Virus-Induced Gene Silencing of Plastidial Soluble Inorganic Pyrophosphatase Impairs Essential Leaf Anabolic Pathways and Reduces Drought Stress Tolerance inNicotiana benthamiana

Abstract

The role of pyrophosphate in primary metabolism is poorly understood. Here, we report on the transient down-regulation of plastid-targeted soluble inorganic pyrophosphatase in Nicotiana benthamiana source leaves. Physiological and metabolic perturbations were particularly evident in chloroplastic central metabolism, which is reliant on fast and efficient pyrophosphate dissipation. Plants lacking plastidial soluble inorganic pyrophosphatase (psPPase) were characterized by increased pyrophosphate levels, decreased starch content, and alterations in chlorophyll and carotenoid biosynthesis, while constituents like amino acids (except for histidine, serine, and tryptophan) and soluble sugars and organic acids (except for malate and citrate) remained invariable from the control. Furthermore, translation of Rubisco was significantly affected, as observed for the amounts of the respective subunits as well as total soluble protein content. These changes were concurrent with the fact that plants with reduced psPPase were unable to assimilate carbon to the same extent as the controls. Furthermore, plants with lowered psPPase exposed to mild drought stress showed a moderate wilting phenotype and reduced vitality, which could be correlated to reduced abscisic acid levels limiting stomatal closure. Taken together, the results suggest that plastidial pyrophosphate dissipation through psPPase is indispensable for vital plant processes.Pyrophosphate (PP i ) is a key metabolite generated in the activation of several polymerization steps (Geigenberger et al., 1998;Stitt, 1998;Rojas-Beltrán et al., 1999;Farré et al., 2001;Sonnewald, 2001; Ló pezMarqués et al., 2004), and its removal is essential to prevent the inhibition of thermodynamically unfavorable reactions (Geigenberger et al., 1998; Ló pezMarqués et al., 2004). PP i is generally removed by inorganic pyrophosphatases, which hydrolyze PP i to orthophosphate (P i ). Pyrophosphatases are ubiquitous in plant cells and found both as soluble forms in the cytosol and plastid and as membrane-bound forms on the tonoplast (Rea and Poole, 1993;Baltscheffsky et al., 1999;Maeshima, 2000), mitochondria (Vianello and Macrì, 1999), and chloroplast (Jiang et al., 1997). In Arabidopsis (Arabidopsis thaliana), six soluble pyrophosphatase (sPPase) isoforms have been identified to date (Schulze et al., 2004). Five (AtPPa1, -2, -3, -4, and -5) are far more similar to each other than to AtPPa6 (Schulze et al., 2004) and have been shown to be localized to the cytosol using GFP fusions (Ergen, 2006). In potato (Solanum tuberosum), two sPPase genes, StPPa1 and StPPa2, which are similar to AtPPa1, have also been identified and demonstrated to be present in the cytosol using immunogold labeling (Rojas-Beltrán et al., 1999). In addition to sPPases, several other cytosolic enzymes can remove PP i , including the soluble enzymes pyrophosphate:Fru 6-P phosphotransferase and UDP-Glc pyrophosphorylase. Due to a lack of adverse phenotypic alterations found when altering the expression of these enzymes in auto...

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