The AP2/ERF Transcription Factor TINY Modulates Brassinosteroid-Regulated Plant Growth and Drought Responses in Arabidopsis

Abstract

APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) family transcription factors have well-documented functions in stress responses, but their roles in brassinosteroid (BR)-regulated growth and stress responses have not been established. Here, we show that the Arabidopsis (Arabidopsis thaliana) stress-inducible AP2/ERF transcription factor TINY inhibits BRregulated growth while promoting drought responses. TINY-overexpressing plants have stunted growth, increased sensitivity to BR biosynthesis inhibitors, and compromised BR-responsive gene expression. By contrast, tiny tiny2 tiny3 triple mutants have increased BR-regulated growth and BR-responsive gene expression. TINY positively regulates drought responses by activating drought-responsive genes and promoting abscisic acid-mediated stomatal closure. Global gene expression studies revealed that TINY and BRs have opposite effects on plant growth and stress response genes. TINY interacts with and antagonizes BRASSINOSTERIOID INSENSITIVE1-ETHYL METHANESULFONATE SUPRESSOR1 (BES1) in the regulation of these genes. Glycogen synthase kinase 3-like protein kinase BR-INSENSITIVE2 (BIN2), a negative regulator in the BR pathway, phosphorylates and stabilizes TINY, providing a mechanism for BR-mediated downregulation of TINY to prevent activation of stress responses under optimal growth conditions. Taken together, our results demonstrate that BR signaling negatively regulates TINY through BIN2 phosphorylation and TINY positively regulates drought responses, as well as inhibiting BR-mediated growth through TINY-BES1 antagonistic interactions. Our results thus provide insight into the coordination of BR-regulated growth and drought responses.

Development ofmPing-based Activation Tags for Crop Insertional Mutagenesis

“…TINY2 belongs to the APETALA2/ethylene response factor transcription factor family. There are three TINY homologs in Arabidopsis, and triple knockouts grow larger than wild-type, while overexpression lines display stunted growth (Xie et al, 2019). There are two additional soybean genes with high sequence similarity to Glyma.08G035000, and phylogenetic analysis revealed four other related soybean genes cluster closer with AtTINY1 and AtTINY2 demonstrating that Glyma.08G035000 is only part of a larger TINY-like gene family (Supplemental Figure 7).…”

“…Our soybean plants overexpressing Glyma.08G035000 did not display any reduction in stature or other obvious visual phenotypes under normal growing conditions. This may be attributed to subfunctionalization of the Glyma.08G035000 gene as well as the difference in magnitude of upregulation by mmPing20F [6.2-fold compared to the ~200-fold increase in TINY-overexpression Arabidopsis lines] (Xie et al, 2019). AtTINY recognizes the DRE promoter element (A/GCCGAC) and negatively regulates brassinosteroid-mediated growth through suppression of genes that respond to brassinosteroid application (Xie et al, 2019).…”

“…This may be attributed to subfunctionalization of the Glyma.08G035000 gene as well as the difference in magnitude of upregulation by mmPing20F [6.2-fold compared to the ~200-fold increase in TINY-overexpression Arabidopsis lines] (Xie et al, 2019). AtTINY recognizes the DRE promoter element (A/GCCGAC) and negatively regulates brassinosteroid-mediated growth through suppression of genes that respond to brassinosteroid application (Xie et al, 2019). The number of downregulated genes in TINY-like overexpression lines relative to wild type was smaller (27 genes) in soybean lines compared to that of Arabidopsis (2247 genes) (Xie et al, 2019).…”

“…AtTINY recognizes the DRE promoter element (A/GCCGAC) and negatively regulates brassinosteroid-mediated growth through suppression of genes that respond to brassinosteroid application (Xie et al, 2019). The number of downregulated genes in TINY-like overexpression lines relative to wild type was smaller (27 genes) in soybean lines compared to that of Arabidopsis (2247 genes) (Xie et al, 2019). Additionally, we did not detect significant enrichment for DRE elements in the promoter regions of downregulated genes in our study, suggesting that Glyma.08G035000 has different target genes than AtTINY2, or the magnitude of Glyma.08G035000 upregulation was not sufficient to suppress its target genes.…”

Robotic Assay for Drought (RoAD): An Automated Phenotyping System for Brassinosteroid and Drought Response

“…Our results demonstrate the utility of phenotyping BR-mediated growth responses across different developmental stages, phenotypic traits, and genotypes. Brassinosteroids have extensive cross-talk with drought and several mechanisms impinge on BES1 to balance BR-regulated growth responses with drought survival Nolan et al, 2017a;Chen et al, 2017;Xie et al, 2019). Gain-of-function bes1-D mutants have reduced survival during drought, whereas loss-of-function bri1-301 mutants display increased drought survival Nolan et al, 2017a).…”