RESUMO
The EL2 gene of rice (Oryza sativa), previously classified as early response gene against the potent biotic elicitor N-acetylchitoheptaose and encoding a short polypeptide with unknown function, was identified as a novel cell cycle regulatory gene related to the recently reported SIAMESE (SIM) gene of Arabidopsis thaliana. Iterative two-hybrid screens, in vitro pull-down assays, and fluorescence resonance energy transfer analyses showed that Orysa; EL2 binds the cyclin-dependent kinase (CDK) CDKA1;1 and D-type cyclins. No interaction was observed with the plant-specific B-type CDKs. The amino acid motif ELERFL was identified to be essential for cyclin, but not for CDK binding. Orysa;EL2 impaired the ability of Orysa; CYCD5;3 to complement a budding yeast (Saccharomyces cerevisiae) triple CLN mutant, whereas recombinant protein inhibited CDK activity in vitro. Moreover, Orysa;EL2 was able to rescue the multicellular trichome phenotype of sim mutants of Arabidopsis, unequivocally demonstrating that Orysa;EL2 operates as a cell cycle inhibitor. Orysa;EL2 mRNA levels were induced by cold, drought, and propionic acid. Our data suggest that Orysa;EL2 encodes a new type of plant CDK inhibitor that links cell cycle progression with biotic and abiotic stress responses.
Assuntos
Ciclo Celular/fisiologia , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Desidratação/metabolismo , Oryza/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Temperatura Baixa , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Desidratação/genética , Mutação de Sentido Incorreto , Oligossacarídeos/metabolismo , Oligossacarídeos/farmacologia , Oryza/genética , Propionatos/farmacologia , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Kip-related proteins (KRPs) play a major role in the regulation of the plant cell cycle. We report the identification of five putative rice (Oryza sativa) proteins that share characteristic motifs with previously described plant KRPs. To investigate the function of KRPs in rice development, we generated transgenic plants overexpressing the Orysa;KRP1 gene. Phenotypic analysis revealed that overexpressed KRP1 reduced cell production during leaf development. The reduced cell production in the leaf meristem was partly compensated by an increased cell size, demonstrating the existence of a compensatory mechanism in monocot species by which growth rate is less reduced than cell production, through cell expansion. Furthermore, Orysa;KRP1 overexpression dramatically reduced seed filling. Sectioning through the overexpressed KRP1 seeds showed that KRP overproduction disturbed the production of endosperm cells. The decrease in the number of fully formed seeds was accompanied by a drop in the endoreduplication of endosperm cells, pointing toward a role of KRP1 in connecting endocycle with endosperm development. Also, spatial and temporal transcript detection in developing seeds suggests that Orysa;KRP1 plays an important role in the exit from the mitotic cell cycle during rice grain formation.
