Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds II. Scutellum as the Site of Sucrose Synthesis

In a close parallel to the developmental pattern of α-amylase activity, a rapid increase of maltase activity occurred in the endosperm tissue of germinating rice seeds after about 4 days of the seed imbibition. The overall pattern of the 2 hydrolytic enzyme activities strongly suggest that amylolytic breakdown is the major metabolic route of starch utilization in the germinating rice seeds. Results of the chemical analyses of sugar constituents as well as the measurements of sucrose synthetase activity show that the scutellum is the site of sucrose synthesis in the germinating rice seeds. It is thus supported that glucose derived from the reserve starch in endosperm is transported to scutellum, where it is converted to sucrose. Sucrose is further mobilized to the growing tissues, shoots and roots.     

Characterization of a rice sucrose-phosphate synthase-encoding gene

A rice genomic clone (spsl) coding for sucrose phosphate synthase (SPS) was isolated and sequenced. Rice spsl contains 13 exons and 12 introns, an unusually long 366-bp leader region with a highly organized primary structure and a promoter region with no obvious homology with eukaryotic promoter consensus sequences. Southern blot analysis showed that SPS is encoded by a single-copy gene in the rice genome. Comparison of the rice, maize, potato and spinach SPS deduced amino acid (aa) sequences showed that these enzymes have a well conserved region comprising their first 700 aa, and a variable C-terminal region. Analysis of rice spsl expression showed that mRNA levels change during leaf development. SPS activity and mRNA were undetectable in roots.     

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.     

Growth rate,photosynthetic activity,and leaf development of Brazil pine seedlings (Araucaria angustifolia[Bert.] O. Ktze.)

Seedlings of Brazil pine, a large-seeded South American conifer, were grown in a climate chamber to investigate vertical growth pattern and the time course of leaf development. We examined shoot growth, photosynthetic performance and markers of leaf maturation such as contents of soluble sugars and activities of sucrose-phosphate synthase (SPS), neutral invertase (nI) and sucrose synthase (Susy). The daily increment of shoot length showed an optimum curve during the first 70 days after germination. The low growth rate during the first 20 days of development correlated with net CO² emission of the seedling. Analyses of leaf maturation markers in older seedlings revealed low sucrose/hexose ratios and high activities of nI and Susy in the uppermost leaves. Although the SPS/Susy ratio was low in these leaves the extractable SPS activity did not change significantly among leaves of different age. The photosynthetic light compensation points of young leaves were about 2-fold higher than those of mature leaves and their photosynthetic capacity was only 50% as high. Our results indicate that a rapid maturation of leaves of Brazil pine seedlings may reduce the respiratory loss of carbohydrates and that the mobilisation of seed storage compounds supports initial shoot growth under light-limiting conditions which may occur in the forest-grassland succession zone.     

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.     

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.     

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.     

棉纤维蔗糖合酶基因SS3上游调控序列的克隆及其表达分析

棉纤维蔗糖合酶基因SS3在棉纤维发育过程中起着重要作用.采用YADE技术克隆了该基因5′上游1717bp的调控区,该调控区含有典型的启动子核心元件TATA box ,以及TATC box、G box、GCN4 -motif、Prolamin box、Skn 1 likemotif、TCA element、HSE和O2 site等各种顺式调控元件和其他一些反应元件.将此序列和报告基因GUS融合在烟草、棉花中表达.组织化学分析结果显示棉花SuSyR序列启动GUS基因在烟草的子房、胎座、种子以及在棉花花蕾与棉铃中表达.在棉花花蕾蕾长为3mm、6mm、9mm和15mm花蕾中表达主要存在于雄蕊及雄蕊管、胎座等器官;在棉铃中,1DPA棉铃的花柱、花药、子房及胚珠中出现了蓝色,6DPA棉铃的子房及胚珠被染成蓝色,在2 0DPA的棉铃中蓝色只出现在胚珠及其纤维中、在胚珠中只有珠心被染成蓝色,在4 0DPA胚珠中只有纤维呈蓝色.研究结果揭示,棉花的SuSyR调控序列启动GUS基因主要在子房、胚珠和纤维等器官和主叶脉、茎微管束等输导组织中表达,在棉花中尤为明显,表明棉纤维蔗糖合酶基因SS3除参与棉花蕾铃发育、纤维素的合成外,还参与了光合产物的运输与分配过程.     

Sequence-specific interaction between S1F, a spinach nuclear factor, and a negative cis-element conserved in plastid-related genes.

The nuclear gene rps1 coding for the spinach plastid ribosomal protein CS1 exhibits both a constitutive and leaf-specific expression pattern. In contrast to other chloroplast-related genes like rbcS and cab, the leaf induction of rps1 expression is light-independent. These unique features of rps1 expression provide good models to study the mechanisms regulating plastid development and differentiation in higher plants. We report on the identification of a spinach leaf nuclear factor, designated S1F, interacting with the rps1 promoter. The S1F binding site is conserved in the promoter region of many plastid-related genes, including rbcS, cab, and rpl21. A binding activity similar to S1F was detected in nuclear extract from dark-grown de-differentiated soybean suspension cells. Through site-specific mutagenesis and transient expression in soybean cell protoplasts, we show that the S1F binding site is a negative element down-regulating the promoter activity of rps1. A ligated tetramer of S1F site was able to repress activity of the cauliflower mosaic virus 35 S promoter extending the negative function of the S1F binding site on promoter activity.