Changes in carbohydrate metabolism and assimilate export in starch-excess mutants of Arabidopsis

The aim of this work was to investigate the effects on carbohydrate metabolism of a reduction in the capacity to degrade leaf starch in Arabidopsis. The major roles of leaf starch are to provide carbon for sucrose synthesis, respiration and, in developing leaves, for biosynthesis and growth. Wild-type plants were compared with plants of a starch-excess mutant line (sex4) deficient in a chloroplastic isoform of endoamylase. This mutant has a reduced capacity for starch degradation, leading to an imbalance between starch synthesis and degradation and the gradual accretion of starch as the leaves age. During the night the conversion of starch into sucrose in the mutant is impaired; the leaves of the mutant contained less sucrose than those of the wild type and there was less movement of ¹⁴C-label from starch to sucrose in radio-labelling experiments. Furthermore, the rate of assimilate export to the roots during the night was reduced in the mutant compared with the wild type. During the day however, photosynthetic partitioning was altered in the mutant, with less photosynthate partitioned into starch and more into sugars. Although the sucrose content of the leaves of the mutant was similar to the wild type during the day, the rate of export of sucrose to the roots was increased more than two-fold. The changes in carbohydrate metabolism in the mutant leaves during the day compensate partly for its reduced capacity to synthesize sucrose from starch during the night.     

Regulation of photosynthetic carbon assimilation at the cellular level: a review

In green leaves and a number of algae, photosynthetically derived carbon is ultimately converted into two carbohydrate end-products, sucrose and starch. Drainage of carbon from the Calvin cycle proceeds via triose phosphate, fructose 6-phosphate and glycollate. Gluconeogenesis in photosynthetic cells is controlled by light, inorganic phosphate and phosphorylated sugars. Light stimulates the production of dihydroxyacetone phosphate, the initial substrate for sucrose and starch synthesis, and inhibits the degradative pathways in the chloroplast. Phosphate inactivates reactions of synthesis and activates reactions of degradation. Among the phosphorylated sugars a special role is allocated to fructose 2,6-bisphosphate, which is present in the cytoplasm at very low concentrations and inhibits sucrose synthesis directly by inactivating pyrophosphatedependent phosphofructokinase. The synthesis of sucrose plays a central role in the partitioning of photosynthetic carbon. The cytoplasmic enzymes, fructose bisphosphate phosphatase and sucrose phosphate synthase are likely key points of regulation. The regulation is carried out by several effector metabolites. Fructose 2,6-bisphosphate is likely to be the main coordinator of the rate of sucrose synthesis, hence of photosynthetic carbon partitioning between sucrose and starch.Paper presented at the FESP meeting (Strasbourg, 1984)     

Photosynthetic carbon partitioning: its regulation and possibilities for manipulation

The regulation of photosynthetic sucrose synthesis and partitioning is reviewed with particular emphasis on the role of fructose-2,6-bisphosphate and sucrose phosphate synthase. It is concluded that a hierachy of regulatory mechanisms exist, which allows partitioning to be changed without this necessarily leading to a reduction in the rate of photosynthesis. Nevertheless, experimental conditions can be defined in which photosynthesis is limited by the rate of end-product synthesis. These systems can be used to screen for genetic mutants or specific inhibitors, and to investigate a shift in sink demand or environmental factors that specifically act on carbon partitioning and/or the rate of endproduct synthesis.     

枸杞蔗糖磷酸合成酶基因的克隆及组织表达分析

利用RT-PCR及RACE技术,从药用植物枸杞中克隆了1个编码蔗糖磷酸合成酶(SPS)基因的全长cDNA,命名为LbSPS(GenBank登录号KC834608)。序列分析表明:LbSPS基因长3 677bp,开放阅读框为3 165bp,编码1 033个氨基酸,分子量为118.457 5kD,理论等电点6.05。系统进化分析显示,LbSPS编码的氨基酸序列与甜瓜、马铃薯、番茄等蔗糖磷酸合成酶基因编码氨基酸序列一致性为66%~98%。qRT-PCR分析显示,LbSPS基因在枸杞花中表达量最高,叶中表达水平较低。该研究为进一步了解LbSPS在枸杞生长发育、逆境胁迫等过程中的生物学功能奠定了基础。     

Localisation of sucrose-phosphate synthase and starch in leaves of C4 plants

The activity and intercellular distribution of sucrose-phosphate synthase (SPS; EC 2.4.1.14) were determined in fully expanded leaves from a range of C₄ plants. In Zea mays L. and Atriplex spongiosa F. Muell., SPS was located almost exclusively in the mesophyll cells. In other species, SPS was found in both cell types, with the activity in the bundle sheath cells ranging from 5% of the total leaf activity in Echinochloa crus-galli (L.) Beauv. to 35% in Sorghum bicolor Moench. At the end of the light period, starch was found only in the bundle sheath cells in all of the species examined. There appears to be little correlation between C₄-acid decarboxylation type and the location of sucrose and starch synthesis in the leaves of C₄ plants. Received: 18 October 1996 / Accepted: 20 November 1996     

拟南芥吲哚—3—甘油磷酸合酶免疫分析

吲哚３甘油磷酸合酶（ＩＧＳ,ｉｎｄｏｌｅ３ｇｌｙｃｅｒｏｌｐｈｏｓｐｈａｔｅｓｙｎｔｈａｓｅ,ＥＣ４．１．１．４８）在色氨酸与吲哚乙酸的生物合成途径中,催化生成吲哚３甘油磷酸。研究该基因的表达调控,对于阐明高等植物是如何调控色氨酸及生长素合成是十分重要的。利用已克隆的ＩＧＳｃＤＮＡ,构建了谷胱甘肽转移酶（ＧＳＴ,ｇｌｕｔａｔｈｉｏｎｅＳｔｒａｎｓｆｅｒａｓｅ,ＥＣ２．５．１．１８）与吲哚３甘油磷酸合酶融合蛋白的表达质粒,并将其导入到在异丙基βＤ硫代半乳糖苷（ＩＰＴＧ）诱导下能高效表达的ＩＧＳ基因缺陷菌株ｔｒｐＣ９８００λＫＣ大肠杆菌中。高表达的融合蛋白通过谷胱甘肽琼脂糖（ｇｌｕｔａｔｈｉｏｎｅａｇａｒｏｓｅ）亲和层析和ＳＤＳ聚丙烯酰胺凝胶电泳纯化后,用以免疫兔子制备抗血清。免疫印迹法分析表明拟南芥（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）四种常用生态型只合成一种分子量约为４０ｋＤ的吲哚３甘油磷酸合酶蛋白。在Ａｇ＋、紫外线等逆境条件下,ＩＧＳ含量都有较大幅度的增加,这说明ＩＧＳ可能与植物的防御反应紧密相关。     

拟南芥CHS基因表达的实时荧光定量PCR检测

在简要介绍实时荧光定量PCR反应和定量原理的基础上,采用TaqMan荧光定量PCR技术,研究了UV-B辐射对拟南芥(Arabidopsis thaliana)CHS(查耳酮合成酶基因)表达的诱导,获得了与传统Northern杂交一致的结果.实时荧光定量PCR用于基因表达的定量检测,具有特异性强、自动化程度高、高效快捷,避免使用放射性同位素,能同时对多个样品中的起始模板进行准确定量等特点,因此该方法已逐渐被广泛用于基因表达的定量分析.     

Mutants for photosystem I subunit D of Arabidopsis thaliana: effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions

In Arabidopsis thaliana, the D-subunit of photosystem I (PSI-D) is encoded by two functional genes, PsaD1 and PsaD2, which are highly homologous. Knock-out alleles for each of the loci have been identified by a combination of forward and reverse genetics. The double mutant psad1-1 psad2-1 is seedling-lethal, high-chlorophyll-fluorescent and deficient for all tested PSI subunits, indicating that PSI-D is essential for photosynthesis. In addition, psad1-1 psad2-1 plants show a defect in the accumulation of thylakoid multiprotein complexes other than PSI. Of the single-gene mutations, psad2 plants behave like wild-type (WT) plants, whereas psad1-1 markedly affects the accumulation of PsaD mRNA and protein, and photosynthetic electron flow. Additional effects of the psad1-1 mutation include a decrease in growth rate under greenhouse conditions and downregulation of the mRNA expression of most genes involved in the light phase of photosynthesis. In the same mutant, a marked decrease in the levels of PSI and PSII polypeptides is evident, as well as a light-green leaf coloration and increased photosensitivity. Increased dosage of PsaD2 in the psad1-1 background restores the WT phenotype, indicating that PSI-D1 and PSI-D2 have redundant functions.     

Classification of Genes Differentially Expressed during Water-deficit Stress in Arabidopsis thaliana: an Analysis using Microarray and Differential Expression Data

Many changes in gene expression occur in response to water-deficitstress. A challenge is to determine which changes support plantadaptation to conditions of reduced soil water content and whichoccur in response to lesions in metabolic and cellular functions.Microarray methods are being employed to catalogue all of thechanges in gene expression that occur in response to specificwater-deficit conditions. Although these methods do not measurethe amount or activities of specific proteins that functionin the water-deficit response, they do target specific biochemicaland cellular events that should be detailed in further work.Potential functions of approx. 130 genes of Arabidopsis thalianathat have been shown to be up-regulated are tabulated here.These point to signalling events, detoxification and other functionsinvolved in the cellular response to water-deficit stress. Asmicroarray techniques are refined, plant stress biologists willbe able to characterize changes in gene expression within thewhole genome in specific organs and tissues subjected to differentlevels of water-deficit stress.     

橄榄果实发育成熟过程中糖积累与相关酶活性的关系

以可溶性总糖含量差异明显的2个橄榄品种为试验材料,测定果实发育成熟过程中蔗糖、葡萄糖、果糖、可溶性总糖含量及蔗糖代谢相关酶活性的动态变化,并对果实糖积累与酶活性进行相关性分析,以明确不同橄榄品种果实糖积累差异的生理基础,为进一步在代谢与分子水平探讨橄榄果实糖积累机制提供依据。结果表明:(1)蔗糖快速积累期是橄榄品种间果实蔗糖积累差异的关键时期,并影响成熟时果实可溶性总糖含量的高低,其中‘马坑22’蔗糖快速积累期较长,增长幅度较大,成熟时可溶性糖含量高;成熟时‘马坑22’、‘檀头23’果实内己糖与蔗糖比分别为0.668、0.904。(2)在蔗糖快速积累期内,‘马坑22’酸性转化酶(AI)活性低于‘檀头23’,为其蔗糖积累创造条件,而中性转化酶活性高于后者则有利于其增加果实库强;两品种蔗糖磷酸合成酶(SPS)活性变化差异不大,说明SPS不是蔗糖积累的关键酶;‘马坑22’蔗糖合成酶(SuSy)合成方向活性在花后144~186d增幅显著高于‘檀头23’,说明SuSy为果实蔗糖积累的关键酶。(3)‘马坑22’蔗糖快速积累主要依靠SuSy合成方向活性变化促进蔗糖合成,‘檀头23’蔗糖快速积累主要依靠SuSy分解方向活性变化促进蔗糖直接进入果实。     

Control of carbohydrate metabolism in a starchless mutant of Arbidopsis thaliana

The biochemical regulation of photosynthate partitioning was investigated in a starchless mutant (TC7) of Arabidopsts thaliana (L.) Henyh, that was deficient in chloroplast phosphoglucomutase (Caspar et al. 1985. Plant Physiol. 79: 11–17). Plants were raised at 20°C with a 20 h light and 4 h dark period, so that the growth rates of the mutant and wild type were similar. Two or 3 isoforms of phosphoglucomutase were separated by ion-exchange chromatography using mutant and wild type leaf preparations, respectively. Initial rate kinetics of all isoforms were similar. Light-saturated photosynthetic oxygen evolution rates of the mutant and wild type were 224 and 302 nmol g^-1 chlorophyll h^-1, respectively. Starch, sucrose and hexose concentrations were unchanged in wild type leaves after a dark to light transition, whereas sucrose and hexose increased in mutant leaves. Hexose-phosphates accumulated in both genotypes in the light, although the steady-state leaf concentrations of glucose 6-phosphate were 3-fold higher in mutant than in wild type samples. Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate were lower in the mutant than in the wild type at the end of the dark period when mutant leaves were depleted of carbohydrates. Levels of UTP were lower in the mutant than in the wild type, possibly indicating that growth conditions had induced phosphate limited photosynthesis. These results are discussed in relation to the regulation of photosynthetic carbon metabolism.     

The effect of water stress on photosynthetic carbon metabolism in four species grown under field conditions

Abstract. The effect of gradually-developing water-stress has been studied in Lupinus albus L., Helianthus annuus L., Vitis vinifera cv. Rosaki and Eucalyptus globulus Labill. Water was withheld and diurnal rhythms were investigated 4–8d later, when the predawn water deficit was more negative than in watered plants, and the stomata closed almost completely early during the photoperiod. The contribution of ‘stomatal’ and ‘non-stomatal’ components to the decrease of photosynthetic rate was investigated by (1) comparing the changes of the rate of photosynthesis in air with the changes of stomatal conductance and (2) measuring photosynthetic capacity in saturating irradiance and 15% CO₂. Three species (lupin, eucalyptus and sunflower) showed larger changes of stomatal conductance than photosynthesis in air, and showed little or no decrease of photosynthetic capacity in saturating CO₂. Photosynthesis in air also recovered fully overnight after watering the plants in the evening. In grapevines, stomatal conductance and photosynthesis in air changed in parallel, there was a marked decrease of photosynthetic capacity, and photosynthesis and stomatal conductance did not recover overnight after watering water-stressed plants. Relative water content remained above 90% in grapevine. We conclude that non-stomatal components do not play a significant role in lupins, sunflower or eucalyptus, but could in grapevine. The effect of water-stress on partitioning of photosynthate was investigated by measuring the amounts of sucrose and starch in leaves during a diurnal rhythm, and by measuring the partitioning of ¹⁴C-carbon dioxide between sucrose and starch. In all four species, starch was depleted in water-stressed leaves but sucrose was maintained at amounts similar to, or higher than, those in watered plants. Partitioning into sucrose was increased in lupins and eucalyptus, and remained unchanged in grapevine and sunflower. It is concluded that water-stressed leaves in all four species maintain high levels of soluble sugars in their leaves, despite having lower rates of field photosynthesis, decreased rates of export, and low amounts of starch in their leaves.