Analysis of the expression, subcellular and tissue localisation of phosphoglucan, water dikinase (PWD/GWD3) in Solanum tuberosum L.: a bioinformatics approach for the comparative analysis of two a-glucan, water dikinases (GWDs) from Solanum tuberosum L. Abstract There are several important factors affecting the rate of starch decomposition in plants, including the circadian clock, the regulation of gene expression, the regulation of enzyme activities and starch phosphorylation by glucan, water dikinase activities (GWDs). One isoform of glucan, water dikinase named GWD3 or PWD (EC 2.7.9.5) was isolated for the first time from Arabidopsis thaliana, and now we report its isolation and identification in Solanum tuberosum L. leaves and tubers. We compare StGWD3 sequence to the other GWDs sequences using bioinformatics tools and propose also structural models for the starch-binding domains in StGWD3 and StGWD1. The StGWD3 gene expression and protein were localised in different heterotrophic and autotrophic potato tissues and organs using in situ RT-PCR and immunolocalisation methods, respectively. Diurnal changes in the transcript abundance of StGWD3 in leaves were analysed using quantitative real-time PCR and they appeared to be typical for most genes involved in starch degradation in chloroplasts.Keywords Starch degradation Á Cold-sweetening Á Potato Electronic supplementary material The online version of this article

Exposure to low temperatures is one of the most important factors that generate abiotic stress in plants, and the rapid accumulation of soluble sugars belongs to significant metabolic responses to cold stress. The accumulation of soluble sugars may be at least partially triggered by an increased rate of starch degradation. The analysis of transcript profiles and starch degrading enzyme activities in leaves of two potato cultivars was performed during a 12-h exposure to 2 °C. An induction of β-amylase expression and activity as well as an accumulation of reducing sugars were observed in cv. Desiree. No accumulation of reducing sugars and no significant changes in the β-amylase activity were initially observed in cv. Russet Burbank. Surprisingly, an increased α-amylase activity was observed in the last hours of the experiment, which was accompanied by an increased amount of reducing sugars. The results indicate that the leaves of Desiree and Russet Burbank potatoes growing under cold stress may degrade starch via different pathways.

Plants are often challenged by an array of unfavorable environmental conditions. During cold exposure, many changes occur that include, for example, the stabilization of cell membranes, alterations in gene expression and enzyme activities, as well as the accumulation of metabolites. In the presented study, the carbohydrate metabolism was analyzed in the very early response of plants to a low temperature (2 °C) in the leaves of 5-week-old potato plants of the Russet Burbank cultivar during the first 12 h of cold treatment (2 h dark and 10 h light). First, some plant stress indicators were examined and it was shown that short-term cold exposure did not significantly affect the relative water content and chlorophyll content (only after 12 h), but caused an increase in malondialdehyde concentration and a decrease in the expression of NDA1, a homolog of the NADH dehydrogenase gene. In addition, it was shown that the content of transitory starch increased transiently in the very early phase of the plant response (3–6 h) to cold treatment, and then its decrease was observed after 12 h. In contrast, soluble sugars such as glucose and fructose were significantly increased only at the end of the light period, where a decrease in sucrose content was observed. The availability of the monosaccharides at constitutively high levels, regardless of the temperature, may delay the response to cold, involving amylolytic starch degradation in chloroplasts. The decrease in starch content, observed in leaves after 12 h of cold exposure, was preceded by a dramatic increase in the transcript levels of the key enzymes of starch degradation initiation, the α-glucan, water dikinase (GWD-EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD-EC 2.7.9.5). The gene expression of both dikinases peaked at 9 h of cold exposure, as analyzed by real-time PCR. Moreover, enhanced activities of the acid invertase as well as of both glucan phosphorylases during exposure to a chilling temperature were observed. However, it was also noticed that during the light phase, there was a general increase in glucan phosphorylase activities for both control and cold-stressed plants irrespective of the temperature. In conclusion, a short-term cold treatment alters the carbohydrate metabolism in the leaves of potato, which leads to an increase in the content of soluble sugars.

The aim of this article is to present the current literature concerning the expression analysis and methods of functional characteristics of genes. The progress in the analysis of gene expression within cells or whole tissues is undisputed and leads to a constant improvement of our understanding of the function of particular gene. The traditional methods of the functional characteristics of genes such as homology, inactivation and overexpression are more frequently being replaced by microarray and DNA chip analysis, which are extensively supported by bioinformatics tools. Knowledge of the functions and changes in gene expression has applications in medical diagnostics, the pharmaceutical industry and in plant and animal biotechnology.