植物蔗糖磷酸合成酶研究进展

蔗糖磷酸合成酶(Sucrose Phosphate Synthase,以下简称SPS)是植物体内控制蔗糖合成的关键酶。植物体内蔗糖的积累与SPS活性正相关,SPS还参与植物的生长和产量形成,并在植物的抗逆过程中起重要作用。高等植物中至少存在A、B、C三个家族的SPS,而禾本科植物至少存在A、B、C、DIII和DIV五个家族的SPS。不同植物体内不同家族的SPS基因的表达特性不同,它们所发挥的功能也存在差异。SPS的活性在基因表达调控和SPS蛋白磷酸化共价修饰作用两个层面受到植物生长发育、光照、代谢产物、外源物质如激素和糖类等多种因素的复杂调控。转基因研究表明,转SPS基因是提高作物产量和品质、增强作物抗逆性的有效途径,值得深入研究。全面总结了国内外在植物蔗糖磷酸合成酶方面的研究进展,并提出问题与研究展望,期望为进一步研究并利用植物SPS基因改良作物品种提供参考。     

Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions

Prior data indicated that enhanced availability of sucrose, a major product of photosynthesis in source leaves and the carbon source for secondary wall cellulose synthesis in fiber sinks, might improve fiber quality under abiotic stress conditions. To test this hypothesis, a family of transgenic cotton plants (Gossypium hirsutum cv. Coker 312 elite) was produced that over-expressed spinach sucrose-phosphate synthase (SPS) because of its role in regulation of sucrose synthesis in photosynthetic and heterotrophic tissues. A family of 12 independent transgenic lines was characterized in terms of foreign gene insertion, expression of spinach SPS, production of spinach SPS protein, and development of enhanced extractable V _max SPS activity in leaf and fiber. Lines with the highest V _max SPS activity were further characterized in terms of carbon partitioning and fiber quality compared to wild-type and transgenic null controls. Leaves of transgenic SPS over-expressing lines showed higher sucrose:starch ratio and partitioning of ¹⁴C to sucrose in preference to starch. In two growth chamber experiments with cool nights, ambient CO₂ concentration, and limited light below the canopy, the transgenic line with the highest SPS activity in leaf and fiber had higher fiber micronaire and maturity ratio associated with greater thickness of the cellulosic secondary wall.     

Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol

Metabolite levels and carbohydrates were investigated in the leaves of tobacco (Nicotiana tabacum L.) and leaves and tubers of potato (Solanum tuberosum L.) plants which had been transformed with pyrophosphatase from Escherichia coli. In tobacco the leaves contained two- to threefold less pyrophosphate than controls and showed a large increase in UDP-glucose, relative to hexose phosphate. There was a large accumulation of sucrose, hexoses and starch, but the soluble sugars increased more than starch. Growth of the stem and roots was inhibited and starch, sucrose and hexoses accumulated. In potato, the leaves contained two- to threefold less pyrophosphate and an increased UDP-glucose/ hexose-phosphate ratio. Sucrose increased and starch decreased. The plants produced a larger number of smaller tubers which contained more sucrose and less starch. The tubers contained threefold higher UDP-glucose, threefold lower hexose-phosphates, glycerate-3-phosphate and phosphoenolpyruvate, and up to sixfold more fructose-2,6-bisphosphatase than the wild-type tubers. It is concluded that removal of pyrophosphate from the cytosol inhibits plant growth. It is discussed how these results provide evidence that sucrose mobilisation via sucrose synthase provides one key site at which pyrophosphate is needed for plant growth, but is certainly not the only site at which pyrophosphate plays a crucial role.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose 6-phosphate - FW fresh weight - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - 3PGA glycerate-3-phosphate - PFK phosphofructokinase - PFP pyrophosphate: fructose-6-phosphate phosphotransferase - Pi inorganic phosphate - PPi inorganic pyrophosphate - UDPGlc UDP-glucose This research was supported by the Deutsche Forschungsgemein-Schaft (SFB 137) and Sandoz AG (T.J., M.H., M.S.) and by the Bundesminister für Forschung und Technologie (U.S., L.W.).     

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     

Overexpression of sucrose phosphate synthase increases sucrose unloading in transformed tomato fruit

Sucrose unloading and sink activity were examined in tomato plants (Lycopersicon esculentum) overexpression sucrose phosphate synthase (SPS; EC 2.3.1.14). Like the leaves, the fruit of the transformed tomato plants had elevated (2.4-fold) SPS activity. SPS over-expression in tomato fruit did not significantly change acid invertase, and only slightly reduced ADPglc ppase activity, but enhanced sucrose synthase activity by 27%. More importantly, the amount of sucrose unloaded into the fruit was considerably increased. Using [³H]- (fructosyl)-sucrose in in vitro unloading experiments with harvested 20-d-old fruit, 70% more sucrose was unloaded into the transformed fruits compared to the untransformed controls. Furthermore, the turnover of the sucrose unloaded into the fruit of transformed plants was 60% higher than that observed in the untransformed controls. Taken together, these results demonstrate that SPS overexpression increases the sink strength of transformed tomato fruit.     

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.     

Sucrose-phosphate synthase activity and yield analysis of tomato plants transformed with maize sucrose-phosphate synthase

Sucrose synthesis is a major element of the interactions between photosynthesis and plant growth and development. Tomato (Lycopersicon esculentum Mill. cv. UC82B) plants transformed with maize sucrose-phosphate synthase (SPS; EC 2.3.1.14) expressed from either a ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit promoter (SSU) or the cauliflower mosaic virus 35S promoter (35S) were used to study effects of increased sucrose synthesis rates on plant growth. The plants were grown in growth chambers, field plots, and open-top chambers. The 35S plants had a 2 to 3-fold increase in young-leaf SPS activity, a 10 to 20-fold increase in young-root SPS activity and no increase in young-fruit SPS activity. The leaf SPS activity in one of the 35S lines fell to control levels by two months of age. The SSU plants had a 4 to 5-fold increase in leaf SPS activity and no significant increase in root or young-fruit SPS activity. One 35S line, which maintained high leaf SPS activity throughout development, yielded 70–80% more than controls at both normal and elevated CO₂ in open-top chambers in the field and 20–30% more than controls in two additional field trials. The other 35S line and the two SSU lines either yielded less or did not differ from controls under several growth conditions. Since only one of four transformed lines showed an increase in yield, we can not yet conclude that increased leaf SPS activity leads to increased yield. However, increased leaf SPS activity appears to result in increased fruit sugar content since all three lines with increased leaf SPS usually also had increased fruit sugars. Received: 18 November 1996 / Accepted: 22 January 1997     

Overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased activities of enzymes for sucrose-starch interconversions, and increased levels of nucleotides in growing potato tubers

Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U. Sonnewald, 1992, Plant J 2: 571–581). The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L. cv. Desirée) tubers. Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa1 transformants would restrict the mobilisation of sucrose and conversion to starch. Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa1 tubers. Unexpectedly, the rate of degradation of [¹⁴C] sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20–30% in ppa1 tubers compared to wild-type tubers. Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa1 tubers were investigated. (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to threefold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase. They also contained 30–100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase. (ii) The transformants contained higher pools of uridine nucleotides. As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP. (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold. In stored ppa1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase. It is concluded that in growing tubers PPi has a wider significance than just being an energy donor for specific reactions in the cytosol. Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein. Possible ways in which PPi hydrolysis could affect these processes are discussed. Received: 9 July 1997 / Accepted: 3 November 1997     

Alterations in carbohydrate metabolism and enhancement in tuber production in white yam (<Emphasis Type="Italic">Dioscorea rotundata</Emphasis> Poir.) under triadimefon and hexaconazole applications

The present investigation was carried out with an aim to study the effects of two important fungicides/plant growth regulators, triadimefon (TDM) and hexaconazole (HEX) on white yam (Dioscorea rotundata Poir.). Each plant was treated with 1 l of aqueous solution containing 15 mg l^-1 TDM and 10 mg l⁻¹ HEX by soil drenching on 10, 20 and 30 days after planting (DAP). Starch and sugar contents, and the activities of α, β-amylases, sucrose phosphate synthase (SPS), sucrose synthase (SS) and acid invertase (AI) enzymes were estimated from leaf and tuber samples on 45, 60, 75 and 90 DAP. Results showed that TDM and HEX treatments inhibited the α, β-amylase and increased the fresh and dry weights, starch, sugar and sucrose contents and SPS, SS and AI activities in white yam. The data suggests that, the application of triazole fungicides may be a useful tool to increase the tuber quality as well as quantity in white yam plants, apart from their fungicidal properties.     

苹果中磷酸蔗糖合酶家族基因的表达特性及其与蔗糖含量的关系

磷酸蔗糖合酶(sucrose phosphate synthase,SPS)是植物中蔗糖合成的主要限速酶,影响植物的生长发育和果实中蔗糖的含量。为探明苹果中SPS基因家族特性及其在蔗糖合成中的作用,该研究从苹果基因组中分离了MdSPS家族基因,分析了它们的进化关系以及mRNA表达特性与酶活性和蔗糖含量的关系。结果显示:(1)在苹果基因组中有8个SPS家族基因表达,它们分别属于双子叶植物的3个SPS亚家族。(2)荧光定量PCR分析显示,苹果C类的MdSPS6基因和A类的MdSPS1a/b基因是苹果中表达丰度最高的SPS基因成员,其中MdSPS6在苹果成熟果中表达丰度最高,其次是成熟叶片,而MdSPS1a/b在不积累蔗糖的幼果中表达丰度最高。(3)在果实发育过程中,除MdSPS1a/b之外,其它5个苹果MdSPS家族基因均随果实的生长表达丰度增加,与SPS活性和蔗糖含量明显呈正相关关系。研究表明,C类家族MdSPS6是苹果果实发育后期和叶片中蔗糖合成的主要SPS基因。     

Sucrose cycling in heterotrophic plant cell metabolism: first step towards an experimental model

Sucrose is the cornerstone of higher plant metabolism. Produced by photosynthesis, sucrose is the main substrate for respiration and biosynthesis. The emerging idea is that sucrose may act as regulator of its own metabolism, characterized in particular by a permanent process of degradation and formation. This sucrose turnover may control several important physiological functions. Of particular concern is an energy dependent cycle involving the hexokinase. This report presents an experimental approach to define quantitatively physiological states of suspension-cultured plant cells wih reference to their sucrose content and respiration rate. Sucrose depletion of normal cells incubated in a medium devoid of sugar is measured in vivo using ¹³C and respiration is simultaneously recorded. Results obtained with sucrose-storing cells and Arabidopsis thaliana show that respiration rate is closely linked to the available sucrose. Sucrose-depleted cells offer a stable model to study the bioenergetics of the process.     

Elevated sucrose-phosphate synthase activity in transgenic tobacco sustains photosynthesis in older leaves and alters development

Constitutive over-expression of a maize sucrose-phosphate synthase (SPS) gene in tobacco (Nicotiana tabacum) had major effects on leaf carbohydrate budgets with consequences for whole plant development. Transgenic tobacco plants flowered earlier and had greater flower numbers than wild-type plants. These changes were not linked to modified source leaf carbon assimilation or carbon export, although sucrose to starch ratios were significantly higher in leaves expressing the transgene. The youngest and oldest leaves of plants over-expressing SPS had up to 10-fold wild-type maximal extractable SPS activity, but source leaf SPS activities were only 2-3 times greater in these lines than in the wild type. In the oldest leaves, where the expression of the transgene led to the most marked enhancement in SPS activity, photosynthesis was also increased. It was concluded that these increases in the capacity for sucrose synthesis and carbon assimilation, particularly in older leaves, accelerate the whole plant development and increase the abundance of flowers without substantial changes in the overall shoot biomass.     

Agrobacterium rhizogenes-mediated transformation of Taraxacum platycarpum and changes of morphological characters

Transformed hairy roots were efficiently induced from seedlings of Taraxacum platycarpum by infection with Agrobacterium rhizogenes 15834. Root explants produced transformed roots at a higher frequency (76.5±3.5%) as compared to stem (32.7±4.8%) or cotyledon (16.2±5.7%). Hairy roots exhibited active elongation with high branching of roots on growth regulator-free medium. The competence of plant regeneration from non-transformed adventitious roots and transformed hairy roots was compared. The frequency of adventitious shoot formation from transformed roots was much higher (88.5±9.8%) than that of non-transformed roots (31.7 ±9.5%) on hormone-free medium. Rooting of hairy root-derived adventitious shoots occurred easily on growth regulator-free medium but no rooting was observed on non-transformed shoots. The stable introduction of rol genes into Taraxacum plants was confirmed by PCR and Southern hybridization. Transgenic plantlets showed considerable differences in their morphology when compared to the corresponding wild-type (non-transgenic) plants. Plantlets formed from transformed roots had numerous fibrous roots with abundant lateral branches instead of the thickened taproots in non-transformed plants. The differences observed may reflect the modification of morphological root characters by introduction of rol genes.Communicated by M.R. Davey     

Growth enhancement of transgenic poplar plants by overexpression of Arabidopsis thaliana endo-1,4–β-glucanase (cel1)

Poplar (Populus tremula) plants which had been transformed with Arabidopsis thaliana cel1 cDNA and successfully over-expressed the gene, exhibited significant phenotypic alterations which included taller plants, larger leaves, increased stem diameter, wood volume index, dry weight and a higher percentage of cellulose and hemicellulose, compared to the wild-type plants. Transgenic A. thaliana plants over-expressing A. thaliana cel1 exhibited similar levels of cel1 mRNA in the elongation zone of the flowering stem and higher levels in mature leaves when compared with wild-type plants. CEL1 protein levels in the elongation zone of the flowering stem of transgenic plants were similar or slightly higher compared to that of the wild-type plants, whereas mature leaves of transgenic plants contained a higher level of CEL1. These data indicate that in elongating zone of Arabidopsis, CEL1 level is tightly regulated. In contrast to transgenic poplar over-expressing the A. thaliana cel1, no phenotypic difference was found between A. thaliana transgenic and wild-type plants.     

Expression of a cyanobacterial sucrose-phosphate synthase from Synechocystis sp. PCC 6803 in transgenic plants

Sucrose-phosphate synthase (SPS) from the cyanobacterium Synechocystis sp. PCC 6803 lacks all of the Ser residues known to be involved in the regulation of higher plant SPS by protein phosphorylation. The Synechocystis SPS is also not allosterically regulated by glucose 6-phosphate or orthophosphate. To investigate the effects of expressing a potentially unregulated SPS in plants, the Synechocystis sps gene was introduced into tobacco, rice and tomato under the control of constitutive promoters. The Synechocystis SPS protein was expressed at a high level in the plants, which should have been sufficient to increase overall SPS activity 2-8-fold in the leaves. However, SPS activities and carbon partitioning in leaves from transgenic and wild-type plants were not significantly different. The maximal light-saturated rates of photosynthesis in leaves from tomato plants expressing the Synechocystis SPS were the same as those from wild-type plants. Tomato plants expressing the maize SPS showed 2-3-fold increases in SPS activity, increased partitioning of photoassimilate to sucrose and up to 58% higher maximal rates of photosynthesis. To investigate the apparent inactivity of the Synechocystis SPS the enzyme was purified from transgenic tobacco and rice plants. Surprisingly, the purified enzyme was found to have full catalytic activity. It is proposed that some other protein in plant cells binds to the Synechocystis SPS resulting in inhibition of the enzyme.