Defects in peroxisomal 6-phosphogluconate dehydrogenase isoform PGD2 prevent gametophytic interaction in Arabidopsis thaliana [Defekte in der peroxisomalen 6-Phosphogluconat-Dehydrogenase Isoform PGD2 verhindern die gametophytische Interaktion in Arabidopsis thaliana]

Hölscher, C; Lutterbey, MC; Lansing, H; Meyer, T; Fischer, K; von Schaewen, A

Abstract

We studied localization of the 6-phosphogluconate dehydrogenase (PGD) isoforms of 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 peroxisomal targeting motif (PTS1) might also target plastids. Reporter-fusion analyses in protoplasts revealed that with 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 plastidic localization of PGD1 and PGD3 but not PGD2. N-terminal deletions in PGD2 fusions with free C-termini resulted in peroxisomal import upon dimerization; plus PGD2 could be immuno-detected in purified peroxisomes. Repeated selfing of pgd2 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 6PGDH activity in peroxisomes affects the male gametophyte. Tetrad analyses in the qrt1-2 background revealed that pgd2-1 pollen is vital and in-vitro germination normal, but pollen-tube growth inside stylar tissues apparently 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 for OPPP reactions in peroxisomes, possibly to maintain critical levels of nitric oxide and/or jasmonic acid, whose biosyntheses both depend on NADPH provision.