Suppression of Acid Invertase Activity by Antisense RNA Modifies the Sugar Composition of Tomato Fruit

cDNA for an acid invertase (EC 3.2.1.26 [EC] ) of tomato (Lycopersiconesculentum Mill.) fruit was introduced into tomato plants underthe control of the cauliflower mosaic virus 35S promoter inthe antisense orientation. The antisense gene effectively suppressedthe invertase activity in soluble and cell wall fractions fromripening fruits. The sucrose content of fruits of the transformantswas markedly increased, while the hexose content was reduced.These results indicate that acid invertase is one of main determinantsof the sugar composition of tomato fruit. The invertase activityin the cell wall fraction of the leaf tissues of the transformantswas not suppressed to the same extent as that in the solublefraction. Wounding of the control leaf tissues induced invertaseactivity in both soluble and cell wall fractions. The inductionof activity in the soluble fraction was suppressed by the antisensegene, while that in the cell wall fraction was unaffected. Thesefindings suggest that mRNA for some other invertase, in particular,the mRNA for a cell wall-bound invertase, was present in leaves. ¹Present address: Plant Breeding and Genetics Research Laboratory,Japan Tobacco Inc., 700 Higashibara, Toyoda, Iwata, Shizuoka,438 Japan. ²Present address: National Institute of Agrobiological Resources,Kannondai, Tsukuba, Ibaraki, 305 Japan.     

转化酶和己糖激酶调控草莓聚合果内糖积累

以设施栽培的草莓品种‘枥乙女’为试材,用高效液相色谱法分析了果实发育进程中草莓聚合果内不同部位糖含量及相关酶活性的变化。果实成熟过程中草莓聚合果果顶部分含糖量高,中间部位次之,果柄端最低。转化酶活性呈现与糖含量相似的梯度变化。己糖激酶和果糖激酶则表现出与糖梯度相反的变化。上述结果表明,聚合果顶端转化酶活性高,有利于形成蔗糖梯度,从而促进光合产物向果顶端转移;聚合果近果柄端的己糖代谢酶活性高,促进果柄端的己糖消耗,导致果柄端相对低的糖含量。     

Modulation of Allosteric Regulation by E38K and G101N Mutations in the Potato Tuber ADP-glucose Pyrophosphorylase

The higher plant ADP-glucose (ADPG) pyrophosphorylase (AGPase), composed of two small subunits and two large subunits (LSs), produces ADPG, the sole substrate for starch biosynthesis from α-D-glucose 1-phosphate and ATP. This enzyme controls a key step in starch synthesis as its catalytic activity is activated by 3-phosphoglycerate (3-PGA) and inhibited by orthophosphate (Pi). Previously, two mutations in the LS of potato AGPase (PLS), PLS-E38K and PLS-G101N, were found to increase sensitivity to 3-PGA activation and tolerance to Pi inhibition. In the present study, the double mutated enzyme (PLS-E38K/G101N) was evaluated. In a complementation assay of ADPG synthesis in an Escherichia coli mutant defective in the synthesis of ADPG, expression of PLS-E38K/G101N mediated higher glycogen production than wild-type potato AGPase (PLS-WT) and the single mutant enzymes, PLS-E38K and PLS-G101N, individually. Purified PLS-E38K/G101N showed higher sensitivity to 3-PGA activation and tolerance to Pi inhibition than PLS-E38K or PLS-G101N. Moreover, the enzyme activities of PLS-E38K, PLS-G101N, and PLS-E38K/G101N were more readily stimulated by other major phosphate-ester metabolites, such as fructose 6-phosphate, fructose 2,6-bisphosphate, and ribose 5-phosphate, than was that of PLS-WT. Hence, although the specific enzyme activities of the LS mutants toward 3-PGA were impaired to some extent by the mutations, our results suggest that their enhanced allosteric regulatory properties and the broadened effector selectivity gained by the same mutations not only offset the lowered enzyme catalytic turnover rates but also increase the net performance of potato AGPase in vivo in view of increased glycogen production in bacterial cells.     

温州蜜柑果实发育进程中光合产物运输分配及糖积累特性

测定了温州蜜柑果实发育进程中糖的积累动态,并用14C示踪方法研究了果实发育不同阶段源叶的光合产物在果实中的运输分配特性.结果表明(1)果实组织中的糖含量随果实发育持续上升,果皮以积累己糖为主而其它组织则以积累蔗糖为主或略多.(2)在果实完熟之前,汁囊分配到的光合产物均占果实的50%以上,为果实中的光合产物分配中心;而到完熟阶段分配到汁囊的14C光合产物与其它组织接近,不再有明显的分配中心.(3)汁囊中的14C放射性比活度随着果实发育呈下降趋势,尤其在后期下降极为明显.(4)维管束/囊瓣表皮和囊瓣表皮/汁囊的14C放射性比活度的比率都随果实发育进程而增大,但后者的比率要大大高于前者,表明韧皮部后运输是光合产物进入汁囊的限速阶段.(5)输入到汁囊的14C光合产物主要以蔗糖形式存在,说明光合产物是以蔗糖为主体输入汁囊参与糖积累和代谢的.     

百合扦插鳞片控温成球过程中4种物质含量的动态变化

以东方型（火百合Stargazer品种）、亚洲型（新中心Nove Cento品种）和麝香型（新铁炮LillHmformo-longii品种）百合鳞片为试验材料,连续在25℃保留8周、17℃保留4周、4～5℃保留8周后分析温控成球过程中鳞片淀粉、总糖、还原糖和蛋白质等含量的动态变化。结果表明：3个品种百合鳞片在控温成球过程中,4种物质的变化与鳞片控温成球处理的不同阶段有密切关系,火百合品种在整个温控成球过程中所测的4种物质含量变化较平缓,而新中心和新铁炮2个品种在整个温控成球过程中4种物质含量变化较大。     

Two Paralogous Genes Encoding Small Subunits of ADP-glucose Pyrophosphorylase in Maize, <Emphasis Type="Italic">Bt2</Emphasis> and <Emphasis Type="Italic">L2</Emphasis>, Replace the Single Alternatively Spliced Gene Found in Other Cereal Species

Two types of gene encoding small subunits (SSU) of ADP-glucose pyrophosphorylase, a starch-biosynthetic enzyme, have been found in cereals and other grasses. One of these genes encodes two SSU proteins. These are targeted to different subcellular compartments and expressed in different organs of the plant: the endosperm cytosol and the leaf plastids. The SSU gene encoding two proteins evolved from an ancestral gene encoding a single protein by the acquisition of an alternative first exon. Prior to the work reported here, this type of SSU gene had been found in all grasses examined except maize. In maize, two separate genes, Bt2 and L2, were known to have the same roles as the alternatively spliced gene found in other grasses. The evolutionary origin of these maize genes and their relationship to the SSU genes in other grasses were unclear. Here we show that Bt2 and L2 are paralogous genes that arose as a result of the tetraploidization of the maize genome. Both genes derive from an ancestral alternatively spliced SSU gene orthologous to that found in other grasses. Following duplication, the Bt2 and L2 genes diverged in function. Each took a different one of the two functions of the ancestral gene. Now Bt2 encodes the endosperm cytosolic SSU but does not contribute significantly to leaf AGPase activity. Similarly, L2 has lost the use of one of its two alternative first exons. It can no longer contribute to the endosperm cytosolic SSU but is probably responsible for the bulk of the leaf AGPase SSU. Reviewing Editor: Dr. Patrick Keeling Sequences have been deposited in GenBank under accession numbers AY727927, DQ118037, and DQ118038.     

Modification of maize starch by thermal processing in glacial acetic acid

Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) methods were used to determine if corn starch–glacial acetic acid mixtures can be melted and thermally processed at reasonable temperatures. DSC studies showed that the melting temperature of dry starch was reduced from about 280 to 180°C in the presence of >30% acetic acid. Glass transition temperatures varied from 110 to 40°C at 15 and 45% acetic acid, respectively. XRD showed the loss of native starch crystallinity and the formation of V-type complexes. Addition of 10% water decreased the melting temperatures to 140–150°C while addition of a base (sodium acetate) had little effect. Some possible applications of processing starch in glacial acetic acid will be discussed.     

Increased fatty acid production in potato by engineering of acetyl-CoA carboxylase

In contrast to oil seeds, potato (Solanum tuberosum L.) is characterized by a high amount of starch stored in the tubers. To assess the capacity for oil synthesis in potato tubers, the changes in lipid content and flux into lipid synthesis were explored in transgenic potatoes altered in carbohydrate or lipid metabolism. A strong decrease in the amount of starch observed in antisense lines for ADP-glucose pyrophosphorylase or plastidic phosphoglucomutase had no effect on storage-lipid content. Similarly, potato lines over-expressing the Arabidopsis thaliana (L.) Heynh. plastidic ATP/ADP transporter that contained an increased amount of starch were not altered in oil content, indicating that the plastidic ATP level is not limiting fatty acid synthesis in potato tubers. However, over-expression of the acetyl-CoA carboxylase from Arabidopsis in the amyloplasts of potato tubers led to an increase in fatty acid synthesis and a more than 5-fold increase in the amount of triacylglycerol. Taken together, these data demonstrate that potato tubers have the capacity for storage-lipid synthesis and that malonyl-CoA, the substrate for elongation during fatty acid synthesis, represents one of the limiting factors for oil accumulation.Abbreviations AATP Plastidic ADP/ATP transporter - ACCase Acetyl-CoA:carboxylase - DGAT Acyl-CoA:diacylglycerol acyltransferase - FW Fresh weight - TLC Thin-layer chromatography - WT Wild typeSource for transgenic plant material. Upon request, transgenic potato lines altered in ACCase activity can be obtained from Peter Dörmann. For potato lines with alterations in AATP transporter activity, please refer to H. Ekkehard Neuhaus. Transgenic AGP and PGM lines are available from A. Fernie (Max-Planck-Institute of Molecular Plant Physiology, Golm, Germany).     

甜菜(Beta vulgaris L.)叶绿体转化体系建立及抗虫和抗除草剂植株的获得

为建立外源基因甜菜叶绿体转化体系,利用分子生物学方法构建了包含有编码苏云金芽孢杆菌晶体蛋白基因Bt cry1Ac和编码膦丝菌素乙酰转移酶基因bar的甜菜叶绿体转化载体pSKARBt/bar,以甜菜叶绿体基因组中atpB/rbcL做同源片段,以甜菜叶绿体16S启动子和终止子为调控基因,以bar基因为筛选标记基因．基因枪法转化甜菜叶柄,经筛选获得抗性转基因植株．对转基因植株进行外源基因Bt cry1Ac和bar的PCR检测、DNA印迹分析,结果表明：外源基因Bt cry1Ac和bar确已导入到甜菜叶绿体基因组中．转基因植株除草剂抗性鉴定及其离体叶片虫试鉴定结果表明：转基因植株具有较强的杀虫活性和抗除草剂特性,表达了相应的蛋白质．研究结果还表明：bar基因在植物叶绿体转化中,既可以用作抗性基因,又可用作转化体筛选的标记基因．建立了甜菜叶绿体转化体系．     

Detection of barbiturate-induced surface changes in cerebrocortical synaptosomes by phase partitioning in an aqueous two-phase system

The barbiturates tested in this work (barbital, phenobarbital, thiopental and pentobartial) modify the partition of synaptosomes in a Dextran T500-poly(ethylene glycol) 4000 two-phase system. Under adequate experimental conditions, the drugs increase the partition into the upper phase and this effect appears to be due to an action on the biological material and not on the interface potential of the system. This conclusion can be drawn from the fact that synaptosomes preincubated with low concentrations (0.1 mM) of barbital and pentobarbital maintained an increased partition into the upper phase. The extent of the effect observed appeared to be inversely proportional to the hydrophobicity of the drugs since phenobarbital and barbital showed a higher effect than thiopental and pentobarbital. Dithionite-induced anoxia, rotenone and ouabain also induced a similar increase of partition of synaptosomes into the upper phase, suggesting that the surface changes detected by phase partitioning modification of synaptosomes could be somehow related to the bioenergetic maintenance of the membrane ATPase.     

Expression of an <Emphasis Type="Italic">Escherichia coli</Emphasis> phosphoglucomutase in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) results in minor changes in tuber metabolism and a considerable delay in tuber sprouting

The aim of this work was to evaluate the influence of elevating the cytosolic activity of phosphoglucomutase (PGM; EC 5.4.2.2) on photosynthesis, growth and heterotrophic metabolism. Here we describe the generation of novel transgenic plants expressing an Escherichia coli phosphoglucomutase (EcPGM) under the control of the 35S promoter. These lines were characterised by an accumulation of leaf sucrose, despite displaying no alterations in photosynthetic carbon partitioning, and a reduced tuber starch content. Determinations of the levels of a wide range of other metabolites revealed dramatic reductions in maltose and other sugars in leaves of the transformants, as well as a modification of the pattern of organic and amino acid content in tubers of these lines. Intriguingly, the transgenics also displayed a dramatically delayed rate of sprouting and significantly enhanced rate of respiration, however, it is important to note that the severity of these traits did not always correlate with the level of transgene expression. These results are discussed in the context of current understanding of the control of respiration and the breaking of tuber dormancy.     

Antisense inhibition of sorbitol synthesis leads to up-regulation of starch synthesis without altering CO<Subscript>2</Subscript> assimilation in apple leaves

Sorbitol is a primary end-product of photosynthesis in apple (Malus domestica Borkh.) and many other tree fruit species of the Rosaceae family. Sorbitol synthesis shares a common hexose phosphate pool with sucrose synthesis in the cytosol. In this study, Greensleeves apple was transformed with a cDNA encoding aldose 6-phosphate reductase (A6PR, EC 1.1.1.200) in the antisense orientation. Antisense expression of A6PR decreased A6PR activity in mature leaves to approximately 15–30% of the untransformed control. The antisense plants had lower concentrations of sorbitol but higher concentrations of sucrose and starch in mature leaves at both dusk and predawn. ¹⁴CO₂ pulse-chase labeling at ambient CO₂ demonstrated that partitioning of the newly fixed carbon to starch was significantly increased, whereas that to sucrose remained unchanged in the antisense lines with decreased sorbitol synthesis. Total activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), sucrose-phosphate synthase (EC 2.4.1.14), and ADP-glucose pyrophosphorylase (EC 2.7.7.27) were not significantly altered in the antisense lines, whereas both stromal and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activities were higher in the antisense lines with 15% of the control A6PR activity. Concentrations of glucose 6-phosphate and fructose 6-phosphate (F6P) were higher in the antisense plants than in the control, but the 3-phosphoglycerate concentration was lower in the antisense plants with 15% of the control A6PR activity. Fructose 2, 6-bisphosphate concentration increased in the antisense plants, but not to the extent expected from the increase in F6P, comparing sucrose-synthesizing species. There was no significant difference in CO₂ assimilation in response to photon flux density or intercellular CO₂ concentration. We concluded that cytosolic FBPase activity in vivo was down-regulated and starch synthesis was up-regulated in response to decreased sorbitol synthesis. As a result, CO₂ assimilation in source leaves was sustained at both ambient CO₂ and saturating CO₂.     

番茄ACC合成酶cDNA克隆及其对果实成熟的反义抑制

利用RT—PcR技术克隆了ACC合成酶多基因家族成员之－LE-ACC2编码区约1．7kb的cDNA,经酶切图谱和序列分析鉴定无误后,反向插入到植物表达载体pBin437中,构建了表达Acc合成酶反望RNA的二元载体。经农杆菌途径转化番茄“丽春”品种后,通过PCR检测从抗卡那霉素再生植株中筛选到6株转基因植株,Southern杂交确证了外源基因是以单拷贝插入到番茄染色体中;对果实乙烯释放的测定结果表明转基因番茄果实的乙烯释放量仅为对照的30％左右,在室温下转基因番茄果实采后保存60 d以上仍然没有变红、软化。以上结果表明其反义RNA在转基因番茄中的表达能有效地抑制乙烯的生物合成从而延缓果实成熟,表现出良好的耐储保鲜特性。对转基因植株子一代(T1)的分析结果进一步表明反义ACC合成酶基因以典型的单基因方式传到子代。通过对子二代的分析已初步筛选到一 个耐储藏的转基因番茄纯合品系。     

通过表达ACC脱氨酶基因控制番茄果实的成熟

乙烯在跃变型果实的成熟过程中起着触发呼吸跃变和促进果实成熟的作用。细菌来源的1-氨基环丙烷-1-羧酸(ACC)脱氨酶能降解乙烯的直接前体ACC,从而抑制植物体内乙烯的合成。我们用PCR方法从假单孢杆菌中克隆到ACC脱氨酶基因并通过农杆菌介导的方法将其转入番茄(Lycopersicun esculentum)中。再生植株经Southern blot检测证明,ACC脱氨酶基因已整合到番茄基因组中并稳定表达。转基因番茄果实成熟期的推迟时间与体内乙烯的抑制程度有相关性。转基因番茄植株乙烯的合成降低80%左右,果实在离体条件下可保鲜75d左右。研究ACC脱氢酶基因在植物体内的作用可阐明高等植物体内乙烯的作用机理并为培育耐贮藏果蔬品种打下基础。     

Sugar Composition of Nectars and Fruits Consumed by Birds and Bats in the Tropics and Subtropics1

Several characteristics of flowers and fruits have been suggested as comprising syndromes of characters that indicate particular classes of pollinators and fruit dispersers. Common phylogenetic history among species, however, may also significantly influence these characters and obscure or enhance perceived patterns of plant syndromes. We analyzed the proportions of glucose, fructose, and sucrose by paper chromatography in the nectar and fruit juice of 525 tropical and subtropical plant species to test whether sugar chemistry was correlated with volant vertebrate pollinator or fruit disperser classes. Samples were taken from Old World and New World species and the calculations kept separate. Kruskal-Wallis tests of family means showed significant deviations in the percent sucrose content among pollinatorl disperser classes. Mann-Whitney U-tests showed significant differences among nectars of all pollinator classes but fruit juices differed only due to the high sucrose content of megachiropteran dispersed fruits. In addition, sign tests of samples occurring within families showed significant correlations between percentage sucrose content and pollinator/disperser classes. Passerine nectars had low sucrose content. In striking contrast, the nectar of hummingbird flowers had very high sucrose content. The Microchiroptera nectars showed hexose richness with a sucrose content somewhat greater than that of passerine flowers. Megachiroptera flowers showed sucrose-rich nectars. The results for fruits were comparable to those for nectars. Passerine fruits were hexose dominated, microchiropteran fruits had a sucrose content similar to passerine fruits, and megachiropteran fruits were sucrose-rich. We speculate on the evolutionary sequence of changes in nectar and fruit juice sugar composition and suggest that future investigations consider the chemistry of other food sources such as pollen and leaves. Only with these additions and other ecological studies can the full interplay of such plant-animal interactions be anticipated.     

Suppression of Aedes aegypti by predatory Toxorhynchites moctezuma in an island habitat

Larval populations of the mosquito Aedes aegypti were suppressed by predatory Toxorhynchites moctezuma mosquito larvae released systematically in a village on Union Island (Saint Vincent and the Grenadines) during March-December 1988. Eggs and larvae of Tx.moctezuma were transported from Trinidad and introduced into all semi-permanent and permanent water-holding containers in the experimental village at Clifton. The semi-isolated village of Ashton served as control. Base-line Ae.aegypti indices (house, ovitrap, Breteau, cistern/tank, drum/barrel, small containers) were obtained for the two villages over a 4-month period prior to the introduction of the predatory Tx.moctezuma mosquito larvae. After sustained releases of predators for 5 months, all indices of Ae.aegypti were lower in the treated village than in the untreated village during the last 3 months of the year.     

Characterisation of anaerobic mixed cultures by partition in an aqueous two-phase system

A method to characterise a dynamic microbial consortium is described. By exploiting differences in surface properties between different cells and between cells of different physiological status, it was possible to develop a partition pattern for a mixed culture under different conditions. The separation method used was partition in aqueous two-phase systems and when using a counter current extraction process one could clearly differentiate the partition profile between resting, active and overloaded biomethanation cultures.