Vacuolar H⁺-Pyrophosphatase and Cytosolic Soluble Pyrophosphatases Cooperatively Regulate Pyrophosphate Levels in Arabidopsis thaliana

Abstract

Inorganic pyrophosphate (PPi) is a phosphate donor and energy source. Many metabolic reactions that generate PPi are suppressed by high levels of PPi. Here, we investigated how proper levels of cytosolic PPi are maintained, focusing on soluble pyrophosphatases (AtPPa1 to AtPPa5; hereafter PPa1 to PPa5) and vacuolar H-pyrophosphatase (H-PPase, AtVHP1/FUGU5) in In planta, five PPa isozymes tagged with GFP were detected in the cytosol and nuclei. Immunochemical analyses revealed a high abundance of PPa1 and the absence of PPa3 in vegetative tissue. In addition, the heterologous expression of each PPa restored growth in a soluble PPase-defective yeast strain. Although the quadruple knockout mutant plant showed no obvious phenotypes, H-PPase and PPa1 double mutants () exhibited significant phenotypes, including dwarfism, high PPi concentrations, ectopic starch accumulation, decreased cellulose and callose levels, and structural cell wall defects. Altered cell arrangements and weakened cell walls in the root tip were particularly evident in and were more severe than in Our results indicate that H-PPase is essential for maintaining adequate PPi levels and that the cytosolic PPa isozymes, particularly PPa1, prevent increases in PPi concentrations to toxic levels. We discuss phenotypes in relation to metabolic reactions and PPi homeostasis.

Excess Pyrophosphate Restrains Pavement Cell Morphogenesis and Alters Organ Flatness in Arabidopsis thaliana

“…The tissue width increased over time in the WT but remained constant in the an-1 fugu5-1 double mutant. Segami et al, 2018). Indeed, the H + -PPase loss-of-function fugu5 mutants of Arabidopsis exhibit a pleiotropic phenotype, as they fail to reactivate cell cycling in cotyledonary palisade tissue following seed imbibition due to inhibition of gluconeogenesis from seed-stored TAG (Ferjani et al, 2011).…”

“…Therefore, PPi exerts an inhibitory effect on specific targets within different cell types. In other words, the function of H + -PPase, the major PPi-hydrolyzing enzyme in plant cells (Ferjani et al, 2014;Segami et al, 2018), goes beyond housekeeping, and the breadth and specificity of its contribution warrants investigation.…”

“…The importance of PPi homeostasis in plant growth and development in Arabidopsis thaliana (Arabidopsis, hereafter) has been intensively investigated using fugu5 mutants, harboring a molecular lesion in the vacuolar-type H + -PPase. For instance, we demonstrated that the H + -PPase is the major PPase in Arabidopsis (Ferjani et al, 2011;Ferjani et al, 2014;Asaoka et al, 2016;Segami et al, 2018). Failure to hydrolyze PPi led to developmental defects at the organism, organ, tissue, and cellular levels.…”

“…Simultaneously, in nearly 200 different metabolic reactions (Heinonen, 2001)including DNA replication, amino acid activation, and protein and cell wall biosynthesis-they produce pyrophosphate (PPi). PPi is a toxic molecule that if not immediately hydrolyzed by solubletype pyrophosphatases (sPPases) and/or membrane-bound pyrophosphatases (H + -PPases) (Ferjani et al, 2014;Segami et al, 2018), irreversibly arrests the above metabolic reaction.…”

Chinese wheat mosaic virus‐derived vsiRNA‐20 can regulate virus infection in wheat through inhibition of vacuolar‐ (H+)‐PPase induced cell death

“…Various proton (H + )‐pumps with different functions are known to localize in specific cellular components (Serrano, ; Segami et al , ). In this study, we analyzed TaVP subcellular location through a transient expression approach and showed that the TaVP‐GFP fusion is localized in the tonoplast (Fig.…”