The main product of the Calvin-Benson cycle, triose-P, is used in the production of sucrose and starch (Figure 1.13). Starch is a carbon reserve, accumulated during the day when there are more sugars available than needed. Starch is made of long branched chains of glucose and is deposited as granules in the chloroplast. Starch synthesis begins in the thylakoid lumen where two triose phosphate molecules are converted to fructose-1,6-biphosphate by aldolase. One phosphate is then removed to leave fructose 6-phosphate, which is used in turn to produce glucose-6- phosphate. The phosphate on C6 is transferred by isomerase to make glucose-1-phosphate. Glucose-1-phosphate is the substrate for ADP-glucose pyrophosphorylase (AGPase) to produce
2x O2 2x Phosphoglycolate
Low levels of ADP-G or the oxidized (inactive) form of AGPase reduce starch accumulation. ADP- G is the starting point for elongation of the nascent starch chain by starch synthases which adds further ADP-glucose units. Starch is composed of amylose and amylopectin. Amylose is a linear glucose polymer chains with α(1-4) glycosidic bonds. Amylopectin is a highly branched glucose polymer with α(1-4) and α(1-6) glycosidic bonds. Debranching enzymes including amylases and amylopectinases break starch down into maltose (two glucose units). Maltose is then exported to the cytosol, or broken into glucose within the chloroplast. Starch breakdown usually occurs at night to provide glucose for glycolysis. In cereals, starch represents 90% of the grain dry weight.
Starch metabolism is regulated not only by the enzymes mentioned above but also by inorganic phosphorus (Pi) levels and the concentrations of certain sugars. Chloroplastic triose-P levels and the cytoplasmic Pi concentration regulate starch synthesis. The triose phosphate/phosphate translocator (TPT) located in the chloroplast membrane requires Pi to export triose-P to the cytosol (Figure 1.13). The TPT regulates movement of carbon from the chloroplast to the cytoplasm as a starting point for various metabolic processes. When cytoplasmic Pi decreases, triose-P is not exported and accumulates in the chloroplast. High triose-P levels stimulate starch synthesis. Lower cytoplasmic Pi levels are associated with increased levels of phosphorylated sugars such as fructose and glucose (Smith and Stitt, 2007; Trevanion, 2002).
Figure 1.13. Sucrose and starch synthesis. Starch synthesis occurs in the thylakoid space where two molecules of triose-P are converted to fructose-1,6-biphosphate (F1,6bP), then to fructose 6-phosphate (F6P), then to glucose 6-phosphate (G6P), and finally to glucose 1-phosphate (G1P). The enzyme ADP-glucose pyrophosphorylase (AGPase), the control point for starch synthesis, produces ADP-glucose (ADP-Glc), the backbone of starch. Sucrose is synthesized in the cytoplasm where UDP-glucose pyrophosphorylase (UGPase) produces UDP-glucose (UDP-G). UDP-G and F6P are combined by sucrose-phosphate synthase (SPS) to produce sucrose- phosphate (Suc-P). Sucrose-phosphatase (SPP) removes the phosphate from Suc-P. Sucrose (Suc) then is exported to other tissues, stored in the vacuole, or used as a source of hexoses. Picture from (Rolland et al., 2006).
Triose-P is exported to the cytoplasm for sucrose synthesis. Sucrose is a dimer (disaccharide) of fructose and glucose that is stored in the vacuole and transported to distal organs with high demand for catabolic processes. Starch and sucrose have identical pathways for synthesis from triose phosphate to glucose-1-phosphate, but diverge thereafter. The starch pathway uses AGPase for subsequent steps, whereas the sucrose pathway uses UDP-glucose pyrophosphorylase (UGPase) to produce UDP-glucose from G1P. UDP-glucose is the substrate for both sucrose and cell wall synthesis. The enzyme sucrose-phosphate synthase (SPS) uses UGP-glucose and fructose-6-phosphate to produce phosphorylated sucrose. Sucrose (dephosphorylated by sucrose-P phosphatase (SPP)) is metabolically inert and for this reason is the main photosynthesis product that is transported throughout the plant.
Sucrose is the substrate for production of several carbohydrate compounds. Two of these are fructans and raffinose. Fructans are fructose polymers, and raffinose is a trisaccharide composed of galactose, glucose and fructose. Fructans are more abundant than starch in 15% of flowering
6-kestotriose, which is composed of one unit of glucose and two units of fructose. Synthesis of 6-kestriose requires the enzyme sucrose:fructan 6-fructosultransferase (6-SFT) to add fructose to sucrose. Degradation of fructans requires the enzyme fructanexohydrolases (FEHs) which hydrolyses the terminal fructose, thus releasing sucrose. For raffinose, the enzyme raffinose synthase adds galactinol to sucrose. The role of fructans and raffinose is mainly as vacuolar carbohydrate reserves. Fructans are also involved in flowering and stress tolerance responses. In wheat, high levels of fructans accumulate in leaf sheaths and stems. Raffinose is a phloem- mobile molecule, like sucrose, which means that it can be transported to demanding tissues (Valluru and Van den Ende, 2008; Van den Ende, 2013).
Sugars are stored in soluble forms such as sucrose, fructans and raffinose, or insoluble as starch. Sugar storage can be for short or long periods of time. In general, starch accumulates in the stems for short periods (hours or days) as a close and rapid sugar reserve for the entire plant. Long term storage (months or years) occurs in fruits and tubers. Sugar reserves change depending on metabolic demand, during organ development, and under stress conditions. For example, starch is used mainly at night, while soluble reserves are used during days and nights. Soluble sugars increase during cold stress for frost protection, because they can work as osmoprotectants to avoid membrane damage. In wheat, synthesis of starch and fructans precursors increases during flowering to provide nutrients to the head.