As in many higher plant species, sucrose plays the main role in growth and development of sugar beets. Because of its non reducing nature, sucrose is considered as the major transport and storage form of sugars in many plant species (9), especially in economically important crop plants such as sugar cane, carrots, melon, sucrose storing tomato varieties and beet roots (fodder beet, red beet, sugar beet).
In general, photosynthetically fixed carbon is converted to sucrose within the cytoplasm of source leaves and then exported through the phloem to photosynthetic growing and non photosynthetic sinks of the plant like sink leaves, flowers, tubers, roots or fruit for sustaining growth and/or for intermediary or long term storage.
Normally the triose phosphate translocator mediates export of the primary photosynthesis products from the chloroplasts into the cytosol (10). This transporter is an integral membrane protein localised at the inner envelope membrane of chloroplasts whereas the outer membrane is considered as largely permeable due to porins. Sequencing results for a full-length clone of the major chloroplast envelope protein gene E29 in potato and spinach, which is assumed to function as the translocator, showed high homology between the two plant species. Expression on the RNA level is restricted to green tissues, is light dependent and cannot be induced by sucrose in darkness (10). Within the cytosol, the primary photosynthesis products are transformed by the well known reactions of the Calvin cycle before they may be subjected to the enzymatic reactions of sucrose synthesis (Calvin cycle and sucrose synthesis just symbolically indicated in fig. 1).
Two distinct enzymes are able to catalyse sucrose synthesis:
i) Sucrose-phosphate-synthase (SPS; EC 184.108.40.206) catalyses the reaction UDP-glucose + Fru-6-P <-> Sucrose-6'-P + UDP + H+
Since sucrose-6'-P is removed from the reaction equilibrium very fast by the subsequent sucrose-phosphatase (SPP) reaction, the SPS reaction is essentially irreversible (11).
ii) Sucrose synthase (SuSy; EC 220.127.116.11) catalyses the reaction UDP-glucose + Fructose <H> Sucrose + UDP + H+
SPS and SuSy have been isolated from wheat seedlings and characterised the first time by Cardini and Leloir (12, 13). In the meantime, the genes encoding SuSy and SPS have been cloned from several species (11, 14, 15, 16) including sugar beet (17, 18). The 3635 bp sugar beet cDNA codes for a 1045 amino acid polypeptide with a predicted molecular mass of 118
kDa. The deduced amino acid sequence shows homologies with SPS from maize (71% identity) and spinach (77% identity)(17). Incubation of detached leaves of sugar beet in light in glucose-containing media led to an accumulation of the SPS transcript, while sucrose feeding reduced the steady-state level of the mRNA (17).
SPS is a low abundance protein (<0.1% of leaf soluble protein) and relatively unstable. In general, the native SPS molecule is reported to be a dimer of 120-138-kDa subunits (16) with hyperbolic substrate saturation profiles for UDP-glucose and Fru-6-P. The enzyme activity (specific activity in spinach is about 150 IU/mg protein) can be influenced by effectors. It is allosterically activated by Glc-6-P in some plant species and inhibited by Pi (19) affecting the affinities for both substrates, Fru-6-P and UDP-Glc (20). SPS activity is also light/dark modulated and controlled by reversible protein phosphorylation with Ser-158 (Ser-162 in maize SPS) as major regulatory phosphorylation site in spinach (see 16, 21 for reviews). Osmotic stress activation is also discussed. Part of the osmotic stress activation of SPS in dark leaves resulted from phosphorylation of serine-424 catalysed by a Ca2+-dependent, 150-kD protein kinase (21, 22). Recent results seem to indicate the occurrence of enzyme complexes between SPS and SPS-kinase possibly facilitating phosphorylation of the low abundance SPS (16). In addition, the possible presence of a sucrose- phosphate synthase (SPS) activating / stabilising factor (SAF) of proteinaceous nature presumed to be lost during SPS purification was investigated in rice leaf extracts (23). Simultaneous studies on the activities of SPS and sucrose-phosphate phosphatase (SPP), closely linked to SPS, led the authors to suggest that SAF could be SPP. The presence of SAF/SPP did not alter the affinity of SPS for its substrates but helped to reverse the Pi inhibition at low Fru-6-P concentrations. Efficient removal of inhibitory sucrose-6'-P via an enzyme complex could be necessary for maximal efficiency of sucrose synthesis.
For sugar beet SuSy, a 2762 bp long cDNA clone (named SBSS 1) encodes for a 822 amino acid polypeptide of a predicted molecular mass of 93.7 kDa. The deduced amino acid sequence showed 65-70% homologies when compared to predicted amino acid sequences of sucrose synthases from other species (18). According to RNA blot analysis SBSS1 was expressed predominantly in taproot under normal growth conditions. Transcription levels were not influenced by sugars. Activity of SuSy may be regulated by phosphorylation of Ser-11 as demonstrated by increase of its apparent affinity for sucrose by in vitro phosphorylation or site directed mutagenesis in mung bean (24). The sucrolytic activity of SuSy seems to exhibit sigmoidal saturation kinetics by sucrose indicating that the enzyme is switched on upon sucrose having reached a certain level (15). SuSy provides precursors for cell wall and starch synthesis.
In rice, three isogenes encode four SuSy isozymes with 808 to 816 amino acids (15). In contrast to SPS, SuSy is considered to be mainly a sucrose cleavage catalysing enzyme (25) although the reaction catalysed by SPS is reported to be reversible as well (16). SuSy exhibits differences in some properties between synthesis and cleavage reaction (26). Sucrose cleavage was inhibited when the enzyme was pretreated with oxidised glutathione or oxidised thioredoxin, dependent on the presence of sucrose and UDP, whereas sucrose synthesis seemed to be unaffected. Sucrose cleavage activity could be restored by incubation with dithiothreitol or reduced glutathione. The observed inhibitory effect on one reaction only suggests a modification in the enzyme affinities for its substrates.
In sugar beet, sucrose is synthesised not only in photosynthetically active leaves but also in non-photosynthetic tissue of the sugar beet taproot (27, 28, 29). In contrast to earlier findings about the absence of SPS activity in growing and sugar storing sugar beet (27, 30) SPS
activity was found in sugar beet petioles and taproots (28, 29). The activity was found to be sufficient to account for the accumulation of sucrose in sugar beet taproots and pointed to an intermediary storage function of the petiole along the long distance transport pathway as suggested earlier by Kursanov (31). This is consistent with the recent finding that RNA blot analysis of sink and source leaves, root and taproot tissue revealed SPS being expressed in an organ-specific manner with a predominance in taproot (17).
3.1. Metabolic control of sucrose synthesis in leaves
Factors favouring photosynthesis may result in enhanced sucrose synthesis and/or starch synthesis provided that RuBP regeneration is not hindered and chloroplast fine structure is not subjected to damaging alterations.
Two sucrose biosynthetic enzymes are believed to possess regulatory properties with respect to the rate of sucrose synthesis: (i) cytosolic FBPase and (ii) sucrose phosphate synthase. Transgenic potato plants with cytosolic FBPase cloned in reversed orientation (between the 35S CaMV promoter and the octopine synthase polyadenylation signal) exhibited reduction of cytosolic FBPase activity to variable degrees (32). A 45% reduction of the cytosolic FBPase activity did not cause any measurable change in metabolite concentrations, growth behaviour or photosynthetic parameters of the transgenic plants. Inhibition of cytosolic FBPase activity below 20% of the wild-type activity resulted in a reduced light-saturated rate of assimilation and a decreased photosynthetic rate with saturating light and CO2. Sucrose synthesis was heavily impaired whereas starch synthesis decreased only by 18-24%. Steady state sucrose concentrations, however, were not affected in source leaves from these transgenic plants. Reduced photosynthetic sucrose biosynthetic capacity was interpreted to be efficiently compensated by changing carbon export strategy, i.e. by presumably exporting hexoses and/or hexosephosphates out of the chloroplasts. As a result, plant growth and potato tuber yield remained unaltered.
The importance of SPS for the regulation of sucrose synthesis is shown by the following examples. Transgenic tomato plants with overexpressed SPS show increased light- and CO2-saturated rates of photosynthesis, increased sucrose to starch ratios in leaves under ambient conditions and increased partitioning at fixed-C into sucrose (33). Plant growth was only enhanced when SPS was overexpressed not only in leaves but also in nonphotosynthetic tissue (34). When transgenic tomato plants were grown and measured under high CO2-conditions, the photosynthetic rates were higher (per unit leaf area) compared with control plants (33). Although increased concentrations of CO2 stimulated photosynthesis, long term exposition to elevated carbon dioxide often led to an attenuation of the potential gain in yield (35). In tobacco and potato, both containing the overexpressed spinach enzyme, SPS was downregulated, presumably by phosphorylation (36) leading to the hypothesis that increased sucrose yields might be obtained in transgenic plants containing SPS with regulatory phosphorylation sites removed (11).
In Arabidopsis, according to the expression pattern and regulation of the gene, SuSy is thought to be involved in the supply of energy for phloem loading in source tissues, and in metabolization of sucrose in sink tissues after unloading (37). For a general discussion of sucrose biosynthesis in leaves and the roles of SPS and SuSy with regard to sink strength, see Stitt et al. (19), Huber et al. (38, 39), Stitt & Sonnewald (36), Huber & Huber (11) Herbers & Sonnewald (40) for reviews and elsewhere in this book.
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