Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field

Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non‐transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress.     

拟南芥肌醇半乳糖苷合成酶与棉子糖合成酶的体外催化活性比较

[目的]基因克隆及原核表达纯化后比较拟南芥的2个肌醇半乳糖苷合成酶及2个棉子糖合成酶的体外催化活性,为微生物法或酶法合成棉子糖尊定基础。[方法]RT-PCR克隆拟南芥的肌醇半乳糖苷合成酶(GolS1及GolS3)与棉子糖合成酶(RafS1及RafS5)的基因,分别构建原核表达菌株,诱导表达纯化获得酶,电泳检测及蛋白定量后进行体外酶催化反应,HPLC分析产物。[结果]克隆到GolS1与GolS3及RafS1与RafS5的基因,原核表纯化获得纯酶,以反应体系中目标产物生成速率衡量,GolS1与GolS3催化速率分别为0.51和0.28mmol/(mg·min),RafS1与RafS5的催化速率分别为0.45和0.21mmol/(mg·min)。[结论]拟南芥的肌醇半乳糖苷合成酶(GolS1及GolS3)与棉子糖合成酶(RafS1及RafS5)基因经异源表达后具有良好酶活,其中GolS1酶活是GolS3的1.82倍,RafS1酶活是RafS5的2.14倍。     

植物肌醇半乳糖苷合酶的生理功能和调控机制

植物肌醇半乳糖苷合酶（galactinol synthase, GolS）是高等植物棉子糖类寡糖合成途径中的关键酶,为棉子糖系列寡糖提供活化的半乳糖基,调控植物体内棉子糖（raffinose, RFO）系列寡糖的生物合成与积累。编码该酶的基因属于糖基转移酶（glycosyltransferases, GTs）GT8基因家族的亚家族。GolS参与合成的最终产物棉子糖家族低聚糖（raffinose family oligosaccharides,RFOs）是植物中重要的碳水化合物存在形式,在细胞内可溶性强,可作为脱水保护剂;还能发挥稳定膜结构的作用。同时,GolS催化合成的直接产物肌醇半乳糖苷（galactinol）和RFOs都能作为羟基自由基捕获分子参与活性氧的清除。因此,GolS参与的代谢途径在植物碳同化物的贮存与运输、生物和非生物逆境响应、种子的脱水效应等生命过程中均发挥了重要作用。GolS基因结构差异与表达模式不同,导致不同GolS基因参与的生物学功能具有很大的差异。研究植物中不同GolS基因的结构特征,组织特异性表达特性及它们响应不同生长发育阶段、环境变化的表达特性,对了解GolS参与的生物学功能具有重要意义。同时,在分子生物学水平上,深入了解调控植物GolS基因的分子调控机制,为通过遗传工程或分子辅助育种等手段,利用GolS改良农林作物的经济性状提供理论支持。本文针对近年来植物中GolS基因的生理功能和调控机制的研究进行了综述。     

植物肌醇半乳糖苷合酶的生理功能和调控机制

植物肌醇半乳糖苷合酶（galactinol synthase, GolS）是高等植物棉子糖类寡糖合成途径中的关键酶,为棉子糖系列寡糖提供活化的半乳糖基,调控植物体内棉子糖（raffinose, RFO）系列寡糖的生物合成与积累。编码该酶的基因属于糖基转移酶（glycosyltransferases, GTs）GT8基因家族的亚家族。GolS参与合成的最终产物棉子糖家族低聚糖（raffinose family oligosaccharides,RFOs）是植物中重要的碳水化合物存在形式,在细胞内可溶性强,可作为脱水保护剂;还能发挥稳定膜结构的作用。同时,GolS催化合成的直接产物肌醇半乳糖苷（galactinol）和RFOs都能作为羟基自由基捕获分子参与活性氧的清除。因此,GolS参与的代谢途径在植物碳同化物的贮存与运输、生物和非生物逆境响应、种子的脱水效应等生命过程中均发挥了重要作用。GolS基因结构差异与表达模式不同,导致不同GolS基因参与的生物学功能具有很大的差异。研究植物中不同GolS基因的结构特征,组织特异性表达特性及它们响应不同生长发育阶段、环境变化的表达特性,对了解GolS参与的生物学功能具有重要意义。同时,在分子生物学水平上,深入了解调控植物GolS基因的分子调控机制,为通过遗传工程或分子辅助育种等手段,利用GolS改良农林作物的经济性状提供理论支持。本文针对近年来植物中GolS基因的生理功能和调控机制的研究进行了综述。     

Structure and regulation of the Arabidopsis thaliana allene oxide synthase gene

Allene oxide synthase (AOS) is encoded by a single intronless gene in Arabidopsis thaliana (L.) Heynh. The promoter region of the AOS gene exhibits, in addition to the elements of a minimal promoter and the presence of general enhancers, cis-elements that, in other promoters, are responsible for stress- and ethylene-responsiveness. Arabidopsis thaliana and Nicotiana tabacum L. were transformed with a chimaeric gene consisting of a 1.9-kb 5′-upstream sequence and the first 95 nucleotides of the AOS coding sequence translationally fused to uid A encoding β-glucuronidase (GUS). Using histochemistry, GUS activity was seen in older leaves, in the bases of petioles and in stipules, during the early stages of carpel development, in maturing pollen grains and at the base of elongated filaments, as well as in abscission-zone scars. A role for jasmonates in floral organ abscission is suggested by these findings. Furthermore, the AOS promoter was activated both locally as well as systemically upon wounding. Jasmonic acid, 12-oxophytodienoic acid and coronatine strongly induced GUS activity. This induction remained confined to the treated leaf when agonists were applied locally to a leaf, suggesting that neither jasmonic acid nor 12-oxophytodienoic acid are physiologically relevant components of the systemic wound signal complex. Rather, the data show that jasmonates behave as local response regulators produced at or around the sites of action in response to appropriate triggers of their synthesis. Received: 21 September 1998 / Accepted: 30 December 1998     

Ectopic expression of nicotianamine synthase genes results in improved iron accumulation and increased nickel tolerance in transgenic tobacco

Heavy metals are essential for basic cellular processes but toxic in higher concentrations. This requires the precise control of their intracellular concentrations, a process known as homeostasis. The metal-chelating, non-proteinogenous amino acid nicotianamine (NA) is a key component of plant metal assimilation and homeostasis. Its precise function is still unknown. Therefore, this article aims to contribute new information on the in vivo function of NA and to evaluate its potential use for plant nutrition and crop fortification. For this purpose, a nicotianamine synthase gene of Arabidopsis thaliana was ectopically expressed in transgenic tobacco plants. The presence of extra copies of the nicotianamine synthase gene co-segregated with up to 10-fold elevated levels of NA in comparison with wild type. The increased NA level led to: (a) a significantly increased iron level in leaves of adult plants; (b) the accumulation of zinc and manganese, but not copper; (c) an improvement of the iron use efficiency in adult plants grown under iron limitation; and (d) an enhanced tolerance against up to 1 m m nickel. Taken together, the data predict that NA may be a useful tool for improved plant nutrition on adverse soils and possibly for enhanced nutritional value of leaf and seed crops.     

转基因改良植物的胁迫耐性

干旱、盐碱和低温等逆境是严重影响栽培植物生产的非生物胁迫因素。导入外源目的的基因已发展成为改良作物对逆境胁迫耐性的新途径。现今已应用于植物胁迫改良的基因包括编码活性氧清除酶类、膜修饰酶类、胁迫诱导蛋白和渗调物质合成酶等基因。     

Transgenic Nicotiana tabacum and Arabidopsis thaliana plants overexpressing allene oxide synthase

 Allene oxide synthase (AOS), encoded by a single gene in Arabidopsis thaliana (L.) Heynh., catalyzes the first step specific to the octadecanoid pathway. Enzyme activity is very low in control plants, but is upregulated by wounding, octadecanoids, ethylene, salicylate and coronatine (D. Laudert and E.W. Weiler, 1998, Plant J 15: 675–684). In order to study the consequences of constitutive expression of AOS on the level of jasmonates, a complete cDNA encoding the enzyme from A. thaliana was constitutively expressed in both  A. thaliana and tobacco (Nicotiana tabacum L.). Overexpression of AOS did not alter the basal level of jasmonic acid; thus, output of the jasmonate pathway in the unchallenged plant appears to be strictly limited by substrate availability. In wounded plants overexpressing AOS, peak jasmonate levels were 2- to 3-fold higher compared to untransformed plants. More importantly, the transgenic plants reached the maximum jasmonate levels significantly earlier than wounded untransformed control plants. These findings suggest that overexpression of AOS might be a way of controlling defense dynamics in higher plants. Received: 10 February 2000 / Accepted: 11 March 2000     

Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants

The expression of the gene Osmyb4, detected at low level in rice (Oryza sativa) coleoptiles grown for 3 days at 29 degrees C, is strongly induced by treatments at 4 degrees C. At sublethal temperatures of 10 and 15 degrees C, its expression in rice seedlings is already evident, but this effect cannot be vicariated by other stresses or ABA treatment. We demonstrate by transient expression that Myb4 transactivates the PAL2, ScD9 SAD and COR15a cold-inducible promoters. The Osmyb4 function in vivo is demonstrated overexpressing its cDNA in Arabidopsis thaliana plants (ecotype Wassilewskija) under the control of the constitutive CaMV 35S promoter. Myb4 overexpressing plants show a significant increased cold and freezing tolerance, measured as membrane or Photosystem II (PSII) stability and as whole plant tolerance. Finally, in Osmyb4 transgenic plants, the expression of genes participating in different cold-induced pathways is affected, suggesting that Myb4 represents a master switch in cold tolerance.     

ACC synthase expression regulates leaf performance and drought tolerance in maize

Ethylene regulates entry into several types of plant developmental cell death and senescence programs besides mediating plant responses to biotic and abiotic stress. The response of cereals to conditions of drought includes loss of leaf function and premature onset of senescence in older leaves. In this study, ACC synthase ( ACS ) mutants, affecting the first step in ethylene biosynthesis, were isolated in maize and their effect on leaf function examined. Loss of ZmACS6 expression resulted in delayed leaf senescence under normal growth conditions and inhibited drought-induced senescence. Zmacs6 leaves continued to be photosynthetically active under both conditions indicating that leaf function was maintained. The delayed senescence phenotype associated with loss of ZmACS6 expression was complemented by exogenous ACC. Surprisingly, elevated levels of foliar chlorophyll, Rubisco, and soluble protein as well as improved leaf performance was observed for all Zmasc6 leaves, including young and fully expanded leaves which were far from initiating senescence. These observations suggest that ethylene may serve to regulate leaf performance throughout its lifespan as well as to determine the onset of natural senescence and mediate drought-induced senescence.