RESUMO
Arabidopsis thaliana reticulate mutants exhibit differential pigmentation of the veinal and interveinal leaf regions, a visible phenotype that often indicates impaired mesophyll development. We performed a metabolomic analysis of one ven6 (venosa6) and three ven3 reticulate mutants that revealed altered levels of arginine precursors, namely increased ornithine and reduced citrulline levels. In addition, the mutants were more sensitive than the wild-type to exogenous ornithine, and leaf reticulation and mesophyll defects of these mutants were completely rescued by exogenous citrulline. Taken together, these results indicate that ven3 and ven6 mutants experience a blockage of the conversion of ornithine into citrulline in the arginine pathway. Consistent with the participation of VEN3 and VEN6 in the same pathway, the morphological phenotype of ven3 ven6 double mutants was synergistic. Map-based cloning showed that the VEN3 and VEN6 genes encode subunits of Arabidopsis carbamoyl phosphate synthetase (CPS), which is assumed to be required for the conversion of ornithine into citrulline in arginine biosynthesis. Heterologous expression of the Arabidopsis VEN3 and VEN6 genes in a CPS-deficient Escherichia coli strain fully restored bacterial growth in minimal medium, demonstrating the enzymatic activity of the VEN3 and VEN6 proteins, and indicating a conserved role for CPS in these distinct and distant species. Detailed study of the reticulate leaf phenotype in the ven3 and ven6 mutants revealed that mesophyll development is highly sensitive to impaired arginine biosynthesis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arginina/biossíntese , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Mutação , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/farmacologia , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Citrulina/genética , Citrulina/metabolismo , Citrulina/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Metanossulfonato de Etila/farmacologia , Células do Mesofilo/metabolismo , Metabolômica , Dados de Sequência Molecular , Morfogênese/genética , Ornitina/genética , Ornitina/metabolismo , Ornitina/farmacologia , Fenótipo , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Alinhamento de SequênciaRESUMO
To investigate the specialization of the two Arabidopsis CDC27 subunits in the anaphase-promoting complex (APC/C), we analyzed novel alleles of HBT/CDC27B and CDC27A, and characterized the expression of complementing HOBBIT (HBT) protein fusions in plant meristems and during the cell cycle. In contrast to other APC/C mutants, which are gametophytic lethal, phenotypes of weak and null hbt alleles indicate a primary role in the control of post-embryonic cell division and cell elongation, whereas cdc27a nulls are phenotypically indistinguishable from the wild type. However, cdc27a hbt double-mutant gametes are non-viable, indicating a redundant requirement for both CDC27 subunits during gametogenesis. Yeast-two-hybrid and pulldown studies with APC/C components suggest that the two Arabidopsis CDC27 subunits participate in several complexes that are differentially required during plant development. Loss-of-function analysis, as well as cyclin B reporter protein accumulation, indicates a conserved role for the plant APC/C in controlling mitotic progression and cell differentiation during the entire life cycle.