Regulation of the oxidative pentose-phosphate pathway (OPPP) in plant cells

“植物细胞中氧化戊糖磷酸途径（OPPP）的调节“我们延长提案的重点在于两个主要主题：1.过氧化物酶体中OPPP亚型的瞬时氧化还原依赖性定位。细胞和代谢方面以及从短暂状态中恢复。我们打算继续进行我们最近出版物所证明的有希望的研究。过氧化物酶体中OPPP的中断导致拟南芥雌雄配子体的相互不育。我们将尝试在受精过程中通过选择性互补来克服这一障碍（使用雄性和雌性特异性启动子，或者RNAi抑制），以使研究过氧化物酶体OPPP缺陷在进一步发育阶段或应激时的程度成为可能。此外，我们正在研究第一个OPPP步骤之间的部分冗余（由异养组织中的G6 PD 2，G6 PD 3和PGL 3，PGL 5表示），即导致过氧化物酶体中瞬时定位的机制-特别是如何调节。除了已确定的氧化还原递质硫氧还蛋白（Trx）和谷氧还蛋白（Grx）外，过氧化物氧还蛋白（Prx）作为H_2O_2传感器是可能的进一步相互作用伙伴。GPT 1对ER的替代初始靶向（通过Grx c1和Trx h7）似乎涉及N-末端脂质修饰，并且在巯基开关旁边的G6 PD-亚型的调节也涉及磷酸化和赖氨酸乙酰化。除了荧光报告基因，我们还将测试不干扰过氧化物酶体靶向的小标签（如HA或TAP），并允许从复杂的蛋白质混合物或完整的分离细胞器中选择性富集。对于以下蛋白质组学分析，怀疑GPT 1（将G6 P底物交换为Ru 5 P产物）与可溶性OPPP组分形成疑似复合物（代谢物概念），其中Trx可作为连接或相互作用平台。还有一个问题是，哪些过程可能需要OPPP作为基质中的NADPH来源。除了在受精过程中形成茉莉酸（JA）和一氧化氮（NO）外，还可以（通过NADPH依赖性还原酶和过氧化物酶）保护免受氧化损伤。目前尚不清楚OPPP是如何从过氧化物酶体中去除的（恢复），我们将随着时间的推移（周转）和压力挑战进行研究。

“Regulation of the oxidative pentose-phosphate pathway (OPPP) in plant cells”Our focus for the prolongation of the proposal lies on two main topics: 1. Transient, redox-dependent localization of OPPP isoforms in peroxisomes.2. Cellular and metabolic aspects as well as recovery from the transient state. We intend to continue the promising studies as proven by our most recent publications. Interruption of the OPPP in peroxisomes resulted in mutual sterility of male and female gametophytes in Arabidopsis. We will try to overcome this block by selective complementation during fertilization (using male and female-specific promoters, alternatively RNAi suppression) to enable studies of the extent of peroxisomal OPPP defects in further developmental stages or upon stress. Moreover, we are investigating partial redundancy among the first OPPP steps (represented by G6PD2, G6PD3 and PGL3, PGL5 in heterotrophic tissues), i.e. mechanisms that lead to transient localization in peroxisomes – especially how this is regulated. Besides identified redox transmitters thioredoxin (Trx) and glutaredoxin (Grx), peroxiredoxins (Prx) as H2O2 sensors are possible further interaction partners. Alternative initial targeting of GPT1 to the ER (via Grx c1 and Trx h7) seems to involve N-terminal lipid modification, and regulation of the G6PD-Isoforms next to thiol switch also phosphorylation and lysine acetylation. Besides fluorescent reporters, we will also test small tags (like HA or TAP) that do not interfere with targeting to peroxisomes, and additionally allow for selective enrichment from complex protein mixtures or intact isolated organelles. For the following proteomics analyses, suspected complex formation of GPT1 (exchanges G6P substrate for Ru5P product) with soluble OPPP components is suspected (Metabolon concept), for which Trx may serve as link or interaction platform. Also in question is, which processes may require the OPPP as NADPH source in the matrix. Aside of formation of jasmonic acid (JA) and nitric oxide (NO) during fertilization, also protection from oxidative damage is conceivable (via NADPH-dependent reductases and peroxidases). Still unknown is how the OPPP is removed from peroxisomes (recovery), which we will study over time (turnover) and upon stress challenges.