Over-expression and analysis of two Vitis vinifera carotenoid biosynthetic genes in transgenic Arabidopsis

[摘要] Plants have evolved photosynthetic systems to efficiently harvest sunlight energy forthe production of carbohydrates, but these systems also are extremely susceptible toan excess of light. To combat the potential damaging effects of light, plants havedeveloped various mechanisms to control and cope with light stress. Thesemechanisms include the movement of either leaves, cells (negative phototaxis) orchloroplasts to adjust the light-capturing potential, the adjustment of thelight-harvesting antenna size through gene expression or protein degradation, theremoval of excess excitation energy either through an alternative electron transportpathway or as heat. However, the latter mechanism based on thermal dissipation,remains the most effective to rid the plant of damaging excess light energy. Thisprocess involves several carotenoid pathway pigments, specifically the de-epoxidisedxanthophyll cycle pigments. The process and extent of thermal dissipation in plantscan be measured and quantified as non-photochemical quenching (NPQ) ofchlorophyll fluorescence by using well-established methodologies. SeveralArabidopsis and Chlamydomonas mutants affected in the xanthophyll cycle havebeen isolated. These mutants have provided evidence for the correlation betweenthe de-epoxidised xanthophyll cycle pigments and NPQ as well as betterunderstanding of the operation of the xanthophyll cycle and the related carotenoidbiosynthetic enzymes. This key photoprotective role of the xanthophyll cycle istherefore a promising target for genetic engineering to enhance environmental stresstolerance in plants. Several genes from the carotenoid biosynthetic pathway ofgrapevine (Vitis vinifera L.) were isolated previously in our laboratory. The main aimof this study was to over-express two xanthophyll cycle genes from grapevine inArabidopsis and to analyse the transgenic population with regards to pigment contentand levels as well as certain photosynthetic parameters. The transgenic lines werecompared with wild type Arabidopsis (untransformed) plants and two xanthophyllcycle mutants under non-limiting conditions as well as a stress condition, specificallya high light treatment to induce possible photodamage and photoinhibition.Transgenic Arabidopsis lines over-expressing the two V. vinifera xanthophyllcycle genes, β-carotene hydroxylase (VvBCH) and zeaxanthin epoxidase (VvZEP),were established following Agrobacterium transformation. In addition to theuntransformed wild type, two NPQ mutants, npq1 (lacking violaxanthin de-epoxidase)and npq2 (lacking zeaxanthin epoxidase), were used as controls throughout thisstudy. The transgenic lines were propagated to a homozygous T3-generation, wherestable integration and expression of the transgenes were confirmed in only 16% and12% for VvBCH and VvZEP lines, respectively. No phenotypical differences could beobserved for the transgenic lines compared to the wild type, but the npq2 mutantshowed a stunted and 'wilty' phenotype, as was previously described. To evaluate the pigment composition of the transgenic lines a reliable andreproducible method was needed to analyse carotenoids from leafy material. To thisend a new high-performance liquid chromatography (HPLC) method was developedfor the quantitative profiling of eight major carotenoids and chlorophyll a and b.Emphasis was placed on baseline separation of the xanthophyll pigments, lutein andzeaxanthin as well as the cis- and trans-forms of violaxanthin and neoxanthin. Themethod effectively distinguished Arabidopsis wild type plantlets from the two NPQmutant lines (npq1 and 2) and could possibly find application for green leafy tissuesamples in general.The carotenoid content of the NPQ mutants were in accordance with previousreports. The lack of zeaxanthin epoxidase activity in the npq2 mutant resulted in theaccumulation of zeaxanthin under both low and high light conditions. This high levelzeaxanthin was found to cause an initial rapid induction of NPQ at low to moderatelight intensities, but this difference disappeared at high light, where zeaxanthinformation induced considerable NPQ in the wild type. Similarly, the npq1 mutant wasunable to de-epoxidise violaxanthin to zeaxanthin under high light conditions, whichresulted in severe inhibition of NPQ induction. Furthermore, these mutant plantletswere shown to be more susceptible to photoinhibition compared to that of the wildtype.The over-expression of VvBCH resulted in a marked increase in thexanthophyll cycle pool pigments (violaxanthin, antheraxanthin and zeaxanthin) andreduced β-carotene levels under both low and high light conditions compared to thatthe wild type, indicating elevated β-carotene hydroxylase activity possibly due toover-expression of the VvBCH gene. Similar to the induction of NPQ in the npq2mutant, the increased levels of zeaxanthin in the VvBCH lines did not offer anyadditional photoprotection. This would suggest that the heightened zeaxanthin levelsobserved for the VvBCH lines do not necessarily enhance photoprotection, howevermay protect the thylakoid membrane against lipid peroxidation as has been shownpreviously. The VvZEP lines however, showed reduce levels of zeaxanthin in highlight conditions to that of the wild type, probably due to the competing epoxidationand de-epoxidation reactions of the xanthophyll cycle. This reduction in zeaxanthinsynthesis in the VvZEP lines resulted in significant reduced NPQ induction comparedthat of the wild type, a phenomenon also observed for the npq1 mutant. Similar tothe npq1 mutant, these lines displayed significantly increased photoinhibition, whichmay be due to photodamage of the reaction centers if one considers the loweredphotosystem II photochemistry efficiency and reaction center openness of these linescompared to the wild type. This may suggest that even small reductions inzeaxanthin amounts can result in an increase in photoinhibition, under high lightconditions.This study and its results provide fundamental information regarding twograpevine-derived carotenoid pathway genes and their possible physiological roles.Moreover, studies like these provide information that is essential when possible biotechnological approaches are planned with this central plant metabolic pathway inmind. The results highlighted the complex regulation of this pathway, necessitatingattention to flux control, simultaneous manipulation of several pathway genes, andthe measurement of other compounds derived from this pathway when evaluating thepossible applications of the carotenoid pathway of plants.

[发布机构] Stellenbosch University