Transport Of Sucrose

Once sucrose is synthesized in mesophyll cells of source leaves, it has to be translocated to sink tissues. This transport occurs through phloem which contains elongated cells joined by sieve plates, consisting of diagonal cell walls perforated by pores. The single cells called sieve elements are surrounded by companion cells. Sieve elements and companion cells, in turn are connected to each other by many plasmodesmata. Photoassimilates generated in mesophyll cells diffuse via plasmodesmata to the phloem parenchyma cells. The further transport of photoassimilates from the phloem parenchyma cells to the sieve tubes can occur symplastically via plasmodesmata without involving translocators or apoplastically in which photoassimilates are first transported from the source cells via the phloem parenchyma cells to the extracellular compartment, the apoplast. This export does not require any energy as the concentration of sucrose is much higher in source cells than in the apoplast. The translocators mediating this transport have not yet been characterized. Further transport of sucrose from the apoplast to the companion cells proceeds via proton symport (Fig. 6), which is driven by proton gradient between the apoplast and the interior of

Fig. 5. Scheme for the regulation of sucrose synthesis by a co-ordinated control of the cytosolic FBPase and SPS. A, feed forward control in response to rising rates of photosynthesis; B, Feed back control when sucrose accumulates.

Sieve element

Sieve element

Proton Translocator
Fig. 6.Schematic representation of the membrane proteins involved in the translocation of sucrose from the chloroplasts of source leaves via the phloem to the sink organs. TPT, Triose-P translocator; SUT, Sucrose translocator; HPT, Hexose-P translocator.

the sieve tube, which is generated by H+-pumping ATPase present in the plasma membrane (115). A number of specialized reviews have already appeared on this subject (115-126).

Molecular studies of metabolite transport across the plasma membrane in plants have been neglected for many years. This has been mainly due to the problems associated with the identification and purification of the respective proteins. Distribution of plasmodesmata and microscopical studies with fluorescent dyes have provided evidence for symplastic transport at least between mesophyll cells (120, 122). Support for the existance of apoplastic transport has come from analysis of plants expressing invertase from the yeast in the apoplast (127-130). If sucrose transport is mediated mainly-through the plasmodesmata, sucrose should not appear in major concentrations in cell wall compartments. This situation was actually observed in several plant species, where apoplastic compartment had less than 5 mM sucrose in contrast to around 10-20 fold higher concentration in the cytosol of the mesophyll and a 100-200 fold higher concentration in the phloem cells. Thus the presence of an invertase in the apoplast should not lead to major effects. However, if the major route of sucrose is carrier mediated, and it has to pass through the apoplast compartment, the invertase should dramatically affect assimilate partitioning, as hexoses are not translocated efficiently into the phloem. In such cases, strong phenotypic effects such as leaf curling and local bleaching, reduced root growth and strong physiological effects such as accumulation of soluble sugars etc are observed. This is what exactly happened when number of investigators (127-130) used the expression of yeast invertase (Suc-2) in the apoplastic space in transgenic tobacco, potato, Arabidopsis and tomato fruits. In all these cases, significant phenotypic changes were observed e.g. transformed tobacco plants showed stunted growth, suppressed root formation and development of pale and necrotic regions in older leaves. These leaves also accumulated high levels of carbohydrates and starch. From these results, the above workers concluded that expression of invertase in the apoplast intrupts the export of photoassimilates. This caused the phenotypic differences observed in these studies. In symplastic loading, sucrose does not enter into apoplastic space and is not accessible to invertase. In these cases, no phenotypic changes were observed in transgenic plants. Since major phenotypic changes were observed in transgenic plants, it favours strongly the transport of sucrose via apoplastic mechanism. The apoplastic transport has recently been confirmed by measuring flux of l^c from CO2 to potato tuber (131). In these experiments, flux and apoplastic sucrose concentration were varied either by changing the light intensity or using transgenic manipulations that specifically affected the source or sink blocks. The elasticity coefficients were used to calculate the flux control coefficients of the source and sink blocks which were 0.8 and 0.2, respectively. The H+-sucrose translocator involved in phloem loading has recently been identified and characterized in several plants (132).

To provide direct evidence for the major route of sucrose loading, it might be useful to clone genes involved both in plasmodesmatal function and carrier-mediated sucrose transport and to study the effect of antisense inhibition of both processes in transgenic plants. One could also modify the symplastic pathway by overexpression of viral movement proteins. This has actually been achieved in transformed tobacco where overexpression increased the size exclusion limit (133). In another experiment, Schulz et al (134) inhibited sucrose-H+/co-transporter activity of potato by antisense repression of StSUTl under control of either a ubiquitously active (CaMV 35S) or a companion cell specific (roi C) promoter in transgenic plants. The transformed plants had reduced levels of the sucrose transporter mRNA and showed a dramatic reduction in root and tuber growth. Regardless of the promoter used, source leaves from transformants showed an altered leaf phenotype and a permanent accumulation of assimilates. Isolation of cDNA clone encoding plant sucrose transporter has further helped the investigation for the importance of this transport protein in phloem loading (135, 136). Transporter genes from spinach (SoSUTl), potato (StSUTl), tomato (LeSUTl), and tobacco (NtSUTl) have hyperbolic proteins having molecular mass of 47 KDa. These proteins structurally belong to the class of metabolite transporters consisting of two sets of six member spanning regions, separated by a large cytoplasmic loop (137). Frommer et al (138) have further shown these polypeptides to be specifically expressed in source leaves of sugar beet.

Energy in the form of ATP for loading and transport of sucrose is supplied by mitochondria. This is supported by the fact that companion cells contain a comparatively higher number of mitochondria. This is further supported by the finding that plasma membrane proton ATPases are specifically expressed in the plants and that antibodies detect the highest amount of the ATPase protein in the phloem (139). Phloem cells also contain a very high activity of sucrose synthase, the enzyme involved in cleavage of sucrose (140, 141). The involvement of sucrose synthase in phloem loading has been demonstrated by blocking ATP synthesis through anaerobiosis etc (140).

Transport in the phloem proceeds by mass flow, driven on one hand by very efficient pumping of sucrose into the sieve tubes and, on the other hand, by its withdrawal at the sites of consumption. By allowing plants to perform photosynthesis in the presence of radioactively labelled active CO2, phloem transport velocities of 30 to 150 cm/h have been measured. This rapid transport proceeds by mass flow as stated above and is driven by many transverse osmotic gradients. The direction of mass flow is governed entirely by consumption of phloem contents which could proceed in an upward direction e.g. from the mature leaf to the growing shoot or flower, or downwards into the roots or storage tubers.

Despite the important role of unloading process in sink organs, this area has virtually remained neglected, mainly because of the fact that the process of unloading varies considerably not only between different species, but even between different tissues within the same plant. Basically, there are again two possibilities for phloem unloading. In symplastic unloading, sucrose reaches the cells of the sink organs directly from the sieve elements via plasmodesmata, whereas, in apoplastic unloading the sucrose is first transported from the sieve tubes to the extracellular compartment and is then taken up in the sink organs. Investigations of the plasmodesmatal frequencies carried out with electron microscope have indicated that in vegetative tissues, phloem unloading occurs symplastically, whereas, in storage tissues, unloading is often apoplastic. In later case, sucrose is taken up from the apoplast into the storage cells via a sucrose carrier and converted there through sucrose synthase (142) and UDPGPPase (143) to fructose and glu-l-P, whereas in the second case, the enzyme invertase hydrolyzes sucrose in the apoplast into glucose and fructose, and these two hexoses are then transported into the cell via hexose-transport systems, where fructokinase and hexokinase convert them into corresponding hexose-P. Respective hexose transporter genes have already been isolated from Arabidopsis and tobacco, some of which are specifically expressed in sink tissues (144, 145). Sonnewald et al (146) produced transgenic potato plants in which neutral invertase derived from yeast was expressed in apoplastic space in a tuber specific manner. This resulted in an increase of up to 3 fold in the individual tuber fresh weight, decrease in the tuber number per plant and up to 30% increase in total tuber yield. However, there was an accumulation of reducing sugars and a decrease in starch content. When the yeast invertase was expressed in the apoplast in a constitutive manner, the plant hardly grew as they were unable to export their assimilates from the source tissues. These results demonstrated that the apoplastic space is involved in sucrose utilization and sucrose hydrolysis might be involved in determining sink strength.

The role of sucrose synthase in determining sink strength has also been investigated by generating transgenic potato plants expressing sucrose synthase antisense RNA corresponding to Sus-4 isoform (147). Although they used constitutive 35S CaMV promoter to drive the expression of antisense RNA, inhibition of sucrose synthase activity was tuber specific. This inhibition of sucrose synthase had no effect on sucrose content but the level of reducing sugars increased and that of starch decreased in developing potato tubers. These changes were accompanied by a decrease in total tuber weight and a reduction in soluble tuber proteins. These data are thus consistent with the assumption that sucrose synthase is also the major determinant of potato tuber sink strength. Antisense repression of even vacoular and cell wall invertase in transgenic carrot affected sucrose partitioning (148). In another case, Sonnewald et al (149) produced transgenic potato plants over expressing yeast invertase specifically in the cytosol of tubers. They expected that the irreversible cleavage of sucrose by a heterologous enzyme would lead to an increased sink strength and starch accumulation. Unexpectedly, there was 10-15% reduction in the starch content of mature tubers of these lines. Biochemical analysis revealed that the tubers contained essentially no sucrose, were unchanged in fructose content but accumulated large quantities of glucose. This led to the hypothesis that the capacity to metabolize glucose in these lines was not sufficient to keep pace with its production. They, therefore, further introduced a second transgene, encoding a glucokinase from Zymomonas mobilis into an invertase expressing transgenic line, with the intention of bringing glucose into the metabolism (150, 151). Transgenic lines thus obtained had up to 3 fold more glucokinase activity than in parent line. However, there was a further dramatic reduction in starch content down to 35 % of wild type levels. Biochemical analysis of growing tubers revealed large increases in the metabolic intermediates of glycolysis, organic acids and amino acids, 2 to 3 fold increases in the maximum catalytic activities of key enzymes of respiratory pathway and 3 to 5 fold increases in CO2 production. Based on these results, the authors concluded that the expression of invertase in potato tubers leads to an increased flux through glycolytic pathway at the expense of starch synthesis and heterologous overexpression of glucokinase enhances this change in partitioning. They further demonstrated a significant stimulation of sucrose synthesis, leading to a rapid cycle of sucrose degradation and synthesis (151). The results discussed here amply demonstrate that unloading processes are not static but can change during development and require a complex control of the expression of different transporters and sucrose metabolizing and synthesizing enzymes.

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