Ferreira et al. BMC Genomics 2010, 11:93 http://www.biomedcentral.com/1471-2164/11/93 Page 5 of 17 Figure 2 Diurnal starch and sucrose content of leaves over a sixteen hour period. A) Starch and B) sucrose content. Error bars indicate standard deviation (n = 3). Interestingly, genes involved in the import of glucose 6- phosphate and ATP into the plastid are co-regulated with ADP-glucose pyrophosphorylase, suggesting that both genes are also involved in transitory starch bio- synthesis in leaves (Figure 3A). To rule out that the observed expression of GPT and NTT in leaf extracts was due to contaminating epidermis rather than meso- phyll cells, cell-specific RNA analysis was performed. To this end epidermis stripes were harvested and expression of GPT and NTT were probed by quantitative real-time PCR (qRT-PCR). As shown in figure 4, GPT and NTT expression in epidermis cells was about ten fold lower compared to whole leaf extracts. This supports the hypothesis that GPT and NTT are indeed required for photosynthetic starch biosynthesis in leaves. However, other cell types such as companion and bundle sheath cells could still contribute to the observed expression pattern. It can also not be excluded that the transporters are exporting substrates from the plastid. Most starch synthases and branching enzymes had a similar pattern to the above mentioned genes in figure 3A with the exception of GBSS which was highest expressed two hours into the light period (Figure 3B). Smith et al. [28] argue and provide evidence that since the enzyme is present within the granule, the protein is degraded together with starch at night and must very quickly be re-synthesised in the morning. Two genes involved in the light reaction of photosynthesis, plasto- cyanin and chlorophyll a/b binding protein, were already up-regulated at the first time point which was taken moments after the lights came on and were highest expressed two hours into the light. Ribulose 1,5 bispho- sphate carboxylase oxygenase (Rubisco) had an expres- sion pattern similar to that of starch biosynthetic genes (Figure 3C). The sucrose cleavage enzymes cell wall- bound invertase and sucrose synthase had very different expression patterns with Susy 4 being much stronger regulated. Susy 4 increased during the light and went down in the dark. Hexokinase 1 was not diurnally regu- lated, whilst hexokinase 2 and fructokinase had a similar pattern of increasing early in the morning and declining at the end of the light period (Figure 3D). For analysis of gene expression during tuber induc- tion, tuber developmental stages were designated according to Kloosterman et al. [26] and the expression of genes were studied by microarray analysis. Stage 1, representing an unswollen stolon, showed very low expression for all starch biosynthetic genes except starch synthase IV which was highest expressed at stage 1, fit- ting with its proposed role in starch granule initiation [22,23]. From stages 3-5 there was an increase in the expression of all starch biosynthetic genes with the exception of starch synthase IV (Figure 5A and 5B). As evidence that stage 1 was before the onset of tuberisa- tion, cell wall-bound invertase expression was very high and sucrose synthase 4, a tuber expressed isoform, low. From stages 3-5 the expression cell wall-bound invertase went down and that of Susy 4 increased (Figure 5C). The expression patterns compared well to earlier experi- ments [29] qRT-PCR is a well accepted method for verifying microarray data and was used to validate the expression patterns of a selected number of genes in independent samples. The expression patterns and levels of Susy 4, GPT and GBSS compared well to the microarray data in both leaves and tubers and confirmed the accuracy of the microarray results (Figure 6). In contrast to starch biosynthesis, the pathway of starch degradation in potato tubers is not well under- stood. Currently it is believed that the major pathway of