Ferreira et al. BMC Genomics 2010, 11:93 http://www.biomedcentral.com/1471-2164/11/93 Page 8 of 17 Figure 6 Quantitative real time PCR confirmation of microarray results. A-C) Relative expression of GPT2, Susy4 and GBSS diurnally in leaves. D-F) Relative expression of GPT2, Susy4 and GBSS in tubers. Error bars represent standard deviation (n = 3). rhythm in Arabidopsis which was maintained under continuous light, but not continuous darkness [42]. Since GBSS is one of the strongest diurnally regulated genes involved in starch biosynthesis [28], it was investi- gated whether this gene is also diurnally regulated in tubers. GBSS expression was measured at different times of the day and under different light regimes. Plants were grown in normal long day conditions and samples were taken from stolon-ends and tubers at dif- ferent time-points of the diurnal cycle. GBSS expression in tubers oscillated during the day and was highest at the end of the light period and lowest 2 hours into the next light period (Figure 8A). Expression was also signif- icantly lower in tubers from plants kept in twenty four hours of darkness compared to tubers from plants kept in a normal light/dark cycle (Figure 8B). As mentioned, sucrose and the circadian clock seem to be important regulators of diurnal gene expression and this is especially true for GBSS. Tenioro et al. [43] showed that GBSS is strongly regulated by the circadian clock and that expression is markedly lower in mutants lacking clock genes LHY and CCA-OX respectively. In a detailed study done on GBSS in snapdragon (Antirrhi- num majus) it was shown that GBSS is diurnally regu- lated in leaves even under continuous light and it was concluded that the regulation is due to the circadian clock. This was not the case in snapdragon roots though, where expression was the same in the middle of the day and in the middle of the night [44]. Also in rice leaves GBSS continues its diurnal cycling under continu- ous light suggesting circadian regulation, but expression can be induced by nitrogen starvation or sucrose feeding and repressed by darkness, indicating the importance of sucrose in its regulation [45]. Moreover, sucrose floating experiments with potato leaves show that GBSS expres- sion can be induced by sucrose [46]. To investigate whether oscillation of GBSS might be due to changes in sucrose supply from the source,