NAD malic enzyme and the control of carbohydrate metabolism in potato tubers

Abstract

Potato (Solanum tuberosum) plants were transformed with a cDNA encoding the 59-kD subunit of the potato tuber NAD-dependent malic enzyme (NADME) in the antisense orientation. Measurements of the maximum catalytic activity of NADME in tubers revealed a range of reductions in the activity of this enzyme down to 40% of wild-type activity. There were no detrimental effects on plant growth or tuber yield. Biochemical analyses of developing tubers indicated that a reduction in NADME activity had no detectable effects on flux through the tricarboxylic acid cycle. However, there was an effect on glycolytic metabolism with significant increases in the concentration of 3-phosphoglycerate and phosphoenolpyruvate. These results suggest that alterations in the levels of intermediates toward the end of the glycolytic pathway may allow respiratory flux to continue at wild-type rates despite the reduction in NADME. There was also a statistically significant negative correlation between NADME activity and tuber starch content, with tubers containing reduced NADME having an increased starch content. The effect on plastid metabolism may result from the observed glycolytic perturbations.

Figure 1

Activity of NADME in tuber extracts from wild-type and transgenic lines. Activity of NADME was measured in tuber extracts from wild-type Desirée (WT), a vector-only transformed line (PA7 control), and a range of independent transgenic lines containing a cDNA encoding the 59-kD subunit of NADME in the antisense orientation, under the control of either the CaMV 35S or the potato tuber patatin promoter. Values are mean ± se (n = 3).

Figure 2

The effect of reduced NADME activity on mitochondrial protein composition. Total mitochondrial protein from tubers of wild-type and line 35S-ME11 was isolated and separated by two-dimensional SDS PAGE, and stained with silver. The complete gels for wild type and 35S-ME11 are shown in A and B, respectively. The boxes indicate the regions containing NADME subunits that are enlarged in C and F. The gel shown in A was transferred onto nitrocellulose and the resulting western blot probed with antibodies specific to the 59- or 62-kD subunits of NADME. Enlarged regions of these blots are shown in D (62-kD subunit) and E (59-kD subunit). The ovals in C and F indicate the position of the NADME subunits.

Figure 3

Relationship between 3PGA content and NADME activity in developing tubers from greenhouse-grown plants. NADME activity and 3PGA content were measured in tubers from a single harvest. The means of these parameters are plotted against each other for wild type and each transgenic line. ▪, Wild type; □, PAT-ME1; ×, 35S-ME1; ○, PAT-ME8; ●, PAT-ME10; ▴, PAT-ME9; ▵, 35S-ME11; ⋄, 35S-ME7; ♦, 35S-ME10. The line was fitted by linear regression and the significance of the relationship was analyzed using a 5% two-tailed significance test on the correlation coefficient.

Figure 4

Relationship between starch content and NADME activity in developing tubers from greenhouse-grown plants. A, NADME activity and starch content were measured in tubers from a single harvest. The means of these parameters are plotted against each other for wild type and each transgenic line. ▪, Wild type; □, PAT-ME1; ×, 35S-ME1; ○, PAT-ME8; ●, PAT-ME10; ▴, PAT-ME9; ▵, 35S-ME11; ⋄, 35S-ME7; ♦, 35S-ME10. The line was fitted by linear regression and the significance of the relationship was analyzed using a 5% two-tailed significance test on the correlation coefficient. B, Tuber slices from freshly harvested developing tubers of wild type and line 35S-ME11 were stained with iodine solution to compare starch contents.