Ferreira et al. BMC Genomics 2010, 11:93 http://www.biomedcentral.com/1471-2164/11/93 Page 9 of 17 Figure 7 Diurnal expression of genes known to be involved in starch degradation. GWD (black), DPE1 (green), DPE2 (light blue), MEX1 (red), Alpha amylase (grey), Isoamylase 3 (dark blue), PCT-BMY (brown) and SEX4 (orange). Values are the mean of two replicates. sucrose content of stolon-ends attached to a tuber was measured. Phloem sucrose content is very high com- pared to those tissues surrounding it [47] and phloem signifies a large proportion of the total stolon tissue [48]. This makes it possible to determine sucrose import to the tuber by measuring sucrose content of the sto- lon-end [3]. Sucrose content differed significantly during the day and was highest at the end of the light and low- est at the end of the dark period. When plants were kept in constant darkness, sucrose content of stolon- ends declined linearly over time indicating that changes in sucrose supply from the leaves contributes to the oscillating expression of GBSS in tubers (Figure 8C). Although GBSS expression in tubers can be linked to diurnal changes in sucrose supply from the source, cau- tion should be exercised in the interpretation of the sig- nificance of this in terms of enzyme activity. As mentioned earlier, the level of GBSS protein does change substantially during the day in Arabidopsis leaves and the reason for this is probably the location of GBSS within the granule [28]. This is also true for algae Chlamydomonas reinhardtii suspension cultures where GBSS expression and enzyme activity correlates with starch levels. The authors state that the correlation is probably due to the fact that the analysis was conducted in suspension cultures, where new cells are produced constantly leading to continuous production of new GBSS protein [49]. This however is not necessarily true for GBSS in other tissues or for other enzymes of starch biosynthesis and several studies have shown that diurnal changes in expression do not lead to changes in protein levels [9,42]. However, it still remains an interesting finding that GBSS expression in tubers follows a diurnal rhythm which declines when sucrose supply from the leaves are reduced. Starch biosynthetic gene expression is influenced by tuber growth velocity As mentioned earlier, tuber initiation and growth rates are not synchronised. Because analysis of tubers growing at different velocities would provide important informa- tion on factors determining growth, it was thought necessary to develop a method to determine growth velocity before harvesting the tubers. For this, X-ray CT was used to determine tuber volume over a time course and then calculate the growth velocity. An overview of the method for calculating tuber volume using X-ray CT is given in figure 9. The accuracy of the growth velocity determination depends largely on the accuracy of volume calculation and it was crucial to establish whether tuber volume could be accurately calculated.