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。     

蔗糖磷酸合成酶研究的新进展

蔗糖磷酸合成酶(sucrose phosphate synthase,SPS)是高等植物体内控制蔗糖合成的关键酶之一,它主要通过异构调节和磷酸化修饰在酶水平调节蔗糖合成。本文简要介绍SPS家族的成员、SPS蛋白上的3个磷酸化位点,以及SPS的生物学功能、SPS与磷酸蔗糖磷酸酶的关系等。     

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。     

河套蜜瓜果实发育过程中糖积累与蔗糖代谢相关酶的关系

以河套蜜瓜为试材,采用外部形态观测与内部生理指标测定相结合的方法,对其果实发育过程中果实生长模式以及果实中蔗糖、果糖、葡萄糖和淀粉含量以及蔗糖代谢相关酶活性进行测定,以揭示河套蜜瓜果实生长发育过程中糖的代谢积累与相关酶的关系.结果显示:(1)河套蜜瓜果实生长速率呈单"S"曲线,果实发育早期以积累葡萄糖为主,进入成熟期后蔗糖积累量迅速增加,最终由蔗糖和己糖共同构成果实品质.(2)在河套蜜瓜果实成熟期前,蔗糖磷酸合成酶(SPS)活性维持较低水平,进入成熟期后,SPS活性迅速升高;蔗糖合成酶(SS)活性在成熟期前为分解活性大于合成活性,成熟期后表现为合成活性大于分解活性;在整个果实发育期,酸性转化酶(AI)活性较低,中性转化酶(NI)活性始终高于AI.(3)在果实整个发育期,蔗糖含量与蔗糖代谢酶的净活力呈极显著正相关,蔗糖代谢相关酶共同作用决定果实中蔗糖含量.研究表明,在河套蜜瓜果实发育前期,以蔗糖分解代谢为主,且蔗糖合成酶和中性转化酶是催化蔗糖分解的关键酶;果实成熟期间,蔗糖代谢转为合成方向为主,蔗糖合成酶和蔗糖磷酸合成酶在蔗糖积累中起主导作用.     

温度对棉纤维糖代谢相关酶活性的影响

以棉纤维比强度高的科棉1号和中等强度的美棉33B 2个基因型棉花品种为材料,于2005年在江苏南京(长江流域下游棉区)和徐州(黄河流域黄淮棉区)设置不同播期(4月25日和5月25日)试验,研究了不同温度下棉纤维发育过程中蔗糖酶、蔗糖合成酶、磷酸蔗糖合成酶和β-1,3-葡聚糖酶等糖代谢相关酶活性的动态变化特征及其与纤维长度和比强度形成的关系.结果表明：棉纤维伸长发育期,蔗糖酶、β-1,3-葡聚糖酶活性较高;纤维加厚发育期,蔗糖合成酶和磷酸蔗糖合成酶活性上升速度快、活性高,蔗糖酶和β-1,3-葡聚糖酶活性下降速度快.纤维伸长期,蔗糖酶活性升高对纤维的伸长具有明显促进作用;纤维加厚发育期,提高蔗糖合成酶、磷酸蔗糖合成酶活性及加快蔗糖酶和β-1,3-葡聚糖酶活性下降速度有利于提高纤维比强度.科棉1号前期蔗糖酶、β-1,3-葡聚糖酶活性及中后期蔗糖合成酶、磷酸蔗糖合成酶活性均较美棉33B高.在本试验条件下,23.3 ℃是高强纤维形成的适宜温度,23.3 ℃～25.5 ℃是纤维长度形成的适宜温度.     

氮对水稻叶蔗糖磷酸合成酶的影响及其与同化物积累和产量的关系

蔗糖磷酸合成酶是植物体内蔗糖合成、碳同化与分配的一个关键调节酶。以3个水稻品种‘两优培九’、‘汕优63’和‘黄华占’为材料,在低氮和高氮处理下分析了水稻幼穗分化至抽穗不同阶段叶片蔗糖磷酸合成酶(sucrose phosphate synthase,SPS)的基因表达和活性及其与植株同化物积累和产量形成的关系。研究结果表明,所有SPS基因(Os SPS1、Os SPS2、Os SPS6、Os SPS8和Os SPS11)的相对表达量均随植株生育进程推进而下降;SPS活性的变化趋势与SPS基因表达量一致。与高氮处理相比,低氮处理下SPS基因的相对表达量和SPS活性增加。相关分析表明,SPS基因的相对表达量和SPS活性均与叶片非结构性碳水化合物(non-structural carbohydrates,NSC)浓度显著正相关,Os SPS1表达和SPS活性状态均与每穗颖花数和籽粒产量显著正相关,Os SPS2、Os SPS6和Os SPS8表达均与结实率和千粒重显著正相关。因此,适当减少氮肥施用有利于提高SPS基因表达和活性,进而增加植株NSC积累和促进产量形成。     

光合碳在叶片淀粉和蔗糖间分配的调节

叶片光合作用中产生的三碳糖在淀粉和蔗糖之间的分配受许多因素控制,蔗糖形成速率是决定性因素。蔗糖形成的调节酶是果糖1,6—二磷酸酯酶(F1,6P_2ase)和磷酸蔗糖合成酶(SPS),调节作用是通过无机磷(Pi)、磷酸二羟丙酮(DHAP)、磷酸己糖(己糖—P)、果糖1,6—二磷酸(F1,6P_2)和果糖2,6—二磷酸(F2,6P_2)之间的复杂的调节关系进行的。其中F2,6P_2起着关键作用,它以极低的浓度调节生糖和酵解作用,既参与蔗糖合成又参与反馈抑制。     

套袋对梨果实发育过程中糖组分及其相关酶活性的影响

以翠冠和黄金梨为试材,测定套袋和未套袋(对照)梨果实发育时期果实中蔗糖、葡萄糖、果糖和山梨醇含量以及蔗糖代谢相关酶酸性转化酶(AI)、中性转化酶(NI)、蔗糖合成酶(SS)和蔗糖磷酸合成酶(SPS)的活性,并对果实中糖组分积累与酶活性的关系进行了分析.结果表明:(1)两梨品种套袋果实在发育过程中蔗糖、葡萄糖、果糖、山梨醇和糖代谢相关酶活性变化趋势与对照基本一致,套袋果实糖含量均低于对照但差异不显著,而各相关酶活性在两类果实间差异表现各异.(2)在梨果实发育早期,果实中以分解酶类为主,糖分积累低;发育后期以合成酶类为主,糖分积累多.(3)两品种套袋和对照果实AI活性与葡萄糖含量均呈显著或极显著正相关,SS合成方向活性与蔗糖含量均为极显著正相关,且翠冠对照果SPS活性与蔗糖含量呈极显著正相关.可见,套袋通过提高果实发育早期转化酶(Inv)活性,降低果实后期蔗糖磷酸合成酶(SPS)、蔗糖合成酶(SS)的活性来影响糖分积累,从而影响梨果品质.     

‘台农1号’芒果果实发育过程中的糖分积累与相关酶活性研究

以‘台农1号’芒果为材料,测定了果实生长发育过程中淀粉、蔗糖、葡萄糖和果糖含量以及淀粉酶、蔗糖代谢相关酶———酸性转化酶(AI)、中性转化酶(NI)、蔗糖合成酶(SS)和蔗糖磷酸合成酶(SPS)的活性,并对果实中糖组分与酶活性的关系进行了分析.结果显示,(1)台农1号芒果果实属于单S型生长曲线,发育前期主要积累淀粉、葡萄糖和果糖,果实成熟软化时,淀粉酶活性降至最低,淀粉水解,蔗糖快速积累.(2)酸性转化酶活性在果实整个发育过程中维持最高,完熟时略有降低;蔗糖磷酸合成酶在果实发育前期略有降低,完熟时升至最高;蔗糖合成酶和中性转化酶活性在整个发育期一直很低且较稳定.(3)淀粉含量与淀粉酶活性呈显著正相关,与SPS活性呈极显著负相关,蔗糖、葡萄糖含量均与SPS、SS呈显著、极显著的正相关;果糖含量与SS呈极显著的正相关.研究表明,芒果成熟时淀粉分解、酸性转化酶活性的降低,且蔗糖合成酶和蔗糖磷酸合成酶活性的增加是引起果实蔗糖积累的主要因子.     

套袋梨贮藏过程中糖代谢及相关酶活性变化特征

以‘翠冠’梨为材料,研究了套双层遮光纸袋梨果实贮藏过程中蔗糖、果糖、葡萄糖、山梨醇及糖代谢中酶活性的变化规律。结果表明,贮藏套袋梨果实中果糖、葡萄糖、山梨醇和蔗糖含量都低于未套袋对照;套袋梨果实中山梨醇脱氢酶活性在贮藏的前5d都低于对照,贮藏10d后活性均高于对照,且与山梨醇含量呈现极显著正相关;贮藏套袋梨果实中蔗糖磷酸合酶(SPS)及蔗糖合酶(SS)分解和合成方向活性都是前期低于对照,贮藏后期都高于对照,且蔗糖含量与蔗糖磷酸合酶和蔗糖合酶(分解方向)活性都呈显著正相关;贮藏的套袋梨果实和对照中的山梨醇含量与果糖含量均呈极显著负相关,蔗糖含量与葡萄糖含量呈极显著负相关,即在贮藏过程中山梨醇可能转化为果糖,而蔗糖则转化成葡萄糖。     

Decreased expression of sucrose phosphate synthase strongly inhibits the water stress-induced synthesis of sucrose in growing potato tubers

Water stress stimulates sucrose synthesis and inhibits starch synthesis in wild-type tubers. Antisense and co-suppression potato transformants with decreased expression of sucrose–phosphate synthase (SPS) have been used to analyse the importance of SPS for the regulation of this water-stress induced change in partitioning. (i) In the absence of water stress, a 70–80% decrease in SPS activity led to a 30–50% inhibition of sucrose synthesis and a slight (10–20%) increase of starch synthesis in tuber discs in short-term labelling experiments with low concentrations of labelled glucose. Similar changes were seen in short-term labelling experiments with intact tubers attached to well-watered plants. Provided plants were grown with ample light and water, transformant tubers had a slightly lower water and sucrose content and a similar or even marginally higher starch content than wild-type tubers. (ii) When wild-type tuber slices were incubated with labelled glucose in the presence of mannitol to generate a moderate water deficit (between –0.12 and –0.72 MPa), there was a marked stimulation of sucrose synthesis and inhibition of starch synthesis. A similar stimulation was seen in labelling experiments with wild-type tubers that were attached to water-stressed wild-type plants. These changes were almost completely suppressed in transformants with a 70–80% reduction of SPS activity. (iii) Decreased irrigation led to an increase in the fraction of the dry-matter allocated to tubers in wild-type plants. This shift in allocation was prevented in transformants with reduced expression of SPS. (iv) The results show that operation of SPS and the sucrose cycle in growing potato tubers may lead to a marginal decrease in starch accumulation in non-stressed plants. However, SPS becomes a crucial factor in water-stressed plants because it is required for adaptive changes in tuber metabolism and whole plant allocation.     

In vitro and in vivo inhibition of sucrose synthesis by sucrose

The inhibitory effects of sucrose on rates of sucrose synthesis by sucrose phosphate synthase (SPS) from the maize scutellum and on net rates of sucrose production in maize scutellum slices from added glucose or fructose were studied. Scutellum extracts were prepared by freezing and thawing scutellum slices in buffer. The extracts contained SPS and sucrose phosphate phosphatase, but were free of sucrose synthase. SPS activity was calculated from measurement of UDP formation in the presence of UDPG, fructose-6-P and sucrose. The ranges of metabolite concentrations used were those estimated to be in scutellum slices after incubation in water or fructose for periods up to 5 hr. UDPG and fructose-6-P also were added at concentrations that saturated SPS. At saturating substrate levels, sucrose inhibition of SPS was less than that when tissue levels of substrates were used. With tissue levels of substrates and sucrose concentrations up to ca 166 mM, sucrose inhibitions of sucrose synthesis in vitro by SPS were similar to those observed in vivo. However, as the sucrose concentration rose above 166 mM, SPS activity was not inhibited further, whereas there was a further sharp decline in sucrose production by the slices. It is concluded that sucrose synthesis in vivo is controlled by sucrose inhibition of SPS over a considerable range of internal sucrose concentrations.     

Engineering plants for elevated CO(2): a relationship between starch degradation and sugar sensing

In the future, plants will have additional CO(2) for photosynthesis. However, plants do not take maximal advantage of this additional CO(2) and it has been hypothesized that end product synthesis limitations and sugar sensing mechanisms are important in regulating plant responses to increasing CO(2). Attempts to increase end product synthesis capacity by engineering increased sucrose-phosphate synthase activity have been generally, but not universally, successful. It was found that plants benefited from a two- to three-fold increase in SPS activity but a 10-fold increase did not increase yield. Despite the success in increasing yield, increasing SPS did not increase photosynthesis. However, carbon export from chloroplasts was increased during the day and reduced at night (when starch provides carbon for sucrose synthesis. We develop here a hypothesis that starch degradation is closely sensed by hexokinase because a newly discovered pathway required for starch to sucrose conversion that involves maltose is one of few metabolic pathways that requires hexokinase activity.     

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.     

Nodule-enhanced expression of a sucrose phosphate synthase gene member (MsSPSA) has a role in carbon and nitrogen metabolism in the nodules of alfalfa (Medicago sativa L.)

Sucrose phosphate synthase (SPS) catalyzes the first step in the synthesis of sucrose in photosynthetic tissues. We characterized the expression of three different isoforms of SPS belonging to two different SPS gene families in alfalfa (Medicago sativa L.), a previously identified SPS (MsSPSA) and two novel isoforms belonging to class B (MsSPSB and MsSPSB3). While MsSPSA showed nodule-enhanced expression, both MsSPSB genes exhibited leaf-enhanced expression. Alfalfa leaf and nodule SPS enzymes showed differences in chromatographic and electrophoretic migration and differences in V _max and allosteric regulation. The root nodules in legume plants are a strong sink for photosynthates with its need for ATP, reducing power and carbon skeletons for dinitrogen fixation and ammonia assimilation. The expression of genes encoding SPS and other key enzymes in sucrose metabolism, sucrose phosphate phosphatase and sucrose synthase, was analyzed in the leaves and nodules of plants inoculated with Sinorhizobium meliloti. Based on the expression pattern of these genes, the properties of the SPS isoforms and the concentration of starch and soluble sugars in nodules induced by a wild type and a nitrogen fixation deficient strain, we propose that SPS has an important role in the control of carbon flux into different metabolic pathways in the symbiotic nodules.     

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.     

Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning.

We isolated a complementary DNA sequence for the enzyme sucrose phosphate synthase (SPS) from maize utilizing a limited amino acid sequence. The 3509-bp cDNA encodes a 1068-amino acid polypeptide. The identity of the cDNA was confirmed by the ability of the cloned sequence to direct sucrose phosphate synthesis in Escherichia coli. Because no plant-specific factors were necessary for enzymatic activity, we can conclude that SPS enzyme activity is conferred by a single gene product. Sequence comparisons showed that SPS is distantly related to the enzyme sucrose synthase. When expressed from a ribulose bisphosphate carboxylase small subunit promoter in transgenic tomatoes, total SPS activity was boosted up to sixfold in leaves and appeared to be physiologically uncoupled from the tomato regulation mechanism. The elevated SPS activity caused a reduction of starch and increase of sucrose in the tomato leaves. This result clearly demonstrates that SPS is involved in the regulation of carbon partitioning in the leaves.