Mihály Kondrák

The effects of enhanced methionine synthesis on amino acid and anthocyanin content of potato tubers

BackgroundPotato is a staple food in the diet of the worlds population and also being used as animal feed. Compared to other crops, however, potato tubers are relatively poor in the essential amino acid, methionine. Our aim was to increase the methionine content of tubers by co-expressing a gene involved in methionine synthesis with a gene encoding a methionine-rich storage protein in potato plants.ResultsIn higher plants, cystathionine γ-synthase (CgS) is the first enzyme specific to methionine biosynthesis. We attempted to increase the methionine content of tubers by expressing the deleted form of the Arabidopsis CgS (CgSΔ90), which is not regulated by methionine, in potato plants. To increase the incorporation of free methionine into a storage protein the CgSΔ90 was co-transformed with the methionine-rich 15-kD β-zein. Results demonstrated a 2- to 6-fold increase in the free methionine content and in the methionine content of the zein-containing protein fraction of the transgenic tubers. In addition, in line with higher methionine content, the amounts of soluble isoleucine and serine were also increased. However, all of the lines with high level of CgSΔ90 expression were phenotypically abnormal showing severe growth retardation, changes in leaf architecture and 40- to 60% reduction in tuber yield. Furthermore, the colour of the transgenic tubers was altered due to the reduced amounts of anthocyanin pigments. The mRNA levels of phenylalanine ammonia-lyase (PAL), the enzyme catalysing the first step of anthocyanin synthesis, were decreased.ConclusionEctopic expression of CgSΔ90 increases the methionine content of tubers, however, results in phenotypic aberrations in potato. Co-expression of the 15-kD β-zein with CgSΔ90 results in elevation of protein-bound methionine content of tubers, but can not overcome the phenotypical changes caused by CgSΔ90 and can not significantly improve the nutritional value of tubers. The level of PAL mRNA and consequently the amount of anthocyanin pigments are reduced in the CgSΔ90 transgenic tubers suggesting that methionine synthesis and production of anthocyanins is linked.

Effects of yeast trehalose-6-phosphate synthase 1 on gene expression and carbohydrate contents of potato leaves under drought stress conditions

BackgroundThe development of drought-tolerant, elite varieties of potato (Solanum tuberosum L.) is a challenging task, which might be achieved by introducing transgenic lines into breeding. We previously demonstrated that strains of the White Lady potato cultivar that express the yeast trehalose-6-phosphate synthase ( TPS1) gene exhibit improved drought tolerance.ResultsWe investigated the responses of the drought-sensitive potato cultivar White Lady and the drought-tolerant TPS1 transgenic variant to prolonged drought stress at both the transcriptional and metabolic levels. Leaf mRNA expression profiles were compared using the POCI microarray, which contains 42,034 potato unigene probes. We identified 379 genes of known function that showed at least a 2-fold change in expression across genotypes, stress levels or the interaction between these factors. Wild-type leaves had twice as many genes with altered expression in response to stress than TPS1 transgenic leaves, but 112 genes were differentially expressed in both strains. We identified 42 transcription factor genes with altered expression, of which four were uniquely up-regulated in TPS1 transgenic leaves. The majority of the genes with altered expression that have been implicated in photosynthesis and carbohydrate metabolism were down-regulated in both the wild-type and TPS1 transgenic plants. In agreement with this finding, the starch concentration of the stressed leaves was very low. At the metabolic level, the contents of fructose, galactose and glucose were increased and decreased in the wild-type and TPS1 transgenic leaves, respectively, while the amounts of proline, inositol and raffinose were highly increased in both the wild-type and TPS1 transgenic leaves under drought conditions.ConclusionsTo our knowledge, this study is the most extensive transcriptional and metabolic analysis of a transgenic, drought-tolerant potato line. We identified four genes that were previously reported as drought-responsive in non-transgenic Andean potato cultivars. The substantial increases in proline, inositol and raffinose contents detected in both the wild-type and TPS1 transgenic leaves appears to be a general response of potatoes to drought stress. The four transcription factors uniquely up-regulated in TPS1 transgenic leaves are good candidates for future functional analyses aimed at understanding the regulation of the 57 genes with differential expression in TPS1 transgenic leaves.

Transcriptome analysis of potato leaves expressing the trehalose-6-phosphate synthase 1 gene of yeast.

Transgenic lines of the potato cultivar White Lady expressing the trehalose-6-phosphate synthase (TPS1) gene of yeast exhibit improved drought tolerance, but grow slower and have a lower carbon fixation rate and stomatal density than the wild-type. To understand the molecular basis of this phenomenon, we have compared the transcriptomes of wild-type and TPS1-transgenic plants using the POCI microarray containing 42,034 potato unigene probes. We show that 74 and 25 genes were up-, and down-regulated, respectively, in the mature source leaves of TPS1-transgenic plants when compared with the wild-type. The differentially regulated genes were assigned into 16 functional groups. All of the seven genes, which were assigned into carbon fixation and metabolism group, were up-regulated, while about 42% of the assigned genes are involved in transcriptional and post-transcriptional regulation. Expression of genes encoding a 14-3-3 regulatory protein, and four transcription factors were down-regulated in the TPS1-transgenic leaves. To verify the microarray results, we used RNA gel blot analysis to examine the expression of eight genes and found that the RNA gel blot and microarray data correlated in each case. Using the putative Arabidopsis orthologs of the assigned potato sequences we have identified putative transcription binding sites in the promoter region of the differentially regulated genes, and putative protein-protein interactions involving some of the up- and down-regulated genes. We have also demonstrated that starch content is lower, while malate, inositol and maltose contents are higher in the TPS1-transgenic than in the wild-type leaves. Our results suggest that a complex regulatory network, involving transcription factors and other regulatory proteins, underpins the phenotypic alterations we have observed previously in potato when expressing the TPS1 gene of yeast.

Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato

The cDNA for a 73-mer peptide containing two locust serine proteinase inhibitors was cloned, fused to the constitutive CaMV35S promoter and introduced into potato by Agrobacterium-mediated transformation. From 23 independent transgenic lines, three with high mRNA level and proteinase inhibitory activity were propagated in vitro and transferred to pots. The peptide from the leaves was identified by its N-terminal sequence and by Ki values against chymotrypsin and trypsin. Colorado potato beetle larvae reared on transgenic plants grew slightly but significantly more slowly than those on control plants. This supports the notion that expression of multifunctional proteinase inhibitors of insect origin might be a good strategy to improve insect resistance in plants.

Soil Drench Treatment with ß-Aminobutyric Acid Increases Drought Tolerance of Potato

The non-protein amino acid β-aminobutyric acid (BABA) is known to be a priming agent for a more efficient activation of cellular defence responses and a potent inducer of resistance against biotic and abiotic stresses in plants. Nevertheless, most of the studies on priming have been carried out in Arabidopsis. In potato, the effect of BABA was demonstrated only on biotic stress tolerance. We investigated the effect of BABA on the drought tolerance of potato and found that soil drenched with BABA at a final concentration of 0.3 mM improves the drought tolerance of potato. Water loss from the leaves of the primed plants is attenuated and the yield is increased compared to the unprimed drought-stressed plants. The metabolite composition of the tubers of the BABA-treated plants is less affected by drought than the tuber composition of the non-treated plants. Nitric oxide and ROS (reactive oxygen species) production is increased in the BABA-treated roots but not in the leaves. In the leaves of the BABA-treated plants, the expression of the drought-inducible gene StDS2 is delayed, but the expression of ETR1, encoding an ethylene receptor, is maintained for a longer period under the drought conditions than in the leaves of the non-treated, drought-stressed control plants. This result suggests that the ethylene-inducible gene expression remains suppressed in primed plants leading to a longer leaf life and increased tuber yield compared to the non-treated, drought-stressed plants. The priming effect of BABA in potato, however, is transient and reverts to an unprimed state within a few weeks.

Influence of the StubSNF1 kinase complex and the expression of the yeast TPS1 gene on growth and tuber yield in potato

Expression of the yeast trehalose-6-phosphate synthase-1 (TPS1) gene in potato results in growth aberrations and arrest of development. Recent studies have shown that this phenomenon could be related to the inhibitory effect of trehalose-6-phosphate on SnRK1s, a family of sucrose non-fermenting-1 (SNF1)-related protein kinases that link metabolic and stress signalling in plants. SnRK1s are heterotrimeric enzymes similar to yeast SNF1 and mammalian AMP-activated protein kinases (AMPKs). Previously, we showed that antisense repression of StubGAL83, one of the three subunits of the potato SnRK1 complex, results in a delay in rooting and increases sensitivity to salt stress. Here we report that StubGAL83 is a positive regulator of SNF1 kinase activity in potato and that repression of the kinase subunit of the SnRK1 complex, StubSNF1, reduces growth and tuber yield in potato plants. Co-repression of StubGAL83 and StubSNF1 at a certain level, however, can result in larger plants and increased tuber yield. We found that repression of StubGAL83, but not repression of StubSNF1 attenuated growth aberrations caused by TPS1 expression. We provide evidence that the increased plant size and yield in StubGAL83-StubSNF1 co-repressed plants as well as the attenuation of aberrations caused by TPS1 expression are related to increased nitrate reductase activity.