Alternative splicing of Arabidopsis G6PD5 recruits NADPH-producing OPPP reactions to the endoplasmic reticulum

Abstract

Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme of the oxidative pentose-phosphate pathway (OPPP). The OPPP mainly provides NADPH and sugar-phosphate building blocks for anabolic pathways and is present in all eukaryotes. In plant cells, the irreversible part of the OPPP is found in several compartments. Among the isoforms catalyzing the first OPPP step in Arabidopsis, G6PD1 to G6PD4 target plastids (with G6PD1 being also directed to peroxisomes), whereas G6PD5 and G6PD6 operate in the cytosol. We noticed that alternative splice forms G6PD5.4 and G6PD5.5 encode N-terminally extended proteoforms. Compared to G6PD5.1, RT-PCR signals differed and fluorescent reporter fusions expressed in Arabidopsis protoplasts accumulated in distinct intracellular sites. Co-expression with organelle-specific markers revealed that the G6PD5.4 and G6PD5.5 proteoforms label different subdomains of the endoplasmic reticulum (ER), and analysis of C-terminal roGFP fusions showed that their catalytic domains face the cytosol. In g6pd5-1 g6pd6-2 mutant protoplasts lacking cytosolic G6PDH activity, the ER-bound proteoforms were both active and thus able to form homomers. Among the Arabidopsis 6-phosphogluconolactonases (catalyzing the second OPPP step), we noticed that isoform PGL2 carries a C-terminal CaaX motif that may be prenylated for membrane attachment. Reporter-PGL2 fusions co-localized with G6PD5.4 in ER subdomains, which was abolished by Cys-to-Ser exchange in the 256CSIL motif. Among the Arabidopsis 6-phosphogluconate dehydrogenases (catalyzing the third OPPP step), S-acylated peptides were detected for all three isoforms in a recent palmitoylome, with dual cytosolic/peroxisomal PGD2 displaying three sites. Co-expression of GFP-PGD2 diminished crowding of OFP-G6PD5.4 at the ER, independent of PGL2's presence. Upon pull-down of GFP-G6PD5.4, not only unlabeled PGD2 and PGL2 were enriched, but also enzymes that depend on NADPH provision at the ER, indicative of physical interaction with the OPPP enzymes. When membrane-bound G6PD5.5 and 5.4 variants were co-expressed with KCR1 (ketoacyl-CoA reductase, involved in fatty acid elongation), ATR1 (NADPH:cytochrome-P450 oxidoreductase), or pulled C4H/CYP73A5 (cinnamate 4-hydroxylase) as indirectly (via ATR) NADPH-dependent cytochrome P450 enzyme, co-localization in ER subdomains was observed. Thus, alternative splicing of G6PD5 can direct the NADPH-producing OPPP reactions to the cytosolic face of the ER, where they may operate as membrane-bound metabolon to support several important biosynthetic pathways of plant cells.

ZmG6PDH1 in glucose-6-phosphate dehydrogenase family enhances cold stress tolerance in maize

“…Members of the G6PDH gene family play vital roles in stress adaptation in various model plants ( Yang et al., 2019 ; Linnenbrügger et al., 2022 ). ZmG6PDH transcriptional profiles were examined in response to low temperature (4°C), alkali (150 mM NaHCO 3 ), salt (200 mM NaCl), and drought (20% PEG) stress treatment conditions to explore the potential roles of these genes in maize abiotic stress responses.…”

“…G6PDH gene family members have been characterized in many plant species, including soybean ( Zhao et al., 2020 ), strawberry ( Zhang et al., 2020 ), wheat ( Tian et al., 2021 ), tomato ( Landi et al., 2016 ), barley ( Zhao et al., 2015 ), and Arabidopsis ( Yang et al., 2019 ; Linnenbrügger et al., 2022 ), where they function as critical regulators of growth and development. The different G6PDH isoforms are classified according to their subcellular localizations, with each regulated by distinct mechanisms and playing different roles in plant metabolic activity ( Feng et al., 2020 ; Landi et al., 2021 ).…”

Metabolic priming in G6PDH isoenzyme‐replaced tobacco lines improves stress tolerance and seed yields via altering assimilate partitioning

“…Of note, salt-induced proline biosynthesis in the light is governed by bZIP transcription factor HY5 in Arabidopsis, leading to elevated PC5S levels (proline Anabolic pathways should generally profit from elevated NADPH provision-also when distributed across different compartments. After all, flux through the cytosolic OPPP constantly feeds C5-sugar phosphates into the plastid stroma via metabolite transporters in the inner envelope membrane: Xu5P mainly by XPT (Eicks et al, 2002) and Ru5P also by GPT1 (Baune et al, 2020; for schemes see Linnenbr€ ugger et al, 2022). Thus, the cP2 approach Figure 9.…”

“…Anabolic pathways should generally profit from elevated NADPH provision—also when distributed across different compartments. After all, flux through the cytosolic OPPP constantly feeds C5‐sugar phosphates into the plastid stroma via metabolite transporters in the inner envelope membrane: Xu5P mainly by XPT (Eicks et al., 2002) and Ru5P also by GPT1 (Baune et al., 2020; for schemes see Linnenbrügger et al., 2022). Thus, the cP2 approach impacts the complete PPP cycle in chloroplasts, showing as fast photosynthetic induction due to metabolic pools shared with the reversible reactions of the Calvin cycle (Scharte et al., 2009; this work).…”

“…The latter may reflect redox activation of acetyl‐CoA carboxylase that catalyses the first committed step in the biosynthesis of long‐chain fatty acids (16:0 and 18:0) in the stroma. After export from plastids, extension to very‐long‐chain fatty acids (C20 to C24) occurs at the cytosolic face of the ER, consuming NADPH in the cytosol together with desaturation by extra‐plastidial FAD enzymes (Shanklin & Cahoon, 1998)—also indirectly, via ER‐bound cytochrome P450 enzymes (Linnenbrügger et al., 2022; for reviews on lipid biosynthesis and transport in plants, see Li et al., 2016 and Rawsthorne, 2002). In fact, levels of polyunsaturated fatty acids (PUFAs, involving ER desaturases FAD2 and FAD3 in Arabidopsis; Lou et al., 2014) correlated with the growth rate of cell cultures from Arabidopsis and Sycamore (Meï et al., 2015).…”