Coordinated regulation of vegetative phase change by brassinosteroids and the age pathway in Arabidopsis.

Vegetative phase change in plants is regulated by a gradual decline in the level of miR156 and a corresponding increase in the expression of its targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. Gibberellin (GA), jasmonic acid (JA), and cytokinin (CK) regulate vegetative phase change by affecting genes in the miR156-SPL pathway. However, whether other phytohormones play a role in vegetative phase change remains unknown. Here, we show that a loss-of-function mutation in the brassinosteroid (BR) biosynthetic gene, DWARF5 (DWF5), delays vegetative phase change, and the defective phenotype is primarily attributable to reduced levels of SPL9 and miR172, and a corresponding increase in TARGET OF EAT1 (TOE1). We further show that GLYCOGEN SYNTHASE KINASE3 (GSK3)-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2) directly interacts with and phosphorylates SPL9 and TOE1 to cause subsequent proteolytic degradation. Therefore, BRs function to stabilize SPL9 and TOE1 simultaneously to regulate vegetative phase change in plants.

Reducing brassinosteroid signalling enhances grain yield in semi-dwarf wheat.

Modern green revolution varieties of wheat (Triticum aestivum L.) confer semi-dwarf and lodging-resistant plant architecture owing to the Reduced height-B1b (Rht-B1b) and Rht-D1b alleles1. However, both Rht-B1b and Rht-D1b are gain-of-function mutant alleles encoding gibberellin signalling repressors that stably repress plant growth and negatively affect nitrogen-use efficiency and grain filling2-5. Therefore, the green revolution varieties of wheat harbouring Rht-B1b or Rht-D1b usually produce smaller grain and require higher nitrogen fertilizer inputs to maintain their grain yields. Here we describe a strategy to design semi-dwarf wheat varieties without the need for Rht-B1b or Rht-D1b alleles. We discovered that absence of Rht-B1 and ZnF-B (encoding a RING-type E3 ligase) through a natural deletion of a haploblock of about 500 kilobases shaped semi-dwarf plants with more compact plant architecture and substantially improved grain yield (up to 15.2%) in field trials. Further genetic analysis confirmed that the deletion of ZnF-B induced the semi-dwarf trait in the absence of the Rht-B1b and Rht-D1b alleles through attenuating brassinosteroid (BR) perception. ZnF acts as a BR signalling activator to facilitate proteasomal destruction of the BR signalling repressor BRI1 kinase inhibitor 1 (TaBKI1), and loss of ZnF stabilizes TaBKI1 to block BR signalling transduction. Our findings not only identified a pivotal BR signalling modulator but also provided a creative strategy to design high-yield semi-dwarf wheat varieties by manipulating the BR signal pathway to sustain wheat production.

Genome-wide identification and expression analysis of BZR gene family and associated responses to abiotic stresses in cucumber (Cucumis sativus L.).

BACKGROUND: BRASSINAZOLE-RESISTANT (BZR) is a class of specific transcription factor (TFs) involved in brassinosteroid (BR) signal transduction. The regulatory mechanism of target genes mediated by BZR has become one of the key research areas in plant BR signaling networks. However, the functions of the BZR gene family in cucumber have not been well characterized. RESULTS: In this study, six CsBZR gene family members were identified by analyzing the conserved domain of BES1 N in the cucumber genome. The size of CsBZR proteins ranges from 311 to 698 amino acids and are mostly located in the nucleus. Phylogenetic analysis divided CsBZR genes into three subgroups. The gene structure and conserved domain showed that the BZR genes domain in the same group was conserved. Cis-acting element analysis showed that cucumber BZR genes were mainly involved in hormone response, stress response and growth regulation. The qRT-PCR results also confirmed CsBZR response to hormones and abiotic stress. CONCLUSION: Collectively, the CsBZR gene is involved in regulating cucumber growth and development, particularly in hormone response and response to abiotic stress. These findings provide valuable information for understanding the structure and expression patterns of BZR genes.

Exogenous 24-epibrassinolide ameliorates tolerance to high-temperature by adjusting the biosynthesis of pigments, enzymatic, non-enzymatic antioxidants, and diosgenin content in fenugreek.

High-temperature stress is widely considered a main plant-growth-limiting factor. The positive effects of 24-epibrassinolide (EBR) as analogs of brassinosteroids (BRs) in modulating abiotic stresses have led this hormone to be referred to as a growth regulator in plants. The current study highlights the influence of EBR on enhancing tolerance to high-temperature and altering the diosgenin content in fenugreek. Different amounts of EBR (4, 8, and 16 µM), harvesting times (6, and 24 h), as well as temperature regimes (23 °C, and 42 °C) were, used as treatments. EBR application under normal temperature and high-temperature stress resulted in decreased malondialdehyde content and electrolyte leakage percentage, while the activity of antioxidant enzymes improved significantly. Exogenous EBR application possibly contributes to activating the nitric oxide, H2O2, and ABA-dependent pathways, enhancing the biosynthesis of abscisic acid and auxin, and regulating the signal transduction pathways, which raises fenugreek tolerance to high-temperature. The SQS (eightfold), SEP (2.8-fold), CAS (11-fold), SMT (17-fold), and SQS (sixfold) expression, considerably increased following EBR application (8 µM) compared to the control. Compared to the control, when the short-term (6 h) high-temperature stress was accompanied by EBR (8 µM), a sixfold increase in diosgenin content was achieved. Our findings highlight the potential role of exogenous 24-epibrassinolide in mitigating the high-temperature stress in fenugreek by stimulating the biosynthesis processes of enzymatic and non-enzymatic antioxidants, chlorophylls, and diosgenin. In conclusion, the current results could be of utmost importance in breeding or biotechnology-based programs of fenugreek and also in the researches related to the engineering of the biosynthesis pathway of diosgenin in this valuable plant.

HD-ZIP III-dependent local promotion of brassinosteroid synthesis suppresses vascular cell division in Arabidopsis root apical meristem.

Spatiotemporal control of cell division in the meristem is vital for plant growth. In the stele of the root apical meristem (RAM), procambial cells divide periclinally to increase the number of vascular cell files. Class III homeodomain leucine zipper (HD-ZIP III) proteins are key transcriptional regulators of RAM development and suppress the periclinal division of vascular cells in the stele; however, the mechanism underlying the regulation of vascular cell division by HD-ZIP III transcription factors (TFs) remains largely unknown. Here, we performed transcriptome analysis to identify downstream genes of HD-ZIP III and found that HD-ZIP III TFs positively regulate brassinosteroid biosynthesis-related genes, such as CONSTITUTIVE PHOTOMORPHOGENIC DWARF (CPD), in vascular cells. Introduction of pREVOLUTA::CPD in a quadruple loss-of-function mutant of HD-ZIP III genes partly rescued the phenotype in terms of the vascular defect in the RAM. Treatment of a quadruple loss-of-function mutant, a gain-of-function mutant of HD-ZIP III, and the wild type with brassinosteroid and a brassinosteroid synthesis inhibitor also indicated that HD-ZIP III TFs act together to suppress vascular cell division by increasing brassinosteroid levels. Furthermore, brassinosteroid application suppressed the cytokinin response in vascular cells. Together, our findings suggest that the suppression of vascular cell division by HD-ZIP III TFs is caused, at least in part, by the increase in brassinosteroid levels through the transcriptional activation of brassinosteroid biosynthesis genes in the vascular cells of the RAM. This elevated brassinosteroid level suppresses cytokinin response in vascular cells, inhibiting vascular cell division in the RAM.

The receptor-like kinase EMS1 and BRI1 coordinately regulate stamen elongation via the transcription factors BES1/BZR1 in Arabidopsis.

Plants possess a large family of receptor kinase proteins to mediate cell-to-cell and cell-to-environment communication, and these regulations are essential for plant growth and development as well as resistance to biotic or abiotic stresses. EMS1 is a receptor kinase which involved in tapetum cell fate determination during anther development, while brassinosteroid (BR) receptor, BRI1, controls most aspects of plant growth and development. Although EMS1 and BRI1 are known to regulate independent biological processes, they interact with identical components of the downstream signaling pathways. However, the biological processes other than the tapetum development controlled by the EMS1 signal are not clear. Here, we report that EMS1 signaling-related mutants exhibited an insufficient stamen elongation phenotype, similar to BR signaling mutants. Transgenic expression of BRI1 restored the short filament phenotype of ems1. Conversely, co-expression of EMS1 and TPD1 also restored the short filaments of BRI1 mutants, bri1. Genetic experiments confirmed that EMS1 and BRI1 regulate filament elongation through their downstream transcription factors BES1/BZR1. Molecular analysis suggested that the decrease in BR signaling output in filaments of the ems1 mutant caused deficient filament development. Moreover, in vitro and in vivo experiments proved BES1 interacts with filament-specific transcription factor MYB21. Together, we found that the two receptor-like kinases (RLKs) EMS1 and BRI1 are cooperatively involved in the regulation of filament elongation via the transcription factors BES1/BZR1. These results indicated that the biological processes regulated by EMS1 and BRI1 in plants are both independent and interactive, which provides us with insights into multidimensional molecular control of the RLK pathway.

Brassinosteroid gene regulatory networks at cellular resolution in the Arabidopsis root.

Brassinosteroids are plant steroid hormones that regulate diverse processes, such as cell division and cell elongation, through gene regulatory networks that vary in space and time. By using time series single-cell RNA sequencing to profile brassinosteroid-responsive gene expression specific to different cell types and developmental stages of the Arabidopsis root, we identified the elongating cortex as a site where brassinosteroids trigger a shift from proliferation to elongation associated with increased expression of cell wall-related genes. Our analysis revealed HOMEOBOX FROM ARABIDOPSIS THALIANA 7 (HAT7) and GT-2-LIKE 1 (GTL1) as brassinosteroid-responsive transcription factors that regulate cortex cell elongation. These results establish the cortex as a site of brassinosteroid-mediated growth and unveil a brassinosteroid signaling network regulating the transition from proliferation to elongation, which illuminates aspects of spatiotemporal hormone responses.

Grape cytochrome P450 CYP90D1 regulates brassinosteroid biosynthesis and increases vegetative growth.

Vine vigor or vegetative growth is an important factor related to berry quality and vinicultural training management, but brassinosteroid (BR)-induced molecular mechanisms underlying vine growth remain unclear. In this study, the hypothesis that the Vitis vinifera CYP90D1 gene VvCYP90D1, one of the genes for BR biosynthesis, plays a critical role in shoot elongation was tested. RNA sequencing analysis of shoots collected from the vigorous cultivar Koshu (KO) and the reference cultivar Pinot Noir (PN) 7 days after bud break showed higher expression levels of various genes in the BR biosynthesis pathway in KO than in PN. The VvCYP90D1 expression level in KO was highest in meristems, followed by internodes and leaves. Cluster analysis of amino acid sequences including those in other plant species showed that the isolated gene belonged to the CYP90D1 group. The vegetative growth and the endogenous BR (brassinolide; BL) content were significantly higher in VvCYP90D1-overexpressing Arabidopsis than in wild type. VvCYP90D1-overexpressing Arabidopsis treated with brassinazole (Brz), a BR biosynthesis inhibitor, showed recovery of vegetative growth. These results indicate that VvCYP90D1 in grapevine has a vegetative growth promoting effect via BR biosynthesis. Our findings on the mechanism of BR-induced grape shoot growth will contribute to the development of new shoot control techniques for grapevine.

Epi-Brassinolide Regulates ZmC4 NADP-ME Expression through the Transcription Factors ZmbHLH157 and ZmNF-YC2.

Maize is a main food and feed crop with great production potential and high economic benefits. Improving its photosynthesis efficiency is crucial for increasing yield. Maize photosynthesis occurs mainly through the C4 pathway, and NADP-ME (NADP-malic enzyme) is a key enzyme in the photosynthetic carbon assimilation pathway of C4 plants. ZmC4-NADP-ME catalyzes the release of CO2 from oxaloacetate into the Calvin cycle in the maize bundle sheath. Brassinosteroid (BL) can improve photosynthesis; however, its molecular mechanism of action remains unclear. In this study, transcriptome sequencing of maize seedlings treated with epi-brassinolide (EBL) showed that differentially expressed genes (DEGs) were significantly enriched in photosynthetic antenna proteins, porphyrin and chlorophyll metabolism, and photosynthesis pathways. The DEGs of C4-NADP-ME and pyruvate phosphate dikinase in the C4 pathway were significantly enriched in EBL treatment. Co-expression analysis showed that the transcription level of ZmNF-YC2 and ZmbHLH157 transcription factors was increased under EBL treatment and moderately positively correlated with ZmC4-NADP-ME. Transient overexpression of protoplasts revealed that ZmNF-YC2 and ZmbHLH157 activate C4-NADP-ME promoters. Further experiments showed ZmNF-YC2 and ZmbHLH157 transcription factor binding sites on the -1616 bp and -1118 bp ZmC4 NADP-ME promoter. ZmNF-YC2 and ZmbHLH157 were screened as candidate transcription factors mediating brassinosteroid hormone regulation of the ZmC4 NADP-ME gene. The results provide a theoretical basis for improving maize yield using BR hormones.

Green means go: Green light promotes hypocotyl elongation via brassinosteroid signaling.

Although many studies have elucidated the mechanisms by which different wavelengths of light (blue, red, far-red, or ultraviolet-B [UV-B]) regulate plant development, whether and how green light regulates plant development remains largely unknown. Previous studies reported that green light participates in regulating growth and development in land plants, but these studies have reported conflicting results, likely due to technical problems. For example, commercial green light-emitting diode light sources emit a little blue or red light. Here, using a pure green light source, we determined that unlike blue, red, far-red, or UV-B light, which inhibits hypocotyl elongation, green light promotes hypocotyl elongation in Arabidopsis thaliana and several other plants during the first 2-3 d after planting. Phytochromes, cryptochromes, and other known photoreceptors do not mediate green-light-promoted hypocotyl elongation, but the brassinosteroid (BR) signaling pathway is involved in this process. Green light promotes the DNA binding activity of BRI1-EMS-SUPPRESSOR 1 (BES1), a master transcription factor of the BR pathway, thus regulating gene transcription to promote hypocotyl elongation. Our results indicate that pure green light promotes elongation via BR signaling and acts as a shade signal to enable plants to adapt their development to a green-light-dominant environment under a canopy.

Signaling by the EPFL-ERECTA family coordinates female germline specification through the BZR1 family in Arabidopsis.

In most flowering plants, the female germline is initiated in the subepidermal L2 layer of ovule primordia forming a single megaspore mother cell (MMC). How signaling from the L1 (epidermal) layer could contribute to the gene regulatory network (GRN) restricting MMC formation to a single cell is unclear. We show that EPIDERMAL PATTERNING FACTOR-like (EPFL) peptide ligands are expressed in the L1 layer, together with their ERECTA family (ERf) receptor kinases, to control female germline specification in Arabidopsis thaliana. EPFL-ERf dependent signaling restricts multiple subepidermal cells from acquiring MMC-like cell identity by activating the expression of the major brassinosteroid (BR) receptor kinase BRASSINOSTEROID INSENSITIVE 1 and the BR-responsive transcription factor BRASSINOZOLE RESISTANT 1 (BZR1). Additionally, BZR1 coordinates female germline specification by directly activating the expression of a nucleolar GTP-binding protein, NUCLEOSTEMIN-LIKE 1 (NSN1), which is expressed in early-stage ovules excluding the MMC. Mutants defective in this GRN form multiple MMCs resulting in a strong reduction of seed set. In conclusion, we uncovered a ligand/receptor-like kinase-mediated signaling pathway acting upstream and coordinating BR signaling via NSN1 to restrict MMC differentiation to a single subepidermal cell.

Genome-Wide Identification of BES1 Gene Family in Six Cucurbitaceae Species and Its Expression Analysis in Cucurbita moschata.

The BES1 (BRI1-EMSSUPPRESSOR1) gene family play a vital role in the BR (brassinosteroid) signaling pathway, which is involved in the growth and development, biotic, abiotic, and hormone stress response in many plants. However, there are few reports of BES1 in Cucurbita moschata. In this study, 50 BES1 genes were identified in six Cucurbitaceae species by genome-wide analysis, which could be classified into 3 groups according to their gene structural features and motif compositions, and 13 CmoBES1 genes in Cucurbita moschata were mapped on 10 chromosomes. Quantitative real-time PCR analysis showed that the CmoBES1 genes displayed differential expression under different abiotic stress and hormone treatments. Subcellular localization showed that the most of CmoBES1 proteins localized in nucleus and cytoplasm, and transactivation assay indicated 9 CmoBES1 proteins played roles as transcription factors. Our analysis of BES1s diversity, localization, and expression in Curcubitaceae contributes to the better understanding of the essential roles of these transcription factors in plants.

Brassinosteroids and gibberellic acid actively regulate the zinc detoxification mechanism of Medicago sativa L. seedlings.

BACKGROUND: Zinc is one of the essential trace elements in plants. There are few studies on the phytohormone to rescue the toxicity of excessive zinc to plants. The aim of this research was to evaluate the alleviating effects of brassinosteroids (BR) and gibberellic acid (GA) on the toxicity of Medicago sativa L. (M. sativa) induced by excessive zinc. RESULTS: After zinc, BR and GA were applied to M. sativa seedlings for 7 weeks, their physiological and biochemical properties and gene expression patterns were evaluated. BR and GA significantly weakened the inhibition effect of zinc stress on growth and biomass of M. sativa. Under zinc stress, the zinc accumulation in M. sativa roots was over 5 times that in shoots. Application of BR and GA reduced zinc accumulation in roots. The content of lipid peroxides in M. sativa decreased and the activity of antioxidant enzymes increased under BR and GA treatments. In addition, BR and GA treatment down-regulated the transcription level of MsZIP1/3/5, the transporters of zinc uptake in root cells. And BR and GA up-regulated the expressions of zinc efflux, chelation, vacuolar storage and long-distance transport related genes: MsZIP7, MsHMA1, MsZIF1, MsMTP1, MsYSL1 and MsNAS1. CONCLUSIONS: Our findings further showed that BR and GA application to M. sativa under zinc stress can reduce zinc accumulation, promote the response of the antioxidant defense system, and actively regulate the mechanism of heavy metal detoxification. Notably, 100 nM BR performed slightly better than 100 nM GA in all aspects of the detoxification of M. sativa by excessive zinc.

BRASSINOSTEROID INSENSITIVE1 internalization can occur independent of ligand binding.

The brassinosteroid (BR) hormone and its plasma membrane (PM) receptor BR INSENSITIVE1 (BRI1) are one of the best-studied receptor-ligand pairs for understanding the interplay between receptor endocytosis and signaling in plants. BR signaling is mainly determined by the PM pool of BRI1, whereas BRI1 endocytosis ensures signal attenuation. As BRs are ubiquitously distributed in the plant, the tools available to study the BRI1 function without interference from endogenous BRs are limited. Here, we designed a BR binding-deficient Arabidopsis (Arabidopsis thaliana) mutant based on protein sequence-structure analysis and homology modeling of members of the BRI1 family. This tool allowed us to re-examine the BRI1 endocytosis and signal attenuation model. We showed that despite impaired phosphorylation and ubiquitination, BR binding-deficient BRI1 internalizes similarly to the wild type form. Our data indicate that BRI1 internalization relies on different endocytic machineries. In addition, the BR binding-deficient mutant provides opportunities to study non-canonical ligand-independent BRI1 functions.

The regulatory module MdBZR1-MdCOL6 mediates brassinosteroid- and light-regulated anthocyanin synthesis in apple.

The anthocyanin content is an important indicator of the nutritional value of most fruits, including apple (Malus domestica). Anthocyanin synthesis is coordinately regulated by light and various phytohormones. In this study on apple, we revealed the antagonistic relationship between light and brassinosteroid (BR) signaling pathways, which is mediated by BRASSINAZOLE-RESISTANT 1 (MdBZR1) and the B-box protein MdCOL6. The exogenous application of brassinolide inhibited the high-light-induced anthocyanin accumulation in red-fleshed apple seedlings, whereas increases in the light intensity decreased the endogenous BR content. The overexpression of MdBZR1 inhibited the anthocyanin synthesis in apple plants. An exposure to a high-light intensity induced the degradation of dephosphorylated MdBZR1, resulting in functional impairment. MdBZR1 was identified as an upstream repressor of MdCOL6, which promotes anthocyanin synthesis in apple plants. Furthermore, MdBZR1 interacts with MdCOL6 to attenuate its ability to activate MdUFGT and MdANS transcription. Thus, MdBZR1 negatively regulates MdCOL6-mediated anthocyanin accumulation. Our study findings have clarified the molecular basis of the integration of light and BR signals during the regulation of anthocyanin biosynthesis, which is an important process influencing fruit quality.

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.

SUMO/deSUMOylation of the BRI1 brassinosteroid receptor modulates plant growth responses to temperature.

Brassinosteroids (BRs) are a class of steroid molecules perceived at the cell surface that act as plant hormones. The BR receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) offers a model to understand receptor-mediated signaling in plants and the role of post-translational modifications. Here we identify SUMOylation as a new modification targeting BRI1 to regulate its activity. BRI1 is SUMOylated in planta on two lysine residues, and the levels of BRI1 SUMO conjugates are controlled by the Desi3a SUMO protease. Loss of Desi3a leads to hypersensitivity to BRs, indicating that Desi3a acts as a negative regulator of BR signaling. Besides, we demonstrate that BRI1 is deSUMOylated at elevated temperature by Desi3a, leading to increased BRI1 interaction with the negative regulator of BR signaling BIK1 and to enhanced BRI1 endocytosis. Loss of Desi3a or BIK1 results in increased response to temperature elevation, indicating that BRI1 deSUMOylation acts as a safety mechanism necessary to keep temperature responses in check. Altogether, our work establishes BRI1 deSUMOylation as a molecular crosstalk mechanism between temperature and BR signaling, allowing plants to translate environmental inputs into growth response.

Propiconazole-induced brassinosteroid deficiency reduces female fertility by inhibiting female gametophyte development in woodland strawberry.

KEY MESSAGE: In woodland strawberry, a brassinosteroid biosynthesis inhibitor propiconazole induced typical brassinosteroid-deficient phenotypes and decreased female fertility due to attenuated female gametophyte development. Brassinosteroids (BRs) play roles in various aspects of plant development. We investigated the physiological roles of BRs in the woodland strawberry, Fragaria vesca. BR-level-dependent phenotypes were observed using a BR biosynthetic inhibitor, propiconazole (PCZ), and the most active natural BR, brassinolide (BL). Endogenous BL and castasterone, the active BRs, were below detectable levels in PCZ-treated woodland strawberry. The plants were typical BR-deficient phenotypes, and all phenotypes were restored by treatment with BL. These observations indicate that PCZ is an effective inhibitor of BR in woodland strawberry. Only one gene for each major step of BR biosynthesis in Arabidopsis is encoded in the woodland strawberry genome. BR biosynthetic genes are highly expressed during the early stage of fruit development. Emasculated flowers treated with BL failed to develop fruit, implying that BR is not involved in parthenocarpic fruit development. Similar to BR-deficient and BR-insensitive Arabidopsis mutants, female fertility was lower in PCZ-treated plants than in mock-treated plants due to failed attraction of the pollen tube to the ovule. In PCZ-treated plants, expression of FveMYB98, the homologous gene for Arabidopsis MYB98 (a marker for synergid cells), was downregulated. Ovules were smaller in PCZ-treated plants than in mock-treated plants, and histological analysis implied that the development of more than half of female gametophytes was arrested at the early stage in PCZ-treated plants. Our findings explain how BRs function during female gametophyte development in woodland strawberry.

Mapping the signaling network of BIN2 kinase using TurboID-mediated biotin labeling and phosphoproteomics.

Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.