RESUMO
Seed dormancy governs germination timing, with both evolutionary and applied consequences. Despite extensive studies on the hormonal and genetic control of these processes, molecular mechanisms directly linking dormancy and germination remain poorly understood. By screening a collection of lines overexpressing Arabidopsis transcription factors, we identified ERF50 as a key gene to control dormancy and germination. To study its regulation, we measured seed-related physiological parameters in loss-of-function mutants and carried out transactivation, protein interaction and ChIP-PCR analyses. We found direct ERF50-mediated repression of DOG1 and activation of EXPA2 transcription, which results in enhanced seed germination. Although ERF50 expression is increased by DOG1 in dormant seeds, ERF50 germination-promoting activity is blocked by RGL2. The physiological, genetic and molecular evidence gathered here supports that ERF50 controls germination timing by regulating DOG1 levels to leverage its role as enhancer of seed germination, via RGL2 antagonism on EXPA2 expression. Our results highlight the central role of ERF50 as a feedback regulator to couple and fine-tune seed dormancy and germination.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Retroalimentação Fisiológica , Regulação da Expressão Gênica de Plantas , Germinação , Dormência de Plantas , Sementes , Fatores de Transcrição , Germinação/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Sementes/crescimento & desenvolvimento , Sementes/fisiologia , Sementes/genética , Dormência de Plantas/genética , Fatores de Tempo , Ligação ProteicaRESUMO
A key component of seed germination is the interplay of mechanical forces governing embryo growth and the surrounding restraining endosperm tissue. Endosperm cell separation is therefore thought to play a critical role in the control of this developmental transition. Here we demonstrate that in Arabidopsis thaliana seeds, endosperm cell expansion is a key component of germination. Endosperm cells expand to accommodate embryo growth prior to germination. We show that this is an actively regulated process supported by spatiotemporal control of the cell expansion gene EXPANSIN 2 (EXPA2). The NAC transcription factors NAC25 and NAC1L were identified as upstream regulators of EXPA2 expression, gibberellin-mediated endosperm expansion, and seed germination. The DELLA protein RGL2 repressed activation of the EXPA2 promoter by NAC25/NAC1L. Taken together, our findings uncover a key role of the GA/DELLA-NAC25/NAC1L-EXPA2 network in regulating endosperm cell expansion to control the seed-to-seedling transition.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Endosperma/metabolismo , Giberelinas/metabolismo , Sementes/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Endosperma/genética , Endosperma/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Germinação , Sementes/genética , Sementes/metabolismoRESUMO
Molecular interactions are an integral part of the regulatory mechanisms controlling gene expression. The yeast one- and two-hybrid systems (Y1H/Y2H) have been widely used by many laboratories to detect DNA-protein (Y1H) and protein-protein interactions (Y2H). The development of efficient cloning systems have promoted the generation of large open reading frame (ORF) clone collections (libraries) for several organisms. Functional analyses of such large collections require the establishment of adequate protocols. Here, we describe a simple straightforward procedure for high-throughput screenings of arrayed libraries with DNA or protein baits that can be carried out by one person with minimal labor and not requiring robotics. The protocol can also be scaled up or down and is compatible with several library formats. Procedures to make yeast stocks for long-term storage (tube and microplate formats) are also provided.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Biblioteca Gênica , Ensaios de Triagem em Larga Escala/métodos , Mapas de Interação de Proteínas , Proteínas/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Animais , DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Ligação Proteica , Proteínas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Since their original description more than 25 years ago, the yeast one- and two-hybrid systems (Y1H/Y2H) have been used by many laboratories to detect DNA-protein (Y1H) and protein-protein interactions (Y2H). These systems use yeast cells (Saccharomyces cerevisiae) as a eukaryotic "test tube" and are amenable for most labs in the world. The development of highly efficient cloning methods has fostered the generation of large collections of open reading frames (ORFs) for several organisms that have been used for yeast screenings. Here, we describe a simple mating based method for high-throughput screenings of arrayed ORF libraries with DNA (Y1H) or protein (Y2H) baits not requiring robotics. One person can easily carry out this protocol in approximately 10 h of labor spread over 5 days. It can also be scaled down to test one-to-one (few) interactions, scaled up (i.e., robotization) and is compatible with several library formats (i.e., 96, 384-well microtiter plates).