Lack of Vacuolar H+ -Pyrophosphatase and Cytosolic Pyrophosphatases Causes Fatal Developmental Defects in Arabidopsis thaliana

Abstract

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,

Roles of type II H+-PPases and PPsPase1/PECP2 in early developmental stages and PPi homeostasis of Arabidopsis thaliana

“…Mutants and all transgenic plants were in the Columbia-0 (Col-0) background, and Arabidopsis thaliana Col-0 was used as the wild type (WT). fugu5-1 and fugu5-3 were isolated and characterized as the loss-of-function mutant of the vacuolar type-I H + -PPase (Horiguchi et al, 2006b;Ferjani et al, 2007;Ferjani et al, 2011;Fukuda et al, 2016;Segami et al, 2018;Fukuda et al, 2020;Gunji et al, 2020;Gunji et al, 2022). pecp2-1 and pecp2-3 (previously described as ppspase1-1 or ps2-1, and ppspase1-3, respectively), pecp1-1, the fugu5-1 pecp2-1 pecp1-1 triple homozygous mutant (Hanchi et al, 2018), and the vhp2;1 and vhp2;2-2 (Segami et al, 2010) loss-of-function mutants were previously reported.…”

“…Increased PPi levels in fugu5 are associated with inhibited hypocotyl elongation in etiolated seedlings (Ferjani et al, 2011). Seedlings grown in the light also display an altered cotyledon shape, as well as modifications of their palisade tissues, adaxial-side pavement cell complexity and stomatal distribution (Ferjani et al, 2007;Ferjani et al, 2011;Asaoka et al, 2019;Fukuda et al, 2020;Gunji et al, 2020;Gunji et al, 2022). These characteristics can be rescued by expressing IPP1, a soluble yeast pyrophosphatase (Ferjani et al, 2011;Segami et al, 2018;Gunji et al, 2020;Gunji et al, 2022).…”

“…As a proxy for direct PPi quantification, one powerful substitute consists in revealing alterations of PPi homeostasis based on plant phenotypic changes (Ferjani et al, 2011;Segami et al, 2018;Asaoka et al, 2019;Fukuda et al, 2020;Gunji et al, 2020;Tabeta et al, 2021). Thanks to the in-depth characterization of Arabidopsis mutants showing altered PPi homeostasis, specific plant anatomical parameters can be quantified to reveal subtle alterations in parameters linked to PPi concentration.…”

“…This phenotype is strongly aggravated when combined with a mutation in the cytosolic sPPase PPa1 gene, whereas combination with ppa2 through 5 did not enhance the fugu5 phenotype. Using fugu5 ppa1 double mutants was necessary to validate the in vivo PPase activity of PPa1, since the effects of a single ppa1 mutation can be masked by the activity of the vacuolar (type-I) FUGU5 V-PPase (Segami et al, 2018;Fukuda et al, 2020). Combinations of fugu5 and candidate PPases are therefore capable of revealing the importance of genes in relation to PPi homeostasis, in a highly quantifiable manner.…”

“…Combinations of fugu5 and candidate PPases are therefore capable of revealing the importance of genes in relation to PPi homeostasis, in a highly quantifiable manner. Other parameters such as cell complexity or cell wall composition were subsequently identified as also altered in the fugu5 mutant in response to PPi levels (Segami et al, 2018;Fukuda et al, 2020;Gunji et al, 2020;Gunji et al, 2022). Altogether, the quantification of these parameters makes it possible to unambiguously predict a PPi homeostasis defect for novel mutants.…”