GRAS transcription factor PINNATE-LIKE PENTAFOLIATA2 controls compound leaf morphogenesis in Medicago truncatula.

The milestone of compound leaf development is the generation of separate leaflet primordia during the early stages, which involves two linked but distinct morphogenetic events: leaflet initiation and boundary establishment for leaflet separation. Although some progress in understanding the regulatory pathways for each event have been made, it is unclear how they are intrinsically coordinated. Here, we identify the PINNATE-LIKE PENTAFOLIATA2 (PINNA2) gene encoding a newly identified GRAS transcription factor in Medicago truncatula. PINNA2 transcripts are preferentially detected at organ boundaries. Its loss-of-function mutations convert trifoliate leaves into a pinnate pentafoliate pattern. PINNA2 directly binds to the promoter region of the LEAFY orthologue SINGLE LEAFLET1 (SGL1), which encodes a key positive regulator of leaflet initiation, and downregulates its expression. Further analysis revealed that PINNA2 synergizes with two other repressors of SGL1 expression, the BEL1-like homeodomain protein PINNA1 and the C2H2 zinc finger protein PALMATE-LIKE PENTAFOLIATA1 (PALM1), to precisely define the spatiotemporal expression of SGL1 in compound leaf primordia, thereby maintaining a proper pattern of leaflet initiation. Moreover, we showed that the enriched expression of PINNA2 at the leaflet-to-leaflet boundaries is positively regulated by the boundary-specific gene MtNAM, which is essential for leaflet boundary formation. Together, these results unveil a pivotal role of the boundary-expressed transcription factor PINNA2 in regulating leaflet initiation, providing molecular insights into the coordination of intricate developmental processes underlying compound leaf pattern formation.

WOX family transcriptional regulators modulate cytokinin homeostasis during leaf blade development in Medicago truncatula and Nicotiana sylvestris.

The plant-specific family of WUSCHEL (WUS)-related homeobox (WOX) transcription factors is key regulators of embryogenesis, meristem maintenance, and lateral organ development in flowering plants. The modern/WUS clade transcriptional repressor STENOFOLIA/LAMINA1(LAM1), and the intermediate/WOX9 clade transcriptional activator MtWOX9/NsWOX9 antagonistically regulate leaf blade expansion, but the molecular mechanism is unknown. Using transcriptome profiling and biochemical methods, we determined that NsCKX3 is the common target of LAM1 and NsWOX9 in Nicotiana sylvestris. LAM1 and NsWOX9 directly recognize and bind to the same cis-elements in the NsCKX3 promoter to repress and activate its expression, respectively, thus controlling the levels of active cytokinins in vivo. Disruption of NsCKX3 in the lam1 background yielded a phenotype similar to the knockdown of NsWOX9 in lam1, while overexpressing NsCKX3 resulted in narrower and shorter lam1 leaf blades reminiscent of NsWOX9 overexpression in the lam1 mutant. Moreover, we established that LAM1 physically interacts with NsWOX9, and this interaction is required to regulate NsCKX3 transcription. Taken together, our results indicate that repressor and activator WOX members oppositely regulate a common downstream target to function in leaf blade outgrowth, offering a novel insight into the role of local cytokinins in balancing cell proliferation and differentiation during lateral organ development.

CsMYBL2 homologs modulate the light and temperature stress-regulated anthocyanin and catechins biosynthesis in tea plants (Camellia sinensis).

Anthocyanin and catechin production in tea (Camellia sinensis) leaves can positively affect tea quality; however, their regulatory mechanisms are not fully understood. Here we report that, while the CsMYB75- or CsMYB86-directed MYB-bHLH-WD40 (MBW) complexes differentially activate anthocyanin or catechin biosynthesis in tea leaves, respectively, CsMYBL2a and CsMYBL2b homologs negatively modified the light- and temperature-induced anthocyanin and catechin production in both Arabidopsis and tea plants. The MBW complexes activated both anthocyanin synthesis genes and the downstream repressor genes CsMYBL2a and CsMYBL2b. Overexpression of CsMYBL2b, but not CsMYBL2a, repressed Arabidopsis leaf anthocyanin accumulation and seed coat proanthocyanin production. CsMYBL2b strongly and CsMYBL2a weakly repressed the activating effects of CsMYB75/CsMYB86 on CsDFR and CsANS, due to their different EAR and TLLLFR domains and interactions with CsTT8/CsGL3, interfering with the functions of activating MBW complexes. CsMYBL2b and CsMYBL2a in tea leaves play different roles in fine-tuning CsMYB75/CsMYB86-MBW activation of biosynthesis of anthocyanins and catechins, respectively. The CsbZIP1-CsmiR858a-CsMYBL2 module mediated the UV-B- or cold-activated CsMYB75/CsMYB86 regulation of anthocyanin/catechin biosynthesis by repressing CsMYBL2a and CsMYBL2b. Similarly, the CsCOP1-CsbZIP1-CsPIF3 module, and BR signaling as well, mediated the high temperature repression of anthocyanin and catechin biosynthesis through differentially upregulating CsMYBL2b and CsMYBL2a, respectively. The present study provides new insights into the complex regulatory networks in environmental stress-modified flavonoid production in tea plant leaves.

Sorghum SbGhd7 is a major regulator of floral transition and directly represses genes crucial for flowering activation.

Molecular genetic understanding of flowering time regulation is crucial for sorghum development. GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (SbGhd7) is one of the six classical loci conferring photoperiod sensitivity of sorghum flowering. However, its functions remain poorly studied. The molecular functions of SbGhd7 were characterized. The gene regulatory network controlled by SbGhd7 was constructed and validated. The biological roles of SbGhd7 and its major targets were studied. SbGhd7 overexpression (OE) completely prevented sorghum flowering. Additionally, we show that SbGhd7 is a major negative regulator of flowering, binding to the promoter motif TGAATG(A/T)(A/T/C) and repressing transcription of the major florigen FLOWERING LOCUS T 10 (SbFT10) and floral activators EARLY HEADING DATE (SbEhd1), FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (SbFKF1) and EARLY FLOWERING 3 (SbELF3). Reinforcing the direct effect of SbGhd7, SbEhd1 OE activated the promoters of three functional florigens (SbFT1, SbFT8 and SbFT10), dramatically accelerating flowering. Our studies demonstrate that SbGhd7 is a major repressor of sorghum flowering by directly and indirectly targeting genes for flowering activation. The mechanism appears ancient. Our study extends the current model of floral transition regulation in sorghum and provides a framework for a comprehensive understanding of sorghum photoperiod response.

The STF/WOX1 MD is required for physical interaction with MtWOX9 and leaf blade outgrowth in Medicago truncatula.

Plant-specific WUSCHEL-related homeobox (WOX) family transcription factors play critical roles in maintaining meristems and lateral organ development. The WUS clade member STF/LAM1 physically interacts with the intermediate clade member WOX9. This interaction contributes to their antagonistical functions on leaf blade outgrowth by competing for the same cis-elements in the promoter of their common target in M. truncatula and N. sylvestris. Here, we identified the main interaction domains of STF and MtWOX9 in Medicago, shedding light on the mechanism of WOX gene function. The middle domain of STF and MtWOX9 are both critical for the interaction, while the conserved motif of STF in the C-terminal domain is also required. Deletion of the middle domain of STF partially rescued the leaf blade phenotypes of the stf null mutant, indicating that the middle domain plays an essential role during leaf blade expansion. This finding provides a new insight that the versatility of WOX function is not only caused by the conserved DNA binding and repression domains but also by the middle domain that recruits different partners.

Diverse roles of MYB transcription factors in regulating secondary metabolite biosynthesis, shoot development, and stress responses in tea plants (Camellia sinensis).

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.

Multiplex CRISPR/Cas9-mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality.

Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.

The MIO1-MtKIX8 module regulates the organ size in Medicago truncatula.

Plant organ size is an important agronomic trait tightly related to crop yield. However, the molecular mechanisms underlying organ size regulation remain largely unexplored in legumes. We previously characterized a key regulator F-box protein MINI ORGAN1 (MIO1)/SMALL LEAF AND BUSHY1 (SLB1), which controls plant organ size in the model legume Medicago truncatula. In order to further dissect the molecular mechanism, MIO1 was used as the bait to screen its interacting proteins from a yeast library. Subsequently, a KIX protein, designated MtKIX8, was identified from the candidate list. The interaction between MIO1 and MtKIX8 was confirmed further by Y2H, BiFC, split-luciferase complementation and pull-down assays. Phylogenetic analyses indicated that MtKIX8 is highly homologous to Arabidopsis KIX8, which negatively regulates organ size. Moreover, loss-of-function of MtKIX8 led to enlarged leaves and seeds, while ectopic expression of MtKIX8 in Arabidopsis resulted in decreased cotyledon area and seed weight. Quantitative reverse-transcription PCR and in situ hybridization showed that MtKIX8 is expressed in most developing organs. We also found that MtKIX8 serves as a crucial molecular adaptor, facilitating interactions with BIG SEEDS1 (BS1) and MtTOPLESS (MtTPL) proteins in M. truncatula. Overall, our results suggest that the MIO1-MtKIX8 module plays a significant and conserved role in the regulation of plant organ size. This module could be a good target for molecular breeding in legume crops and forages.

The MYB Activator WHITE PETAL1 Associates with MtTT8 and MtWD40-1 to Regulate Carotenoid-Derived Flower Pigmentation in Medicago truncatula.

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.

Multidimensional Gene Regulatory Landscape of Motor Organ Pulvinus in the Model Legume Medicago truncatula.

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.

Sinorhizobium meliloti succinylated high-molecular-weight succinoglycan and the Medicago truncatula LysM receptor-like kinase MtLYK10 participate independently in symbiotic infection.

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.

The 3-ketoacyl-CoA synthase WFL is involved in lateral organ development and cuticular wax synthesis in Medicago truncatula.

KEY MESSAGE: 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.

The geometry of the compound leaf plays a significant role in the leaf movement of Medicago truncatula modulated by mtdwarf4a.

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 MtDWARF4A (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.

WOX9 functions antagonistic to STF and LAM1 to regulate leaf blade expansion in Medicago truncatula and Nicotiana sylvestris.

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.

The WOX family transcriptional regulator SlLAM1 controls compound leaf and floral organ development in Solanum lycopersicum.

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.

The F-box protein MIO1/SLB1 regulates organ size and leaf movement 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.

HSI2/VAL1 and HSL1/VAL2 function redundantly to repress DOG1 expression in Arabidopsis seeds and seedlings.

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.

SMALL LEAF AND BUSHY1 controls organ size and lateral branching by modulating the stability of BIG SEEDS1 in Medicago truncatula.

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.

Lateral Leaflet Suppression 1 (LLS1), encoding the MtYUCCA1 protein, regulates lateral leaflet development in Medicago truncatula.

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.

A study of male fertility control in Medicago truncatula uncovers an evolutionarily conserved recruitment of two tapetal bHLH subfamilies in plant sexual reproduction.

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.