Immunomodulation of polyamine biosynthesis in tobacco plants has a significant impact on polyamine levels and generates a dwarf phenotype

Ornithine decarboxylase (ODC) is a cytosolic enzyme that catalyses the direct decarboxylation of l-ornithine to putrescine, one of the rate-limiting steps of polyamine biosynthesis in plants. In the present study, an ODC-specific murine single-chain antibody fragment (scFvODC1) was generated by phage display technology. To evaluate the effect of the recombinant antibody fragment on ODC activity and polyamine levels, we produced transgenic tobacco plants that accumulated scFvODC1 in the cytosol. Expression levels of up to 4% total soluble protein (TSP) were achieved, resulting in the inhibition of up to 90% of endogenous ODC activity. A significant reduction in putrescine, spermidine and spermine levels was observed in transgenic lines producing high levels of scFvODC1. Furthermore, these lines showed developmental abnormalities and a dwarf phenotype. We show that the immunomodulation of enzyme activity is a powerful approach that can be used to alter complex and tightly controlled metabolic pathways, allowing specific steps in the pathway to be blocked and the resulting physiological effects to be investigated.     

Improvement of plant abiotic stress tolerance through modulation of the polyamine pathway

Polyamines(mainly putrescine(Put),spermidine(Spd),and spermine(Spm))have been widely found in a range of physiological processes and in almost all diverse environmental stresses.In various plant species,abiotic stresses modulated the accumulation of polyamines and related gene expression.Studies using loss-of-function mutants and transgenic overexpression plants modulating polyamine metabolic pathways confirmed protective roles of polyamines during plant abiotic stress responses,and indicated the possibility to improve plant tolerance through genetic manipulation of the polyamine pathway.Additionally,putative mechanisms of polyamines involved in plant abiotic stress tolerance were thoroughly discussed and crosstalks among polyamine,abscisic acid,and nitric oxide in plant responses to abiotic stress were emphasized.Special attention was paid to the interaction between polyamine and reactive oxygen species,ion channels,amino acid and carbon metabolism,and other adaptive responses.Further studies are needed to elucidate the polyamine signaling pathway,especially polyamine-regulated downstream targets and the connections between polyamines and other stress responsive molecules.     

Improvement of plant abiotic stress tolerance through modulation of the polyamine pathway FA简

Polyamines （mainly putrescine （Put）, spermidine （Spd）, and spermine （Spin）） have been widely found in a range of physiological processes and in almost all diverse environ- mental stresses. In various plant species, abiotic stresses modulated the accumulation of polyamines and related gene expression. Studies using loss-of-function mutants and transgenic overexpression plants modulating polyamine metabolic pathways confirmed protective roles of polyamines during plant abiotic stress responses, and indicated the possibility to improve plant tolerance through genetic manipulation of the polyamine pathway. Additionally, puta- tive mechanisms of polyamines involved in plant abiotic stress tolerance were thoroughly discussed and crosstalks among polyamine, abscisic acid, and nitric oxide in plant responses to abiotic stress were emphasized. Special attention was paid to the interaction between polyamine and reactive oxygen species, ion channels, amino acid and carbon metabolism, and other adaptive responses. Further studies are needed to elucidate the polyamine signaling pathway, especially polyamine-regulated downstream tar- gets and the connections between polyamines and other stress responsive molecules.     

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。     

Maize Rabl7 overexpression in Arabidopsis plants promotes osmotic stress tolerance

Rabl7 is a Late Embryogenesis Abundant (LEA) protein from maize, which accumulates largely during embryogenesis and also in vegetative tissues when subjected to stress conditions. We have analysed the effect of Rab 17 expression under a constitutive promoter in vegetative tissues of transgenic Arabidopsis thaliana plants. These transgenic plants have higher sugar and proline contents, and also higher water loss rate under water stress. In addition, these plants are more tolerant than non-transformed controls to high salinity and recover faster from mannitol treatment. Our results point to a protective effect of Rabl7 protein in vegetative tissues under osmotic stress conditions.     

Metabolic engineering of ketocarotenoid formation in higher plants

Although higher plants synthesize carotenoids, they do not possess the ability to form ketocarotenoids. In order to generate higher plants capable of synthesizing combinations of ketolated and hydroxylated carotenoids the genes responsible for the carotene 4,4' oxygenase and 3,3' hydroxylase have been transformed into tomato and tobacco. The gene products were produced as a polyprotein. Subsequent cleavage of the polyprotein, targeting of the two enzymes to the plastid and enzyme activities have been shown for both gene products. Metabolite profiling has shown the formation of ketolated carotenoids from beta-carotene and its hydroxylated intermediates in tobacco and tomato leaf. In the nectary tissues of tobacco flowers a quantitative increase (10-fold) as well as compositional changes were evident, including the presence of astaxanthin, canthaxanthin and 4-ketozeaxanthin. Interestingly, in this tissue the newly formed carotenoids resided predominantly as esters. These data are discussed in terms of metabolic engineering of carotenoids and their sequestration in higher plant tissues.     

Characteristics of oxidative stress in potato plants with modified carbohydrate metabolism

Effects of sugars on the development of hypothermia-induced oxidative stress were studied in leaves of two potato genotypes (Solanum tuberosum L., cv. Désirée): with normal carbohydrate metabolism and a genotype with increased sugar content modified by insertion of yeast-derived invertase gene. It was found that generation of proceeds more actively in transformed plants than in control plants. On the contrary H₂O₂ concentration and the catalese and peroxidase activities were lower. At the same time, the activities of superoxide dismutase were similar in plants of both genotypes. A short-term incubation of plants at ?7°C confirmed that a higher freezing tolerance of transformed plants was due to low-molecular-weight components of antioxidant protection system rather than to enzymatic component. Literature data and experimental results suggest that the protective effect of sugars is caused by their ability to scavenge ROS nonspecifically under stress conditions     

Transgenic approaches to increase dehydration-stress tolerance in plants

Plant productivity is strongly influenced by abiotic stress conditions induced by drought, high salt and low temperature. Plants respond to these conditions with an array of biochemical and physiological adaptations, at least some of which are the result of changes in gene expression. Transgenic approaches offer a powerful means of gaining valuable information to better understand the mechanisms governing stress tolerance. They also offer new opportunities to improve dehydration-stress tolerance in crops by incorporating a gene involved in stress protection into species that lack them. In this review, we discuss progress made towards understanding the molecular elements involved in dehydration-stress responses that have been used to improve salt or drought tolerance following several transgenic approaches. Further, we discuss various strategies being used to produce transgenic plants with increased tolerance to dehydration stress. These include the overproduction of enzymes responsible for biosynthesis of osmolytes, late-embryogenesis-abundant proteins and detoxification enzymes. At this time, there is a need for a careful appraisal of the genes to be selected and promoter elements to be used, because constitutive expression of these genes may not be desirable in all applications. In this context, the advantages and limitations of transgenic approaches currently being used are discussed together with the importance of using stress-inducible promoters and the introduction of multiple genes for the improvement of dehydration-stress tolerance.     

Metabolic engineering of ketocarotenoid biosynthesis in higher plants

Ketocarotenoids such as astaxanthin and canthaxanthin have important applications in the nutraceutical, cosmetic, food and feed industries. Astaxanthin is derived from β-carotene by 3-hydroxylation and 4-ketolation at both ionone end groups. These reactions are catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. The hydroxylation reaction is widespread in higher plants, but ketolation is restricted to a few bacteria, fungi, and some unicellular green algae. The recent cloning and characterization of β-carotene ketolase genes in conjunction with the development of effective co-transformation strategies permitting facile co-integration of multiple transgenes in target plants provided essential resources and tools to produce ketocarotenoids in planta by genetic engineering. In this review, we discuss ketocarotenoid biosynthesis in general, and characteristics and functional properties of β-carotene ketolases in particular. We also describe examples of ketocarotenoid engineering in plants and we conclude by discussing strategies to efficiently convert β-carotene to astaxanthin in transgenic plants.     

Plasticity of polyamine metabolism associated with high osmotic stress in rape leaf discs and with ethylene treatment

In rape leaf discs the response to osmotic stress has been found to be associated with increases in putrescine and 1,3-diaminopropane (an oxidation product of spermidine and/or spermine) and decreases in spermidine titers. In contrast, agmatine and spermine titers showed small changes while cadaverine accumulated massively. Similar results were observed in whole rape seedlings subjected to drought conditions. -DL-difluoromethylarginine (DFMA), a specific irreversible inhibitor of arginine decarboxylase, strongly inhibited polyamine accumulation in unstressed rape leaf discs, which suggested that the arginine decarboxylase pathway is constitutively involved in putrescine biosynthesis. In leaf discs treated under high osmotic stress conditions, both DFMA and DFMO (-DL-difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase) inhibited the accumulation of polyamines. Although the stressed discs treated with DFMA had a lower concentration of putrescine than those treated with DFMO, we propose that under osmotic stress the synthesis of putrescine might involve both enzymes. DFMA, but not DFMO, was also found to inhibit cadaverine formation strongly in stressed explants. The effects on polyamine biosynthesis and catabolism of cyclohexylamine, the spermidine synthase inhibitor, aminoguanidine, the diamine-oxidase inhibitor and -aminobutyric acid, a product of putrescine oxidation via diamine oxidase or spermidine oxidation via polyamine oxidase were found to depend on environmental osmotic challenges. Thus, it appears that high osmotic stress did not block spermidine biosynthesis, but induced a stimulation of spermidine oxidation. We have also demonstrated that in stressed leaf discs, exogenous ethylene, applied in the form of (2-chloroethyl) phosphonic acid or ethephon, behaves as an inhibitor of polyamine synthesis with the exception of agmatine and diaminopropane. In addition, in stressed tissues, when ethylene synthesis was inhibited by aminooxyacetic acid or aminoethoxyvinylglycine, S-adenosylmethionine utilization in polyamine synthesis was not promoted. The relationships between polyamine and ethylene biosynthesis in unstressed and stressed tissues are discussed.     

Transgenic plants: An insight into oxidative stress tolerance mechanisms

Environmental stresses considerably limit plant productivity. At the molecular level the negative effect of stress is often mediated by reactive oxygen species-initiated oxidative damage. Hence, it was hypothesised that increased tolerance to several environmental constraints could be achieved through enhanced tolerance to oxidative stress. In recent years much effort has been undertaken to improve oxidative stress tolerance by transforming plants with native or bacterial genes coding either for reactive oxygen species-scavenging enzymes or for enzymes modulating the cellular antioxidant capacity. This review deals with data on transgenic plants with altered antioxidant capacity and focuses on the new insight into the antioxidant defence mechanism given by this type of experimental model.     

Development of selection marker-free transgenic potato plants with enhanced tolerance to oxidative stress

A binary vector devoid of a plant selection-marker gene (designated as pSSA-F) was constructed to overcome bio-safety concerns about genetically modified plants. This vector carried chloroplast-targeted superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible(SWPA2) promoter, and was utilized to transform potato (Solanum tuberosum L.). Integration of these foreign genes into transgenic plants was primarily performed via PCR with genomic DNA. Twelve marker-free transgenic lines were obtained by inoculating stem explants. The maximum transformation efficiency was 6.25% and averaged 2.2%. Successful integration of the SOD and APX genes rendered transgenic plants tolerant to methyl viologen-mediated oxidative stress at the leaf-disc and whole-plant levels. Our findings suggest that this technique for developing selection marker-free transgenic plants is feasible and can be employed with other crop species.     

谷胱甘肽在植物抗逆中的作用

在简要总结谷胱甘肽(GSH)的结构、分布、代谢和调控的基础上,概述了GSH在植物抗逆性方面的 作用,认为GSH通过植物体内螯合肽合成酶催化下聚合形成植物螯合肽来抵抗重金属的胁迫,作为抗氧化剂 参与低温伤害的保护,以亲核进攻一结合反应方式进行生物解毒等。讨论了GSH在植物抗逆性功能中的机 制,并就GSH今后在该方面的研究前景进行了展望。     

代谢转基因植物的研究现状与展望

代谢转基因是通过基因工程技术对细胞内的代谢途径进行遗传修饰,进而完成细胞特性改造。代谢修饰转基因植物是一个极具商业前景的领域,在医药、环境、农业等方面已有许多成功应用的实例。综合调控代谢的基因工程策略,讨论了代谢转基因植物的研究现状,我国农业生产中存在的主要问题和代谢转基因技术对我国农业发展的意义和前景。