Molecular and physiological characterization of brassinosteroid receptor BRI1 mutants in Sorghum bicolor
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ABSTRACT: * The high sequence and structural similarities between BRI1 brassinosteroid receptors of Arabidopsis (AtBRI1) and sorghum (SbBRI1) prompted us to study the functionally conserved roles of BRI in both organisms. * Introducing sorghum SbBRI1 in Arabidopsis bri1 mutants restores defective growth and complements developmental phenotypes to WT levels. * Sorghum mutants for SbBRI1 receptors show defective BR sensitivity and results in impaired growth and development throughout the entire sorghum life cycle. Embryonic analysis of sorghum primary roots traced the root growth and development at the early stages, revealing the role of SbBRI1 in BR perception during cell division and BR sensing. RNA-seq of SbBRI1 mutants support the roles of SbBRI1 in cell wall biosynthesis and remodeling. * Together, these results uncover that sorghum SbBRI1 receptor protein play functionally conserved roles in plant growth and development.
Project description:The brassinosteroid (BR) plant hormones regulate numerous developmental processes, including those determining stem height, leaf angle, and grain size that have agronomic relevance in cereals. Indeed, barley (Hordeum vulgare) varieties containing uzu alleles that impair BR perception through mutations in the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) exhibit a semi-dwarf growth habit and more upright leaves suitable for high-density planting. We used forward and reverse genetic approaches to develop novel BRI1 alleles in wheat (Triticum aestivum L.) and investigated their potential for crop improvement. The combination of ethyl methanesulfonate-induced mutations introducing premature stop codons in all three homoeologous TaBRI1 genes resulted in severe dwarfism, malformed leaves and sterility as observed in bri1 mutants in other species. Double mutants had reduced flag leaf angles (FLAs) conferring a more upright canopy but exhibited no differences in height or grain weight. In a forward genetics screen using a double mutant, we identified two BR-insensitive lines with reduced height and FLA that contained amino acid substitutions in conserved regions of BRI-A1. The less severe mutant had a 56% reduction in FLA and was 35% shorter than the wild-type although seed set, seed area and grain weights were also reduced. The most severe mutants contained elevated levels of bioactive BRs and increased expression of BR-biosynthesis genes consistent with reduced feedback suppression of biosynthesis. Our study gives a better understanding of BRI1 function in wheat and provides mutants that could potentially be explored for improving grain yields when sown at high density.
Project description:Drought is a critical issue in modern agriculture, therefore there is a need to create crops with drought resilience. The complexity of plant responses to abiotic stresses, particularly in the field of brassinosteroid (BR) signaling, has been the subject of extensive research. In this study, we unveil compelling insights indicating that the BRASSINOSTEROID INSENSITIVE 1 (BRI1) receptor in Arabidopsis and Sorghum plays a critical role as a negative regulator of drought responses. Introducing untargeted mutation in the sorghum BRI1 receptor (SbBRI1) effectively enhances the plant ability to withstand osmotic and drought stress. Through DNA Affinity Purification sequencing (DAP-Seq) we show that the sorghum BRI1-EMS-SUPPRESSOR 1 (SbBES1) transcription factor, a downstream player of the BR signaling, binds to a conserved G-box binding motif, and it is responsible for regulating BR homeostasis, as its Arabidopsis ortholog AtBES1. We further characterized the drought tolerance of sorghum bri1 mutants and decipher SbBES1-mediated regulation of phenylpropanoid pathway. Our findings suggest that SbBRI1 signaling serves as a dual purpose: under normal conditions, it regulates lignin biosynthesis by SbBES1, but during drought conditions, BES1 becomes less active, allowing the activation of the flavonoid pathway. This adaptive shift improves the photosynthetic rate and photoprotection, reinforcing crop adaptation to drought.
Project description:Molecular genetic analyses support a central role of BZR1 in Brassinosteroid (BR) regulation of plant development. The dominant bzr1-1D mutation, which stabilizes the BZR1 protein, completely suppresses the de-etiolated phenotype of the null bri1-116 mutant grown in the dark. Using microarray analysis, we identified genes differentially expressed in bri1-116 compared to wild type and genes differentially expressed in the bzr1-1D;bri1-116 double mutant compared to the bri1-116 single mutant. Consistent with the phenotypic suppression of bri1-116 by bzr1-1D, about 80% of the genes affected in bri1-116 were affected oppositely by bzr1-1D
Project description:Molecular genetic analyses support a central role of BZR1 in Brassinosteroid (BR) regulation of plant development. The dominant bzr1-1D mutation, which stabilizes the BZR1 protein, completely suppresses the de-etiolated phenotype of the null bri1-116 mutant grown in the dark. Using microarray analysis, we identified genes differentially expressed in bri1-116 compared to wild type and genes differentially expressed in the bzr1-1D;bri1-116 double mutant compared to the bri1-116 single mutant. Consistent with the phenotypic suppression of bri1-116 by bzr1-1D, about 80% of the genes affected in bri1-116 were affected oppositely by bzr1-1D BZR1 regulated genes were generated from comparing genes differentially expressed by bzr1-1D;bri1-116 and bri1-116. Genes affected by BRI1 were generated from comparing differentially expressed genes of bri1-116 and Col control. ANOVA was used to find genes whose expression was different between bzr1-1D;bri1-116 and bri1-116 or between bri1-116 and Col samples [see Supplementary file below].
Project description:Molecular genetic analyses support important roles for the AtGATA2 gene in brassinolide (BR) and light regulation of plant development. The overexpression line 6-9 of AtGATA2 suppresses the etiolated phenotype of Col-0 grown in the dark. Using microarray analysis, we identified genes differentially expressed in the AtGATA2ox transgenic line 6-9 compared to wild type, and genes differentially expressed in the AtGATA2ox transgenic line 6-9 compared to the bri1-116 single mutant. Consistent with the phenotypic similarity of bri1-116 and AtGATA2, overall about 93% of the co-regulated genes were affected in the same way by GATA2-ox and bri1-116. AtGATA2-regulated genes have been identified by comparing genes differentially expressed by the Col control and bri1-116, which could find the crosslink between light and BR regulations of plant development.
Project description:Molecular genetic analyses support important roles for the AtGATA2 gene in brassinolide (BR) and light regulation of plant development. The overexpression line 6-9 of AtGATA2 suppresses the etiolated phenotype of Col-0 grown in the dark. Using microarray analysis, we identified genes differentially expressed in the AtGATA2ox transgenic line 6-9 compared to wild type, and genes differentially expressed in the AtGATA2ox transgenic line 6-9 compared to the bri1-116 single mutant. Consistent with the phenotypic similarity of bri1-116 and AtGATA2, overall about 93% of the co-regulated genes were affected in the same way by GATA2-ox and bri1-116.
Project description:bri1-5 is a weak mutant of Brassinosteroid Insensitive 1 (BRI1). Suppressors by activation tagging bri1-1D, brs1-1D and bak1-1D can recover bri1-5 phenotype. We use microarray to investigate which pathways or functional categories have been transcriptionally regulated by bri1-1D, brs1-1D and bak1-1D. Whole seedlings from wild-type (WS2), bri1-5, bri1-5/brs1-1D, bri1-5/bak1-1D, bri1-5/bri1-1D. Three biological replicates for each genotype.
Project description:Climate change is having a drastic impact on global agriculture. Indeed stress factors such as elevated temperature, drought and rising atmospheric CO2 reduce arable land surface, crop cultivation and yield and overall sustainable food production on earth. However, plants possess immense innate adaptive plasticity and a more in-depth understanding of the underlying molecular mechanisms is crucial to strategize for sustaining populations under worsening climate change. Brassinosteroids (BRs) are constitutive plant growth regulators that also control plant adaptation to abiotic stress. Downstream components of the BR biosynthetic pathway, BES1/BZR1 play central role in thermomorphogenesis, but involvement of the BR receptors is not well understood. Here, we show that the BRL3 receptor is essential for plant adaptation to warmer environment. The brl3 mutants lack thermal responsiveness and the BRL3 overexpression causes hyper-thermomorphogenesis response. BRL3 activates canonical BRI1 pathway upon elevated temperature. Further, phloem-specific expression of BRL3 completely rescues the growth adaptation defects of the brl3 mutant. This ability of BRL3 represents a previously unknown thermoresponsive mechanism specifically from phloem and uncouples the roles of BR receptors in generic growth vs adaptation to changing climate conditions.
Project description:bri1-5 is a weak mutant of Brassinosteroid Insensitive 1 (BRI1). Suppressors by activation tagging bri1-1D, brs1-1D and bak1-1D can recover bri1-5 phenotype. We use microarray to investigate which pathways or functional categories have been transcriptionally regulated by bri1-1D, brs1-1D and bak1-1D.
Project description:Brassinosteroids (BRs) are growth-promoting steroid hormones in plants. BRs affect plant growth by regulating panels of downstream genes. Much effort has been made to establish BR-regulated gene expression networks, but these published expression networks poorly overlap. To address this, here we build an optimal BR-regulated gene expression network. 7- and 24-day-old seedlings of constitutive photomorphogenesis and dwarfism (cpd) mutant and bri1-701 (brassinosteroid-insensitive 1) brl1 (BRI1-like receptor genes 1) brl3 (BRI1-like receptor genes 3) triple mutant seedlings were treated with brassinolide (BL), and RNA sequencing (RNA-seq) was used to detect differentially expressed genes (DEGs). By this approach, we generated a transcriptomic database of 4498 DEGs and identified 110 transcription factors specifically respond to BR at different stage. Moreover, we found that among the identified BR-responsive transcription factors, ABSCISIC ACID-INSENSlTIVE4 (ABI4), an ethylene response factor (ERF) transcription factor, inhibits BR-regulated growth. Compared to wild-type plants, the abi4-102 mutant was less sensitive to brassinazole (BRZ) and more sensitive to BR. Next, we performed a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) assay and found that ABI4 directly binds to the BAK1 (BRI1 Associated receptor Kinase 1) promoter and inhibits transcription. These results provide new insights into BR-responsive gene functions in regulating plant growth at different stages and may serve as a basis for predicting gene function, selecting candidate genes, and improving the understanding of BR regulatory pathways.