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.

In plants, the effective mobilization of seed nutrient reserves is crucial during germination and for seedling establishment. The Arabidopsis H+-PPase-loss-of-function fugu5 mutants exhibit a reduced number of cells in the cotyledons. This leads to enhanced post-mitotic cell expansion, also known as compensated cell enlargement (CCE). While decreased cell numbers have been ascribed to reduced gluconeogenesis from triacylglycerol, the molecular mechanisms underlying CCE remain ill-known. Given the role of indole 3-butyric acid (IBA) in cotyledon development, and because CCE in fugu5 is specifically and completely cancelled by ech2, which shows defective IBA-to-indoleacetic acid (IAA) conversion, IBA has emerged as a potential regulator of CCE. Here, to further illuminate the regulatory role of IBA in CCE, we used a series of high-order mutants that harbored a specific defect in IBA-to-IAA conversion, IBA efflux, IAA signaling, or vacuolar type H+-ATPase (V-ATPase) activity and analyzed the genetic interaction with fugu5–1. We found that while CCE in fugu5 was promoted by IBA, defects in IBA-to-IAA conversion, IAA response, or the V-ATPase activity alone cancelled CCE. Consistently, endogenous IAA in fugu5 reached a level 2.2-fold higher than the WT in 1-week-old seedlings. Finally, the above findings were validated in icl–2, mls–2, pck1–2 and ibr10 mutants, in which CCE was triggered by low sugar contents. This provides a scenario in which following seed germination, the low-sugar-state triggers IAA synthesis, leading to CCE through the activation of the V-ATPase. These findings illustrate how fine-tuning cell and organ size regulation depend on interplays between metabolism and IAA levels in plants.

Excess PPi triggers developmental defects in a cell-autonomous manner. The level of inorganic pyrophosphate (PPi) must be tightly regulated in all kingdoms for the proper execution of cellular functions. In plants, the vacuolar proton pyrophosphatase (H+-PPase) has a pivotal role in PPi homeostasis. We previously demonstrated that the excess cytosolic PPi in the H+-PPase loss-of-function fugu5 mutant inhibits gluconeogenesis from seed storage lipids, arrests cell division in cotyledonary palisade tissue, and triggers a compensated cell enlargement (CCE). Moreover, PPi alters pavement cell (PC) shape, stomatal patterning, and functioning, supporting specific yet broad inhibitory effects of PPi on leaf morphogenesis. Whereas these developmental defects were totally rescued by the expression of the yeast soluble pyrophosphatase IPP1, sucrose supply alone canceled CCE in the palisade tissue but not the epidermal developmental defects. Hence, we postulated that the latter are likely triggered by excess PPi rather than a sucrose deficit. To formally test this hypothesis, we adopted a spatiotemporal approach by constructing and analyzing fugu5-1 PDF1pro::IPP1, fugu5-1 CLV1pro::IPP1, and fugu5-1 ICLpro::IPP1, whereby PPi was removed specifically from the epidermis, palisade tissue cells, or during the 4 days following seed imbibition, respectively. It is important to note that whereas PC defects in fugu5-1 PDF1pro::IPP1 were completely recovered, those in fugu5-1 CLV1pro::IPP1 were not. In addition, phenotypic analyses of fugu5-1 ICLpro::IPP1 lines demonstrated that the immediate removal of PPi after seed imbibition markedly improved overall plant growth, abolished CCE, but only partially restored the epidermal developmental defects. Next, the impact of spatial and temporal removal of PPi was investigated by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF MS). Our analysis revealed that the metabolic profiles are differentially affected among all the above transgenic lines, and consistent with an axial role of central metabolism of gluconeogenesis in CCE. Taken together, this study provides a conceptual framework to unveil metabolic fluctuations within leaf tissues with high spatio–temporal resolution. Finally, our findings suggest that excess PPi exerts its inhibitory effect in planta in the early stages of seedling establishment in a tissue- and cell-autonomous manner.