Functional analyses of cytosolic glucose-6-phosphate dehydrogenases and their contribution to seed oil accumulation in Arabidopsis

Abstract

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in the supply of reduced nicotine amide cofactors for biochemical reactions and in modulating the redox state of cells. In plants, identification of its role is complicated due to the presence of several isoforms in the cytosol and plastids. Here we focus on G6PDHs in the cytosol of Arabidopsis (Arabidopsis thaliana) using single and double mutants disrupted in the two cytosolic G6PDHs. Only a single G6PDH isoform remained in the double mutant and was present in chloroplasts, consistent with a loss of cytosolic G6PDH activity. The activities of the cytosolic isoforms G6PD5 and G6PD6 were reciprocally increased in single mutants with no increase of their respective transcript levels. We hypothesized that G6PDH plays a role in supplying NADPH for oil accumulation in developing seeds in which photosynthesis may be light limited. G6PDH activity in seeds derived from G6PD6 and a plastid G6PDH isoform and showed a similar temporal activity pattern as oil accumulation. Seeds of the double mutant but not of the single mutants had higher oil content and increased weight compared to those of the wild type, with no alteration in the carbon to nitrogen ratio or fatty acid composition. A decrease in total G6PDH activity was observed only in the double mutant. These results suggest that loss of cytosolic G6PDH activity affects the metabolism of developing seeds by increasing carbon substrates for synthesis of storage compounds rather than by decreasing the NADPH supply specifically for fatty acid synthesis.

Figure 1.

Cytosolic localization of G6PD5 and G6PD6. Onion cells were bombarded with either pCAMBIA1302 inserted with the coding sequence of G6PD5 (A) or G6PD6 (B) or the vector alone (C). D, Zymogram with isolated chloroplasts shows enrichment in the G6PDH band that is neither G6PD5 nor G6PD6. The arrow indicates origin and direction of electrophoresis. Arrowheads indicate the position of G6PDH isoforms previously identified (Wakao and Benning, 2005), and the plastidic isoform. Lm, Standard G6PDH from Leuconostoc mesenteroides.

Figure 2.

PCR genotypes and zymogram phenotypes of single and double mutants for G6PD5 and G6PD6. A, Morphological phenotypes of the single and double mutants. B, PCR genotyping. 5w, 6w, wild-type allele-specific PCR for G6PD5 and G6PD6, respectively; 5t, 6t, T-DNA insertion-specific PCR for G6PD5 and G6PD6, respectively. C, G6PDH zymogram patterns in bud tissues of different G6PDH genotypes. The arrow indicates origin and direction of electrophoresis. G5, G6PD5; g5, g6pd5; G6, G6PD6; g6, g6pd6; Dbl, double mutant. D, G6PDH zymograms of single and double mutants rescued by cosmid complementation. The arrow indicates origin and direction of electrophoresis. Bud tissues were used for visualization of the three bands. Lm, Standard G6PDH from L. mesenteroides.

Figure 3.

Activity and gene expression of cytosolic isoforms in single and double mutants and the effect of Suc. A, Zymogram of seedlings grown on presence and absence of Suc. The arrow indicates origin and direction of electrophoresis. g5, g6pd5; g6, g6pd6; Dbl, double. B, Levels of mRNA of G6PD5 and G6PD6 analyzed by quantitative RT-PCR in the different genotypes. Top section shows mRNA levels only in −Suc samples. Bars indicate sd from technical triplicates. Experiment was repeated three times with similar results. Graph shows one representative data set. The relative level of mRNA was normalized to wild-type-Suc values defined as 1.

Figure 4.

G6PDH activity in developing seeds. Measured by zymogram (A) and by liquid assay (B). The arrow indicates origin and direction of electrophoresis. Lm, Standard G6PDH from L. mesenteroides.

Figure 5.

Developing pds1 seeds. A, Siliques of wild-type and PDS1/pds1 plants. B, White seeds accumulate oil at a lower rate than wild type. Ten white or green seeds were pooled and the oil content was measured. Bars indicate sd of five technical replicates. C, G6PDH activity in wild-type and white seeds measured by zymogram. Arrow indicates direction of electrophoresis and origin.

Figure 6.

Oil in mature seeds from a PDS1/pds1 plant. A, Distribution of single seed oil content from wild-type and PDS1/pds plants. B, Average composition (mol%) of C_16-18 and C_20-24 FAs in seeds with different total oil content.

Figure 7.

G6PDH activity in wild type, single, and double mutants during seed development. G6PDH activity in whole siliques during seed development was detected by zymogram (A) and liquid assay (B). Lm, Standard G6PDH from L. mesenteroides.