第115回 Plant Science Seminar

Pyrophosphate hydrolysis by the vacuolar
H+-PPase promotes gluconeogenesis
in germinating oilseeds

Dr. Ali Ferjani
Department of Biology, Tokyo Gakugei University

日時 ： １０月10日 (金) １６：３０~１８：００
場所 ： 理学部 ５号館 ８階 ８１３号室
Upon germination, oil-seed plants rely on triacylglycerol (TAG) reserves to sustain their growth during aheterotrophic period that requires β-oxidation, the glyoxylate cycle, and gluconeogenesis activities to convert TAG tosucrose (Suc), which provides energy to developing tissues in the form of ATP. Besides, macromolecular syntheses
in proliferating young tissues of the embryo require huge amounts of ATP, which is often hydrolyzed to AMP plus PPi.
The loss of the pyrophosphatase (PPase) activity causes growth arrest, and/or developmental defects in manyorganisms, such as Escherichia coli, yeast, and Caenorhabditis elegans. In Arabidopsis, the proton-pumping vacuolar
pyrophosphatase (H+-PPase) uses the energy from PPi hydrolysis to acidify the vacuole. The Arabidopsis fugu5mutants, previously characterized as having defects in cotyledon development (Ferjani et al., 2007), are lacking theAVP1 H +-PPase (Ferjani et al., 2011). The fugu5 mutants failed to sustain postgerminative heterotrophic growth,which recovered upon Suc supply, or specific PPi removal by the cytosolic IPP1 from yeast under the control of theArabidopsis AVP1 promoter in the AVP1pro:IPP1 transgenic lines. This demonstrated that the major function of H+PPase in seedling development is the removal of inhibitory PPi rather than vacuolar acidification. Following
quantification of sucrose and PPi amounts in etiolated seedlings of the wild type versus fugu5, we concluded thatelevated level of PPi is most likely to inhibit gluconeogenesis, but the mechanism or metabolic targets of PPi inhibition
remained unclear.
Here, our profiling of major metabolites that occur during TAG mobilization showed that the amounts of Glu-1-P and
UDP-Glc were 2.0- and 0.5-fold in fugu5 versus the wild type, while other intermediate metabolites were basicallyunaffected. This result suggested that UDP-Glucose Pyrophosphorylase (UGPase) is specifically inhibited bycytosolic PPi accumulation in the fugu5 mutant background. To confirm these findings, we used two independent
AVP1pro:IPP1 transgenic lines, in which IPP1 actively hydrolyzes cytosolic PPi, but has no effect on vacuolar
acidification. Importantly, the above metabolic defects were reversed in AVP1pro:IPP1, where Glc-1-P and UDP-Glc
were 1.0- and > 2.0-fold compared to the wild type. It is also noticeable that the amounts of UDP-Glc in AVP1pro:IPP1
were > 4.0-fold higher than in fugu5 muants. It is well established from biochemical in vitro experiments that UGPase
equilibrates UDP-Glc and its precursor Glc-1-P. Thus, given the readily reversible nature of the Glc-1-P/UDP-Glcreaction, the metabolic target of PPi overaccumulation in fugu5 is UGPase. PPi metabolism is enigmatic in all livingorganism, including plants. Our recent reports have supported a pivotal role of the vacuolar H+-PPase in maintaining
cytosolic PPi homeostasis (Ferjani et al., 2011; 2014). Also, we proposed gluconeogenesis, which exclusively occur
in the cytosol, as a potential target of inhibitory high PPi levels (Ferjani et al., 2011). Here, the pinpoint target of PPi
has been identified as UGPase, a crucial enzyme that during germination acts prior to Suc production from TAG.
Taken together, our results represent an unprecedented breakthrough in this field, which has identified a major
missing piece of the modern biochemistry.

Plant Science Seminar
北大内で植物科学を研究している（特に）若い人たちの交流促進の場を提供するためのセミナーです。Aliさんは内藤カンファレンスで来札されます。その後にこちらにも寄ってお話していただきます。生化学の教科書ではenigmaと紹介されている難問を解いてくれるはずです。

世話人： 川出 健介（内線：4474）