Table_1_Tissue-targeted inorganic pyrophosphate hydrolysis in a fugu5 mutant reveals that excess inorganic pyrophosphate triggers developmental defects in a cell-autonomous manner.XLSX

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.

过量无机焦磷酸（inorganic pyrophosphate, PPi）以细胞自主方式诱发发育缺陷。无机焦磷酸的水平在所有生物界中均需严格调控，以保障细胞功能的正常执行。在植物中，液泡质子焦磷酸酶（H+-PPase）在PPi稳态维持中发挥关键作用。我们此前的研究证实，H+-PPase功能缺失型突变体fugu5中过量的胞质PPi会抑制种子贮藏脂质来源的糖异生作用，阻滞子叶栅栏组织的细胞分裂，并诱发补偿性细胞增大（CCE）。此外，PPi会改变表皮细胞（pavement cell, PC）的形态、气孔模式建成与功能，证实了PPi对叶片形态发生具有特异性且广泛的抑制作用。尽管通过表达酵母可溶性焦磷酸酶IPP1可完全挽救上述发育缺陷，但仅添加蔗糖仅能抵消栅栏组织中的CCE，却无法修复表皮发育缺陷。因此我们推测，表皮发育缺陷更可能由过量PPi而非蔗糖缺乏诱发。为正式验证这一假说，我们采用时空调控策略，构建并分析了fugu5-1 PDF1pro::IPP1、fugu5-1 CLV1pro::IPP1以及fugu5-1 ICLpro::IPP1三个转基因株系，分别实现仅在表皮、栅栏组织细胞中，或种子吸胀后的4天内清除PPi。值得注意的是，fugu5-1 PDF1pro::IPP1株系中的PC缺陷可完全恢复，但fugu5-1 CLV1pro::IPP1株系则无法恢复。此外，对fugu5-1 ICLpro::IPP1株系的表型分析显示，种子吸胀后即时清除PPi可显著改善植株整体生长状况，消除CCE，但仅能部分修复表皮发育缺陷。随后，我们通过毛细管电泳-飞行时间质谱（capillary electrophoresis time-of-flight mass spectrometry, CE-TOF MS）研究了时空清除PPi所产生的影响。分析结果显示，上述所有转基因株系的代谢谱均受到不同程度的影响，且这一结果与糖异生的中心代谢过程在CCE中的核心作用相符。综上，本研究建立了一套可在高时空分辨率下解析叶片组织内代谢波动的概念框架。最后，我们的研究结果表明，过量PPi在植物体内通过组织特异性与细胞自主方式，在幼苗建成早期发挥抑制作用。