RESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arginina/biosíntesis , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/metabolismo , Mutación , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arabidopsis/anatomía & histología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/farmacología , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/genética , Citrulina/genética , Citrulina/metabolismo , Citrulina/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Metanosulfonato de Etilo/farmacología , Células del Mesófilo/metabolismo , Metabolómica , Datos de Secuencia Molecular , Morfogénesis/genética , Ornitina/genética , Ornitina/metabolismo , Ornitina/farmacología , Fenotipo , Hojas de la Planta/anatomía & histología , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Alineación de SecuenciaRESUMEN
Leaf development in Arabidopsis thaliana is considered to be a two-step process. In the first step, a leaf primordium is formed that involves a switch from indeterminate to leaf developmental fate in the shoot apical meristem cells. The second step, known as leaf morphogenesis, consists of post-initiation developmental events such as patterned cell proliferation, cell expansion, and cell differentiation. The results are presented of the molecular and genetic analyses of the rotunda2 (ron2) mutants of Arabidopsis, which were isolated based on their wide and serrated vegetative leaf lamina. The RON2 gene was positionally cloned and was identical to LEUNIG (LUG); it encodes a transcriptional co-repressor that has been described to affect flower development. Morphological and histological analyses of expanded leaves indicated that RON2 (LUG) acts at later stages of leaf development by restricting cell expansion during leaf growth. Real-time reverse-transcription polymerase chain reaction was used to quantify the expression of KNOX, WUSCHEL, YABBY3, LEAFY, ASYMMETRIC LEAVES, and GIBBERELLIN OXIDASE genes in expanding and fully expanded rosette leaf laminas of the wild type and ron2 and lug mutants. SHOOTMERISTEMLESS was expressed in wild-type leaves and down-regulated in the mutants. The results indicate that RON2 (LUG) has a function in later stages of leaf development.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Hojas de la Planta/crecimiento & desarrollo , Factores de Transcripción/genética , Secuencia de Aminoácidos , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/química , Tamaño de la Célula , Cromosomas de las Plantas/genética , Secuencia Conservada , Cartilla de ADN , Microscopía de Interferencia/métodos , Datos de Secuencia Molecular , Morfogénesis , Oryza/genética , Fenotipo , Hojas de la Planta/genética , Mapeo Restrictivo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/químicaRESUMEN
Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant (sre) and salobreño (sañ). The sre and sañ mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1, sre1-2, sañ3-1, and sañ3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a K(m) value for xanthoxin of 19 micro M and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC-mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin-->abscisic aldehyde-->ABA transition as the last steps of the major ABA biosynthetic pathway.