Decreased expression of cytosolic pyruvate kinase in potato tubers leads to a decline in pyruvate resulting in an in vivo repression of the alternative oxidase

Abstract

The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.

Figure 1.

Phylogenetic alignment of PK proteins. Protein sequences were deduced from cDNA or EST sequences obtained from the Joint Genome Institute and the National Center for Biotechnology Information. The tree shows a phylogram of PK protein sequences (including partial sequences and gaps, excluding the variable transit peptides and C-terminal extensions) using the JTT evolutionary model of amino acid substitution and rooted with the yeast sequences. Bootstrap values from 100 replicates are indicated. Potato PK proteins are shown in bold. The accession codes and protein sequences are given as supplemental material.

Figure 2.

Generation of plants with reduced PKc in tubers. A, RNAi construct used to transform potato plants, containing a PCR-amplified, 650-bp fragment homologous to PKCYT1 (PK-cyt) in a hairpin orientation under the control of the B33 patatin promoter. B, PK activity in tuber extracts from five resultant transgenic lines plus wild type assayed at pH 6.9 (*, significant difference from wild-type levels, n = six tubers from six different plants). C, Western blot of tuber extracts probed with an anti-PKc antibody. D, Quantitative real-time RT-PCR expression analysis of PK transcripts for PKCYT1, PKPα1, and PKPβ2 genes in tubers from the PKc transgenic lines and wild type (normalized to wild-type expression levels). *, Significant difference from wild-type levels (P < 0.05). Data represent the mean and se (n = four independent tuber RNA extractions from different plants).

Figure 3.

Growth parameters and metabolite levels in wild-type and transgenic tubers with decreased PKc. Plants were grown under greenhouse conditions, and 10-week-old tubers were harvested for analysis. Data show the mean and se (n = six tubers from six different plants per line). *, Significant difference from wild-type levels (P < 0.05).

Figure 4.

Metabolism of [¹⁴C]Glc in wild-type and transgenic tubers with decreased PKc. Tuber discs were cut from freshly harvested 10-week-old tubers and incubated with 2 mm [U-¹⁴C]Glc (specific activity = 11.54 kBq μmol⁻¹) for 2 h, followed by fractionation of the material and determination of label distribution. Data show the mean of three biological replicates per line and error bars represent ses. *, Significant difference from wild-type levels (P < 0.05).

Figure 5.

Measurement of flux through the TCA cycle and OPP pathway in wild-type and transgenic tubers with decreased PKc by incubation of tuber discs in positionally labeled isotopomers of [¹⁴C]Glc. Tuber discs were cut from freshly harvested 10-week-old tubers and incubated in [¹⁴C]Glc labeled in either the C1, C3,4, or C6 position for 6 h. The evolved CO₂ gas was continuously captured in 10% KOH and the amount of label released as CO₂ was measured. TCA cycle flux was determined as the ratio of label evolved as CO₂ from the C3,4 incubation compared to the C1 incubation, while OPP pathway flux was determined as the ratio of label evolved as CO₂ from the C6 incubation compared to the C1 incubation. Data shows the mean of three replicate incubations per Glc isotopomer per line, and error bars represent ses. *, Significant difference from wild-type levels (P < 0.05); n = six tubers from six different plants.

Figure 6.

Measurement of AOX capacity in intact tissue slices (A) and isolated mitochondria (B) from 10-week-old wild-type and transgenic tubers with decreased PKc. Data show the average KCN-insensitive, SHAM-sensitive respiration in the absence (light gray bars) and presence (black bars) of pyruvate. Respiration rates were calculated from oxygen consumption rates measured using a Clark electrode in the presence of KCN (to inhibit the COX), before and after the addition of pyruvate (to stimulate the AOX), followed by SHAM (to inhibit the AOX and calculate residual respiration rates, which were subtracted to obtain KCN-sensitive, SHAM-insensitive respiration). Error bars represent ses (n = 12 tubers from six different plants per line [A] or four mitochondrial preparations per line, representing four independent tuber harvests [B]). *, Significant difference from wild-type levels (P < 0.05).

Figure 7.

Quantification of AOX and COX protein levels in wild-type and PKc transgenic tubers. Protein from isolated tuber mitochondria was electrophoresed under reducing conditions and western blots were probed with antibodies against AOX (A) or COX (B). The graphs show the mean protein levels obtained from quantification of the western blots (n = three tuber mitochondrial isolations per line, representing three independent tuber harvests. Representative western blot results are shown beneath graph bars (bands taken from the same western blot are shown for each antibody). Error bars represent ses. *, Significant difference from wild-type levels (P < 0.05).