Bailey, A. K. | Search bioRxiv

bioRxiv2026

GLABRA2 regulates gene expression via its own EAR-motif mediated recruitment of the TPL/TPR corepressors

The Arabidopsis HD-Zip IV transcription factor GLABRA2 (GL2) displays dual regulatory capabilities, as an activator and repressor of genes that mediate cell-type differentiation of the epidermis. GL2 binds L1 box elements in the promoters of its target genes; however, the mechanisms by which it controls gene expression remain elusive. GL2 contains two putative ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motifs proximal to its N- and C-termini. The N-terminal EAR motif is highly conserved among GL2 orthologs that form a distinct clade of HD-Zip IV transcription factors in monocots and dicots. We demonstrate that deletion or Ala substitution of this N-terminal EAR motif results in a partial loss-of-function phenotypes in trichomes, non-hair root cells, and seed coat mucilage. In contrast, mutations affecting the C-terminal EAR motif display improper nuclear localization, likely due to protein misfolding. Yeast two-hybrid and in planta co-immunoprecipitation assays show that GL2 selectively interacts with the TOPLESS (TPL) and TPL-RELATED (TPR) corepressors via its N-terminal EAR motif. Fusion of the SUPERMAN REPRESSIVE DOMAIN X (SRDX) with the gl2 N-terminal EAR motif mutant (gl2EAR-N) rescues the epidermal defects of gl2 mutants. Transcriptome analysis of mutant and wild-type seedling roots further confirms the role of the GL2 N-terminal EAR motif in tuning gene expression. Our findings support a model whereby GL2 recruits TPL/TPR corepressors via its EAR motif to sequester histone-modifying proteins, resulting in chromatin remodeling required for epidermal development.

plant biology↗

bioRxiv2026

ATML1-GIR1-TPL/TPR transcriptional repression module controls glucosinolates and giant cells in Arabidopsis thaliana sepals

Glucosinolates (GSLs) are sulfur- and nitrogen-containing secondary metabolites that serve as defense compounds in Arabidopsis and other members of the Brassicales. Although the enzymatic pathway that produces GSLs is well-studied, the upstream mechanisms that control their tissue-specific synthesis are poorly understood. We identified a novel repression module that transcriptionally regulates GSL levels in sepals, the modified leaves that protect reproductive tissues within the floral bud. GLABRA2 (GL2) INTERACTING REPRESSOR1 (GIR1) interacts directly with Arabidopsis thaliana MERISTEM LAYER1 (ATML1), an HD-Zip IV transcription factor known to be required for giant cell formation in the sepal epidermis. This interaction requires a predicted Zn finger of GIR1 and the C-terminal START adjacent domain (STAD) of ATML1. The gir1 loss-of-function mutants exhibit excess giant cells, in contrast to atml1 mutants which display fewer giant cells, supporting the role of GIR1 as a negative regulator of ATML1. We confirmed that GIR1 interacts with TOPLESS (TPL) and TOPLESS-RELATED (TPR) corepressors, and coimmunoprecipitation demonstrated that GIR1 acts as an adaptor protein connecting ATML1 and TPL/TPR. RNA sequencing revealed that numerous genes involved in GSL biosynthesis, including the key transcriptional regulator MYB29, are upregulated in gir1 mutants. Consistent with the transcriptomic data, chemical analysis revealed that gir1 mutants display elevated GSL levels in sepals. Mass spectrometry imaging confirmed high GSL accumulation in gir1 sepals compared to wild type and atml1. Overall, our findings uncover a previously unrecognized link between cell expansion and GSL metabolism, suggesting strategies for engineering plants with cell-type specific GSL profiles. Significance StatementPlants belonging to the order Brassicales produce sulfur-containing glucosinolate (GSL) metabolites that serve in defense against herbivory. In cruciferous vegetables such as broccoli and kale, these compounds contribute to their unique flavors and health-promoting attributes. In agriculturally important oilseed crops, they affect the palatability of animal feeds. Here, we identified a novel transcriptional regulatory module that controls GSL biosynthesis in the epidermis of the sepal, the floral organ that protects the reproductive tissues. This regulatory module also controls cell expansion of specific cell types in the sepal, demonstrating a surprising connection between cell growth and a chemical defense pathway in plants. Our results suggest strategies for engineering crops with tissue-specific GSL profiles to fit agronomic needs.

plant biology↗