promoter leading to repression ofDOG1transcription during germination and seedling establishment.DOG1expression is partially de-repressed inhsi2/val1(hsi2-2) but not inhsl1/val2(hsl1-1) knockout mutants and is strongly upregulated in ahsi2/val1 hsl1/val2double mutant, indicating that HSI2/VAL1 and HSL1/VAL2 act redundantly to repressDOG1expression. HSI2/VAL1 and HSL1/VAL2 form homo- and hetero-dimersin vivo, and dimerization is dependent on the HSI2/VAL1 PHD-like domain. Complementation ofhsi2-2with HSI2/VAL1 harboring a disrupted plant homeodomain (PHD)-like domain results in stronger de-repression ofDOG1expression than thehsi2-2knockout, indicating that the PHD-like domain plays a critical role in mediating functional interactions between HSI2/VAL1 and HSL1/VAL2. Both HSI2/VAL1 and HSL1/VAL2 interact with components of polycomb repressive complex 2 (PRC2), including CURLY LEAF and MULTICOPY SUPPRESSOR OF IRA1 (MSI1), along with LIKE HETERCHROMATIN PROTEIN 1 (LHP1), which are involved in the deposition and expansion of histone H3 lysine 27 trimethylation (H3K27me3) marks in repressive chromatin. Thus, HSI2/VAL1 HSL1/VAL2-dependent recruitment of PRC2 leads to silencing ofDOG1through the deposition of H3K27me3.

Organ size is a major agronomic trait that determines grain yield and biomass production in crops. However, the molecular mechanisms controlling organ size, especially in legumes, are poorly understood. Using forward genetic approaches in a Tnt1 insertion mutant population of the model legume Medicago truncatula, we identified SMALL LEAF AND BUSHY1 (SLB1), which is required for the control of organ size and lateral branching. Loss of function of SLB1 led to reduced leaf and flower size but increased lateral branch formation in M. truncatula. SLB1 encodes an F‐box protein, an orthologue of Arabidopsis thaliana STERILE APETALA (SAP), that forms part of an SKP1/Cullin/F‐box E3 ubiquitin ligase complex. Biochemical and genetic analyses revealed that SLB1 controls M. truncatula organ growth and lateral branching by modulating the stability of BIG SEEDS1 (BS1). Moreover, the overexpression of SLB1 increased seed and leaf size in both M. truncatula and soybean (Glycine max), indicating functional conservation. Our findings revealed a novel mechanism by which SLB1 targets BS1 for degradation to regulate M. truncatula organ size and shoot branching, providing a new genetic tool for increasing seed yield and biomass production in crop and forage legumes.

In species with compound leaves, the positions of leaflet primordium initiation are associated with local peaks of auxin accumulation. However, the role of auxin during the late developmental stages and outgrowth of compound leaves remains largely unknown. Using genome resequencing approaches, we identified insertion sites at four alleles of the LATERAL LEAFLET SUPPRESSION1 (LLS1) gene, encoding the auxin biosynthetic enzyme YUCCA1 in Medicago truncatula. Linkage analysis and complementation tests showed that the lls1 mutant phenotypes were caused by the Tnt1 insertions that disrupted the LLS1 gene. The transcripts of LLS1 can be detected in primordia at early stages of leaf initiation and later in the basal regions of leaflets, and finally in vein tissues at late leaf developmental stages. Vein numbers and auxin content are reduced in the lls1-1 mutant. Analysis of the lls1 sgl1 and lls1 palm1 double mutants revealed that SGL1 is epistatic to LLS1, and LLS1 works with PALM1 in an independent pathway to regulate the growth of lateral leaflets. Our work demonstrates that the YUCCA1/YUCCA4 subgroup plays very important roles in the outgrowth of lateral leaflets during compound leaf development of M. truncatula, in addition to leaf venation.

Compound leaves show more complex patterns than simple leaves, and this is mainly because of a specific morphogenetic process (leaflet initiation and arrangement) that occurs during their development. How the relevant morphogenetic activity is established and modulated to form a proper pattern of leaflets is a central question. Here we show that the trifoliate leaf pattern of the model leguminous plant Medicago truncatula is controlled by the BEL1-like homeodomain protein PINNATE-LIKE PENTAFOLIATA1 (PINNA1). We identify PINNA1 as a determinacy factor during leaf morphogenesis that directly represses transcription of the LEAFY (LFY) orthologue SINGLE LEAFLET1 (SGL1), which encodes an indeterminacy factor key to the morphogenetic activity maintenance. PINNA1 functions alone in the terminal leaflet region and synergizes with another determinacy factor, the C2H2 zinc finger protein PALMATE-LIKE PENTAFOLIATA1 (PALM1), in the lateral leaflet regions to define the spatiotemporal expression of SGL1, leading to an elaborate control of morphogenetic activity. This study reveals a framework for trifoliate leaf-pattern formation and sheds light on mechanisms generating diverse leaf forms.

Switchgrass (Panicum virgatum L.), a perennial warm season bunchgrass native to North America, has been a target in the U.S. as a renewable bioenergy crop because of its ability to produce moderate to high biomass yield on marginal soils. Delaying flowering can increase vegetative biomass production by allowing prolonged growth before switching to the reproductive phase. Despite the identification of flowering time as a biomass trait in switchgrass, the molecular regulatory factors involved in controlling floral transition are poorly understood. Here we identified PvFT1, PvAPL1-3 and PvSL1, 2 as key flowering regulators required from floral transition initiation to development of floral organs. PvFT1 expression in leaves is developmentally regulated peaking at the time of floral transition, and diurnally regulated with peak at approximately 2 h into the dark period. Ectopic expression of PvFT1 in Arabidopsis, Brachypodium and switchgrass led to extremely early flowering, and activation of FT downstream target genes, confirming that it is a strong activator of flowering in switchgrass. Ectopic expression of PvAPL1-3 and PvSL1, 2 in Arabidopsis also activated early flowering with distinct floral organ phenotypes. Our results suggest that switchgrass has conserved flowering pathway regulators similar to Arabidopsis and rice.

Highlight The rice WUSCHEL-related homeobox transcription factor OsWOX3A affects homeostasis of gibberellic acid by functioning in the negative feedback regulation of gibberellic acid biosynthesis., The plant-specific WUSCHEL-related homeobox (WOX) nuclear proteins have important roles in the transcriptional regulation of many developmental processes. Among the rice (Oryza sativa) WOX proteins, a loss of OsWOX3A function in narrow leaf2 (nal2) nal3 double mutants (termed nal2/3) causes pleiotropic effects, such as narrow and curly leaves, opened spikelets, narrow grains, more tillers, and fewer lateral roots, but almost normal plant height. To examine OsWOX3A function in more detail, transgenic rice overexpressing OsWOX3A (OsWOX3A-OX) were generated; unexpectedly, all of them consistently exhibited severe dwarfism with very short and wide leaves, a phenotype that resembles that of gibberellic acid (GA)-deficient or GA-insensitive mutants. Exogenous GA3 treatment fully rescued the developmental defects of OsWOX3A-OX plants, suggesting that constitutive overexpression of OsWOX3A downregulates GA biosynthesis. Quantitative analysis of GA intermediates revealed significantly reduced levels of GA20 and bioactive GA1 in OsWOX3A-OX, possibly due to downregulation of the expression of KAO, which encodes ent-kaurenoic acid oxidase, a GA biosynthetic enzyme. Yeast one-hybrid and electrophoretic mobility shift assays revealed that OsWOX3A directly interacts with the KAO promoter. OsWOX3A expression is drastically and temporarily upregulated by GA3 and downregulated by paclobutrazol, a blocker of GA biosynthesis. These data indicate that OsWOX3A is a GA-responsive gene and functions in the negative feedback regulation of the GA biosynthetic pathway for GA homeostasis to maintain the threshold levels of endogenous GA intermediates throughout development.

The Medicago truncatula WUSCHEL-related homeobox (WOX) gene, STENOFOLIA (STF), plays a key role in leaf blade outgrowth by promoting cell proliferation at the adaxial-abaxial junction. STF functions primarily as a transcriptional repressor, but the underlying molecular mechanism is unknown. Here, we report the identification of a protein interaction partner and a direct target, shedding light on the mechanism of STF function. Two highly conserved motifs in the C-terminal domain of STF, the WUSCHEL (WUS) box and the STF box, cooperatively recruit TOPLESS (Mt-TPL) family corepressors, and this recruitment is required for STF function, as deletion of these two domains (STFdel) impaired blade outgrowth whereas fusing Mt-TPL to STFdel restored function. The homeodomain motif is required for direct repression of ASYMMETRIC LEAVES2 (Mt-AS2), silencing of which partially rescues the stf mutant phenotype. STF and LAMINALESS1 (LAM1) are functional orthologs. A single amino acid (Asn to Ile) substitution in the homeodomain abolished the repression of Mt-AS2 and STF's ability to complement the lam1 mutant of Nicotiana sylvestris. Our data together support a model in which STF recruits corepressors to transcriptionally repress its targets during leaf blade morphogenesis. We propose that recruitment of TPL/TPL-related proteins may be a common mechanism in the repressive function of modern/WUS clade WOX genes.

Leaf shape elaboration and organ separation are critical for plant morphogenesis. We characterized the developmental roles of LOBED LEAFLET1 by analyzing a recessive mutant in the model legume Medicago truncatula. An ortholog of Arabidopsis thaliana ARGONAUTE7 (AGO7), Mt-AGO7/LOBED LEAFLET1, is required for the biogenesis of a trans-acting short interfering RNA (ta-siRNA) to negatively regulate the expression of AUXIN RESPONSE FACTORs in M. truncatula. Loss of function in AGO7 results in pleiotropic phenotypes in different organs. The prominent phenotype of the ago7 mutant is lobed leaf margins and more widely spaced lateral organs, suggesting that the trans-acting siRNA3 (TAS3) pathway negatively regulates the formation of boundaries and the separation of lateral organs in M. truncatula. Genetic interaction analysis with the smooth leaf margin1 (slm1) mutant revealed that leaf margin formation is cooperatively regulated by the auxin/SLM1 (ortholog of Arabidopsis PIN-FORMED1) module, which influences the initiation of leaf margin teeth, and the TAS3 ta-siRNA pathway, which determines the degree of margin indentation. Further investigations showed that the TAS3 ta-siRNA pathway and NO APICAL MERISTEM (ortholog of Arabidopsis CUP-SHAPED COTYLEDON) antagonistically regulate both leaf margin development and lateral organ separation, and the regulation is partially dependent on the auxin/SLM1 module.

Nodulation in legumes involves the coordination of epidermal infection by rhizobia with cell divisions in the underlying cortex. During nodulation, rhizobia are entrapped within curled root hairs to form an infection pocket. Transcellular tubes called infection threads then develop from the pocket and become colonized by rhizobia. The infection thread grows toward the developing nodule primordia and rhizobia are taken up into the nodule cells, where they eventually fix nitrogen. The epidermal and cortical developmental programs are synchronized by a yet-to-be-identified signal that is transmitted from the outer to the inner cell layers of the root. Using a new allele of the Medicago truncatula mutant Lumpy Infections, lin-4, which forms normal infection pockets but cannot initiate infection threads, we show that infection thread initiation is required for normal nodule development. lin-4 forms nodules with centrally located vascular bundles similar to that found in lateral roots rather than the peripheral vasculature characteristic of legume nodules. The same phenomenon was observed in M. truncatula plants inoculated with the Sinorhizobium meliloti exoY mutant, and the M. truncatula vapyrin-2 mutant, all cases where infections arrest. Nodules on lin-4 have reduced expression of the nodule meristem marker MtCRE1 and do not express root-tip markers. In addition, these mutant nodules have altered patterns of gene expression for the cytokinin and auxin markers CRE1 and DR5. Our work highlights the coordinating role that bacterial infection exerts on the developing nodule and allows us to draw comparisons with primitive actinorhizal nodules and rhizobia-induced nodules on the nonlegume Parasponia andersonii.

Summary Molecular genetic analyses revealed that the WUSCHEL‐related homeobox (WOX) gene superfamily regulates several programs in plant development. Many different mechanisms are reported to underlie these alterations. The WOX family member STENOFOLIA (STF) is involved in leaf expansion in the eudicot Medicago truncutula. Here, we report that when this gene was ectopically expressed in a locally adapted hard red winter wheat cultivar (Triticum aestivum), the transgenic plants showed not only widened leaves but also accelerated flowering and increased chlorophyll content. These desirable traits were stably inherited in the progeny plants. STF binds to wheat genes that have the (GA)n/(CT)n DNA cis element, regardless of sequences flanking the DNA repeats, suggesting a mechanism for its pleiotropic effects. However, the amino acids between position 91 and 262 in the STF protein that were found to bind with the (GA)n motif have no conserved domain with any other GAGA‐binding proteins in animals or plants. We also found that STF interacted with a variety of proteins in wheat in yeast 2 hybrid assays. We conclude that the eudicot STF gene binds to (GA)n/(CT)n DNA elements and can be used to regulate leaf width, flowering time and chlorophyll content in monocot wheat.

Background Two crossing techniques for hybridization of chickpea have been reported and include pollination after emasculation and pollination without emasculation. Success of crossing with emasculation varied from 5 to 17%; while the success rate varied from 20 to 50% by pollination without emasculation. The important reason for the low success rate of the two procedures could be lack of detailed information on the flowering stages chosen for crossing together with the environment where plants grow. Results We describe a comprehensive method for chickpea crossing where two genotypes, ICCV96029 as female and PI503023 as male parent were used. Leaf shape and seed size were used as morphological markers to select hybrids. For crossing, incision was made along the central line of the keel petal for the removal of anthers and to expose the stigma for placement of pollen from donor parent on its surface. After pollination, style was inserted back gently inside the keel petal and covered by wing petals and standard petals to make a natural sac which prevents drying of internal organs. Alternatively, if the conditions are favorable there is no need to protect the pollinated flower and therefore petal removal method for cross-pollination can be used. Our method showed around 78% crossing success rate which is much higher than the previous results. Conclusions We have shown that the crossing by keel petal incision or petal removal is an effective approach which significantly increases the crossing success rate. Furthermore, our detailed method shows that the flowering stage, selection of parents and temperature play crucial roles in crossing success. Electronic supplementary material The online version of this article (doi:10.1186/s13007-017-0202-6) contains supplementary material, which is available to authorized users.

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod−), 51 mutants with totally ineffective nodules (Nod+ Fix−), 17 mutants with partially ineffective nodules (Nod+ Fix+/−), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/− Fix−), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/− Fix+), and 11 supernodulating mutants (Nod++Fix+/−). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN’T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod− lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging.

WUSCHEL-RELATED HOMEOBOX (WOX) genes are plant specific transcription factors that serve as master switches controlling key developmental programs from embryo apical-basal asymmetric patterning to organizing stem cells and development of lateral organs. Recently, we reported the requirement of a WOX1/MAW-like gene, STENOFOLIA (STF), for blade outgrowth and leaf vascular patterning in Medicago truncatula and Nicotiana sylvestris. The stf mutant in Medicago produces narrow leaves where mediolateral outgrowth of the blade is severely curtailed while proximodistal growth and trifoliate identity remain unaffected. The lam1 mutant in N. sylvestris produces leaves devoid of blade tissue with just 1-2 layers of rudimentary strips and lacks stem elongation. stf and lam1 mutants have narrow petals and are female sterile due to defective ovule development. Morphological analysis of mutants and STF expression patterns suggest that STF regulates blade outgrowth mainly by controlling cell division in the margins of leaf primordium. Both the blade and flower phenotypes of lam1 can be complemented with WUS expressed under the STF promoter suggesting a conserved mechanism in stem cell maintenance and lateral organ development.

Tnt1 (transposable element if Nicotiana tabaccum cell type 1) is one of the very few active LTR retrotransposons used for gene tagging in plants. In the model legume Medicago truncatula, Tnt1 has been effectively used as a gene knock-out tool to generate several very useful mutants. stenofolia (stf) is such a mutant identified by Tnt1 insertion in a WUSCHEL-like homeobox transcription factor. STF is required for blade outgrowth, leaf vascular patterning and female reproductive organ development in barrel medic and woodland tobacco. Using transcript profiling and metabolite analysis, we uncovered that mutant leaves are compromised in steady-state levels of multiple phytohormones, sugar metabolites and derivatives including flavonoids and polyamines. In the lam1 mutant (caused by deletion of the STF ortholog in Nicotiana sylvestris), while glucose, fructose, mannose, galactose, myo-inositol and aromatic aminoacids are dramatically reduced, sucrose is comparable to wild-type levels, and glutamine, proline, putrescine, nicotine and sorbitol are highly increased. We demonstrated that both stf and lam1 mutants accumulate reduced levels of free auxin and ABA in their leaves, and ectopic expression of STF in tobacco leads to auxin and cytokinin overproduction phenotypes including formation of tumors on the roots and crown. These data suggest that STF mediated integration of metabolic and hormonal signals are required for lateral organ morphogenesis and elaboration.

Legumes form endosymbiotic associations with nitrogen-fixing bacteria and arbuscular mycorrhizal (AM) fungi which facilitate nutrient uptake. Both symbiotic interactions require a molecular signal exchange between the plant and the symbiont, and this involves a conserved symbiosis (Sym) signaling pathway. In order to identify plant genes required for intracellular accommodation of nitrogen-fixing bacteria and AM fungi, we characterized Medicago truncatula symbiotic mutants defective for rhizobial infection of nodule cells and colonization of root cells by AM hyphae. Here, we describe mutants impaired in the interacting protein of DMI3 (IPD3) gene, which has been identified earlier as an interacting partner of the calcium/calmodulin-dependent protein, a member of the Sym pathway. The ipd3 mutants are impaired in both rhizobial and mycorrhizal colonization and we show that IPD3 is necessary for appropriate Nod-factor-induced gene expression. This indicates that IPD3 is a member of the common Sym pathway. We observed differences in the severity of ipd3 mutants that appear to be the result of the genetic background. This supports the hypothesis that IPD3 function is partially redundant and, thus, additional genetic components must exist that have analogous functions to IPD3. This explains why mutations in an essential component of the Sym pathway have defects at late stages of the symbiotic interactions.

FLOWERING LOCUS T (FT) genes encode proteins that function as the mobile floral signal, florigen. In this study, we characterized five FT-like genes from the model legume, Medicago (Medicago truncatula). The different FT genes showed distinct patterns of expression and responses to environmental cues. Three of the FT genes (MtFTa1, MtFTb1, and MtFTc) were able to complement the Arabidopsis (Arabidopsis thaliana) ft-1 mutant, suggesting that they are capable of functioning as florigen. MtFTa1 is the only one of the FT genes that is up-regulated by both long days (LDs) and vernalization, conditions that promote Medicago flowering, and transgenic Medicago plants overexpressing the MtFTa1 gene flowered very rapidly. The key role MtFTa1 plays in regulating flowering was demonstrated by the identification of fta1 mutants that flowered significantly later in all conditions examined. fta1 mutants do not respond to vernalization but are still responsive to LDs, indicating that the induction of flowering by prolonged cold acts solely through MtFTa1, whereas photoperiodic induction of flowering involves other genes, possibly MtFTb1, which is only expressed in leaves under LD conditions and therefore might contribute to the photoperiodic regulation of flowering. The role of the MtFTc gene is unclear, as the ftc mutants did not have any obvious flowering-time or other phenotypes. Overall, this work reveals the diversity of the regulation and function of the Medicago FT family.