Regulator Of Brassinosteroids Signaling Research Articles

A computational analysis revealed BES1 transcription factor and β-amylase as crosstalk elements in Upland cotton species (Gossypium sp.)

Cotton is a resilient and multipurpose crop, meeting major of the world’s textile needs while also yielding byproducts like edible oil and animal feed. Starch plays a crucial role in cotton fabric production. It enhances fabric strength by forming a protective film around cotton fibers, making them more resistant to wear and tear. BES1 (brassinosteroid insensitive 1) is a key regulator in brassinosteroid signaling. It controls thousands of target genes involved in development processes. Interestingly, two β-amylase proteins (BAM7 and BAM8) are part of the BES1 family, despite their primary function as β-amylases. β-Amylase (BAM) and BES1 are two gene families with functional and regulatory roles in controlling shoot growth and development by mediating brassinosteroid effects. They share similar domains and participate in various biological processes, tolerance and responses to stresses like salt and drought. In a computational analysis comparing Arabidopsis and Gossypium species, BAM and BES1 were characterized. BES1 genes were grouped into four clusters based on the comparison of the two species. Two clusters corresponded to BAM7 and BAM8, while the other two clusters were associated with BES1. The conserved nucleotide domain sequence is GCTGGATGG. Short tandem repeats include TG and TTG, which can serve as molecular markers. BES1 is specifically linked to cellulose and fiber production and holds promise as a candidate for plant selection and breeding in Gossypium (cotton).

A multifaceted crosstalk between brassinosteroid and gibberellin regulates the resistance of cucumber to Phytophthora melonis.

Cucumber plants are highly susceptible to the hemibiotroph oomycete Phytophthora melonis. However, the mechanism of resistance to cucumber blight remains poorly understood. Here, we demonstrated that cucumber plants with impairment in the biosynthesis of brassinosteroids (BRs) or gibberellins (GAs) were more susceptible to P. melonis. By contrast, increasing levels of endogenous BRs or exogenously application of 24-epibrassinolide enhanced the resistance of cucumber plants against P. melonis. Furthermore, we found that both knockout and overexpression of the BR biosynthesis gene CYP85A1 reduced the endogenous GA3 content compared with that of wild-type plants under the condition of inoculation with P. melonis, and the enhancement of disease resistance conferred by BR was inhibited in plants with silencing of the GA biosynthetic gene GA20ox1 or KAO. Together, these findings suggest that GA homeostasis is an essential factor mediating BRs-induced disease resistance. Moreover, BZR6, a key regulator of BR signaling, was found to physically interact with GA20ox1, thereby suppressing its transcription. Silencing of BZR6 promoted endogenous GA biosynthesis and compromised GA-mediated resistance. These findings reveal multifaceted crosstalk between BR and GA in response to pathogen infection, which can provide a new approach for genetically controlling P. melonis damage in cucumber production.

A transthyretin-like protein acts downstream of miR397 and LACCASE to regulate grain yield in rice.

Increasing grain yield is a major goal of breeders due to the rising global demand for food. We previously reported that the miR397-LACCASE (OsLAC) module regulates brassinosteroid (BR) signaling and grain yield in rice (Oryza sativa). However, the precise roles of laccase enzymes in the BR pathway remain unclear. Here, we report that OsLAC controls grain yield by preventing the turnover of TRANSTHYRETIN-LIKE (OsTTL), a negative regulator of BR signaling. Overexpressing OsTTL decreased BR sensitivity in rice, while loss-of-function of OsTTL led to enhanced BR signaling and increased grain yield. OsLAC directly binds to OsTTL and regulates its phosphorylation-mediated turnover. The phosphorylation site Ser226 of OsTTL is essential for its ubiquitination and degradation. Overexpressing the dephosphorylation-mimic form of OsTTL (OsTTLS226A) resulted in more severe defects than did overexpressing OsTTL. These findings provide insight into the role of an ancient laccase in BR signaling and suggest that the OsLAC-OsTTL module could serve as a target for improving grain yield.

BRZ-INSENSITIVE-LONG HYPOCOTYL8 inhibits kinase-mediated phosphorylation to regulate brassinosteroid signaling.

Glycogen synthase kinase 3 (GSK3) is an evolutionarily conserved serine/threonine protein kinase in eukaryotes. In plants, the GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) functions as a central signaling node through which hormonal and environmental signals are integrated to regulate plant development and stress adaptation. BIN2 plays a major regulatory role in brassinosteroid (BR) signaling and is critical for phosphorylating/inactivating BRASSINAZOLE-RESISTANT 1 (BZR1), also known as BRZ-INSENSITIVE-LONG HYPOCOTYL 1 (BIL1), a master transcription factor of BR signaling, but the detailed regulatory mechanism of BIN2 action has not been fully revealed. In this study, we identified BIL8 as a positive regulator of BR signaling and plant growth in Arabidopsis (Arabidopsis thaliana). Genetic and biochemical analyses showed that BIL8 is downstream of the BR receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and promotes the dephosphorylation of BIL1/BZR1. BIL8 interacts with and inhibits the activity of the BIN2 kinase, leading to the accumulation of dephosphorylated BIL1/BZR1. BIL8 suppresses the cytoplasmic localization of BIL1/BZR1, which is induced via BIN2-mediated phosphorylation. Our study reveals a regulatory factor, BIL8, that positively regulates BR signaling by inhibiting BIN2 activity.

Brassinosteroid decreases cadmium accumulation via regulating gibberellic acid accumulation and Cd fixation capacity of root cell wall in rice (Oryza sativa)

The precise mechanism behind the association between plants' reactions to cadmium (Cd) stress and brassinosteroid (BR) remains unclear. In the current investigation, Cd stress quickly increased the endogenous BR concentration in the rice roots. Exogenous BR also increased the hemicellulose level in the root cell wall, which in turn increased its capacity to bind Cd. Simultaneously, the transcription level of genes responsible for root Cd absorption was decreased, including Natural Resistance-Associated Macrophage Protein 1/5 (OsNRAMP1/5) and a major facilitator superfamily gene called OsCd1. Ultimately, the increased expression of Heavy Metal ATPase 3 (OsHMA3) and the decreased expression of OsHMA2, which was in charge of separating Cd into vacuoles and translocating Cd to the shoots, respectively, led to a decrease in the amount of Cd that accumulated in the rice shoots. In contrast, transgenic rice lines overexpressing OsGSK2 (a negative regulator in BR signaling) accumulated more Cd, while OsGSK2 RNA interference (RNAi) rice line accumulated less Cd. Furthermore, BR increased endogenous Gibberellic acid (GA) level, and applying GA could replicate its alleviative effect. Taken together, BR decreased Cd accumulation in rice by mediating the cell wall’s fixation capacity to Cd, which might relied on the buildup of the GA.

The protein phosphatase qGL3/OsPPKL1 self-regulates its degradation to orchestrate brassinosteroid signaling in rice

Brassinosteroids (BRs) are a class of phytohormones that regulate plant growth and development. In previous studies, we cloned and identified PROTEIN PHOSPHATASE WITH KELCH-LIKE1 (OsPPKL1) as the causal gene for the quantitative trait locus GRAIN LENGTH3 (qGL3) in rice (Oryza sativa). We also showed that qGL3/OsPPKL1 is mainly located in the cytoplasm and nucleus and negatively regulates BR signaling and grain length. Because qGL3 is a negative regulator of BR signaling, its turnover is critical for rapid response to changes in BRs. Here, we demonstrate that qGL3 interacts with the WD40-domain-containing protein WD40-REPEAT PROTEIN48 (OsWDR48), which contains a nuclear export signal (NES). The NES signal is crucial for the cytosolic localization of OsWDR48 and also functions in the self-turnover of qGL3. We show that OsWDR48 physically interacts with and genetically acts through qGL3 to modulate BR signaling. Moreover, qGL3 may indirectly promote the phosphorylation of OsWDR48 at the Ser-379 and Ser-386 sites. Substitutions of both phosphorylation sites in OsWDR48 to non-phosphorylatable alanine enhanced the strength of the OsWDR48–qGL3 interaction. Furthermore, we found that brassinolide can promote the accumulation of non-phosphorylated OsWDR48, leading to strong interaction intensity between qGL3 and OsWDR48. Taken together, our results show that OsWDR48 facilitates qGL3 retention and induces degradation of qGL3 in the cytoplasm. These findings suggest that qGL3 self-modulates its turnover by binding to OsWDR48 to regulate its cytoplasmic localization and stability, leading to efficient orchestration of BR signal transduction in rice.

Unveiling a new regulator: Vacuolar V-ATPase mediates brassinosteroid signaling in Arabidopsis

Plant growth and development are intricately regulated by hormones such as auxin, gibberellins, jamonates, cytokinins, and brassinosteroids (BR). BRs are a group of plant steroid hormones that play a vital role in plant growth and response to the environment. BRs regulate many biological processes including cell elongation, cell division, and male fertility as well as abiotic and biotic stress responses. To facilitate modulation of these biological processes, induction of BR signaling results in massive alterations in the transcriptome, proteome, and phosphoproteome (Clark et al., 2021). BRs are perceived by the plasma membrane-bound leucine-rich repeat receptor BRASSINOSTEROID INSENSITIVE1 (BRI1), causing a conformational change in the receptor and promoting interaction with the co-receptor BRI1-ASSOCIATED KINASE1 (BAK1). BR perception initiates a downstream kinase signaling cascade inactivating the GSK3-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2), which is a negative regulator of BR signaling, ultimately resulting in dephosphorylation and activation of BRASSINAZOLE RESISTANT 1 (BZR1) and BRI1-EMS-SUPPRESSOR1 (BES1) (Wang et al., 2016; Nolan et al., 2020; Wang et al., 2021).

Cell type–specific attenuation of brassinosteroid signaling precedes stomatal asymmetric cell division

In Arabidopsis thaliana, brassinosteroid (BR) signaling and stomatal development are connected through the SHAGGY/GSK3-like kinase BR INSENSITIVE2 (BIN2). BIN2 is a key negative regulator of BR signaling but it plays a dual role in stomatal development. BIN2 promotes or restricts stomatal asymmetric cell division (ACD) depending on its subcellular localization, which is regulated by the stomatal lineage-specific scaffold protein POLAR. BRs inactivate BIN2, but how they govern stomatal development remains unclear. Mapping the single-cell transcriptome of stomatal lineages after triggering BR signaling with either exogenous BRs or the specific BIN2 inhibitor, bikinin, revealed that the two modes of BR signaling activation generate spatiotemporally distinct transcriptional responses. We established that BIN2 is always sensitive to the inhibitor but, when in a complex with POLAR and its closest homolog POLAR-LIKE1, it becomes protected from BR-mediated inactivation. Subsequently, BR signaling in ACD precursors is attenuated, while it remains active in epidermal cells devoid of scaffolds and undergoing differentiation. Our study demonstrates how scaffold proteins contribute to cellular signal specificity of hormonal responses in plants.

The brassinosteroid signaling component SlBZR1 promotes tomato fruit ripening and carotenoid accumulation.

The plant hormone ethylene is essential for climacteric fruit ripening, although it is unclear how other phytohormones and their interactions with ethylene might affect fruit ripening. Here, we explored how brassinosteroids (BRs) regulate fruit ripening in tomato (Solanum lycopersicum) and how they interact with ethylene. Exogenous BR treatment and increased endogenous BR contents in tomato plants overexpressing the BR biosynthetic gene SlCYP90B3 promoted ethylene production and fruit ripening. Genetic analysis indicated that the BR signaling regulators Brassinazole-resistant1 (SlBZR1) and BRI1-EMS-suppressor1 (SlBES1) act redundantly in fruit softening. Knocking out SlBZR1 inhibited ripening through transcriptome reprogramming at the onset of ripening. Combined transcriptome deep sequencing and chromatin immunoprecipitation followed by sequencing identified 73 SlBZR1-repressed targets and 203 SlBZR1-induced targets involving major ripening-related genes, suggesting that SlBZR1 positively regulates tomato fruit ripening. SlBZR1 directly targeted several ethylene and carotenoid biosynthetic genes to contribute to the ethylene burst and carotenoid accumulation to ensure normal ripening and quality formation. Furthermore, knock-out of Brassinosteroid-insensitive2 (SlBIN2), a negative regulator of BR signaling upstream of SlBZR1, promoted fruit ripening and carotenoid accumulation. Taken together, our results highlight the role of SlBZR1 as a master regulator of tomato fruit ripening with potential for tomato quality improvement and carotenoid biofortification.

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.

Brassinosteroid signaling positively regulates abscisic acid biosynthesis in response to chilling stress in tomato.

Brassinosteroids (BRs) and abscisic acid (ABA) are essential regulators of plant growth and stress tolerance. Although the antagonistic interaction of BRs and ABA is proposed to ensure the balance between growth and defense in model plants, the crosstalk between BRs and ABA in response to chilling in tomato (Solanum lycopersicum), a warm-climate horticultural crop, is unclear. Here, we determined that overexpression of the BR biosynthesis gene DWARF (DWF) or the key BR signaling gene BRASSINAZOLE-RESISTANT1 (BZR1) increases ABA levels in response to chilling stress via positively regulating the expression of the ABA biosynthesis gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE1 (NCED1). BR-induced chilling tolerance was mostly dependent on ABA biosynthesis. Chilling stress or high BR levels decreased the abundance of BRASSINOSTEROID-INSENSITIVE2 (BIN2), a negative regulator of BR signaling. Moreover, we observed that chilling stress increases BR levels and results in the accumulation of BZR1. BIN2 negatively regulated both the accumulation of BZR1 protein and chilling tolerance by suppressing ABA biosynthesis. Our results demonstrate that BR signaling positively regulates chilling tolerance via ABA biosynthesis in tomato. The study has implications in production of warm-climate crops in horticulture.

Ammonium protects rice against rice stripe virus by activating HDA703/OsBZR1-mediated BR signaling

Ammonium (NH4+) is a major inorganic nitrogen source for plants and also as a signal regulates plant growth and defense. Brassinosteroids (BRs) are a class of steroid hormones that control plant developmental and physiological processes through its signaling pathway. Rice is a kind of NH4+-preferring plant which responds to virus infection involving in the regulation of BR biosynthesis and signaling. However, the BR-mediated regulatory mechanisms in rice-virus interactions are not fully understood. In addition, it remains unknown whether there is a direct link between NH4+ and BRs in regulating rice response to virus. HDA703, a histone deacetylase and OsBZR1, a transcription factor, are two positive regulator of BR signaling and interact with each other. In this study, we show that rice plants grown with NH4+ as the sole N source have enhanced resistance to rice stripe virus (RSV), one of the most devastating viruses of rice, than those grown with NO3- as the sole N source. We also show that in contrast to NO3-, NH4+ does not affect BR biosynthesis but promotes BR signaling by upregulating the expression of HDA703 and promoting the accumulation of OsBZR1 in rice shoots. We further show that BR biosynthesis and signaling is required for rice defense against RSV and BR-mediated resistance to RSV attributes to activating HDA703/OsBZR1 module, then decreasing the expression of Ghd7, a direct target of HDA703/OsBZR1. Consistently, increase of the expression of HDA703 or decrease of the expression of Ghd7 enhances rice resistance to RSV. Together, our study reveals that activation of HDA703/OsBZR1-Ghd7 signaling cascade is an undescribed mechanism conferring BR-mediated RSV resistance and NH4+ protects rice against RSV by activating HDA703/OsBZR1-Ghd7-mediated BR signaling in rice.

Brassinosteroids modulate autophagy through phosphorylation of RAPTOR1B by the GSK3-like kinase BIN2 in Arabidopsis

ABSTRACT Macroautophagy/autophagy is a conserved recycling process that maintains cellular homeostasis during environmental stress. Autophagy is negatively regulated by TOR (target of rapamycin), a nutrient-regulated protein kinase that in plants is activated by several phytohormones, leading to increased growth. However, the detailed molecular mechanisms by which TOR integrates autophagy and hormone signaling are poorly understood. Here, we show that TOR modulates brassinosteroid (BR)-regulated plant growth and stress-response pathways. Active TOR was required for full BR-mediated growth in Arabidopsis thaliana. Autophagy was constitutively up-regulated upon blocking BR biosynthesis or signaling, and down-regulated by increasing the activity of the BR pathway. BIN2 (brassinosteroid-insensitive 2) kinase, a GSK3-like kinase functioning as a negative regulator in BR signaling, directly phosphorylated RAPTOR1B (regulatory-associated protein of TOR 1B), a substrate-recruiting subunit in the TOR complex, at a conserved serine residue within a typical BIN2 phosphorylation motif. Mutation of RAPTOR1B serine 916 to alanine, to block phosphorylation by BIN2, repressed autophagy and increased phosphorylation of the TOR substrate ATG13a (autophagy-related protein 13a). By contrast, this mutation had only a limited effect on growth. We present a model in which RAPTOR1B is phosphorylated and inhibited by BIN2 when BRs are absent, activating the autophagy pathway. When BRs signal and inhibit BIN2, RAPTOR1B is thus less inhibited by BIN2 phosphorylation. This leads to increased TOR activity and ATG13a phosphorylation, and decreased autophagy activity. Our studies define a new mechanism by which coordination between BR and TOR signaling pathways helps to maintain the balance between plant growth and stress responses.

The U-box ubiquitin ligase TUD1 promotes brassinosteroid-induced GSK2 degradation in rice

Brassinosteroids (BRs) are a class of steroid hormones with great potential for use in crop improvement. De-repression is usually one of the key events in hormone signaling. However, how the stability of GSK2, the central negative regulator of BR signaling in rice (Oryza sativa), is regulated by BRs remains elusive. Here, we identify the U-box ubiquitin ligase TUD1 as a GSK2-interacting protein by yeast two-hybrid screening. We show that TUD1 is able to directly interact with GSK2 and ubiquitinate the protein. Phenotypes of the tud1 mutant are highly similar to those of plants with constitutively activated GSK2. Consistent with this finding, GSK2 protein accumulates in the tud1 mutant compared with the wild type. In addition, inhibition of BR synthesis promotes GSK2 accumulation and suppresses TUD1 stability. By contrast, BRs can induce GSK2 degradation but promote TUD1 accumulation. Furthermore, the GSK2 degradation process is largely impaired in tud1 in response to BR. In conclusion, our study demonstrates the role of TUD1 in BR-induced GSK2 degradation, thereby advancing our understanding of a critical step in the BR signaling pathway of rice.

Structure of maize BZR1-type β-amylase BAM8 provides new insights into its noncatalytic adaptation

Plant β-amylase (BAM) proteins play an essential role in growth, development, stress response, and hormone regulation. Despite their typical (β/α)8 barrel structure as active catalysts in starch breakdown, catalytically inactive BAMs are implicated in diverse yet elusive functions in plants. The noncatalytic BAM7/8 contain N-terminal BZR1 domains and were shown to be involved in the regulation of brassinosteroid signaling and possibly serve as sensors of yet an uncharacterized metabolic signal. While the structures of several catalytically active BAMs have been reported, structural characterization of the catalytically inactive BZR1-type BAMs remain unknown. Here, we determine the crystal structure of β-amylase domain of Zea mays BAM8/BES1/BZR1-5 and provide comprehensive insights into its noncatalytic adaptation. Using structural-guided comparison combined with biochemical analysis and molecular dynamics simulations, we revealed conformational changes in multiple distinct highly conserved regions resulting in rearrangement of the binding pocket. Altogether, this study adds a new layer of understanding to starch breakdown mechanism and elucidates the acquired adjustments of noncatalytic BZR1-type BAMs as putative regulatory domains and/or metabolic sensors in plants.

OsSLA1 functions in leaf angle regulation by enhancing the interaction between OsBRI1 and OsBAK1 in rice.

Leaf angle is an important trait in plants. Here, we demonstrate that the leucine-rich repeat receptor-like kinase OsSLA1 plays an important role in leaf angle regulation in rice (Oryza sativa). OsSLA1 mutant plants exhibited a small leaf angle phenotype due to changes of adaxial cells in the lamina joint. GUS staining revealed that OsSLA1 was highly expressed in adaxial cells of the lamina joint. The OsSLA1 mutant plants were insensitive to exogenous epibrassinolide (eBL) and showed upregulated expression of DWARF and CPD, but downregulated expression of BU1, BUL1, and ILI1, indicating that brassinosteroid (BR) signal transduction was blocked. Fluorescence microscopy showed that OsSLA1 was localized to the plasma membrane and nearby periplasmic vesicles. Further study showed that OsSLA1 interacts with OsBRI1 and OsBAK1 via its intracellular domain and promotes the interaction between OsBRI1 and OsBAK1. In addition, phosphorylation experiments revealed that OsSLA1 does not possess kinase activity, but that it can be phosphorylated by OsBRI1 in vitro. Knockout of OsSLA1 in the context of d61 caused exacerbation of the mutant phenotype. These results demonstrate that OsSLA1 regulates leaf angle formation via positive regulation of BR signaling by enhancing the interaction of OsBRI1 with OsBAK1.

Overexpression of a BRASSINAZOLE RESISTANT 1 homolog attenuates drought tolerance by suppressing the expression of PLETHORA-LIKE 1 in Setaria italica

Two potential BRASSINAZOLE RESISTANT 1 (BZR1) homologs were downregulated by brassinosteroids (BRs) in Setaria italica roots. Functional analysis showed that BR regulates the dephosphorylation and nuclear localization of SiBZR1 and that SiBZR1 binds conserved BZR1-recognizing cis elements. In comparison with the wild type, SiBZR1-overexpressing S. italica seedlings were more sensitive to BR-inhibited primary root growth and drought stress, indicating that SiBZR1 is a positive regulator of BR signaling and a negative regulator of drought tolerance in S. italica. PLETHORA-LIKE 1 (SiPLT-L1) was found to be a direct target gene of SiBZR1 in S. italica roots. The expression of SiPLT-L1 was downregulated by SiBZR1. SiPLT-L1-overexpressing S. italica was less sensitive to BR-inhibited root growth and more tolerant to drought stress, possibly owing to the upregulation of drought-inducible Dehydrin-family genes.

Regulation of Brassinosteroid Signaling and Salt Resistance by SERK2 and Potential Utilization for Crop Improvement in Rice.

The complex roles of the steroid hormone brassinosteroids (BRs) in many different yield- and stress-related traits make it difficult to utilize the hormones for crop improvement. Here, we show that SERK2 as a BR signaling component is a potentially useful candidate for BR manipulation in rice. We generated multiple mutant alleles of SERK2 by CRISPR/Cas9 editing and show that knockout of SERK2 results in a compact structure accompanied with increased grain size. SERK2 is localized on plasma membrane and can interact with OsBRI1, the BR receptor, suggesting its conserved role as co-receptor in BR signaling. Consistently, the mutant has impaired BR sensitivity compared to wild type. Notably, the mutant is highly sensitive to salt stress as evaluated by plant survival rate as well as transcriptome analysis, whereas has slightly increased sensitivity to ABA, the stress hormone. By contrast, overexpression of SERK2 significantly enhances grain size and salt stress resistance, importantly, without affecting plant architecture. Furthermore, while salt suppresses SERK2 transcription, the protein is greatly induced by salt stress. Taken together, we propose that the adverse condition induces SERK2 accumulation to enhance early BR signaling on plasma membrane in favor of the anti-stress response. Our results illustrate the great potentials of specific BR components such as SERK2 for crop improvement by utilizing flexible strategies.

Multiple origins of Indian dwarf wheat by mutations targeting the TREE domain of a GSK3-like kinase for drought tolerance, phosphate uptake, and grain quality.

Multiple origins of Indian dwarf wheat were due to two mutations targeting the same TREE domain of a GSK3-like kinase, and these mutations confer to enhanced drought tolerance and increased phosphate and nitrogen accumulation for adaptation to the dry climate of Indian and Pakistan. Indian dwarf wheat, featured by the short stature, erect leaves, dense spikes, and small, spherical grains, was a staple crop in India and Pakistan from the Bronze Age until the early 1900s. These morphological features are controlled by a single locus Sphaerococcum 1 (S1), but the genetic identity of the locus and molecular mechanisms underlying the selection of this wheat type are unknown. In this study, we showed that the origin of Indian dwarf wheat was due to two independent missense mutations targeting the conserved TREE domain of a GSK3-like kinase, which is homologous to the Arabidopsis BIN2 protein, a negative regulator in brassinosteroid signaling. The S1 protein is involved in brassinosteroid signaling by physical interaction with the wheat BES1/BZR1 proteins. The dwarf alleles are insensitive to brassinosteroid, upregulates brassinosteroid biosynthetic genes, significantly enhanced drought tolerance, facilitated phosphate accumulation, and increased high molecular weight glutenins. It is the enhanced drought tolerance and accumulation of nitrogen and phosphate that contributed to the adaptation of such a small-grain form of wheat to the dry climate of India and Pakistan. Thus, our research not only identified the genetic events underlying the origin of the Indian dwarf wheat, but also revealed the function of brassinosteroid in the regulation of drought tolerance, phosphate homeostasis, and grain quality.

Brassinosteroid Signaling Converges With Auxin-Mediated C3H17 to Regulate Xylem Formation in Populus.

Brassinosteroid (BR) signaling has long been reported to have an effect on xylem development, but the detailed mechanism remains unclear, especially in tree species. In this study, we find PdC3H17, which was demonstrated to mediate xylem formation driven by auxin in our previous report, is also involved in BR-promoted xylem development. Y1H analysis, EMSA, and transcription activation assay confirmed that PdC3H17 was directly targeted by PdBES1, which is a key transcriptional regulator in BR signaling. Tissue specificity expression analysis and in situ assay revealed that PdC3H17 had an overlapping expression profile with PdBES1. Hormone treatment examinations verified that xylem phenotypes in PdC3H17 transgenic plants, which were readily apparent in normal condition, were attenuated by treatment with either brassinolide or the BR biosynthesis inhibitor propiconazole. The subsequent quantitative real-time polymerase chain reaction (qRT-PCR) analyses further revealed that BR converged with PdC3H17 to influence transcription of downstream xylem-related genes. Additionally, the enhancement of xylem differentiation by auxin in PdC3H17 overexpression plants was significantly attenuated compared with wild-type and dominant negative plants due to BR deficiency, which suggested that the BR- and auxin-responsive gene PdC3H17 acted as an mediation of these two hormones to facilitate xylem development. Taken together, our results demonstrate that BR signaling converges with auxin-mediated PdC3H17 to regulate xylem formation in Populus and thus provide insight into the regulation mechanism of BRs and the crosstalk with auxin signaling on xylem formation.