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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., (© 2023 Scandinavian Plant Physiology Society.)

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., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

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., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

The WUSCHEL related homeobox (WOX) genes play key roles in stem cell maintenance, embryonic patterning, and lateral organ development. WOX genes have been categorized into three clades--ancient, intermediate, and modern/WUS--based on phylogenetic analysis, but a functional basis for this classification has not been established. Using the classical bladeless lam1 mutant of Nicotiana sylvestris as a genetic tool, we examined the function of the Medicago truncatula WOX gene, STENOFOLIA (STF), in controlling leaf blade outgrowth. STF and LAM1 are functional orthologs. We found that the introduction of mutations into the WUS-box of STF (STFm1) reduces its ability to complement the lam1 mutant. Fusion of an exogenous repressor domain to STFm1 restores complementation, whereas fusion of an exogenous activator domain to STFm1 enhances the narrow leaf phenotype. These results indicate that transcriptional repressor activity mediated by the WUS-box of STF acts to promote blade outgrowth. With the exception of WOX7, the WUS-box is conserved in the modern clade WOX genes, but is not found in members of the intermediate or ancient clades. Consistent with this, all members of the modern clade except WOX7 can complement the lam1 mutant when expressed using the STF promoter, but members of the intermediate and ancient clades cannot. Furthermore, we found that fusion of either the WUS-box or an exogenous repressor domain to WOX7 or to members of intermediate and ancient WOX clades results in a gain-of-function ability to complement lam1 blade outgrowth. These results suggest that modern clade WOX genes have evolved for repressor activity through acquisition of the WUS-box.

FLOWERING LOCUS C (FLC) in Arabidopsis encodes a dosage dependent repressor of flowering. We isolated five FLC-related sequences from Brassica napus (BnFLC1-5). Expression of each of the five sequences in Arabidopsis delayed flowering significantly, with the delay in flowering time ranging from 3 weeks to more than 7 months, relative to the flowering time of 3 weeks in untransformed Ler. In the reciprocal experiment, expression of Arabidopsis FLC (AtFLC) in an early flowering B. napus cultivar delayed flowering by 2-6 weeks, confirming the requirement of this gene for floral repression. In B. napus, we show that late flowering and responsiveness to vernalization correlate with the level of BnFLC mRNA expression. The different BnFLC genes show differential expression in leaves, stems and shoot tips, but expression is not detectable in roots. Vernalization dramatically reduces the level of BnFLC transcript and restores early flowering in the winter cultivar Colombus. We conclude that BnFLC genes confer winter requirement in B. napus and account for the major vernalization-responsive flowering time differences in the different cultivars of B. napus in a manner analogous to that of AtFLC in Arabidopsis ecotypes.