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

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“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.