RESUMO
We have isolated PcGA3ox1, a cDNA clone from developing runner bean (Phaseolus coccineus) seeds that shows significant amino acid homology with the gibberellin (GA) 3-oxidases. A recombinant fusion protein of PcGA3ox1 converted GA20 and GA9 to GA1 and GA4, respectively. In situ hybridization results showed that transcripts of this gene accumulate specifically within the suspensor of globular-stage embryos. PcGA3ox1 mRNA begins to accumulate in the epidermal cells of the embryo proper and is also detectable in the endosperm during the transition from globular- to heart-stage embryos. PcGA3ox1 transcripts were localized exclusively in the cotyledons from the early cotyledonary stage up to the cotyledonary stage. Transcripts of the previously cloned GA 2-oxidase (PcGA2ox1) from developing seeds of runner bean were found primarily within the suspensor neck region from the late globular stage up to the heart stage. PcGA2ox1 mRNA was detectable in the whole suspensor from the early cotyledonary stage, and was found in the inner layer of integuments at the cotyledonary stage. Soluble enzyme preparations made from suspensors and embryos at two stages of embryogenesis (the heart and cotyledonary stages) were incubated with [14C]GA20 and [14C]GA1. Only young suspensor preparations converted GA20 to GA1 and GA5. Both suspensor preparations converted GA1 to GA8. Both embryo preparations converted GA20 to GA1, but were unable to convert GA1 to GA8.
Assuntos
Regulação da Expressão Gênica de Plantas , Oxigenases de Função Mista/genética , Phaseolus/embriologia , Phaseolus/genética , Sementes/embriologia , Sementes/genética , Sistema Livre de Células , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Escherichia coli/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Genes de Plantas/genética , Genoma de Planta , Hibridização In Situ , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Filogenia , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
The polymorphic barley (Hordeum vulgare) Mla locus harbors allelic race-specific resistance (R) genes to the powdery mildew fungus Blumeria graminis f sp hordei. The highly sequence-related MLA proteins contain an N-terminal coiled-coil structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR region. Using transgenic barley lines expressing epitope-tagged MLA1 and MLA6 derivatives driven by native regulatory sequences, we show a reversible and salt concentration-dependent distribution of the intracellular MLA proteins in soluble and membrane-associated pools. A posttranscriptional process directs fourfold greater accumulation of MLA1 over MLA6. Unexpectedly, in rar1 mutant plants that are compromised for MLA6 but not MLA1 resistance, the steady state level of both MLA isoforms is reduced. Furthermore, differential steady state levels of MLA1/MLA6 hybrid proteins correlate with their requirement for RAR1; the RAR1-independent hybrid protein accumulates to higher levels and the RAR1-dependent one to lower levels. Interestingly, yeast two-hybrid studies reveal that the LRR domains of RAR1-independent but not RAR1-dependent MLA isoforms interact with SGT1, a RAR1 interacting protein required for the function of many NB-LRR type R proteins. Our findings implicate the existence of a conserved mechanism to reach minimal NB-LRR R protein thresholds that are needed to trigger effective resistance responses.
