甜高粱茎秆不同节间糖分累积与相关酶活性的变化

为了进一步了解甜高粱茎秆糖分代谢的规律,利用高效液相色谱等方法测定了考利、拉马达和MN-2747等3个甜高粱品种成熟期6个节间果糖、葡萄糖和蔗糖含量以及中性转化酶(NI)、可溶性酸性转化酶(SAI)、蔗糖磷酸合成酶(SPS)和蔗糖合成酶(SS)的酶活性,并对其变化规律和相关性进行了分析。结果表明:不同品种间,果糖、葡萄糖和蔗糖含量变化范围较大,分别为2.32～4.34mg/g、2.30～4.14mg/g和35.92～95.92mg/g。随着节间的变化,3个品种果糖和葡萄糖均呈现"U"型变化趋势,而蔗糖无明显的变化规律,只是略有增高的趋势。3个品种成熟期茎秆中NI、SAI、SPS和SS酶活性普遍较低,随着节间的提高均呈现降低的趋势。节间蔗糖含量与SAI酶活性呈显著负相关(R=-0.71,P0.01),与NI、SPS和SS酶活性无明显相关性。SAI可能为甜高粱茎秆糖分代谢的关键调控酶。     

The role of sucrose-metabolizing enzymes in pear fruit that differ in sucrose accumulation

In the paper, the soluble sugar composition and activities of enzymes metabolizing sucrose: invertase (β-fructosidase, EC 3.2.1.26), sucrose synthase (SS; EC 2.4.1.13) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) were investigated during fruit development of two pear species: Pyrus bretschneideri Rehd. cv. ‘Yali’ and P. pyrifolia Nakai cv. ‘Aikansui’, characterized as low and high sucrose types, respectively. It was found that, at the end of fruit development of ‘Aikansui’, the level of sucrose was five times higher than in ‘Yali’ in the same period. It was coincident with the significantly higher activities of SS (synthesis) and SPS and lower activities of invertase (vacuolar and cell wall-bound acid invertase and neutral invertase). The high correlation was found between sucrose level and SS (synthesis) and SPS activities in ‘Aikansui’ pears.     

Differential regulation of grain sucrose accumulation and metabolism in Coffea arabica (Arabica) and Coffea canephora (Robusta) revealed through gene expression and enzyme activity analysis

* Coffea arabica (Arabica) and Coffea canephora (Robusta) are the two main cultivated species used for coffee bean production. Arabica genotypes generally produce a higher coffee quality than Robusta genotypes. Understanding the genetic basis for sucrose accumulation during coffee grain maturation is an important goal because sucrose is an important coffee flavor precursor. * Nine new Coffea genes encoding sucrose metabolism enzymes have been identified: sucrose phosphate synthase (CcSPS1, CcSPS2), sucrose phosphate phosphatase (CcSP1), cytoplasmic (CaInv3) and cell wall (CcInv4) invertases and four invertase inhibitors (CcInvI1, 2, 3, 4). * Activities and mRNA abundance of the sucrose metabolism enzymes were compared at different developmental stages in Arabica and Robusta grains, characterized by different sucrose contents in mature grain. * It is concluded that Robusta accumulates less sucrose than Arabica for two reasons: Robusta has higher sucrose synthase and acid invertase activities early in grain development - the expression of CcSS1 and CcInv2 appears to be crucial at this stage and Robusta has a lower SPS activity and low CcSPS1 expression at the final stages of grain development and hence has less capacity for sucrose re-synthesis. Regulation of vacuolar invertase CcInv2 activity by invertase inhibitors CcInvI2 and/or CcInvI3 during Arabica grain development is considered.     

‘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.     

Relationship between sucrose accumulation and activities of sucrose-phosphatase, sucrose synthase, neutral invertase and soluble acid invertase in micropropagated sugarcane plants

The activities of sucrose-phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) regulates sucrose activity in sugarcane were studied. Micropropagated sugarcane plants were obtained from callus cultures of four Mexican commercially available sugarcane varieties characterized by differences in sugar production, and activities of SPS, SUSY, NI, SAI and concentrations of sucrose were monitored in the sugarcane stem. The results indicated that sucrose accumulation was positively and significantly related to an increase in activity of SPS and SUSY and negatively to a reduction in activity of SAI and NI (P<0.05). SPS explained most of the variations found for sucrose accumulation and least for NI. The relationship between activity of SPS, SUSY, NI and SAI in sugarcane stem was similar in each variety.     

宁夏枸杞果实糖积累和蔗糖代谢相关酶活性的关系

通过对枸杞果实发育过程中果实生长模式、蔗糖、果糖、葡萄糖和淀粉含量及糖代谢相关酶活性的测定,研究了宁夏枸杞果实生长发育过程中糖的代谢积累与相关酶活性的关系.结果表明:(1)宁夏枸杞果实发育呈双S"曲线,果实主要以积累己糖为主.(2)蔗糖磷酸合成酶(SPS)活性在果实发育初期处于下降的趋势,在花后19d开始上升,果实转色后又逐渐下降;蔗糖合成酶(SS)活性总体表现为SS分解方向的活性大于SS合成方向的活性,说明枸杞果实发育过程中,SS的活性主要以分解方向的为主;酸性转化酶(AI)和中性转化酶(NI)的活性随果实发育呈上升趋势,但在果实成熟后期有所下降,且AI和NI活性高于合成酶类的活性,较高的转化酶类活性促进了果实内部己糖的积累.(3)在枸杞果实生长发育中,葡萄糖和果糖含量与AI和NI均呈极显著正相关,而与其它酶不具有相关性.说明AI和NI在宁夏枸杞果实的糖代谢中起着主要的调控作用.     

Sucrose accumulation in sweet sorghum stem internodes in relation to growth

Sweet sorghum (Sorghum bicolor L. Moench) stems of different cultivars (NK 405. Keller and Tracy) reveal a different pattern of sucrose accumulation with respect to in-ternodal sugar content and distribution. The onset of sucrose storage is not necessarily associated with the reproductive stage of the plant, as was hitherto assumed, but obviously occurs after cessation of internodai elongation as was postulated for the sugarcane stem. For at least two of the three cultivars, ripening is an internode to internode process beginning at the lowermost culm parts. Intensive growth of the internodes, combined with a high hexose content in stern parenchyma, shows a strong positive correlation (r |Mg 0.94) to the activity of sucrose synthase (SuSy; EC 2.4.13), but not to invertase (EC 3.2.1.26) which is not present as soluble (neutral and acid) or cell wall-bound, salt-extractable enzyme in the three culsivars investigated. Sucrose synthase measured in sucrose cleavage and synthesis direction reveals divergent activity rates and sensitivity towards exogenously applied Mg²⁺ ions and pH. SuSy activity is connected to the increase of internodai sucrose content in so far as (1) its decline is a prerequisite for the onset of sucrose accumulation and (2) it remains at a constant low level during sucrose storage. Sucrose phosphate synthase (SPS; EC 2.4.1.14) activity in the sorghum stem is low compared to SuSy and uniformly distributed over all inter-nodes. Only source leaves of sorghum show a considerable SPS activity, but neither stem nor leaf SPS reveal a positive correlation to the increase of internodai sucrose content. Sucrose phosphate phosphatase (SPP; EC 3.1.3.24) amounts lo only 24–30% of the respective SPS activity but follows the same distribution pattern. None of the enzymes under study proves to be responsible for the extent of sucrose storage in the stem, so other phenomena such as transport processes within the stern tissue require further investigation.     

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.     

Interconversion of free sugars in relation to activities of enzymes catalyzing synthesis and cleavage of sucrose in growing stem tissues of sorghum

Free sugar interconversion and activities of soluble acidic (pH 4.8) and neutral (pH 7.5) invertases, sucrose synthase (synthesis) and sucrose phosphate synthase were investigated in the growing nodes and internodes of sorghum (Sorghum vulgare). The results were substantiated with incorporation of 14C from supplied sucrose and hexoses into endogenous sugars of these stem tissues. With the advancement in plant growth, the content of total free sugars in apical nodes and internodes increased till 70 DAS (flowering stage) followed by a decline. In the corresponding basal tissues, the sugar build-up continued even beyond this stage of plant growth. Compared with basal stem tissues, the apical ones contained high activities of soluble invertases and a low proportion amongst free sugars of sucrose. The activities of sucrose-hydrolyzing enzymes were higher as compared with those of sucrose-synthesizing ones in both nodes and internodes and with the growth of plant, the activity of neutral invertase increased in these tissues. More 14C from supplied sucrose and hexoses appeared in extracted sugars from cut discs of apical nodes and internodes in comparison with their basal counterparts. 14C from supplied sucrose appeared in glucose, fructose and from supplied hexoses appeared in sucrose. The results suggest that in apical nodes and internodes, where a rapid cell division and cell expansion occur, sucrose is obligatorily inverted to meet the increased requirement of hexoses and there is a compartmentalized synthesis and cleavage of sucrose in the nodes and internodes of growing sorghum plant.     

Occurrence of sucrose and sucrose metabolizing enzymes in achlorophyllous algae

The presence of sucrose and the enzymes related to sucrose metabolism, i.e. sucrose synthase (SS) (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13), sucrose phosphate synthase (SPS) (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was demonstrated in Prototheca zopfii, a colorless alga. The levels of enzyme activities were lower than those obtained in Chlorella vulgaris, which is generally considered the photosynthetic counterpart of P. zopfii. Whem enzyme activities were measured in bleached cells of C. vulgaris, the levels were of the same order than those found in P. zopfii. These results would indicate that the sucrose metabolizing enzymes are not related to the algae ability to carry on photosynthesis.     

Expression analysis of genes associated with sucrose accumulation in sugarcane (Saccharum spp. hybrids) varieties differing in content and time of peak sucrose storage

Sucrose synthesis/accumulation in sugarcane is a complex process involving many genes and regulatory sequences that control biochemical events in source–sink tissues. Among these, sucrose synthase (SuSy), sucrose phosphate synthase (SPS), soluble acid (SAI) and cell wall (CWI) invertases are important. Expression of these enzymes was compared in an early (CoJ64) and late (BO91) maturing sugarcane variety using end‐point and qRT‐PCR. Quantitative RT‐PCR at four crop stages revealed high CWI expression in upper internodes of CoJ64, which declined significantly in both top and bottom internodes with maturity. In BO91, CWI expression was high in top and bottom internodes and declined significantly only in top internodes as the crop matured. Overall, CWI expression was higher in CoJ64 than in BO91. During crop growth, there was no significant change in SPS expression in bottom internodes in CoJ64, whereas in BO91 it decreased significantly. Apart from a significant decrease in expression of SuSy in mature bottom internodes of BO91, there was no significant change. Similar SAI expression was observed with both end‐point and RT‐PCR, except for significantly increased expression in top internodes of CoJ64 with maturity. SAI, being a major sucrose hydrolysing enzyme, was also monitored with end‐point PCR expression in internode tissues of CoJ64 and BO91, with higher expression of SAI in BO91 at early crop stages. Enzyme inhibitors, e.g. manganese chloride (Mn⁺⁺), significantly suppressed expression of SAI in both early‐ and late‐maturing varieties. Present findings enhance understanding of critical sucrose metabolic gene expression in sugarcane varieties differing in content and time of peak sucrose storage. Thus, through employing these genes, improvement of sugarcane sucrose content is possible.     

枇杷果实发育过程中糖积累及相关酶活性变化研究

以'青种'、'霸红'和'鸡蛋白'3个枇杷品种为材料,测定不同果实发育时期果实中蔗糖、葡萄糖和果糖含量以及蔗糖代谢相关酶即酸性转化酶(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,并对果实中糖积累与酶活性的关系进行了分析.结果表明:在果实膨大期(5月3日)之前,3种枇杷果实的蔗糖、葡萄糖和果糖积累缓慢,之后则迅速积累,存在着明显的转折点;果实成熟(5月23日)之后糖分积累速度趋于平稳.3种枇杷果实在发育过程中转化酶、蔗糖合成酶和蔗糖磷酸合成酶的活性变化与3种糖积累的动态变化趋势相一致.NI和AI活性在果实膨大期之前都较低且没有明显的变化,之后均快速上升;SS和SPS的活性在果实膨大期之前都很低且几乎无变化,随后'鸡蛋白'的活性迅速上升至果实成熟之后便缓慢上升,而'青种'和'霸红'随果实成熟度的增加而升高,但均低于'鸡蛋白'.可见,枇杷果实膨大期是糖分积累代谢活跃期,其糖积累受蔗糖代谢相关酶综合调控.     

A role for 'futile cycles' involving invertase and sucrose synthase in sucrose metabolism of tomato fruit.

Current concepts of the factors determining sink strength and the subsequent regulation of carbohydrate metabolism in tomato fruit are based upon an understanding of the relative roles of sucrose synthase, sucrose phosphate synthase and invertase, derived from studies in mutants and transformed plants. These enzymes participate in at least four futile cycles that involve sugar transport between the cytosol, vacuole and apoplast. Key reactions are (1) the continuous rapid degradation of sucrose in the cytosol by sucrose synthase (SuSy), (2) sucrose re-synthesis via either SuSy or sucrose phosphate synthase (SPS), (3) sucrose hydrolysis in the vacuole or apoplast by acid invertase, (4) subsequent transport of hexoses to the cytosol where they are once more converted into sucrose, and (5) rapid synthesis and breakdown of starch in the amyloplast. In this way futile cycles of sucrose/hexose interchange govern fruit sugar content and composition. The major function of the high and constant invertase activity in red tomato fruit is, therefore, to maintain high cellular hexose concentrations, the hydrolysis of sucrose in the vacuole and in the intercellular space allowing more efficient storage of sugar in these compartments. Vacuolar sugar storage may be important in sustaining fruit cell growth at times when less sucrose is available for the sink organs because of exhaustion of the carbohydrate pools in source leaves.     

Calcium-mediated conversion of sucrose to starch in relation to the activities of amylases and sucrose-metabolizing enzymes in sorghum grains raised through liquid culture

Detached ears of sorghum (Sorghum vulgare) were cultured in complete liquid medium containing Ca2+(0, 3, 10 and 30 mM) and effect of this ion on the conversion of sucrose to starch with respect to the activities of amylases, sucrose synthase, sucrose phosphate synthase and soluble invertases were studied in developing grains. Presence of 3 mM Ca2+ in culture medium enhanced both accumulation of starch and activity of alpha-amylase in grain but without having any influence on the activity of beta-amylase. However, with 10 and 30 mM Ca2+, the accumulation of starch and activities of both amylases decreased and with advancement in culturing period, starch accumulation was further decreased. Irrespective of its concentration, Ca2+ enhanced the activities of sucrose synthase (synthesis), sucrose-phosphate synthase, soluble acid invertase and soluble-neutral invertase. Increase in the concentration of Ca2+ in culture medium was concomitant with an elevation in relative proportion of sucrose in the grain reflecting a net balance in per cent increase with Ca2+ in the activities of sucrose-synthesizing enzymes over sucrose-hydrolysing ones. Based on the results, it is suggested that assimilation of Ca2+ by grain is essential for maintaining high activity of alpha-amylase to generate starch primers required for the conversion of sucrose to starch during grain filling in sorghum.     

Kinetic model of sucrose accumulation in maturing sugarcane culm tissue

Biochemically, it is not completely understood why or how commercial varieties of sugarcane (Saccharum officinarum) are able to accumulate sucrose in high concentrations. Such concentrations are obtained despite the presence of sucrose synthesis/breakdown cycles (futile cycling) in the culm of the storage parenchyma. Given the complexity of the process, kinetic modelling may help to elucidate the factors governing sucrose accumulation or direct the design of experimental optimisation strategies. This paper describes the extension of an existing model of sucrose accumulation (Rohwer, J.M., Botha, F.C., 2001. Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data. Biochem. J. 358, 437-445) to account for isoforms of sucrose synthase and fructokinase, carbon partitioning towards fibre formation, and the glycolytic enzymes phosphofructokinase (PFK), pyrophosphate-dependent PFK and aldolase. Moreover, by including data on the maximal activity of the enzymes as measured in different internodes, a growth model was constructed that describes the metabolic behaviour as sugarcane parenchymal tissue matures from internodes 3-10. While there was some discrepancy between modelled and experimentally determined steady-state sucrose concentrations in the cytoplasm, steady-state fluxes showed a better fit. The model supports a hypothesis of vacuolar sucrose accumulation against a concentration gradient. A detailed metabolic control analysis of sucrose synthase showed that each isoform has a unique control profile. Fructose uptake by the cell and sucrose uptake by the vacuole had a negative control on the futile cycling of sucrose and a positive control on sucrose accumulation, while the control profile for neutral invertase was reversed. When the activities of these three enzymes were changed from their reference values, the effects on futile cycling and sucrose accumulation were amplified. The model can be run online at the JWS Online database (http://jjj.biochem.sun.ac.za/database/uys).     

Sucrose Phosphate Synthase and Acid Invertase as Determinants of Sucrose Concentration in Developing Muskmelon (Cucumis melo L.) Fruits

Fruits of orange-fleshed and green-fleshed muskmelon (Cucumis melo L.) were harvested at different times throughout development to evaluate changes in metabolism which lead to sucrose accumulation, and to determine the basis of differences in fruit sucrose accumulation among genotypes. Concentrations of sucrose, raffinose saccharides, hexoses and starch, as well as activities of the sucrose metabolizing enzymes sucrose phosphate synthase (SPS) (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. Sucrose synthase and neutral invertase activities were relatively low (1.7 ± 0.3 micromole per hour per gram fresh weight and 2.2 ± 0.2, respectively) and changed little throughout fruit development. Acid invertase activity decreased during fruit development, (from as high as 40 micromoles per hour per gram fresh weight) in unripe fruit, to undetectable activity in mature, ripened fruits, while SPS activity in the fruit increased (from 7 micromoles per hour per gram fresh weight) to as high as 32 micromoles per hour per gram fresh weight. Genotypes which accumulated different amounts of sucrose had similar acid invertase activity but differed in SPS activity. Our results indicate that both acid invertase and SPS are determinants of sucrose accumulation in melon fruit. However, the decline in acid invertase appears to be a normal function of fruit maturation, and is not the primary factor which determines sucrose accumulation. Rather, the capacity for sucrose synthesis, reflected in the activity of SPS, appears to determine sucrose accumulation, which is an important component of fruit quality.     

Molecular characterization and expression patterns of sucrose transport-related genes in sweet sorghum under defoliation

Sucrose is the principal form of photosynthesis products, and long-distance transport of sucrose requires sucrose transporters (SUTs) to perform loading and unloading functions. SUTs play an important role in plant growth, development and reproduction. In this study, five unique sucrose transporter (SbSUT) genes that contain full-length cDNA sequences were cloned from sweet sorghum, and these SbSUT genes were clustered into four different clades: SUT1, SUT3, SUT4 and SUT5. Heterologous expression of SbSUTs in yeast demonstrated that they were functional sucrose transporters. Tissue-specific expression profiles showed that sorghum SUT genes had different tissue-specific expression patterns, suggesting that sorghum SUT genes may play an important role in plant growth and developmental processes. After defoliation, expression patterns of SbSUT1, SbSUT2 and SbSUT4 were different in leaf sheaths, leaves and roots. Taken together, the results indicate that the above mentioned five unique sucrose transporter genes may play important roles in performing sucrose loading and unloading functions and that they exhibit different expression in response to leaf blade removal.