Modification of Sugar Composition in Strawberry Fruit by Antisense Suppression of an ADP-glucose Pyrophosphorylase

To modulate the soluble sugar content of strawberry fruits, we generated transgenic plants that incorporated an antisense cDNA of ADP-glucose pyrophosphorylase (AGPase) small subunit (FagpS) under the control of the strawberry fruit-dominant ascorbate peroxidase (APX) promoter (cv. Anther). Several independent transgenic lines were obtained and grown in the greenhouse for analysis of agronomic traits. Most transgenic fruit did not show significant differences in weight and hardness compared to control fruit. However, the starch content in fruit was decreased to 27–47% and the total soluble sugar content was increased to 16–37% in transgenic plants (analyzed by the HPLC of sugar composition at four different stages of fruit development). The sugar contents of fruits in transgenic lines were particularly higher than control fruits at the red stage. The results were consistent with northern analysis, which showed that the levels of AGPase mRNA drastically were reduced in the red stage of fruits in all the transgenic plants. In other tissues of transgenic plants, the FagpS mRNA expression level was similar to control plants. Our studies indicate that fruit-specific down-regulation of the AGPase gene might be an effective strategy for increasing sugar and decreasing starch content in strawberry.     

Effect of Antisense Suppression of Endopolygalacturonase Activity on Polyuronide Molecular Weight in Ripening Tomato Fruit and in Fruit Homogenates

Fruit of tomato (Lycopersicon esculentum Mill.) in which endopolygalacturonase (PG) activity had been suppressed to <1% of wild-type levels were slightly firmer than nontransgenic controls later in ripening. Enzymically inactive cell walls were prepared from these ripening fruit using Tris-buffered phenol. When extracted with chelator followed by Na2CO3, the amounts of pectin solubilized from cell walls of nontransgenic control or from transgenic antisense PG fruit were similar. Size-exclusion chromatography analysis showed that, relative to controls, in antisense PG fruit polyuronide depolymerization was delayed in the chelator-soluble fraction throughout ripening and reduced in the Na2CO3-soluble fraction at the overripe stage. Reduced pectin depolymerization rather than altered extractability thus may have contributed to enhanced fruit firmness. Substantially larger effects of suppressed PG activity were detected in tomato fruit homogenates processed to paste. In control paste the majority of the polyuronide was readily soluble in water and was very highly depolymerized. In antisense PG paste the proportion of polyuronide solubilized by water was reduced, and polyuronides retained a high degree of polymerization. The suppression of fruit PG activity thus has a small effect on polyuronide depolymerization in the fruit but a much larger effect in paste derived from these fruit. This indicates that in the cell wall PG-mediated degradation of polyuronide is normally restricted but that in tissue homogenates or in isolated cell walls this restriction is removed and extensive pectin disassembly results unless PG is inactivated.     

Size and Levels of mRNA for Acid Invertase in Ripe Tomato Fruit

Poly(A)⁺RNA was isolated from ripe tomato fruit and translatedin a wheat germ cell-free translation system. A 74-kDa polypeptidewas detected as a putative precursor of acid invertase by immunoprecipitationwith antiserum raised against SDS-treated acid invertase (denaturedform) from tomato fruit. The molecular mass of the mRNA foracid invertase was estimated to be about 8 ? 10⁵ Da (2.4 k nucleotides)by sucrose density gradient centrifugation. Mature, green tomatofruit contained very low levels of invertase mRNA. When mature,green tomato fruit were stored at 22?C, levels of invertasemRNA per gram fresh weight increased to a maximum after fourdays and then declined. (Received May 24, 1989; Accepted May 2, 1990)     

Intercellular Localization of Acid Invertase in Tomato Fruit and Molecular Cloning of a cDNA for the Enzyme

The localization of acid invertase (AI, EC 3.2.1.26 [EC] ) in tomatofruits was studied. AI was localized in the intercellular fraction(cell wall fraction). A cDNA encoding a wall-bound form of AIfrom tomato fruits was cloned and its nucleotide sequence wasdetermined. The cloned cDNA was 2363 base pairs long and containedan open reading frame of 1908 base pairs which encoded a polypeptideof 636 amino acids. RNA blot analysis indicated that the mRNAfor the acid invertase was about 2.5 kb in length. The levelsof the mRNA were low at the mature green stage but increasedduring ripening of fruit. (Received July 13, 1992; Accepted December 3, 1992)     

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。     

番茄叶片糖与转化酶的日变化研究

为探讨转化酶在叶片糖分含量和光合作用日变化中的作用,测定了番茄(Lycopersicon esculentumL.)叶片光合速率、转化酶活性、淀粉、蔗糖、葡萄糖和果糖含量,并分别分析了12:00前后它们间的相关关系.结果表明:番茄日间叶片净光合速率在10:00和16:00出现一大一小两个高峰;6:00~18:00叶片中淀粉和果糖呈现持续升高,而蔗糖和葡萄糖为先升后降趋势;光合速率同叶片蔗糖含量和胞质转化酶活性存在高度正相关,淀粉和果糖含量同光合速率未表现出显著相关性.由此可知,胞质转化酶在蔗糖代谢方面有明显的作用;果糖可能是通过抑制胞壁转化酶活性,促进了蔗糖外运.     

Carbohydrate Composition and Acid Invertase Activity in Rice Leaves Infected with Pyricularia oryzae

Ethanol-soluble carbohydrates in healthy and blast-infected leaves and also cultures of Pyricularia oryzae were analyzed using gas chromatography. Arabitol, mannitol, and trehalose occurred in infected leaf tissue, but not in healthy controls. Cultures of P. oryzae contained mannitol and trehalose, but not arabitol and sucrose. The presence of arabitol only in blast-infected rice leaves suggests that arabitol synthesis may be induced in infected leaf tissue as a result of the rice-blast fungus interaction, but may not be within blast fungus itself. The amounts of glucose, fructose, and sucrose in infected leaves were slightly increased until 5 days, and greatly enhanced at 7 days after inoculation, There were no differences in amounts of these sugars between the cultivars Nakdong and Dobong. At 7 days after inoculation, increases in amounts of arabitol, mannitol, and trehalose were pronounced in the susceptible cultivar Nakdong than in the moderately susceptible cultivar Dobong. The increased amounts of glucose and fructose in infected plants of the two cultivars were closely correlated with the presence of a very active invertase.     

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。     

Photosynthetic Activity of Ripening Tomato Fruit

Gas exchanges, chlorophyll (Chl) a fluorescence and carboxylation activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) and phosphoenolpyruvate carboxylase (PEPC) were determined in tomato (Lycopersicon esculentum Mill.) fruits picked at different developmental stages (immature, red-turning, mature, and over-ripe). The fruits did not show signs of CO₂ fixation. However, photochemical activity was detectable and an effective electron transport was observed, the values of Chl fluorescence parameters in green fruits being similar to those determined in the leaves. The RuBPCO activity, which was similar to those recorded in the leaves at the immature stage of the fruit, decreased as the fruit ripened. PEPC activity was always higher than RuBPCO activity.     

Posttranslational Elevation of Cell Wall Invertase Activity by Silencing Its Inhibitor in Tomato Delays Leaf Senescence and Increases Seed Weight and Fruit Hexose Level

Invertase plays multiple pivotal roles in plant development. Thus, its activity must be tightly regulated in vivo. Emerging evidence suggests that a group of small proteins that inhibit invertase activity in vitro appears to exist in a wide variety of plants. However, little is known regarding their roles in planta. Here, we examined the function of INVINH1, a putative invertase inhibitor, in tomato (Solanum lycopersicum). Expression of a INVINH1:green fluorescent protein fusion revealed its apoplasmic localization. Ectopic overexpression of INVINH1 in Arabidopsis thaliana specifically reduced cell wall invertase activity. By contrast, silencing its expression in tomato significantly increased the activity of cell wall invertase without altering activities of cytoplasmic and vacuolar invertases. Elevation of cell wall invertase activity in RNA interference transgenic tomato led to (1) a prolonged leaf life span involving in a blockage of abscisic acid–induced senescence and (2) an increase in seed weight and fruit hexose level, which is likely achieved through enhanced sucrose hydrolysis in the apoplasm of the fruit vasculature. This assertion is based on (1) coexpression of INVINH1 and a fruit-specific cell wall invertase Lin5 in phloem parenchyma cells of young fruit, including the placenta regions connecting developing seeds; (2) a physical interaction between INVINH1 and Lin5 in vivo; and (3) a symplasmic discontinuity at the interface between placenta and seeds. Together, the results demonstrate that INVINH1 encodes a protein that specifically inhibits the activity of cell wall invertase and regulates leaf senescence and seed and fruit development in tomato by limiting the invertase activity in planta.     

反义酸性转化酶基因低温贮藏马铃薯块茎还原糖和淀粉含量的影响

对2个含有酸性转化酶(AcInv)反义基因的转基因马铃薯品系及对照品种进行低温贮藏(4℃)及室温还暖处理.随低温贮藏时间的延长,供试材料均表现出还原糖含量升高,总淀粉含量下降的趋势.低温处理40 d时,"Ac转Atlantic"和"Ac转甘农薯2号"的还原糖含量比未转基因品种低23%和18%.总淀粉含量分别比未处理前下降约1.0%和1.3%,支链淀粉含量分别下降约1.4%和1.7%,淀粉直/支比明显低于对照,分别为0.29和0.38.块茎的石蜡切片显示,转基因块茎中深蓝色淀粉颗粒明显少于未转基因对照.另外,对低温贮藏的块茎室温还暖后,2个转基因品系的还原糖含量仍低于对照品种.实验结果证明反义AcInv基因对低温贮藏下块茎还原糖和淀粉含量具有下向调节作用.     

外源5-氨基乙酰丙酸对设施番茄果实发育及糖酸品质的影响北大核心CSCD

番茄果实糖酸类物质的含量及比例直接影响其风味品质,前期研究表明,适宜浓度的外源5-氨基乙酰丙酸(ALA)能够促进果实的成熟并提高其芳香品质。该试验为探究外源ALA对番茄果实发育及其糖酸品质的影响,以番茄‘原味1号’(Solanum lycopersicum cv.Yuanwei No.1)品种为试材,于第4穗果授粉后10 d果实表面喷施0、100和200 mg·L^(-1)的ALA溶液,分析ALA对番茄果实形态、果皮色泽及果实不同部位组织中糖、酸类物质组分及含量的影响。结果表明:(1)外源ALA溶液能显著促进番茄果实横径、纵径的增加,提高果实单果重,还显著降低果实硬度,促进果实软化,提升果实口感,并提高了果实V_(C)和可溶性固形物含量。(2)果实不同部位组织(包括果肉、小柱和隔膜)糖类物质组分含量测定结果显示,外源ALA处理能够显著提高果实可溶性总糖含量(包括果糖、葡萄糖和蔗糖),并有利于糖类物质向果肉中积累。(3)在有机酸类物质中,除酒石酸含量增加外,外源ALA处理均能不同程度地降低果实各部位组织中酸类物质含量,从而显著提高番茄果实果肉部位糖酸比,提升果实糖酸品质。研究发现,在番茄果实发育过程中外源施用200 mg·L^(-1) ALA不仅能够促进果实发育及着色,提高单果重,提升果实的外观品质,还有利于果实糖酸品质的形成。     

Expression Patterns,Activities and Sugar Metabolism Regulation of Sucrose Phosphate Synthase,Sucrose Synthase,Neutral Invertase and Soluble Acid Invertase in Different Goji Cultivars during Fruit Development

Russian Journal of Plant Physiology - Sugars are crucial factors that contribute to fruit flavor. To uncover the regulatory mechanism of sugar metabolism in developing fruit, we isolated four...     

Response of Tomato Sugar and Acid Metabolism and Fruit Quality under Different High Temperature and Relative Humidity Conditions

The combined stress of high temperature and high relative air humidity is one of the most serious agrometeorological disasters that restricts the production capacity of protected agriculture. However, there is little information about the precise interaction between them on tomato fruit quality. The objectives of this study were to explore the effects of the combined stress of high temperature and relative humidity on the sugar and acid metabolism and fruit quality of tomato fruits, and to determine the best relative air humidity for fruit quality under high temperature environments. Four temperature treatments (32°C, 35°C, 38°C, 41°C), three relative air humidity (50%, 70%, 90%) and four duration (3, 6, 9, 12 d) orthogonal experiments were conducted, with 28°C, 50% as control. The results showed that under high temperature and relative air humidity, the activity of sucrose metabolizing enzymes in young tomato fruits changed, which reduced fruits soluble sugar content; in addition, enzyme activities involved phosphopyruvate carboxylase (PEPC), mitochondria aconitase (MDH) and citrate synthetase (CS) increased which increased the content of organic acids (especially malic acid). Eventually, vitamin C, total sugar and sugar-acid ratio decreased significantly, while the titratable acid increased, resulting in a decrease in fruit flavor quality and nutritional quality in ripe fruit. Specifically, a temperature of 32°C and a relative air humidity of 70% were the best cultivation conditions for tomato reproductive growth period under high temperature. Our results indicating that fruit quality reduced under high temperature at the flowering stage, while increasing the relative air humidity to 70% could alleviate this negative effect. Our results are benefit to better understand the interaction between microclimate parameters under specific climatic conditions in the greenhouse environment and their impact on tomato flavor quality.     

转反义PG基因番茄果实细胞结构变化的研究

经细胞学观察发现 ,转反义PG基因番茄果实在不同成熟期及存放前后 ,其果皮外面几层细胞的厚度都比未转基因的厚 1～ 5 μm ,细胞结构、细胞质和细胞核等的状态都有明显区别。尤以贮存后更为明显 ,未转基因果实的果皮细胞结构解体、细胞质凝聚、细胞核变的模糊程度都比转基因的严重。经外源乙烯处理后 ,转基因和未转基因果实的细胞结构也有相似的变化。结果表明 :反义PG基因的转入降低了PG活性 ,并且减弱了外源乙烯的作用 ,延缓了果实的衰老 ,提高了耐贮性能 ,从而起到果实保鲜作用     

Regulation of Growth and Invertase Activity by Kinetin and Gibberellic Acid in Developing Avena Internodes

Evidence presented here indicates that there is a complex interaction between kinetin, gibberellin, and sucrose in the regulation of turnover of invertase in vivo. The synthesis of invertase is maintained in the presence of GA₃ and sucrose over relatively long periods of time. Kinetin, on the other hand, inhibits the full development of invertase activity seen in the sucrose and gibberellin control treatments. Moreover, the peak in invertase activity occurs earlier with kinetin treatment. During invertase turnover, once the peak is reached, kinetin enhances the rate of decay of enzyme activity relative to synthesis. The regulatory significance of invertase in intercalary growth and possible modes of kinetin action in this process are discussed.     

6-BA对'全球红'葡萄果实发育过程中糖分含量和转化酶活性的影响

经处理的‘全球红’葡萄果实发育过程中的蔗糖、葡萄糖、果糖和总糖的含量变化趋势与未经6-BA处理的基本上一致,采收时经6-BA处理的果实中糖含量比未经6-BA处理的有不同程度的提高,而且随着6-BA处理浓度的升高而增加,以30mg&#183;L^-16-BA处理的最为显著,经6-BA处理的葡萄果实发育过程中的转化酶活性也有不同程度的提高。