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.     

Antisense acid invertase (TIV1) gene alters soluble sugar composition and size in transgenic tomato fruit.

Invertase (beta-fructosidase, EC 3.2.1.26) hydrolyzes sucrose to hexose sugars and thus plays a fundamental role in the energy requirements for plant growth and maintenance. Transgenic plants with altered extracellular acid invertase have highly disturbed growth habits. We investigated the role of intracellular soluble acid invertase in plant and fruit development. Transgenic tomato (Lycopersicon esculentum Mill.) plants expressing a constitutive antisense invertase transgene grew identically to wild-type plants. Several lines of transgenic fruit expressing a constitutive antisense invertase gene had increased sucrose and decreased hexose sugar concentrations. Each transgenic line with fruit that had increased sucrose concentrations also had greatly reduced levels of acid invertase in ripe fruit. Sucrose-accumulating fruit were approximately 30% smaller than control fruit, and this differential growth correlated with high rates of sugar accumulation during the last stage of development. These data suggest that soluble acid invertase controls sugar composition in tomato fruit and that this change in composition contributes to alterations in fruit size. In addition, sucrose-accumulating fruit have elevated rates of ethylene evolution relative to control fruit, perhaps as a result of the smaller fruit size of the sucrose-accumulating transgenic lines.     

Sucrose Metabolism in Netted Muskmelon Fruit during Development

Sugar content and composition are major criteria used in judging the quality of netted muskmelon (Cucumis melo L. var reticulatus) fruit. Sugar composition and four enzymes of sucrose metabolism were determined in `Magnum 45' muskmelon fruit at 10-day intervals beginning 10 days after pollination (DAP) until full-slip (35 DAP). Sugar content increased in both outer (green) mesocarp and inner (orange) mesocarp between 20 and 30 DAP. The major proportion of total increase in sugar was attributed to sucrose accumulation. The large increase in sucrose relative to glucose and fructose was accompanied by a dramatic decrease in acid invertase activity, which was highest in both tissues at 10 and 20 DAP, and increases in sucrose phosphate synthase (SPS) and sucrose synthase activities. The green tissue had a lower proportion of total sugar as sucrose, greater invertase activity, and less SPS activity than the orange tissue. Changes in relative sucrose content were highly correlated with changes in enzyme activity. The results strongly suggest that increases in the proportion of sucrose found in melon fruit were associated with a decline in acid invertase activity and an increase in SPS activity approximately 10 days before full-slip. Therefore, these enzymes apparently play a key role in determining sugar composition and the quality of muskmelon fruit.     

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 synthase in carrot (Daucus carota L.) affects growth rather than sucrose partitioning

To unravel the roles of sucrose synthase in carrot, we reduced its activity in transgenic carrot plants by an antisense approach. For this purpose, the cDNA for the main form of carrot sucrose synthase was expressed in antisense orientation behind the 35S promoter of cauliflower mosaic virus. In independent antisense plant lines grown in soil, sucrose synthase activity was reduced in tap roots but not in leaves. In the sink organs, sucrose utilization was markedly decreased and higher levels of sucrose but lower levels of UDP-glucose, glucose, fructose, starch and cellulose were found. The phenotype of the antisense plants clearly differed from that of control plants. Both leaves and roots were markedly smaller, and the antisense line with the lowest sucrose synthase activity also developed the smallest plants. In most of the plant lines, the leaf-to-root dry weight ratios were not changed, suggesting that sucrose synthase in carrot is a major determinant of plant growth rather than of sucrose partitioning. In contrast to the acid invertases, which are critical for partitioning of assimilated carbon between source leaves and tap roots (Tang et al., Plant Cell 11: 177–189 (1999)), sucrose synthase appears to be the main sucrose-cleaving activity, feeding sucrose into metabolism.     

Suppression of Acid Invertase Activity by Antisense RNA Modifies the Sugar Composition of Tomato Fruit

cDNA for an acid invertase (EC 3.2.1.26 [EC] ) of tomato (Lycopersiconesculentum Mill.) fruit was introduced into tomato plants underthe control of the cauliflower mosaic virus 35S promoter inthe antisense orientation. The antisense gene effectively suppressedthe invertase activity in soluble and cell wall fractions fromripening fruits. The sucrose content of fruits of the transformantswas markedly increased, while the hexose content was reduced.These results indicate that acid invertase is one of main determinantsof the sugar composition of tomato fruit. The invertase activityin the cell wall fraction of the leaf tissues of the transformantswas not suppressed to the same extent as that in the solublefraction. Wounding of the control leaf tissues induced invertaseactivity in both soluble and cell wall fractions. The inductionof activity in the soluble fraction was suppressed by the antisensegene, while that in the cell wall fraction was unaffected. Thesefindings suggest that mRNA for some other invertase, in particular,the mRNA for a cell wall-bound invertase, was present in leaves. ¹Present address: Plant Breeding and Genetics Research Laboratory,Japan Tobacco Inc., 700 Higashibara, Toyoda, Iwata, Shizuoka,438 Japan. ²Present address: National Institute of Agrobiological Resources,Kannondai, Tsukuba, Ibaraki, 305 Japan.     

Grafting helps improve photosynthesis and carbohydrate metabolism in leaves of muskmelon

The most important quality for muskmelon (Cucumis melo L.) is their sweetness which is closely related to the soluble sugars content. Leaves are the main photosynthetic organs in plants and thus the source of sugar accumulation in fruits since sugars are translocated from leaves to fruits. The effects of grafting muskmelon on two different inter-specific (Cucurbita maxima×C. moschata) rootstocks was investigated with respect to photosynthesis and carbohydrate metabolism. Grafting Zhongmi1 muskmelon on RibenStrong (GR) or Shengzhen1 (GS) rootstocks increased chlorophyll a, chlorophyll b and chlorophyll a+b content and the leaf area in middle and late developmental stages of the plant compared to the ungrafted Zhongmi1 check (CK). Grafting enhanced the net photosynthesis rate, the stomatal conductance, concentration of intercellular CO(2) and transpiration rate. Grafting influenced carbohydrates contents by changing carbohydrate metabolic enzymes activities which was observed as an increase in acid invertase and neutral invertase activity in the functional leaves during the early and middle developmental stages compared to CK. Grafting improved sucrose phosphate synthase and stachyose synthase activities in middle and late developmental stages, thus translocation of sugars (such as sucrose, raffinose and stachyose) in GR and GS leaves were significantly enhanced. However, compared with CK, translocation of more sugars in grafted plants did not exert feedback inhibition on photosynthesis. Our results indicate that grafting muskmelon on inter-specific rootstocks enhances photosynthesis and translocation of sugars in muskmelon leaves.     

Soluble acid invertase determines the hexose-to-sucrose ratio in cold-stored potato tubers

Cold storage of potato (Solanum tuberosum L.) tubers is known to cause accumulation of reducing sugars. Hexose accumulation has been shown to be cultivar-dependent and proposed to be the result of sucrose hydrolysis via invertase. To study whether hexose accumulation is indeed related to the amount of invertase activities, two different approaches were used: (i) neutral and acidic invertase activities as well as soluble sugars were measured in cold-stored tubers of 24 potato cultivars differing in the cold-induced accumulation of reducing sugars and (ii) antisense potato plants with reduced soluble acid invertase activities were created and the soluble sugar accumulation in cold-stored tubers was studied. The cold-induced hexose accumulation in tubers from the different potato cultivars varied strongly (up to eightfold). Large differences were also detected with respect to soluble acid (50-fold) and neutral (5-fold) invertase activities among the different cultivars. Although there was almost no correlation between the total amount of invertase activity and the accumulation of reducing sugars there was a striking correlation between the hexose/sucrose ratio and the extractable soluble invertase activitiy. To exclude the possibility that other cultivar-specific features could account for the obtained results, the antisense approach was used to decrease the amount of soluble acid invertase activity in a uniform genetic background. To this end the cDNA of a cold-inducible soluble acid invertase (EMBL nucleicacid database accession no. X70368) was cloned from the cultivar Desirée, and transgenic potato plants were created expressing this cDNA in the antisense orientation under control of the constitutive 35S cauliflower mosaic virus promotor. Analysis of the harvested and cold-stored tubers showed that inhibition of the soluble acid invertase activity leads to a decreased hexose and an increased sucrose content compared with controls. As was already found for the different potato cultivars the hexose/sucrose ratio decreased with decreasing invertase activities but the total amount of soluble sugars did not significantly change. From these data we conclude that invertases do not control the total amount of soluble sugars in coldstored potato tubers but are involved in the regulation of the ratio of hexose to sucrose.The authors are grateful to Heike Deppner and Christiane Prüßner for tuber harvest and technical assistance during the further analysis. We thank Andrea Knospe for taking care of tissue culture, Birgit Schäfer for patient photographic work, Hellmuth Fromme and the greenhouse personnel for attending plant growth and development and Astrid Basner for elucidating the sequence of clone INV-19. The work was supported by the Bundesministerium für Forschung und Technologie (BMFT).     

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.     

Cloning and characterization of a sucrose synthase-encoding gene from muskmelon

A full-length cDNA clone encoding sucrose synthase (SS; EC 2.4.1.13) was isolated from muskmelon (Cucumis melo L.) by RT-PCR and RACE. The clone, designated as CmSS1, contains 2,585 nucleotides with an open reading frame of 2,412 nucleotides. The deduced 804 amino acid sequence showed high identities with other plant sucrose synthase. Real time PCR analysis indicated that CmSS1 expression differed among root, stem, leaf, flower and fruit tissues. The analysis during fruit development indicated that CmSS1 mRNA showed its maximum level at 5 days after pollination (DAP) and decreased gradually during fruit development until its minimum level in mature fruit. The sucrose content was very low in fruit before 20 DAP but increased dramatically between 20 and 30 DAP during fruit development. However, SS activities in both direction of sucrose synthesis and sucrose cleavage were very low and changed little during fruit development, suggesting that SS may play little role in determining sucrose accumulation during muskmelon fruit.     

网纹甜瓜发育果实糖分积累与蔗糖代谢参与酶的关系

随着网纹甜瓜果实的发育,果实中葡萄糖和果糖的含量增加,蔗糖的快速积累发生在果实发育的中后期,高蔗糖积累型果实中蔗糖积累速率明显快于低蔗糖积累型.蔗糖磷酸合成酶活性在果实发育的前期短暂下降, 而后稳步上升,在果实发育的中后期高蔗糖积累型果实中该酶的活性显著高于低蔗糖积累型果实;随着果实发育,蔗糖合成酶的分解活性降低而合成活性升高.酸性和中性转化酶在未成熟果实中活性较高,而在成熟果实中很低; 高蔗糖积累型果实中酸性转化酶活性显著低于同期低蔗糖积累型果实.合成蔗糖的酶活性小于分解蔗糖的酶活性时蔗糖几乎没有积累.根据这些结果推测,转化酶活性的下降、蔗糖磷酸合成酶活性的增加以及蔗糖合成酶分解活性的下降和合成活性的增加,是引起果实蔗糖积累的主要内在因子.     

Acid and Neutral Invertases in the Mesocarp of Developing Muskmelon (Cucumis melo L. cv Prince) Fruit

Acid and neutral invertases were found in the mesocarp of developing muskmelon (Cucumis melo L. cv Prince) fruit and the activities of these enzymes declined with maturation of the fruit, concomitantly with the accumulation of sucrose. Neutral invertase was only present in the soluble fraction and acid invertase was present in both the soluble and cell-wall fractions. The cell-wall fraction contained three types of acid invertase: a NaCl-released invertase; an EDTA-released invertase, and a tightly bound invertase that still remained on the cell wall after treatment with NaCl and EDTA. The soluble acid and neutral invertases could be separated from one another by chromatography on DEAE-cellulose and they exhibited clear differences in their properties, namely, in their pH optima, substrate specificity, K_m values for sucrose, and inhibition by metal ions. The EDTA-released invertase and the soluble acid invertase were similar with regard to their chromatographic behavior on DEAE-cellulose, but the NaCl-released invertase was different because it was adsorbed to a column of CM-cellulose. The soluble acid invertase and two cell-wall bound invertases had very similar characteristics with regard to optimal pH and temperature, K_m value for sucrose, and substrate specificity.     

Manipulation of sink-source relations in transgenic plants

Since 1980, the use of transgenic plants in modern plant science has become a powerful tool to study whole plant physiology. In this review, we try to summarize the data obtained in the field of photoassimilate partitioning. Attempts to study sink-source interactions concern factors which might limit sink strength and source capacity. Transgenic plants have been used to manipulate the sucrose to starch ratio in order to produce plants with higher sucrose levels in their source leaves. Alterations in partitioning were achieved by manipulating Calvin cycle enzymes, transport proteins and sucrose biosynthetic enzymes. The ability of sink tissues to attract photoassimilates has been altered by either increasing or decreasing sucrose hydrolytic activities. The increase of sucrose hydrolysis was achieved by creating transgenic potato plants with tuber specific yeast-derived invertase. Decreased sucrose utilization was achieved by antisense inhibition of sucrose synthase in potato tubers.     

Impaired photoassimilate partitioning caused by phloem-specific removal of pyrophosphate can be complemented by a phloem-specific cytosolic yeast-derived invertase in transgenic plants.

Constitutive expression of the Escherichia coli ppa gene encoding inorganic pyrophosphatase resulted in sugar accumulation in source leaves and stunted growth of transgenic tobacco plants. The reason for this phenotype was hypothesized to be reduced sucrose utilization and loading into the phloem. To study the role of PPi in phloem cells, a chimeric gene was constructed using the phloem-specific rolC promoter of Agrobacterium rhizogenes to drive the expression of the ppa gene. Removal of cytosolic PPi in those cells resulted in photoassimilate accumulation in source leaves, chlorophyll loss, and reduced plant growth. From these data, it was postulated that sucrose hydrolysis via sucrose synthase is essential for assimilate partitioning. To bypass the PPi-dependent sucrose synthase step, transgenic plants were produced that express various levels of the yeast suc2 gene, which encodes cytosolic invertase, in their phloem cells. To combine the phloem-specific expression of the ppa gene and the suc2 gene, crosses between invertase- and pyrophosphatase-containing transgenic plants were performed. Analysis of their offspring revealed that invertase can complement the phenotypic effects caused by the removal of PPi in phloem cells.     

The changes in invertase activity and the content of sugars in the course of adaptation of potato plants to hypothermia

The pattern of changes in the activity of various forms of invertase (acid soluble, alkaline, and acid insoluble) and the content of sugars (glucose, fructose, and sucrose) in the course of plant adaptation to prolonged (6 days) hypothermia (5°C) was investigated in the leaves of potato plants (Solanum tuberosum L., cv. Desiree) produced in vitro. We used the wild-type plants as a control and transformed plants with carbohydrate metabolism modified by inserting the yeast gene for invertase (apoplastic enzyme). In the course of adaptation to hypothermia, the activity of acid invertase was shown to rise and the content of sucrose and glucose to increase in the leaves of both genotypes. The greatest activity of acid invertases by the third day of cold acclimation corresponded to the peak level of sugars; in transformed plants, these characteristics exceeded those in the control plants. The transformed plants were more cold resistant than the control plants as suggested by the lack of disturbance of ion permeability of their membranes. It was concluded that owing to accumulation of low-molecular carbohydrates in the course of cold acclimation caused by activation of acid invertase cold resistant plants better adapt to temperature drop.     

Dynamic characteristics of sugar accumulation and related enzyme activities in sweet and non-sweet watermelon fruits

Sugar content largely determines watermelon fruit quality. We compared changes in sugar accumulation and activities of carbohydrate enzymes in the flesh (central portion) and mesocarp of elite sweet watermelon line 97103 (Citrullus lanatus subsp. vulgaris) and exotic non-sweet line PI296341-FR (C. lanatus subsp. lanatus) to elucidate the physiological and biochemical mechanisms of sugar accumulation in watermelon fruit. The major translocated sugars, raffinose and stachyose, were more unloaded into sweet watermelon fruit than non-sweet fruit. During the fruit development, acid α-galactosidase activity was much higher in flesh of 97103 than in mesocarp of 97103, in flesh and mesocarp of PI296341-FR fruit. Insoluble acid invertase activity was higher in 97103 flesh than in 97103 mesocarp, PI296341-FR flesh or mesocarp from 18 days after pollination (DAP) to 34 DAP. Changes in soluble acid invertase activity in 97103 flesh were similar to those in PI296341-FR flesh and mesocarp from 18 DAP to full ripening. Sucrose synthase and sucrose phosphate synthase activities in 97103 flesh were significantly higher than those in 97103 mesocarp and PI296341-FR fruits from 18 to 34 DAP. Only insoluble acid invertase and sucrose phosphate synthase activities were significantly positively correlated with sucrose content in 97103 flesh. Therefore, phloem loading, distribution and metabolism of major translocated sugars, which are controlled by key sugar metabolism enzymes, determine fruit sugar accumulation in sweet and non-sweet watermelon and reflect the distribution diversity of translocated sugars between subspecies.     

Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions

In higher plants sucrose plays a central roles with respect to both short-term storage and distribution of photoassimilates formed in the leaf. Sucrose is synthesized in the cytosol, transiently stored in the vacuole and exported via the apoplast. In order to elucidate the role of the different compartments with respect to sucrose metabolism, a yeast-derived invertase was directed into the cytosol and vacuole of transgenic tobacco plants. This was in addition to the targeting of yeast-derived invertase into the apoplast described previously. Vacuolar targeting was achieved by fusing an N-terminal portion (146 amino acids long) of the vacuolar protein patatin to the coding region of the mature invertase protein. Transgenic tobacco plants expressing the yeast-derived invertase in different subcellular compartments displayed dramatic phenotypic differences when compared to wild-type plants. All transgenic plants showed stunted growth accompanied by reduced root formation. Starch and soluble sugars accumulated in leaves indicating that the distribution of sucrose was impaired in all cases. Expression of cytosolic yeast invertase resulted in the accumulation of starch and soluble sugars in both very young (sink) and older (source) leaves. The leaves were curved, indicating a more rapid cell expansion or cell division at the upper side of the leaf. Light-green sectors with reduced photosynthetic activity were evenly distributed over the leaf surface. With the apoplastic and vacuolar invertase, the phenotypical changes induced only appear in older (source) leaves. The development of bleached and/or necrotic sectors was linked to the source state of a leaf. Bleaching followed the sink to source transition, starting at the rim of the leaf and moving to the base. The bleaching was paralleled by the inhibition of photosynthesis.     

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