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 phosphate synthase, a key enzyme for sucrose biosynthesis in plants: protein purification from corn leaves and immunological detection

We have purified the protein for the enzyme sucrose phosphate synthase (SPS) from corn (Zea mays) leaves. Partially purified SPS protein was used to generate specific monoclonal antibodies. The following immunoaffinity chromatography allowed the isolation of pure SPS protein. The apparent molecular mass of the SPS polypeptide is 138 kilodaltons. By immunoblot, an SPS antigen was found to accumulate in mature leaves. SPS protein levels remain constant during the day/night cycle. The observed diurnal fluctuation of extractable enzyme activity, therefore, must be caused by modification of the specific activity of SPS in vivo.     

Rhythmic growth and carbon allocation in Quercus robur. Sucrose metabolizing enzymes in leaves

The high sucrose phosphate synthase (SPS) capacity and the low soluble acid invertase activity of mature leaves of the first flush of leaves remained stable during second flush development. Conversely, fluctuations of sucrose synthase (SS) activity were in parallel with the sucrose requirement of the second flush. Sucrose synthase activity (synthesis direction) in first flush leaves could increase in 'response' to sink demand constituted by the second flush growth. Only the ptotosynthates provided by flush mature leaves were translocated for a current flush, while the starch content of these leaves remained stable. After their emergence, second flush leaves showed an increase in SPS and SS (Synthetic direction) activities. The high sucrose synthesis in second flush leaves was used for leaf expansion. When young leaves were 30% fully expanded (stage II20), SPS activity showed little change whereas SS activity declined rapidly toward and after full leaf expansion. The starch accumulation in the young leaves occured simultaneously with their expansion. Developing leaves showed a high level of acid invertase activity until maximum leaf expansion (stage II1). In first and second flush leaves, changes in acid invertase activity correlated positively with changes in reducing sugar concentrations. Alkaline invertase and sucrose synthase (cleavage direction) activities showed similar changes with low values when compared with those of acid invertase activity, especially in second flush leaves. The present results suggest that soluble acid invertase was the primary enzyme responsible for sucrose catabolism in the expanding common oak leaf.     

Sucrose-Phosphate Synthase,Sucrose Synthase,and Invertase in Sugar Beet Leaves

The activity of sucrose-phosphate synthase (SPS) in sugar beet (Beta vulgaris L.) leaves was shown to exceed considerably the synthesizing activity of sucrose synthase (SS). The rise in SPS activity was related to the daylight period; i.e., it was associated with the rate of photosynthesis. The highest SPS activity was characteristic of fully expanded source leaves. In young developing leaves (leaves expanded to less than half of their final size), which represent the sink organs, the SPS activity was 2.5 times lower. At all stages of leaf development, the synthesizing SS activity was rather low. The diurnal change of SS activity was independent of photosynthesis and showed a slight rise from 6:00–8:00 p.m. Under field conditions, the highest SPS activity was found in leaves in the terminal stage of their development (105-day-old plants); the synthesizing activity of SS showed little changes during this period. The activity of soluble acid invertase was characteristic of young leaves. In mature leaves, the activity of this enzyme correlated with the daylight period. These changes occurred on the background of low sucrose content in leaves. The regulation of SPS, SS, and invertase activity is discussed. It is supposed that compartmentation of these enzymes in the photosynthesizing cell is important for transport, metabolism, and the osmotic function of sucrose in leaves.     

Hormonal Control of Sucrose Phosphate Synthase and Sucrose Synthase in Sugar Beet

We studied the effects of synthetic analogs of phytohormones (benzyladenine, IAA, and GA) on the activities of the enzymes catalyzing sucrose synthesis and metabolism, sucrose phosphate synthase (SPS, EC 2.4.1.14) and sucrose synthase (SS, EC 2.4.1.13), and on the content of chlorophyll and protein during the sugar-beet (Beta vulgaris L.) ontogeny. Plant spraying with phytohormonal preparations activated SPS in leaves; direct interaction between phytohormones and the enzyme also increased its activity. The degree of this activation differed during the ontogeny and in dependence on the compound used for treatment. Analogs of phytohormones maintained high protein level in leaves, retarded chlorophyll breakdown, and, thus, prolonged leaf functional activity during development. Phytohormonal preparations practically did not affect the SS activity both after plant treatment and at their direct interaction with the enzyme. It is supposed that the SS activity in sugar-beet roots is controlled by sucrose synthesized in leaves rather than by phytohormones. The effects of hormones on leaf metabolism were mainly manifested in growth activation.     

Possible control of maize leaf sucrose-phosphate synthase activity by light modulation

Sucrose phosphate synthase (SPS) activity was measured in extracts of maize (Zea mays L.) and soybean (Glycine max L. [Merr.]) leaves over a single day/night cycle. There was a 2- to 3-fold postillumination increase in extractable enzyme activity in maize leaves, whereas the activity of soybean SPS was only about 30% higher in extracts prepared from light- compared to dark-adapted leaves. Alterations in extractable maize leaf SPS activity correlated with light/dark transitions suggesting that the enzyme may be light modulated. Diurnal variations of extractable maize leaf SPS activity were also observed in a greenhouse experiment. A transition from high (light) to low (dark) extractable SPS activity occurred near the light compensation point for photosynthesis (about 20 micromole photons per square meter per second). Further increases in irradiance did not increase extractable SPS activity. Substrate affinities for uridine 5′-diphosphoglucose (Michaelis constant = 3.5 and 5.1 millimolar) and fructose-6 phosphate (half maximal concentration = 1.0 and 2.5 millimolar) were lower for partially purified SPS obtained from light compared to dark acclimated maize leaves. Light-induced changes in extractable SPS activity were stable for at least one column chromatography step. The above results indicate that light-induced changes in SPS activity may be important in controlling the photosynthetic production of sucrose.     

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。     

Light Modulation and Localization of Sucrose Phosphate Synthase Activity between Mesophyll Cells and Bundle Sheath Cells in C(4) Species

Experiments were conducted with several Panicum species (representing the different C₄ subtypes) to examine the light modulation of sucrose phosphate synthase (SPS) activity and the effect of illumination on the distribution of SPS activity between mesophyll cells (MC) and bundle sheath cells (BSC). Activity of SPS in the light decreased in the order: C₄ > C₃-C₄ intermediate > C₃. In illuminated leaves, SPS activities were similar among the three C₄ subtypes, but SPS activity was higher for NAD-malic enzyme (NAD-ME) species with centripetal chloroplasts in BSC (NAD-ME(P) species) than for NAD-ME species with centrifugal chloroplasts in BSC (NAD-ME(F) species). Transfer of plants into darkness for 30 minutes resulted in decreased SPS activity for all species tested except Panicum bisulcatum (C₃ species) and Panicum virgatum (NAD-ME(P) species) which showed little or no change. All C₄ subtypes had some SPS activity both in MC and BSC. In the light, SPS activity was mainly in the MC for NADP-ME, NAD-ME(F) and phosphoenolpyruvate carboxykinase species, while it was mainly in the BSC for NAD-ME(P) species. In the dark, for all C₄ subtypes, SPS activity in the MC was decreased to a greater extent than that in the BSC. It is intriguing that NAD-ME(F) and NAD-ME(P) species differed in the activity and distribution of SPS activity between MC and BSC, although they are otherwise identical in the photosynthetic carbon assimilation pathway. Diurnal changes in SPS activity in the MC and BSC were also examined in maize leaves. SPS activity in the MC in maize leaves was high and relatively constant throughout the middle of the light period, dropped rapidly after sunset and increased again prior to the light period. On the other hand, SPS activity in the BSC was lower and changed more coincidently with light intensity than that in the MC. The results suggested that light activation of SPS activity located in the BSC may require higher irradiance for saturation than the SPS in the MC. We conclude that SPS may function in both MC and BSC for sucrose synthesis in the light, particularly at high light intensity, while in the dark, the major function may be in the BSC during starch degradation.     

Sucrose metabolism of perennial ryegrass in relation to cold acclimation

Sugar metabolism is one of the important factors involved in winter hardiness and since the discovery of sucrose biosynthesis, considerable advances have been made in understanding its regulation and crucial role. This investigation examined the changes in activities of sucrose metabolizing enzymes and sugar content during cold hardening of perennial ryegrass (Lolium perenne L.). Changes in acid invertase (AI), sucrose synthase (SS) and sucrose phosphate synthase (SPS) along with all the three soluble sugars glucose, fructose and sucrose were measured in leaves and stem base tissue during cold acclimation. Although fructans were the predominant carbohydrate the changes in glucose, fructose and sucrose were significant. All the three soluble sugars in both leaf and stem tissues started to decrease from the first day and continued up to day 7 and thereafter started to increase until day 28. AI in the soluble fraction showed a higher activity than that in the cell wall bound fraction. In both the leaf and stem bases soluble AI activity increased during the first week and after that it started to decrease gradually. On the other hand both the SS and SPS increased gradually throughout the acclimation period. Sucrose content was negatively correlated with AI and positively correlated with SS and SPS accounting well for the relation between the substrate and enzyme activity. These results suggest that AI, SS and SPS in ryegrass are regulated by cold acclimation and play an important role in sugar accumulation and acquisition of freezing tolerance.     

Comparative studies of the light modulation of nitrate reductase and sucrose-phosphate synthase activities in spinach leaves

We recently obtained evidence that the activity of spinach (Spinacia oleracea L.) leaf nitrate reductase (NR) responds rapidly and reversibly to light/dark transitions by a mechanism that is strongly correlated with protein phosphorylation. Phosphorylation of the NR protein appears to increase sensitivity to Mg²⁺ inhibition, without affecting activity in the absence of Mg²⁺. In the present study, we have compared the light/dark modulation of sucrose-phosphate synthase (SPS), also known to be regulated by protein phosphorylation, and NR activities (assayed with and without Mg²⁺) in spinach leaves. There appears to be a physiological role for both enzymes in mature source leaves (production of sucrose and amino acids for export), whereas NR is also present and activated by light in immature sink leaves. In mature leaves, there are significant diurnal changes in SPS and NR activities (assayed under selective conditions where phosphorylation status affects enzyme activity) during a normal day/night cycle. With both enzymes, activities are highest in the morning and decline as the photoperiod progresses. For SPS, diurnal changes are largely the result of phosphorylation/dephosphorylation, whereas with NR, the covalent modification is super-imposed on changes in the level of NR protein. Accumulation of end products of photosynthesis in excised illuminated leaves increased maximum NR activity, reduced its sensitivity of Mg²⁺ inhibition, and prevented the decline in activity with time in the light seen with attached leaves. In contrast, SPS was rapidly inactivated in excised leaves. Overall, NR and SPS share many common features of control but are not identical in terms of regulation in situ.     

Companion-Cell Specific Localization of Sucrose Synthase in Zones of Phloem Loading and Unloading

An immunohistochemical approach was used in maize (Zea mays) and citrus (Citrus paradisi) to address the previously noted association between sucrose synthase and vascular bundles and to determine the localization of the low but detectable levels of sucrose synthase that remain in leaves after the import-export transition. Sucrose synthase protein was immunolocalized at the light microscope level using paraffin sections reacted with rabbit sucrose synthase polyclonal antisera and gold-conjugated goat anti-rabbit immunoglobulin G. Immunolabel was specifically observed in phloem companion cells of minor and intermediate veins in mature leaves of both species. Similar localization was apparent in the midrib of mature citrus leaves, with additional labeling in selected files of phloem parenchyma cells. A clear companion-cell specificity was evident in the phloem unloading zone of citrus fruit, where high activity of sucrose synthase has been demonstrated in vascular bundles during periods of rapid import. Sucrose synthase protein was not associated with adjacent cells surrounding the vascular strands in this tissue. The companion-cell specificity of sucrose synthase in phloem of both importing and exporting structures of these diverse species implies that this may be a widespread association and underscores its potential importance to the physiology of vascular bundles.     

Effects of Elevated Sucrose-Phosphate Synthase Activity on Photosynthesis,Assimilate Partitioning,and Growth in Tomato (Lycopersicon esculentum var UC82B)

The expression of a sucrose-phosphate synthase (SPS) gene from maize (Zea mays, a monocotyledon) in tomato (Lycopersicon esculentum, a dicotyledon) resulted in marked increases in extractable SPS activity in the light and the dark. Diurnal modulation of the native tomato SPS activity was found. However, when the maize enzyme was present the tomato leaf cells were unable to regulate its activation state. No detrimental effects were observed and total dry matter production was unchanged. However, carbon allocation within the plants was modified such that in shoots it increased, whereas in roots it decreased. There was, therefore, a change in the shoot:root dry weight ratio favoring the shoot. This was positively correlated with increased SPS activity in leaves. SPS was a major determinant of the amount of starch in leaves as well as sucrose. There was a strong positive correlation between the ratio of sucrose to starch and SPS activity in leaves. Therefore, SPS activity is a major determinant of the partitioning of photosynthetically fixed carbon in the leaf and in the whole plant. The photosynthetic rate in air was not significantly increased as a result of elevated leaf SPS activity. However, the light- and CO2-saturated rate of photosynthesis was increased by about 20% in leaves expressing high SPS. In addition, the temporary enhancement of the photosynthetic rate following brief exposures to low light was increased in the high SPS plants relative to controls. We conclude that the level of SPS in the leaves plays a pivotal role in carbon partitioning. Furthermore, high SPS levels have the potential to boost photosynthetic rates under favorable conditions.     

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。     

Regulation of Maize Leaf Nitrate Reductase Activity Involves Both Gene Expression and Protein Phosphorylation

Nitrate reductase (NR; EC 1.6.6.1) activity increased at the beginning of the photoperiod in mature green maize (Zea mays L.) leaves as a result of increased enzyme protein level and protein dephosphorylation. In vitro experiments suggested that phosphorylation of maize leaf NR affected sensitivity to Mg2+ inhibition, as shown previously in spinach. When excised leaves were fed 32P-labeled inorganic phosphate, NR was phosphorylated on seryl residues in both the light and dark. Tryptic peptide mapping of NR labeled in vivo indicated three major 32P-phosphopeptide fragments, and labeling of all three was reduced when leaves were illuminated. Maize leaf NR mRNA levels that were low at the end of the dark period peaked within 2 h in the light and decreased thereafter, and NR activity generally remained high. It appears that light signals, rather than an endogenous rhythm, account primarily for diurnal variations in NR mRNA levels. Overall, regulation of NR activity in mature maize leaves in response to light signals appears to involve control of gene expression, enzyme protein synthesis, and reversible protein phosphorylation.     

不同反光膜对设施葡萄光合特性和叶片糖代谢的影响

以‘京秀’葡萄为试材,研究了蓝膜、红膜、铝膜3种反光膜对设施葡萄光合特性和叶片糖代谢的影响。结果显示:红膜和蓝膜处理的葡萄叶片的净光合速率均显著高于对照(不铺设反光膜);在果实发育前期,各处理的叶绿素含量和叶绿素b/a值均大于对照,而后期均低于对照;蓝膜和红膜处理叶片的蔗糖含量较高,而蓝膜处理叶片中的蔗糖合酶(SS)和蔗糖磷酸合酶(SPS)活性最高;各铺膜处理均比对照提高了葡萄果实鲜量和可溶性糖含量、降低可滴定酸含量,且蓝膜处理显著优于红膜和铝膜。研究表明,在设施栽培条件下,地面铺设蓝色反光膜可显著提高葡萄叶片的光合速率和叶片中碳水化合物的关键合成酶活性,并显著提高葡萄果实品质。     

Phosphate Deficiency in Maize. IV. Changes in Amounts of Sucrose Phosphate Synthase during the Course of Phosphate Deprivation

With low-P treatment of maize, the level of sucrose phosphatesynthase (SPS) protein decreased to 15% of the control, whilethe "V_max" activity stayed relatively high (100–80% ofthe control) and the substrate limiting activity increased about2 fold in the leaves. These results suggest that leaf phosphatestatus has dual effects on the amount of SPS protein and theactivation state of SPS. (Received January 20, 1992; Accepted April 14, 1993)     

Isolation and characterization of multiple forms of maize leaf sucrose-phosphate synthase

Sucrose-phosphate synthase SPS; (EC 2.4.1.14) from maize (Zea mays L. cv. Pioneer 3184) leaves was partially purified and kinetically characterized. Maize SPS was activated by glucose-6-phosphate (G-6-P) due to an increase in Vmax and a decrease in the Km for UDP-glucose. The UDP-glucose saturation profile was biphasic; thus two Km values for UDP-glucose were calculated. Inhibition by inorganic phosphate was observed only in the presence of G-6-P. Chromatography of partially purified maize leaf extracts on hydroxyapatite resolved two forms of SPS activity, which differed in their affinity for UDP-glucose and in the degree of activation by G-6-P. SPS was partially purified from maize leaves that were harvested in the light and in the dark. The light enzyme had a higher specific activity than the enzyme isolated from dark harvested leaves, and this difference persisted during enzyme purification. The apparent molecular weight (Stokes radius) of the light enzyme was 547 kDa, which was greater than that of the dark enzyme (457 kDa). Light and dark SPS differed in their affinities for UDP-glucose in the absence G-6-P. Both the light and the dark SPS were activated by G-6-P; the Km for UDP-glucose of the light enzyme was lowered by G-6-P, while the Km for UDP-glucose for the dark enzyme remained unchanged. These results suggest that light activation involves a conformational change that results in differences in maximum velocity, substrate affinities and regulation by metabolites. Chromatography of either the light or dark SPS on hydroxyapatite yielded two peaks of enzyme activity, suggesting that the occurrence of the two activity peaks was not due to an interconversion of the light and dark forms.     

Regulation of sucrose phosphate synthase by gibberellins in soybean and spinach plants

Exogenous applications of gibberellins (GAs) increased the extractable activity of leaf sucrose phosphate synthase (SPS) in soybean (Glycine max [L.]) and spinach (Spinacia oleracea [L.]). The response to GA applications was detectable within 2 h postapplication and was still observed 6 h, 24 h, and 7 d after treatment. When paclobutrazol, a GA biosynthesis inhibitor, was applied to intact soybean and spinach plants, decreased extractable SPS activity resulted within 24 h following the treatment. Different methods of GA application (spray, injection, capillary wick, and excised leaf systems) produced similar effects on SPS activity of soybean leaves. Protein synthesis in soybean leaves appeared to be necessary for GA-promoted SPS activity because gibberellic acid only partially reversed the inhibitory effect of pretreatment with cycloheximide. Levels of SPS protein from crude extracts of spinach plants were measured by a dot blot technique using monoclonal antibodies against SPS. Application of gibberellic acid to spinach leaves increased levels of SPS protein 2 h, 24 h, and 7 d after treatment. The results suggest that, in both soybean and spinach, GA is one of the endogenous hormonal factors that regulate the steady-state level of SPS protein and, hence, its activity.