Genetic engineering of a zeaxanthin-rich potato by antisense inactivation and co-suppression of carotenoid epoxidation.

Zeaxanthin is an important dietary carotenoid but its abundance in our food is low. In order to provide a better supply of zeaxanthin in a staple crop, two different potato (Solanum tuberosum L.) varieties were genetically modified. By transformation with sense and antisense constructs encoding zeaxanthin epoxidase, zeaxanthin conversion to violaxanthin was inhibited. Both approaches (antisense and co-suppression) yielded potato tubers with higher levels of zeaxanthin. Depending on the transgenic lines and tuber development, zeaxanthin content was elevated 4 to 130-fold reaching values up to 40 microg/g dry weight. As a consequence of the genetic manipulation, the amount of violaxanthin was diminished dramatically and in some cases the monoepoxy intermediate antheraxanthin accumulated. Between one and eight copies of the sense or antisense epoxidase gene fragments were integrated into the genome. In addition, most of the transformants with higher zeaxanthin levels showed also increased total carotenoid contents (up to 5.7-fold) and some of them exhibited reduced amounts of lutein. The increase in total carotenoids suggests that the genetic modification affects the regulation of the whole carotenoid biosynthetic pathway in potato tubers. Northern blot analysis demonstrated that upregulation of carotenogenesis in the transgenics is accompanied by substantial higher phytoene synthase transcript levels in 6-week-old tubers and a very slight increase of the beta-carotene hydroxylase transcript. The amount of the deoxyxylulose 5-phosphate synthase mRNA was very similar in wild type and transformed tubers. Abscisic acid content of tubers remained unchanged whereas alpha-tocopherol was 2 to 3 fold elevated in the transformants.

Expression of an abscisic acid-binding single-chain antibody influences the subcellular distribution of abscisic acid and leads to developmental changes in transgenic potato plants.

Potato (Solanum tuberosum L. cv. Désirée) plants were transformed to express a single-chain variable-fragment antibody against abscisic acid (ABA), and present in the endoplasmic reticulum at to up to 0.24% of the soluble leaf protein. The resulting transgenic plants were only able to grow normally at 95% humidity and moderate light. Four-week-old plants accumulated ABA to high extent, were retarded in growth and their leaves were smaller than those of control plants. Leaf stomatal conductivity was increased due to larger stomates. The subcellular concentrations of ABA in the chloroplast, cytoplasm and vacuole, and the apoplastic space of leaves were determined. In the 4-week-old transgenic plants the concentration of ABA not bound to the antibody was identical to that of control plants and the stomates were able to close in response to lower humidity of the atmosphere. A detailed analysis of age-dependent changes in plant metabolism showed that leaves of young transformed plants developed in ABA deficiency and leaves of older plants in ABA excess. Phenotypic changes developed in ABA deficiency partly disappeared in older plants.

Ontogenetic changes of potato plants during acclimation to elevated carbon dioxide.

Transgenic potato plants (Solanum tuberosum cv. Desirée) with an antisense repression of the chloroplastic triosephosphate translocator were compared with wild-type plants. Plants were grown in chambers with either an atmosphere with ambient (400 mu bar) or elevated (1000 mu bar) CO2. After 7 weeks, the rate of CO2 assimilation between wild-type and transgenic plants in both CO2 concentrations was identical, but the tuber yield of both plant lines was increased by about 30%, when grown in elevated CO2. One explanation is that plants respond to the elevated CO2 only at a certain growth stage. Therefore, growth of wild-type plants was analysed between the second and the seventh week. Relative growth rate and CO2 assimilation were stimulated in elevated CO2 only in the second and the third weeks. During this period, the carbohydrate content of leaves grown with elevated CO2 was lower than that of leaves grown with ambient CO2. In plants grown in elevated CO2, the rate of CO2 assimilation started to decline after 5 weeks, and accumulation of carbohydrates began after 7 weeks. From this observation it was concluded that acclimation of potato plants to elevated CO2 is the result of accelerated development rather than of carbohydrate accumulation causing down-regulation of photosynthesis. For a detailed analysis for the cause of the stimulation of growth after 2 weeks, the contents of phosphorylated intermediates of wild-type plants and transgenics were measured. Stimulation of CO2 assimilation was accompanied by changes in the contents of phosphorylated intermediates, resulting in an increase in the amount of dihydroxyacetone phosphate, the metabolite which is exported from the chloroplast into the cytosol. An increase of dihydroxyacetone phosphate was found in wild-type plants in elevated CO2 when compared with ambient CO2 and in triosephosphate translocator antisense plants in ambient CO2, but not in the transgenic plants when grown in elevated CO2. These plants were not able to increase dihydroxyacetone phosphate further to cope with the increased CO2 supply. From these changes in phosphorylated intermediates in wild-type and transgenic plants it was concluded that starch and sucrose synthesis pathways can replace each other only at moderate carbon flux rates.

The role of transient starch in acclimation to elevated atmospheric CO2.

Although increased concentrations of CO2 stimulate photosynthesis, this stimulation is often lost during prolonged exposure to elevated carbon dioxide, leading to an attenuation of the potential gain in yield. Under these conditions, a wide variety of species accumulates non-structural carbohydrates in leaves. It has been proposed that starch accumulation directly inhibits photosynthesis, that the rate of sucrose and starch synthesis limits photosynthesis, or that accumulation of sugars triggers changes in gene expression resulting in lower activities of Rubisco and inhibition of photosynthesis. To distinguish these explanations, transgenic plants unable to accumulate transient starch due to leaf mesophyll-specific antisense expression of AGP B were grown at ambient and elevated carbon dioxide. There was a positive correlation between the capacity for starch synthesis and the rate of photosynthesis at elevated CO2 concentrations, showing that the capability to synthesize leaf starch is essential for photosynthesis in elevated carbon dioxide. The results show that in elevated carbon dioxide, photosynthesis is restricted by the rate of end product synthesis. Accumulation of starch is not responsible for inhibition of photosynthesis. Although transgenic plants contained increased levels of hexoses, transcripts of photosynthetic genes were not downregulated and Rubisco activity was not decreased arguing against a role of sugar sensing in acclimation to high CO2.