Compensated Cell Enlargement in fugu5 is Specifically Triggered by Lowered Sucrose Production from Seed Storage Lipids

Abstract

To reveal the logic of size regulation in multicellular organisms, we have used Arabidopsis thaliana as a model organism and its leaves as a model organ. We discovered the existence of a compensatory system, whereby a decrease in leaf cell number often triggers unusual cell enlargement. However, despite the large number of compensation-exhibiting mutants analyzed to date, we have only a limited understanding of the detailed molecular mechanisms triggering the decrease in cell number and subsequent compensated cell enlargement (CCE). CCE in fugu5, the vacuolar type H+-pyrophosphatase loss-of-function mutant, is specific to cotyledons and completely suppressed when sucrose (Suc) is supplied or cytosolic pyrophosphate (PPi) is specifically removed. In addition, several lines of evidence suggest that excess cytosolic PPi in fugu5 impairs gluconeogenesis from triacylglycerol (TAG) to Suc. Here, detailed cellular phenotyping revealed that the loss-of-function mutants icl-2, mls-2 and pck1-2 triggered CCE in cotyledons. All double mutant combinations between fugu5-1 and the above three mutants exhibited compensation, but did not display a further increase in cell size. Importantly, similar phenotypes were observed in icl-2 mls-2, icl-2 pck1-2 and mls-2 pck1-2. Quantification of TAG breakdown and Suc contents further supported our findings. Taken together, we demonstrate that de novo Suc synthesis from TAG is fundamentally important for proper resumption of post-germinative cotyledon development. Moreover, provided that icl-2, mls-2 and pck1-2 are only compromised in Suc biosynthesis de novo from TAG, our findings clearly indicate that lowered Suc production in fugu5, rather than excess cytosolic PPi, is the direct trigger of CCE.

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

“…These 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. The sucrose content in fugu5 seedlings is low due to suppressed gluconeogenesis (Ferjani et al, 2011;Takahashi et al, 2017). The sucrose content per seedling in fugu5-1 ppa1-1 was lower than in the wild type but slightly higher than in the fugu5-1 single mutant ( Figure 5D).…”

“…Under these conditions, sucrose consumption might be markedly reduced in fugu5-1 ppa1-1. This reduced sucrose consumption may exceed the decline in sucrose supply due to the inhibition of gluconeogenesis that occurs in fugu5 (Ferjani et al, 2011;Takahashi et al, 2017). This is a plausible scenario for why the sucrose levels were elevated in fugu5-1 ppa1-1 plants.…”

“…High sucrose levels may result from severe growth suppression. High PPi levels suppress plant growth by inhibiting macromolecule biosynthesis (Maeshima, 2001;Ferjani et al, 2011;Takahashi et al, 2017). Under these conditions, sucrose consumption might be markedly reduced in fugu5-1 ppa1-1.…”

“…Three independent VHP1 loss-of-function mutant lines (the fugu5 mutant series) have been identified based on their gross phenotypes (including oblong-shaped cotyledons) and their cellular phenotypes (namely, compensation; i.e., cell enlargement due to a decrease in cell number) (Ferjani et al, 2007). This phenotype is caused by an increase in PPi levels, which inhibits the gluconeogenesis of seed storage lipids to sucrose (Ferjani et al, 2011;Takahashi et al, 2017). At the postgermination stage of development, fugu5 mutants also exhibit moderately delayed growth, although they appear healthy Fukuda et al, 2016).…”

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

“…Indeed, the fugu5 mutant plants display retarded post-germinative growth and exhibit oblong-shaped cotyledons and compensation in their palisade tissue, such as excessive cell expansion triggered by decreased cell proliferation (Ferjani et al, 2007;Ferjani et al, 2008;Ferjani et al, 2011;Ferjani et al, 2012). In addition, gluconeogenesis, the process that produces sucrose (Suc) from triacylglycerol (TAG) in seed storage lipids, is partially suppressed in fugu5 mutants (Ferjani et al, 2011;Takahashi et al, 2017).…”