Antisense repression of vacuolar and cell wall invertase in transgenic carrot alters early plant development and sucrose partitioning.

To unravel the functions of cell wall and vacuolar invertases in carrot, we used an antisense technique to generate transgenic carrot plants with reduced enzyme activity. Phenotypic alterations appeared at very early stages of development; indeed, the morphology of cotyledon-stage embryos was markedly changed. At the stage at which control plantlets had two to three leaves and one primary root, shoots of transgenic plantlets did not separate into individual leaves but consisted of stunted, interconnected green structures. When transgenic plantlets were grown on media containing a mixture of sucrose, glucose, and fructose rather than sucrose alone, the malformation was alleviated, and plantlets looked normal. Plantlets from hexose-containing media produced mature plants when transferred to soil. Plants expressing antisense mRNA for cell wall invertase had a bushy appearance due to the development of extra leaves, which accumulated elevated levels of sucrose and starch. Simultaneously, tap root development was markedly reduced, and the resulting smaller organs contained lower levels of carbohydrates. Compared with control plants, the dry weight leaf-to-root ratio of cell wall invertase antisense plants was shifted from 1:3 to 17:1. Plants expressing antisense mRNA for vacuolar invertase also had more leaves than did control plants, but tap roots developed normally, although they were smaller, and the leaf-to-root ratio was 1.5:1. Again, the carbohydrate content of leaves was elevated, and that of roots was reduced. Our data suggest that acid invertases play an important role in early plant development, most likely via control of sugar composition and metabolic fluxes. Later in plant development, both isoenzymes seem to have important functions in sucrose partitioning.     

Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.     

Altering flux through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana modifies photosynthetic acclimation at low temperatures and the development of freezing tolerance

To test the hypothesis that the up‐regulation of sucrose biosynthesis during cold acclimation is essential for the development of freezing tolerance, the acclimation responses of wild‐type (WT) Arabidopsis thaliana (Heynh.) were compared with transgenic plants over‐expressing sucrose phosphate synthase (over‐sps) or with antisense repression of either cytosolic fructose‐1,6‐bisphosphatase (antifbp) or sucrose phosphate synthase (antisps). Plants were grown at 23 °C and then shifted to 5 °C. The leaves shifted to 5 °C for 10 d and the new leaves that developed at 5 °C were compared with control leaves on plants at 23 °C. Plants over‐expressing sucrose phosphate synthase showed improved photosynthesis and increased flux of fixed carbon into sucrose when shifted to 5 °C, whereas both antisense lines showed reduced flux into soluble sugars relative to WT. The improved photosynthetic performance by the over‐sps plants shifted to 5 °C was associated with an increase in freezing tolerance relative to WT (?9.1 and ?7.2 °C, respectively). In contrast, both antisense lines showed impaired development of freezing tolerance (? 5.2 and ?5.8 °C for antifbp and antisps, respectively) when shifted to 5 °C. In the new leaves developed at 5 °C the recovery of photosynthesis as typically seen in WT was strongly inhibited in both antisense lines and this inhibition was associated with a further failure of both antisense lines to cold acclimate. Thus, functional sucrose biosynthesis at low temperature in the over‐sps plants reduced the inhibition of photosynthesis, maintained the mobilization of carbohydrates from source leaves to sinks and increased the rate at which freezing tolerance developed. Modification of sucrose metabolism therefore represents an additional approach that will have benefits both for the development of freezing tolerance and over‐wintering, and for the supply of exportable carbohydrate to support growth at low temperatures.     

Antisense inhibition of tomato fruit sucrose synthase decreases fruit setting and the sucrose unloading capacity of young fruit

The role of sucrose synthase (SuSy) in tomato fruit was studied in transgenic tomato (Lycopersicon esculentum) plants expressing an antisense fragment of fruit-specific SuSy RNA (TOMSSF) under the control of the cauliflower mosaic virus 35S promoter. Constitutive expression of the antisense RNA markedly inhibited SuSy activity in flowers and fruit pericarp tissues. However, inhibition was only slight in the endosperm and was undetectable in the embryo, shoot, petiole, and leaf tissues. The activity of sucrose phosphate synthase decreased in parallel with that of SuSy, but acid invertase activity did not increase in response to the reduced SuSy activity. The only effect on the carbohydrate content of young fruit was a slight reduction in starch accumulation. The in vitro sucrose import capacity of fruits was not reduced by SuSy inhibition at 23 days after anthesis, and the rate of starch synthesized from the imported sucrose was not lessened even when SuSy activity was decreased by 98%. However, the sucrose unloading capacity of 7-day-old fruit was substantially decreased in lines with low SuSy activity. In addition, the SuSy antisense fruit from the first week of flowering had a slower growth rate. A reduced fruit set, leading to markedly less fruit per plant at maturity, was observed for the plants with the least SuSy activity. These results suggest that SuSy participates in the control of sucrose import capacity of young tomato fruit, which is a determinant for fruit set and development.     

Acid invertase (sucrose hydrolysis) is not required for sucrose mobilization from the vacuole

The possible involvement of acid invertase (sucrose hydrolysis) as a prerequisite for sucrose mobilization from the vacuole of storage cells was investigated. Sugarcane ( Saccharum officinarum ) stalks, carrot ( Daucus carota ) roots and red beet ( Beta vulgaris ) hypocotyls were planted under greenhouse conditions and allowed to resume growth. The plants, however, were not permitted to become photosynthetically autotrophic by removing the new expanded leaves. Sucrose levels declined significantly in all three tissues without the development of acid invertase (EC 3.2.1.26) during the 21‐day experimental period. Acid invertase and thus sucrose hydrolysis within the vacuole was, therefore, not required for sucrose mobilization.     

Copper-induced alteration in sucrose partitioning and its relationship to the root growth of two Elsholtzia haichowensis Sun populations

Hydroponic culture was used to comparatively investigate the copper (Cu)-induced alteration to sucrose metabolism and biomass allocation in two Elsholtzia haichowensis Sun populations with one from a Cu-contaminated site (CS) and the other from a non-contaminated site (NCS). Experimental results revealed that biomass allocation preferred roots over shoots in CS population, and shoots over roots in NCS population under Cu exposure. The difference in biomass allocation was correlated with the difference in sucrose partitioning between the two populations. Cu treatment (45 μM) significantly decreased leaf sucrose content and increased root sucrose content in CS population as a result of the increased activities of leaf sucrose synthesis enzymes (sucrose phosphate synthetase and sucrose synthase) and root sucrose cleavage enzyme (vacuolar invertase), which led to increased sucrose transport from leaves to roots. In contrast, higher Cu treatment increased sucrose content in leaves and decreased sucrose content in roots in NCS population as a result of the decreased activities of root sucrose cleavage enzymes (vacuolar and cell wall invertases) that led to less sucrose transport from leaves to roots. These results provide important insights into carbon resource partitioning and biomass allocation strategies in metallophytes and are beneficial for the implementation of phytoremediation techniques.     

Decreased expression of two key enzymes in the sucrose biosynthesis pathway, cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, has remarkably different consequences for photosynthetic carbon metabolism in transgenic Arabidopsis thaliana

Photosynthetic carbon metabolism was investigated in antisense Arabidopsis lines with decreased expression of sucrose phosphate synthase (SPS) and cytosolic fructose-1,6-bisphosphatase (cFBPase). In the light, triose phosphates are exported from the chloroplast and converted to sucrose via cFBPase and SPS. At night, starch is degraded to glucose, exported and converted to sucrose via SPS. cFBPase therefore lies upstream and SPS downstream of the point at which the pathways for sucrose synthesis in the day and night converge. Decreased cFBPase expression led to inhibition of sucrose synthesis; accumulation of phosphorylated intermediates; Pi-limitation of photosynthesis; and stimulation of starch synthesis. The starch was degraded to maintain higher levels of sugars and a higher rate of sucrose export during the night. This resembles the response in other species when expression of enzymes in the upper part of the sucrose biosynthesis pathway is reduced. Decreased expression of SPS inhibited sucrose synthesis, but phosphorylated intermediates did not accumulate and carbon partitioning was not redirected towards starch. Sugar levels and sucrose export was decreased during the night as well as during the day. Although ribulose-1,5-bisphosphate regeneration and photosynthesis were inhibited, the PGA/triose-P ratio remained low and the ATP/ADP ratio high, showing that photosynthesis was not limited by the rate at which Pi was recycled during end-product synthesis. Two novel responses counteracted the decrease in SPS expression and explain why phosphorylated intermediates did not accumulate, and why allocation was not altered in the antisense SPS lines. Firstly, a threefold decrease of PPi and a shift of the UDP-glucose/hexose phosphate ratio favoured sucrose synthesis and prevented the accumulation of phosphorylated intermediates. Secondly, there was no increase of AGPase activity relative to cFBPase activity, which would prevent a shift in carbon allocation towards starch synthesis. These responses are presumably triggered when sucrose synthesis is decreased in the night, as well as by day.     

Analysis of carbon metabolism in transgenic<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transformed with the cyanobacterial sucrose phosphate synthase gene

To investigate the effects of sucrose-phosphate synthase (SPS) on carbon partitioning, transgenicArabidopsis plants transformed withSynechocystis SPS were constructed. The integration, copy number and expression level were confirmed by Southern and Northern blot analyses. SPS activity in leaves from the transgenic and wild type plants was not significantly different. The level of sucrose and starch in the leaves of transgenic plant were slightly decreased compared to wild type. The glucose and fructose contents were increased up to two-fold compared to wild type during the light period. It is our speculation that the decreased sucrose level of the transgenic plant might be caused by the high acid invertase. These authors contributed equally to this work     

Effects of nitrogen and phosphorus deficiencies on levels of carbohydrates, respiratory enzymes and metabolites in seedlings of tobacco and their response to exogenous sucrose

A simple method of growing plants in agar was exploited to investigate the effect of long-term nitrogen (N) and phosphorus (P) deficiencies on respiratory metabolism and growth in shoots and roots of Nicotiana tabacum seedlings, and their interaction with exogenously supplied sucrose. Levels of hexose phosphates and 3-phosphoglyceric acid (3-PGA) were low in P-deficient shoots and roots and high in N-deficient shoots and roots. The ratio of hexose phosphates to 3-PGA and levels of fructose-2,6-bisphosphate were high in P-deficient plants and low in N-deficient plants. These data reflect differences in the way metabolism was perturbed, yet both deficiencies were associated with increased root growth relative to shoot growth, starch accumulation in the shoots, and soluble carbohydrate accumulation, especially hexoses, in the roots. Enzymes for sucrose degradation (sucrose synthase, acid and alkaline invertase) and glycolysis (phosphofructokinase, pyrophosphate-dependent phospho-fructokinase and pyruvate kinase) remained unaltered or declined in the shoots and roots. The accumulation of hexoses in roots of N- and P-deficient plants may result from maintenance of high invertase activities relative to sucrose synthase and glycolytic enzymes in the roots. The possibility that hexose accumulation may drive preferential root growth osmotically in N and P deficiencies is discussed. The addition of sucrose to roots to further investigate the interaction of carbohydrates with growth and allocation in low N and low P produced clear effects even though endogenous levels of soluble carbohydrate were already high in the nutrient-deficient plants. In complete nutrition, growth was stimulated, protein content particularly of the roots was increased and there was a preferential increase in activity of sucrose synthase in roots. At low P, enzyme activities in roots were increased, including sucrose synthase, and protein content increased, particularly in the roots, but there was no increase in growth. In N-deficient plants, exogenous sucrose led to decreased protein, Rubisco and chlorophyll content in shoots, in contrast to the other conditions, and a higher protein content and a general increase of catabolic enzyme activities and growth in the roots.     

Activities of Sucrose Synthase and Acid Invertase in Pea Seedling Organs

The organ topography of sucrose synthase and soluble acid invertase in pea seedlings at heterotrophic stage (3–14 days) was studied. Sucrose synthase was most active in the roots, with the highest activity on the 6–8th days. In the leaves, its activity decreased from day 3 to day 14. In the stems, sucrose synthase activity was at an invariantly low level. The patterns of sucrose synthase activity in etiolated and green plants were similar. As distinct from sucrose synthase, invertase activity was the highest in the stem, especially in etiolated plants. The peak of its activity was observed on the 6-8th days. In the leaves, invertase activity was lower but its pattern was the same. In the roots, acid invertase activity decreased from the 3rd day and did not depend on illumination. The conclusion is that differences in sucrose synthase and acid invertase activities in roots, leaves, and stem are determined by differences in the import of hydrolytic products of stored compound from the cotyledons as well as by different demands of these organs for these products for the processes of organ expansion and for the maintenance of organ metabolism.     

Relations between carbohydrate accumulation in leaves, sucrose phosphate synthase activity and photoassimilate transport in chilling treated maize seedlings

Sucrose and starch concentration, sucrose phosphate synthase (SPS) activity in leaves, and long distance transport were studied in maize seedlings treated with moderate chilling (14 °C/12 °C - day/night). Two inbred lines were tested: chilling-tolerant KW1074 and chilling-sensitive CM109. Seedlings were grown in phytotrone on water nutrient until the 4-th leaf appearance. The estimations were done on fully developed 2-nd leaf. Six days after the temperature was lowered, leaves of line KW 1074 plants contained 5-fold more sucrose and starch than the control ones. The same treatment of CM 109 seedlings resulted in accumulation of sucrose and starch by 2-fold and 8.5-fold, respectively. As the result of chilling-treatment, ¹⁴C assimilation rate (P_a), transport speed in the leaf blade (TS₁) and along the plant (TS_m) decreased by about 50 % in both lines. On the other hand, time necessary for radiolabel movement into the phloem loading region (AT) increased strongly, especially in chilling-sensitive line CM 109. It was also noted, that the radioactivity exported from leaves (R₁) and imported by roots (R_m) decreased in line CM 109, and increased slightly in line KW 1074. The activity of SPS extracted from leaves of both lines decreased by about 3.3 when temperature was lowered form 30°C to 10°C. There was no effect of 6 day treatment of chilling on SPS activity. Changes in sucrose and starch concentration, SPS activity as well as differences in transport parameters observed in KW1074 and CM109 seedlings at moderate low temperatures are discussed in terms of mechanism of maize chilling-sensitivity.     

稻纵卷叶螟和白背飞虱序列危害及间隔天数对水稻生理生化的影响

【目的】为了解稻纵卷叶螟Cnaphalocrocis medinalis和白背飞虱Sogatella furcifera的复合危害对水稻产量相关因子和相关酶类的影响。【方法】本文设定两种害虫的先后危害顺序,并通过调整两者开始危害的时间,研究了受害后水稻在灌浆期根、茎和叶片中淀粉和蔗糖含量的变化以及蔗糖合成酶(Sucrose synthase,SS)和蔗糖磷酸合成酶(Sucrose phosphate synthase,SPS)活性的变化。【结果】随着接虫量的增加,水稻不同组织内各生理指标与对照相比均显著下降。随着间隔天数的增加,先接白背飞虱为害要重于先接稻纵卷叶螟。比如叶片中蔗糖含量在先接稻纵卷叶螟的处理中随着间隔天数的增加显著增加,相应的SPS活性显著增加,间隔24 d的处理显著高于间隔6 d和12 d;而先接白背飞虱的处理中蔗糖含量与SPS活性均显著降低,间隔24 d的处理显著低于间隔6 d和12 d的处理。茎部淀粉含量在先接稻纵卷叶螟的处理中随着间隔天数的增加逐渐增加,SS活性显著增加,而先接白背飞虱的处理中淀粉含量和SS活性均显著降低;叶片中淀粉含量均随着间隔天数的增加逐渐降低,而相应的SS活性在先接稻纵卷叶螟的处理中显著增加,在先接白背飞虱的处理中相反。另外,接虫量和间隔天数间有显著的交互作用。【结论】本研究对指导水稻生产中的"两迁害虫"防治具有潜在应用价值。     

Analysis of the sucrose synthase gene family in Arabidopsis

The properties and expression patterns of the six isoforms of sucrose synthase in Arabidopsis are described, and their functions are explored through analysis of T-DNA insertion mutants. The isoforms have generally similar kinetic properties. Although there is variation in sensitivity to substrate inhibition by fructose this is unlikely to be of major physiological significance. No two isoforms have the same spatial and temporal expression patterns. Some are highly expressed in specific locations, whereas others are more generally expressed. More than one isoform is expressed in all organs examined. Mutant plants lacking individual isoforms have no obvious growth phenotypes, and are not significantly different from wild-type plants in starch, sugar and cellulose content, seed weight or seed composition under the growth conditions employed. Double mutants lacking the pairs of similar isoforms sus2 and sus3, and sus5 and sus6, are also not significantly different in these respects from wild-type plants. These results are surprising in the light of the marked phenotypes observed when individual isoforms are eliminated in crop plants including pea, maize, potato and cotton. A sus1/sus4 double mutant grows normally in well-aerated conditions, but shows marked growth retardation and accumulation of sugars when roots are subjected to hypoxia. The sucrose synthase activity in roots of this mutant is 3% or less of wild-type activity. Thus under well-aerated conditions sucrose mobilization in the root can proceed almost entirely via invertases without obvious detriment to the plant, but under hypoxia there is a specific requirement for sucrose synthase activity.     

A comparison of the carbohydrate composition and kinetic properties of sucrose phosphate synthase (SPS) in transgenic tobacco (Nicotiana tabacum) leaves expressing maize SPS protein with untransformed controls

Tobacco plants, transformed with a maize sucrose phosphate synthase (SPS) cDNA clone, had threefold increased SPS activity compared to wild‐type tobacco. Measurement of SPS maximal activity and protein abundance using specific antibodies to the maize protein showed that the specific activity of the maize SPS protein was maintained when expressed in tobacco. Comparison of the kinetic properties of SPS in the transgenic lines compared to either wild‐type maize or tobacco revealed that the heterologously expressed protein had reduced affinity for both substrates (fructose‐6‐phosphate and UDP‐glucose) and reduced sensitivity to allosteric inhibition by inorganic phosphate. Moreover, the extent of light‐induced activation was reduced in the transgenic lines, with smaller changes observed in the K_m for both F6P compared to maize and tobacco wild‐type plants. Increased sucrose concentrations were observed in the transgenic lines at the end of the photoperiod and this was linearly related to SPS activity and associated with a parallel decrease in starch content. This suggests that SPS is a major control point for carbohydrate partitioning between starch and sucrose during photosynthesis.