Analysis of homo- and hetero-dimerization among the three 6-phosphogluconate dehydrogenase isoforms of Arabidopsis.

We recently described that all three 6-phosphogluconate dehydrogenase (6PGDH) isoforms of Arabidopsis (PGD) with similar length show dual localization: PGD1 and PGD3 in the cytosol and in plastids, and PGD2 in the cytosol and in peroxisomes. We set out to investigate heterodimer formation, however due to only weak homodimerization of all Arabidopsis PGD isoforms in yeast cells, we conducted further protein-protein interaction studies in planta to investigate homomer versus heteromer formation and their sub-cellular localization. Bimolecular fluorescence complementation (BiFC) analyses in co-transfected Arabidopsis protoplasts demonstrated that all PGD isoforms may form homo- and heterodimers. Notably, with free N-terminal ends, PGD1-PGD3 heterodimers were detected both in the cytosol and in the plastid stroma, but heterodimers with PGD2 only in the cytosol. This independently confirmed that PGD2 cannot enter plastids. On the other hand, with free C-terminal ends, PGD1-PGD2 and PGD3-PGD2 heterodimers were confined to the cytosol, indicating that only PGD2 homodimers are imported by peroxisomes. Together these findings suggest functional redundancy of PGD1 and PGD3 inside plastids, and relevance of PGD1-PGD2 or PGD3-PGD2 heterodimer formation in the cytosol: this could retain sufficient 6PGDH activity needed for NADPH provision, especially during stress defense and initiation of developmental responses.

Defects in Peroxisomal 6-Phosphogluconate Dehydrogenase Isoform PGD2 Prevent Gametophytic Interaction in Arabidopsis thaliana.

We studied the localization of 6-phosphogluconate dehydrogenase (PGD) isoforms of Arabidopsis (Arabidopsis thaliana). Similar polypeptide lengths of PGD1, PGD2, and PGD3 obscured which isoform may represent the cytosolic and/or plastidic enzyme plus whether PGD2 with a peroxisomal targeting motif also might target plastids. Reporter-fusion analyses in protoplasts revealed that, with a free N terminus, PGD1 and PGD3 accumulate in the cytosol and chloroplasts, whereas PGD2 remains in the cytosol. Mutagenesis of a conserved second ATG enhanced the plastidic localization of PGD1 and PGD3 but not PGD2. Amino-terminal deletions of PGD2 fusions with a free C terminus resulted in peroxisomal import after dimerization, and PGD2 could be immunodetected in purified peroxisomes. Repeated selfing of pgd2 transfer (T-)DNA alleles yielded no homozygous mutants, although siliques and seeds of heterozygous plants developed normally. Detailed analyses of the C-terminally truncated PGD2-1 protein showed that peroxisomal import and catalytic activity are abolished. Reciprocal backcrosses of pgd2-1 suggested that missing PGD activity in peroxisomes primarily affects the male gametophyte. Tetrad analyses in the quartet1-2 background revealed that pgd2-1 pollen is vital and in vitro germination normal, but pollen tube growth inside stylar tissues appeared less directed. Mutual gametophytic sterility was overcome by complementation with a genomic construct but not with a version lacking the first ATG. These analyses showed that peroxisomal PGD2 activity is required for guided growth of the male gametophytes and pollen tube-ovule interaction. Our report finally demonstrates an essential role of oxidative pentose-phosphate pathway reactions in peroxisomes, likely needed to sustain critical levels of nitric oxide and/or jasmonic acid, whose biosynthesis both depend on NADPH provision.