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.

The plant vacuolar H+-pyrophosphatase (H+-PPase) functions as a proton pump coupled with the hydrolysis of pyrophosphate (PPi). Loss-of-function mutants (fugu5s and vhp1) of the H+-PPase of Arabidopsis thaliana show clear morphological phenotypes in the cotyledons, caused by inhibition of gluconeogenesis from seed storage lipids due to excessive accumulation of PPi. In this study, we investigated the phenotypes of the fugu5 and vhp1 mutants during vegetative growth under a specific nitrogen nutritional regime. When nitrate in the culture medium was the sole nitrogen source, growth of the mutant rosette leaves was severely compromised. Interestingly, trypan blue staining revealed notable cell death at the leaf blade–petiole junctions of young leaves, a region known to have meristematic features. Physical contact of the leaf tip with the culture medium also triggered leaf atrophy, suggesting that absorption of some elements through the hydathodes was probably involved in this phenotype. Prevention of such leaf–medium contact resulted in a marked decrease in phosphate content in the shoots, and suppressed leaf atrophy. Furthermore, fugu5 necrotic symptoms were rescued completely by heterologous expression of yeast cytosolic soluble pyrophosphatase IPP1 or uncoupling-type H+-PPases that retained only PPi-hydrolysis activity, indicating that the damage of actively proliferating cells was caused by the loss of the PPi-hydrolyzing function of H+-PPase. Importantly, cell death and growth defects of the fugu5 leaves were suppressed completely by the simple addition of ammonium (>1 mM) to the culture medium. The PPi content in the shoots of fugu5 grown on ammonium-free medium was 70% higher than that of the wild type, and PPi levels were restored to normal upon growth on ammonium-supplemented medium. Together, these findings suggest that the PPi-hydrolyzing activity of H+-PPase is essential to maintain the PPi contents at optimal levels when grown on ammonium-free culture medium, and any direct contact of the leaves with the culture medium may raise PPi levels in the leaves through increased phosphate uptake.

The cytosolic level of inorganic pyrophosphate (PPi) is finely regulated, with PPi hydrolyzed primarily by the vacuolar H +-pyrophosphatase (H +-PPase, VHP1/FUGU5/AVP1) and secondarily by five cytosolic soluble pyrophosphatases (sPPases; PPa1-PPa5) in Arabidopsis thaliana. Loss-of-function mutants of H +-PPase (fugu5s) have been reported to show atrophic phenotypes in their rosette leaves when nitrate is the sole nitrogen source in the culture medium. For this phenotype, two questions remain unanswered: why does atrophy depend on physical contact between shoots and the medium, and how does ammonium prevent such atrophy. To understand the mechanism driving this phenotype, we analyzed the growth and phenotypes of mutants on ammonium-free medium in detail. fugu5-1 showed cuticle defects, cell swelling, reduced β-glucan levels, and vein malformation in the leaves, suggesting cell wall weakening and cell lethality. Based on the observation in the double mutants fugu5-1 ppa1 and fugu5-1 ppa4 of more severe atrophy compared to fugu5-1, the nitrogen-dependent phenotype might be linked to PPi metabolism. To elucidate the role of ammonium in this process, we examined the fluctuations of sPPase mRNA levels and the possibility of alternative PPi-removing factors, such as other types of pyrophosphatase. First, we found that both the protein and mRNA levels of sPPases were unaffected by the nitrogen source. Second, to assess the influence of other PPi-removing factors, we examined the phenotypes of triple knockout mutants of H +-PPase and two sPPases on ammonium-containing medium. Both fugu5 ppa1 ppa2 and fugu5 ppa1 ppa4 had nearly lethal embryonic phenotypes, with the survivors showing striking dwarfism and abnormal morphology. Moreover, fugu5 ppa1 +/− ppa4 showed severe atrophy at the leaf margins. The other triple mutants, fugu5 ppa1 ppa5 and fugu5 ppa2 ppa4, exhibited death of root hairs and were nearly sterile due to deformed pistils, respectively, even when grown on standard medium. Together,