Fig. 3. Schematic representation of the pathway of sucrose and starch synthesis in photosynthetic tissues. The substarte for the pathway of sucrose synthesis is provided by the export of triose phosphates from the chloroplast during photosynthetic CO2 fixation. The enzymes involved are: 1, Phosphohexose isomerase; 2, Phosphoglucomutase; 3, ADP-glucose pyrophosphorylase; 4, Starch synthase; 5, Triose-P translocator; 6, FBPase; 7, Phosphofructo kinase; 8, Pyrophosphate:fru-6-P-l-phosphotransferase; 9, UDP-glucose pyrophosphorylase; 10, Sucrose phosphate synthase; 11, Sucrose phosphate phosphatase.

maintains the pool of triose phosphates , DHAP and GAP at equilibrium within the cytoplasm. Aldolase in the next reaction catalyzes aldol condensation of DHAP and GAP to form fru-l,6-P2, which is then hydrolyzed by the enzyme FBPase cleaving phosphate group from C-l position. This reaction is essentially irreversible and represents the first committed step in the pathway of sucrose biosynthesis. Part of fru-6-P formed as above is then converted to glu-6-P by the enzyme phosphohexose isomerase which is further converted to glu-l-P through the action of phosphoglucomutase. UDP-glucose pyrophosphorylase (UDPGPPase)in the next reaction catalyzes the formation of UDP-glucose, the substrate for sucrose biosynthesis. Contrary to the chloroplast stroma, the cytosol of mesophyll cells does not contain pyrophosphatase (PPase) to withdraw PPi from the equilibrium and therefore, the reaction catalyzed by this enzyme is reversible. However, a PPi dependent fru-6-P-l-phosphotransferase (PFP) present in the cytoplasm catalyzes the reversible production of fru-l,6-P2 and Pi from fru-6-P and PPi (49, 72). Though the exact role of this enzyme is yet to be ascertained in photosynthetic tissues of higher plants, it may help in hydrolyzing PPi, thus favouring the formation of UDP-glucose for sucrose synthesis (72). Sucrose phosphate synthase then catalyzes the formation of sucrose-6-P (Suc-6-P) from UDP-glucose and fru-6-P. SPS is another regulatory enzyme and in conjunction with cytosolic FBPase serves to coordinate the rate of sucrose synthesis with the rate of photosynthesis (74). The last reaction in the sequence is catalyzed by suc-6-P phosphatase hydrolyzing suc-6-P to sucrose and Pi.

4.1. Regulation of sucrose synthesis

In leaves, triose-P is continually removed and Pi is regenerated by sucrose synthesis. This balance needs to be regulated if optimal rates of photosynthesis are to be achieved (69). In photosynthetic leaves, the situation is complex as the cytosol contains pools of triose-P and PGA as well as Pi, all of which turn over within seconds (75) and also compete for transport on the phosphate translocator. Rapid sustained photosynthesis will not occur unless sucrose synthesis is regulated. This allows the rate of CC>2 fixation and Pi recycling to be balanced at a point where the subcellular concentration of triose-P and Pi permit all the partial processes of photosynthesis to operate efficiently (69). Rates of sucrose synthesis are primarily regulated by two enzymes namely cytosolic FBPase and SPS, both of which catalyze irreversible reactions and have tight allosteric regulation by cytosolic metabolites and intermediates in vivo. The rate of sucrose synthesis is also controlled via molecular regulation by changes in the amount of these regulatory enzymic proteins and/or post translational modifications of the pre-existing enzymes.

4.2. Cytosolic FBPase

The cytosolic FBPase is one of the regulatory enzymes in the sucrose biosynthetic pathway and its activity is regulated by both fine and coarse control mechanisms. The enzyme is a tetramer with approximate molecular weight of about 130 KDa (76). In the absence of effector molecules, the Km of the cytosolic FBPase for the substrate fru-l,6-P2 is about 4-6 pM (77).

The enzyme requires Mg2+ and is subject to inhibition by Pi, fru-6-P, AMP and strong inhibition by fru-2,6-P2 (78). AMP inhibition is non-competitive

(allosteric) with fru-l,6-P2 and competitive with Mg2+ (79). The potent inhibitor, fru-2,6-P2 increases the enzyme sensitivity to AMP inhibition and dramatically decreases the substrate affinity, resulting in sigmoidal saturation curves (80). Fru-2,6-P2 exerts its strongest inhibition at low substrate concentrations and acts synergistically with AMP. In the presence of fru-2,6-P2, product inhibition by Pi is increased , while inhibition by fru-6-P does not occur. Kinetic properties of cytosolic FBPase are also altered in the presence of various ions, for example, K+ and Rb+ increase and Li+ decreases its sensitivity to fru-2,6-P2 (81). On the other hand, depending on substrate concentration and presence of other divalent cations, Ca?+ can act either as an inhibitor or activator of the enzyme (82).

The enzyme is relatively conserved among various organisms both at amino acid and nucleotide sequence levels (78). Although localized in different compartments, plastidic and cytosolic FBPase are both nuclear encoded enzymes by single but distinct genes. Despite, many similarities between the two isozymes including catalyzing identical reactions, they differ in molecular structure, kinetics and therefore regulation. For example, a unique sequence insert of 7-15 amino acid residues on the chloroplastic isoenzyme encodes a domain involved in the light regulation (83, 84). The absence of this sequence insert in plant cytosolic FBPase makes it light insensitive in response to ferredoxin/thioredoxin system.

Expression of plant cytosolic FBPase gene is developmentally regulated and appears to be coordinated with the expression of Rubisco and other carbon metabolism enzymes (85). End products repress the transcription of genes encoding regulatory enzymes of photosynthesis and sucrose synthesis. Sugar-repressed photosynthetic genes include Rubisco, plastidic FBPase, cytosolic FBPase and SPS (86, 87). Both expression and activity of the cytosolic FBPase are regulated by environmental factors such as light and drought conditions. However, direct and unequivocal evidence for the precise mechanism of the presumed post-translational modification of plant cytosolic FBPase is yet to be produced (88, 89).

4.3. Sucrose phosphate synthase (SPS)

The native SPS is likely a dimer of 120-138 KDa subunits (90). It is now generally accepted that substrate saturation profiles for UDP-glucose and fru-6-P are hyperbolic rather than sigmoidal and that the enzyme from some species can be allosterically activated by glu-6-P and inhibited by Pi (91, 92). These effectors have a large effect on the affinities of both substrates, fru-6-P and UDP-glucose (93, 94). Alteration of the affinity for substrates and effectors is also involved in the light modulation of SPS that occurs by reversible protein phosphorylation in an analogous manner to the enzyme from photosynthetic tissues. At the substrate concentrations estimated to be present in the cytosol, metabolic control of SPS by the glu-6-P/Pi ratio will play an important role in the fine control of sucrose formation. The enzyme has been cloned from maize (95), spinach (96), potato (97), sugar beet (98), rice (99), sugarcane (100), citrus (101) and Vicia faba (102) etc. In general, the N-terminal portion of the 120 KDa subunit of SPS is highly conserved. With respect to mechanisms for control, it is now clear that SPS is controlled (a) at the level of enzyme protein (e.g. leaf development), (b) by allosteric effectors (glu-6-P and Pi), and (c) by reversible seryl phosphorylation (Fig. 4). There appears to be significant quantitative differences among species in the regulatory properties of SPS in vitro, i.e. the extent of glu-6-P activation and Pi inhibition (103). There are also differences in the modulation of SPS in vivo. Some species exhibit a marked light activation of SPS (designated as class I and class II species), whereas others do not (class III species). The distinctions among the three classes of the plants are quantitative rather than qualitative in nature.

The major phosphorylation site has been identified as Ser 158 (104). Phosphorylation of Ser 158 is both necessary and sufficient for the activation of SPS in vitro. However, in maize Ser 162 is involved in phosphorylation. Studies with maize leaf SPS kinase have identified a single form of enzyme (91). The enzyme is strictly Ca2+ dependent, indicating that cytosolic [Ca2+] may regulate sucrose biosynthesis at least in some species. There is evidence that cytosolic [Ca2+] is reduced in the light relative to the dark. These changes in cytosolic [Ca2+] could contribute to the light activation of SPS in vivo. Another factor that may be important in vivo is glu-6-P, which is not only an allosteric activator of SPS but also an inhibitor of SPS kinase per se. Phospho-SPS is dephosphorylated/activated by a type 2A protein phosphatase (SPS-PP) that is inhibited by Pi (103). In spinach, there is a distinct light activation of SPS-PP that involves an increase in total extractable activity as well as a decrease in sensitivity to Pi inhibition (105). However, the molecular basis for the light activation remains unclear; it could result from either a covalent modification of existing protein or the synthesis of a target/regulatory subunit or modifying enzyme. Regardless of the mechanism, the light modulation of SPS-PP and its regulation by Pi are thought to play an important role in the activation of SPS after a dark-to-light transition. Other potential effectors of SPS-PP include a variety of Pesters (105) and amino acids (70). The inhibition by amino acids may play an important role in feed-back regulation of sucrose synthesis. Activation of SPS also occurs during osmotic stress of leaf tissue in darkness, which may function to facilitate sucrose formation for osmoregulation (106).

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