RESUMO
ANTHER INDEHISCENCE FACTOR (AIF), a NAC-like gene, was identified in Arabidopsis. In AIF:GUS flowers, ß-glucuronidase (GUS) activity was detected in the anther, the upper parts of the filaments, and in the pollen of stage 7-9 young flower buds; GUS activity was reduced in mature flowers. Yellow fluorescent protein (YFP)+AIF-C fusion proteins, which lacked a transmembrane domain, accumulated in the nuclei of the Arabidopsis cells, whereas the YFP+AIF fusion proteins accumulated in the membrane and were absent in the nuclei. Further detection of a cleaved AIF protein in flowers revealed that AIF needs to be processed and released from the endoplasmic reticulum in order to function. The ectopic expression of AIF-C caused a male-sterile phenotype with indehiscent anthers throughout flower development in Arabidopsis. The presence of a repressor domain in AIF and the similar phenotype of indehiscent anthers in AIF-C+SRDX plants suggest that AIF acts as a repressor. The defect in anther dehiscence was due to the down-regulation of genes that participate in jasmonic acid (JA) biosynthesis, such as DAD1/AOS/AOC3/OPR3/OPCL1. The external application of JA rescued the anther indehiscence in AIF-C and AIF-C+SRDX flowers. In AIF-C+VP16 plants, which are transgenic dominant-negative mutants in which AIF is converted to a potent activator via fusion to a VP16-AD motif, the anther dehiscence was promoted, and the expression of DAD1/AOS/AOC3/OPR3/OPCL1 was up-regulated. Furthermore, the suppression of AIF through an antisense strategy resulted in a mutant phenotype similar to that observed in the AIF-C+VP16 flowers. The present data suggest a role for AIF in controlling anther dehiscence by suppressing the expression of JA biosynthesis genes in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Vias Biossintéticas/genética , Ciclopentanos/metabolismo , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Oxilipinas/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Ciclopentanos/farmacologia , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Flores/efeitos dos fármacos , Flores/genética , Flores/ultraestrutura , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes Dominantes , Genes de Plantas , Glucuronidase/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , Oxilipinas/farmacologia , Fenótipo , Infertilidade das Plantas/efeitos dos fármacos , Infertilidade das Plantas/genética , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/ultraestrutura , Transporte Proteico/efeitos dos fármacos , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Suppression of expression of DAF [DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1)-Activating Factor], a gene that encodes a putative RING-finger E3 ligase protein, causes non-dehiscence of the anthers, alters pollen development and causes sterility in 35S:DAF RNAi/antisense Arabidopsis plants. This mutant phenotype correlates with the suppression of DAF but not with expression of the two most closely related genes, DAFL1/2. The expression of DAD1 was significantly reduced in 35S:DAF RNAi/antisense plants, and complementation with 35S:DAF did not rescue the dad1 mutant, indicating that DAF acts upstream of DAD1 in jasmonic acid biosynthesis. This assumption is supported by the finding that 35S:DAF RNAi/antisense plants showed a similar cellular basis for anther dehiscence to that found in dad1 mutants, and that external application of jasmonic acid rescued the anther non-dehiscence and pollen defects in 35S:DAF antisense flowers. We further demonstrate that DAF is an E3 ubiquitin ligase and that its activity is abolished by C132S and H137Y mutations in its RING motif. Furthermore, ectopic expression of the dominant-negative C132S or H137Y mutations causes similar indehiscence of anthers and reduction in DAD1 expression in transgenic Arabidopsis. This result not only confirms that DAF controls anther dehiscence by positively regulating the expression of DAD1 in the jasmonic acid biosynthesis pathway, but also supports the notion that DAF functions as an E3 ubiquitin ligase, and that the conserved RING-finger region is required for its activity.
