The Phosphoenolpyruvate/Phosphate Translocator Is Required for Phenolic Metabolism, Palisade Cell Development, and Plastid-Dependent Nuclear Gene Expression

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.

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“…Consistent with the requirement for PEP in early steps of the shikimate pathway (Li et al. , 1995; Streatfield et al. , 1999; Knappe et al.…”

Section: Introductionsupporting

confidence: 77%

“…, 1997). PEP is synthesized in the cytosol, and serves as a substrate for the synthesis of amino acids and a variety of secondary metabolites within chloroplasts through the shikimate pathway (Streatfield et al. , 1999; Knappe et al.…”

Section: Introductionmentioning

confidence: 99%

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

Mollá‐Morales¹,

Sarmiento-Mañús²,

Robles³

et al. 2010

The Plant Journal

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“…The A. thaliana genome contains two PPT genes, which are expressed in leaves, i.e., PPT1 and PPT2 (Knappe et al, 2003). PPT1, which is defective in the chlorophyll a/b binding protein underexpressed1 ( cue1 ) mutant (Streatfield et al, 1999) has been proposed to provide chloroplasts and non-green plastids with PEP as a precursor for the shikimate pathway. Chloroplasts and some non-green plastids are unable to provide PEP via plastidial glycolysis because of a lack of enolase (Prabhakar et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

The Mysterious Rescue of adg1-1/tpt-2 – an Arabidopsis thaliana Double Mutant Impaired in Acclimation to High Light – by Exogenously Supplied Sugars

Heinrichs¹,

Schmitz²,

Flügge³

et al. 2012

Front. Plant Sci.

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An Arabidopsis thaliana double mutant (adg1-1/tpt-2) defective in the day- and night-path of photoassimilate export from the chloroplast due to a knockout in the triose phosphate/phosphate translocator (TPT; tpt-2) and a lack of starch [mutation in ADP glucose pyrophosphorylase (AGPase); adg1-1] exhibits severe growth retardation, a decrease in the photosynthetic capacity, and a high chlorophyll fluorescence (HCF) phenotype under high light conditions. These phenotypes could be rescued when the plants were grown on sucrose (Suc) or glucose (Glc). Here we address the question whether Glc-sensing hexokinase1 (HXK1) defective in the Glc insensitive 2 (gin2-1) mutant is involved in the sugar-dependent rescue of adg1-1/tpt-2. Triple mutants defective in the TPT, AGPase, and HXK1 (adg1-1/tpt-2/gin2-1) were established as homozygous lines and grown together with Col-0 and Landsberg erecta (Ler) wild-type plants, gin2-1, the adg1-1/tpt-2 double mutant, and the adg1-1/tpt-2/gpt2-1 triple mutant [additionally defective in the glucose 6-phosphate/phosphate translocator 2 (GPT2)] on agar in the presence or absence of 50 mM of each Glc, Suc, or fructose (Fru). The growth phenotype of the double mutant and both triple mutants could be rescued to a similar extent only by Glc and Suc, but not by Fru. All three sugars were capable of rescuing the HCF and photosynthesis phenotype, irrespectively of the presence or absence of HXK1. Quantitative RT-PCR analyses of sugar-responsive genes revealed that plastidial HXK (pHXK) was up-regulated in adg1-1/tpt-2 plants grown on sugars, but showed no response in adg1-1/tpt-2/gin2-1. It appears likely that soluble sugars are directly taken up by the chloroplasts and enter further metabolism, which consumes ATP and NADPH from the photosynthetic light reaction and thereby rescues the photosynthesis phenotype of the double mutant. The implication of sugar turnover and probably signaling inside the chloroplasts for the concept of retrograde signaling is discussed.

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“…The stock numbers of the batches were CS27941, CS27951, CS27942, CS27952, CS27943, CS27953 and CS27944. Previously described alleles used for allelism tests were either purchased from NASC [ tz‐1 (Li and Redei, ), gls1‐30 (Coschigano et al ., ), tir1‐1 (Ruegger et al ., ), esi1‐1 (Chen et al ., ), and ddl‐2 (Morris et al ., )] or kindly provided by the authors who first described them [Hidetoshi Saze, ibm1‐4 (Saze et al ., ); Joanne Chory, cue1‐2 (Streatfield et al ., ); José Alonso, wei8‐1 (Stepanova et al ., ); Edgar Spalding, mdr1‐1 and mdr1‐3 (Noh et al ., ); Scott Poethig, sqn‐1 and sqn‐2 (Berardini et al ., ); Scott Michaels, cdc73‐1 and cdc73‐2 (Yu and Michaels, ); and Hiroyasu Motose, ibo1‐4 (Motose et al ., )]. Seeds were sterilized with bleach and stratified at 4°C for 72 h. Seedlings were grown in vitro as described in Ponce et al .…”

Section: Methodsmentioning

confidence: 99%

Leaf phenomics: a systematic reverse genetic screen for Arabidopsis leaf mutants

et al. 2014

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SUMMARYThe study and eventual manipulation of leaf development in plants requires a thorough understanding of the genetic basis of leaf organogenesis. Forward genetic screens have identified hundreds of Arabidopsis mutants with altered leaf development, but the genome has not yet been saturated. To identify genes required for leaf development we are screening the Arabidopsis Salk Unimutant collection. We have identified 608 lines that exhibit a leaf phenotype with full penetrance and almost constant expressivity and 98 additional lines with segregating mutant phenotypes. To allow indexing and integration with other mutants, the mutant phenotypes were described using a custom leaf phenotype ontology. We found that the indexed mutation is present in the annotated locus for 78% of the 553 mutants genotyped, and that in half of these the annotated T-DNA is responsible for the phenotype. To quickly map non-annotated T-DNA insertions, we developed a reliable, cost-effective and easy method based on whole-genome sequencing. To enable comprehensive access to our data, we implemented a public web application named PhenoLeaf (http://genetics.umh.es/phenoleaf) that allows researchers to query the results of our screen, including text and visual phenotype information. We demonstrated how this new resource can facilitate gene function discovery by identifying and characterizing At1g77600, which we found to be required for proximal-distal cell cycle-driven leaf growth, and At3g62870, which encodes a ribosomal protein needed for cell proliferation and chloroplast function. This collection provides a valuable tool for the study of leaf development, characterization of biomass feedstocks and examination of other traits in this fundamental photosynthetic organ.

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The Phosphoenolpyruvate/Phosphate Translocator Is Required for Phenolic Metabolism, Palisade Cell Development, and Plastid-Dependent Nuclear Gene Expression

Cited by 235 publications

References 34 publications

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

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

The Mysterious Rescue of adg1-1/tpt-2 – an Arabidopsis thaliana Double Mutant Impaired in Acclimation to High Light – by Exogenously Supplied Sugars

Leaf phenomics: a systematic reverse genetic screen for Arabidopsis leaf mutants

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