Analysis of ven3 and ven6 reticulate mutants reveals the importance of arginine biosynthesis in Arabidopsis leaf development

Abstract

SUMMARYArabidopsis 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.

Influence of plastids on light signalling and development

“…The distinct PhANG expression phenotypes of variegated mutants are consistent with multiple plastid-to-nucleus signalling mechanisms affecting development [61,65]. The depletion of essential metabolites during their transport from the bundle sheath cells to the lamina was suggested to cause reticulate phenotypes [66] because many of the mutant alleles that cause reticulate phenotypes disrupt amino acid or purine metabolism [10,[65][66][67]. The finding that scabra3 (sca3) exhibited reticulate phenotypes without directly affecting the biosynthesis of essential metabolites [10] provides evidence that metabolic deficiencies may not underpin all reticulate phenotypes.…”

The Metabolic Status Drives Acclimation of Iron Deficiency Responses in Chlamydomonas reinhardtii as Revealed by Proteomics Based Hierarchical Clustering and Reverse Genetics

“…It is of note that these enzyme are all found in the chloroplast proteome of C. reinhardtii (47) and Arabidopsis thaliana (113,114). Knock-out of the small or large subunit of CPS resulted in a reticulate leaf phenotype that was correlated with a defect in mesophyll development (115), implicating that CPS function and arginine may play a role in these developmental processes. CPS is involved in the conversion of glutamine and bicarbonate into carbamoyl phosphate (CP) and glutamate (116).…”

Arabidopsis Roots and Shoots Show Distinct Temporal Adaptation Patterns toward Nitrogen Starvation

“…results have demonstrated their potential role as regulatory molecules (Hannah et al, 2010;Mollá-Morales et al, 2011). Furthermore, the synthesis and degradation of individual amino acids occur through a variety of enzymatic reactions, with different C components as the substrate or product, respectively.…”