Tissue-targeted inorganic pyrophosphate hydrolysis in a fugu5 mutant reveals that excess inorganic pyrophosphate triggers developmental defects in a cell-autonomous manner

Abstract

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.

Leaf-size control beyond transcription factors: Compensatory mechanisms

“…among transgenic lines, consistent with an axial role in the central metabolism of gluconeogenesis CCE (Gunji et al, 2022). These findings not only provide a conceptual framework to unveil metabolic fluctuations within leaf tissues with high spatiotemporal resolution, but also suggest that excess PPi exerts its inhibitory effect in planta during the early stages of seedling establishment in a tissue-and cellautonomous manner.…”

“…However, the compensatory interplay between cell proliferation and post-mitotic cell expansion during the induction of compensatory cell enlargement (CCE) is ill-known. Because this knowledge on cellular dynamics is instrumental in determining future directions aiming to delineate the holistic mechanisms of compensation, it has been recently investigated using leaves chimeric for the key elements of class I, II, or III compensation ( Kawade et al., 2010 ; Gunji et al., 2022 ) ( Figure 1 ). The above investigations revealed that both cell-autonomous and non-cell autonomous mechanisms are involved in CCE.…”

“…By contrast, epidermal cells trigger CCE in a cell-autonomous manner, preventing cell-to-cell communication across tissue layers ( Nozaki et al., 2020 ). (B) Cell-to-cell communication between epidermal and mesophyll cells is absent in fugu5 , which has class II compensation ( Gunji et al., 2022 ). Given that excess PPi inhibits metabolic reactions, fugu5 -mediated induction of CCE is likely controlled in a cell-autonomous manner.…”

“…The above hypothesis has been formally tested using a spatiotemporal approach by constructing and analyzing transgenic lines in which PPi has been removed from the epidermis, from palisade tissue cells, or during the 4 days following seed imbibition ( Gunji et al., 2022 ). When the yeast PPase IPP1 was expressed in the epidermis or palisade tissue alone, fugu5 phenotypes were independently restored ( Gunji et al., 2022 ) ( Figure 1B ). Furthermore, the immediate removal of excess PPi after seed imbibition suppressed CCE of palisade cells but failed to totally rescue the epidermal development defects ( Gunji et al., 2022 ).…”

“…When the yeast PPase IPP1 was expressed in the epidermis or palisade tissue alone, fugu5 phenotypes were independently restored ( Gunji et al., 2022 ) ( Figure 1B ). Furthermore, the immediate removal of excess PPi after seed imbibition suppressed CCE of palisade cells but failed to totally rescue the epidermal development defects ( Gunji et al., 2022 ). Next, the impacts of spatial and temporal removal of PPi were investigated by capillary electrophoresis time-of-flight mass spectrometry.…”