Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis.

In Arabidopsis thaliana, female gametophyte (FG) development is accompanied by the formation and expansion of the large vacuole in the FG; this is essential for FG expansion, nuclear polar localization, and cell fate determination. Arabidopsis VACUOLELESS GAMETOPHYTES (VLG) facilitates vesicular fusion to form large vacuole in the FG, but the regulation of VLG remains largely unknown. Here, we found that gain-of-function mutation of BRASSINOSTEROID INSENSITIVE2 (BIN2) (bin2-1) increases VLG abundance to induce the vacuole formation at stage FG1, and leads to abortion of FG. Loss-of-function mutation of BIN2 and its homologs (bin2-3 bil1 bil2) reduced VLG abundance and mimicked vlg/VLG phenotypes. Knocking down VLG in bin2-1 decreased the ratio of aberrant vacuole formation at stage FG1, whereas FG1-specific overexpression of VLG mimicked the bin2-1 phenotype. VLG partially rescued the bin2-3 bil1 bil2 phenotype, demonstrating that VLG acts downstream of BIN2. Mutation of VLG residues that are phosphorylated by BIN2 altered VLG stability and a phosphorylation mimic of VLG causes similar defects as did bin2-1. Therefore, BIN2 may function by interacting with and phosphorylating VLG in the FG to enhance its stability and abundance, thus facilitating vacuole formation. Our findings provide mechanistic insight into how the BIN2-VLG module regulates the spatiotemporal formation of the large vacuole in FG development.

Interaction of brassinosteroid and cytokinin promotes ovule initiation and increases seed number per silique in Arabidopsis.

Ovule initiation is a key step that strongly influences ovule number and seed yield. Notably, mutants with enhanced brassinosteroid (BR) and cytokinin (CK) signaling produce more ovules and have a higher seed number per silique (SNS) than wild-type plants. Here, we crossed BR- and CK-related mutants to test whether these phytohormones function together in ovule initiation. We determined that simultaneously enhancing BR and CK contents led to higher ovule and seed numbers than enhancing BR or CK separately, and BR and CK enhanced each other. Further, the BR-response transcription factor BZR1 directly interacted with the CK-response transcription factor ARABIDOPSIS RESPONSE REGULATOR1 (ARR1). Treatments with BR or BR plus CK strengthened this interaction and subsequent ARR1 targeting and induction of downstream genes to promote ovule initiation. Enhanced CK signaling partially rescued the reduced SNS phenotype of BR-deficient/insensitive mutants whereas enhanced BR signaling failed to rescue the low SNS of CK-deficient mutants, suggesting that BR regulates ovule initiation and SNS through CK-mediated and -independent pathways. Our study thus reveals that interaction between BR and CK promotes ovule initiation and increases seed number, providing important clues for increasing the seed yield of dicot crops.

Effective Modulating Brassinosteroids Signal to Study Their Specific Regulation of Reproductive Development and Enhance Yield.

Brassinosteroid (BR) is a family of bioactive steroid hormones that plays vital roles in plant growth and development. The BR-mediated regulation of plant growth and architecture has been well studied. However, relatively few studies have investigated the BR-related regulation of reproductive development because of the difficulties in excluding non-specific regulation and secondary responses from severe vegetative phenotypes and poor nutritional status. Furthermore, differentially regulating the BR signal in vegetative and reproductive organs is problematic. Thus, establishing a method for modulating the BR signal only in reproductive organs or during reproductive developmental stages will be beneficial. Additionally, the utility of BR applications for crop production is limited because of deleterious side-effects, including the associated decrease in the planting density and lodging resistance. Moreover, enhancing the BR signal may lead to feedback inhibition. In this study, we developed a transformation system for modulating the BR signal differentially during reproductive and vegetative developmental stages. This system involves transformations with different combinations of a reproductive tissue-specific promoter, coding sequences that increase or decrease the BR signal, and various genotypic backgrounds with enhanced or decreased BR signals. The enhanced BR signal generated in transformants was targeted to reproductive organs without affecting vegetative organs. This system may be useful for studying the BR-specific regulation of plant reproductive development and shows promise for optimizing seed yield.

Functional characterization of GmBZL2 (AtBZR1 like gene) reveals the conserved BR signaling regulation in Glycine max.

Brassinosteroids (BRs) play key roles in plant growth and development, and regulate various agricultural traits. Enhanced BR signaling leads to increased seed number and yield in Arabidopsis bzr1-1D (AtBZR1(P234L), gain-of-function mutant of the important transcription factor in BR signaling/effects). BR signal transduction pathway is well elucidated in Arabidopsis but less known in other species. Soybean is an important dicot crop producing edible oil and protein. Phylogenetic analysis reveals AtBZR1-like genes are highly conserved in angiosperm and there are 4 orthologues in soybean (GmBZL1-4). We here report the functional characterization of GmBZL2 (relatively highly expresses in flowers). The P234 site in AtBZR1 is conserved in GmBZL2 (P216) and mutation of GmBZL2(P216L) leads to GmBZL2 accumulation. GmBZL2(P216L) (GmBZL2*) in Arabidopsis results in enhanced BR signaling; including increased seed number per silique. GmBZL2* partially rescued the defects of bri1-5, further demonstrating the conserved function of GmBZL2 with AtBZR1. BR treatment promotes the accumulation, nuclear localization and dephosphorylation/phosphorylation ratio of GmBZL2, revealing that GmBZL2 activity is regulated conservatively by BR signaling. Our studies not only indicate the conserved regulatory mechanism of GmBZL2 and BR signaling pathway in soybean, but also suggest the potential application of GmBZL2 in soybean seed yield.