Engineering And Regulating Plant Tocopherol Biosynthetic Pathway

Tocopherols, known as vitamin E, play paramount roles in the daily diet of both humans and animals as important lipid-soluble antioxidants. Tocopherols can be synthesized only in photoautotrophy organisms, including plants and other oxygenic, photosynthetic organisms, and hence human everyday needs of vitamin E are derived from plant materials or supplementary nutritional drugs. Recently, genes required for tocopherol biosynthetic pathway have been identified and cloned with the help of genomics-based approaches and molecular manipulation in the model organisms: Arabidopsis thaliana and Synechocystis sp. PCC 6803.Genes encoding five enzymes involved in tocopherol biosynthesis of Arabidopsis were cloned, and genetic manipulations were put forward with these genes in this study. Strategies were given rise to value the role of each individual gene in one plant species system, attempting to enhance the level of tocopherol content or convert the constitution of tocopherol. In addition, dual-gene transformation were performed in both Arabidopsis and lettuce (Lactuca sativa L. var. logifolia), succeeding in obtaining strains with elevated tocopherol contents andα-tocopherol composition. On the other hand, transgenic lines with target gene(s) overexpression or inhibition could be used to investigate tocopherol physiology in plant. The relationship of lipid-soluble antioxidant tocopherol and water-soluble antioxidant ascorbate, and function of tocopherol in plant defense were analyzed and discussed in this study.1) Systematic value of individual gene encoding enzyme involved in tocopherol biosynthetic pathway under Arabidopsis backgroundIn order to increase tocopherol content and to obtain desired tocopherol composition in plant, genetic manipulation of tocopherol biosynthetic pathway is an effective approach. In this study, five genes(HPPD, HPT, MPBQ MT, TC, andγ-TMT), encoding enzymes involved in tocopherol biosynthesis, were over-expressed in model plant Arabidopsis thaliana, either alone or in couple combinations (γ-TMT+HPT andγ-TMT+MPBQ MT), aiming to value and compare the functions of enzymes played in tocopherol biosynthetic pathway under the same genetic background. Our results suggested that, elevated expression level of biosynthetic pathway gene affected either total tocopherol content or composition, based on the position of he enzyme in tocopherol biosynthetic pathway. It can be divided into two groups because of the different functions -- the enzymes whose function are mainly on increasing total tocopherol contents (such as HPPD and HPT), and the enzymes effectively change the composition of tocopherols (such as MPBQ MT, TC andγ-TMT). In addition, dual genes transformation was predominant compared with single gene transformation strategies. Based on these studies, an appraisable system in sole plant was set up to value and compare the different roles of enzymes played in tocopherol biosynthetic pathway in term of tocopherol content and composition.2) Genetic engineering tocopherol biosynthetic pathway in lettuce (Lactuca sativa L. var. logifolia)In order to increase tocopherol content and to obtain desired tocopherol composition, genetic manipulation of tocopherol biosynthetic pathway can be employed as an effective approach. In this study, genes encoding Arabidopsis homogentisate phytyltransferase (HPT) andγ-tocopherol methyltransferase (γ-TMT) were constitutively over-expressed in lettuce(Lactuca sativa L. var. logifolia), alone or in couple combination. Our results suggested that, over-expression of hpt could increase total flux of tocopherol biosynthetic pathway, while over-expression ofγ-tmt could shift tocopherol composition in favor ofα-tocopherol. Transgenic lettuce lines expressing both hpt andγ-tmt produced significantly higher levels of tocopherol and vitamin E activity compared with non-transgenic control and transgenic lines harboring a single gene(hpt orγ-tmt). The best line produced 64.55μg/g FW total tocopherol content in leaves, which was over eight times of that in the non-transgenic control and more than twice the amount in the highest tocopherol-producing hpt single-gene transgenic line. We conclude that transgenic plants harboring both hpt andγ-tmt lead to increased tocopherol content and elevatedα-/γ-tocopherol ratio, which is more efficient than plants harboring only one of the two genes. The downstream enzymeγ-TMT plays important roles as a pulling force by improving the tocopherol composition, whereas the functioning of the upstream enzyme HPT is increased proportionally. Based on these studies, an effective approach is provided for improving the nutritional value of vegetable by increasing natural vitamin E content.3) Genetic engineering tocopherol biosynthetic pathway influence ascorbate level in transgenic linesWith the point of view of network, elevated tocopherol levels might have effect on other antioxidants in transgenic lines, especially ascorbic acid (AsA). It was found that metabolic engineering of tocopherol biosynthetic pathway affected endogenous AsA pool in leaves, which had negative correlation with tocopherol content and/orα-/γ-tocopherol ratio. Further study suggested that expression levels of genes encoding enzymes of Halliwell-Asada cycle were up-regulated, such as APX, DHAR and MDAR, while AsA biosynthetic pathway genes tested did not show remarkable changes. These findings provided hints on the relationship and regulation between lipid-soluble antioxidant tocopherol and water-soluble antioxidant AsA, which will help to perfect theory in plant physiology and give practical instruction for metabolic engineering.4) Overexpression and inhibition of tocopherol cyclase in Arabidopsis and its function in tocopherol biosynthesis and plant stress physiologyTocopherol functions have been studied most extensively in animal systems, and understanding of plant tocopherol functions pales by comparison. Tocopherols play important roles as antioxidants in many physiology processes. Transgenic lines with altered tocopherol levels and types will be important tools for physiology studies. In this study, expression level of gene encoding tocopherol cyclase was up- or down-regulated by transgenic methods. Changing in phenotype and physiology of transgenic and NC lines were investigated under normal, salt and drought stress state, respectively. Up-regulated of tc expression resulted in elevatingγ-tocopherol proportion, whileα-tocopherol content did not show marked increase compared with NC. RNAi inhibition lines succeeded in down-regulating tc expression level, and led to deficiency of total tocopherols. Decreased total tocopherol content was not lethal under normal growing state because of the complementary function of increased vitamin C level, but the strains were obviously weaker than NC. Tocopherol deficiency increased sensitivity of tc::RNAi transgenic lines toward all tested stress conditions, with altered proline or soluble sugar metabolism and increased lipid peroxidation and electrolyte leakage. However, tc overexpression lines showed an improved performance when challenged with salt or drought stress. These findings primarily confirm that vitamin E has important function in maintaining the balance of redox homeostasis and improving plant stress defense potential, with co-operation of vitamin C.γ-Tocopherol has similar antioxidant function asα-tocopherol.