Ferreira et al. BMC Genomics 2010, 11:93 http://www.biomedcentral.com/1471-2164/11/93 substrate for ADP-glucose production by ADP-glucose pyrophosphorylase (AGPase), which is the first reaction committed to starch biosynthesis. ADP-glucose is then the glycosyl donor for the various starch synthases forming linear glucans which are subsequently branched by branching enzymes to produce starch. For starch production in the tuber photoassimilates, in the form of sucrose, must first be imported from photo- autotrophic tissue via the phloem [2]. There is conflict- ing evidence to whether carbon accumulation is sink or source limited. It has been shown that under normal conditions flux control of tuber starch biosynthesis is mostly source regulated [3]. In another study, reduced photosynthetic activity of potato leaves by silencing cytosolic fructose 1, 6 bisphosphatase did not have an effect on tuber yield or plant growth and it was con- cluded that tuber starch biosynthesis is not source lim- ited [4]. Both studies however emphasize the importance of sucrose supply to the tuber. This idea is further supported by constitutive [5] or phloem-specific [6] antisense inhibition of the sucrose transporter. Sucrose transport activity is essential for apoplastic phloem loading, hence, silencing its expression lead to a reduced phloem loading of sucrose which was accompa- nied by decreased photosynthetic rates and reduced tuber yield of transgenic potato plants. In another study [7], over-expression of a sucrose transporter from spi- nach in potato lead to a reduced sucrose level in leaves and an increased sucrose content of tubers. However, this had no effect on tuber starch content. Even though this did not lead to an increased starch content of tubers, it did provide evidence as to the important role of sucrose as regulator of carbon metabolism [7]. Of particular note was the decrease in plastidial amino acid synthesis even though sucrose was still in abundant supply. There is also conflicting evidence as to whether the supply rate of sucrose from the leaves to tubers is con- stant throughout the diurnal cycle or whether there is a diurnal rhythm. It has been shown that source to sink carbon flux is constant [8]. It can also be argued that for starch biosynthesis in tubers to be unaffected by lim- itations in photosynthetic capacity [4], sucrose supply to the tuber should be constant. It has been shown that there are significant differences of tuber sucrose content at the start and end of the light period and that this has an effect on tuber metabolism [9]. After the formation of tubers they represent the pre- dominant sink in the plant. The onset of tuberisation is marked by many physiological and biochemical changes in the stolons. The major changes which are seen as markers of tuberisation are the switch from apoplastic to symplastic sucrose unloading and the change from hydrolytic to sucrolytic cleavage of sucrose [10]. Sucrose Page 2 of 17 synthase (Susy) activity increases with the development of the tuber and is the major determinant of potato sink strength [11]. After the cleavage of sucrose to UDP-glu- cose and fructose by Susy, the two molecules must be converted to glucose 6-phosphate via different pathways for starch biosynthesis. The major difference between starch biosynthesis in tubers compared to leaves is that hexose-phosphate, in the form of glucose 6-phosphate, and ATP must be imported from the cytosol. Although there is evidence that glucose 1-phosphate can also be transported across the plastid membrane and directly used for starch biosynthesis via starch phosporylase in potato tuber discs [12], it seems that in vivo glucose 6- phosphate is the predominant form of hexose-phosphate transport across the amyloplast membrane. Transport of glucose 6-phosphate and ATP across the amyloplast membrane is facilitated by the glucose 6-phosphate translocator (GPT) [13], and the plastidial ATP/ADP translocator (NTT) [14], respectively. Simultaneous over-expression of these transporters lead to an increase in total starch yield per plant showing that starch bio- synthesis is co-limited by hexose-phosphate and ATP in the plastid [15]. Glucose 6-phosphate is then converted to glucose 1-phosphate by plastidial PGM. Silencing of plastidial PGM lead to dramatic reduction in starch con- tent of the tubers, illustrating not only the importance of this enzyme in starch biosynthesis, but provides com- pelling evidence that starch is synthesised from imported glucose 6-phosphate [16]. As mentioned ear- lier, glucose 1-phosphate and ATP serve as substrates for ADP-glucose synthesis by AGPase and further starch biosynthesis is similar to leaves. Since large scale com- parative transcriptome analysis comparing leaf and tuber starch biosynthesis have not been done, it is not known whether or not the same isoforms of these genes are active in both tissues. The importance of AGPase in starch biosynthesis has been shown on various occasions and reduced activity lead to low starch, high sucrose containing tubers [17]. As far as increasing starch bio- synthesis by over-expression of AGPase is concerned, two studies over-expressing an E. coli AGPase give con- flicting results. Stark et al. [18] could increase starch content by over-expression but Sweetlove et al. [19] showed that increased biosynthesis is accompanied by increased breakdown. There are mainly four soluble starch synthase enzymes responsible for biosynthesis, starch synthase I, II, III, IV and one that is exclusively granule bound, named granule bound starch synthase (GBSS). Soluble starch synthases and the branching enzymes are believed to be responsible for amylopectin synthesis. The major starch synthases are starch synthase II and III and silencing of these two isoforms lead to greatly altered amylopectin structure [20]. Gran- ule bound starch synthase is exclusively responsible for