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

Plants have evolved photosynthetic systems to efficiently harvest sunlight energy for the production of carbohydrates, but these systems also are extremely susceptible to an excess of light.To combat the potential damaging effects of light, plants have developed various mechanisms to control and cope with light stress.These mechanisms include the movement of either leaves, cells (negative phototaxis) or chloroplasts to adjust the light-capturing potential, the adjustment of the light-harvesting antenna size through gene expression or protein degradation, the removal of excess excitation energy either through an alternative electron transport pathway or as heat.However, the latter mechanism based on thermal dissipation, remains the most effective to rid the plant of damaging excess light energy.This process involves several carotenoid pathway pigments, specifically the de-epoxidised xanthophyll cycle pigments.The process and extent of thermal dissipation in plants can be measured and quantified as non-photochemical quenching (NPQ) of chlorophyll fluorescence by using well-established methodologies.Several Arabidopsis and Chlamydomonas mutants affected in the xanthophyll cycle have been isolated.These mutants have provided evidence for the correlation between the de-epoxidised xanthophyll cycle pigments and NPQ as well as better understanding of the operation of the xanthophyll cycle and the related carotenoid biosynthetic enzymes.This key photoprotective role of the xanthophyll cycle is therefore a promising target for genetic engineering to enhance environmental stress tolerance in plants.Several genes from the carotenoid biosynthetic pathway of grapevine (Vitis vinifera L.) were isolated previously in our laboratory.The main aim of this study was to over-express two xanthophyll cycle genes from grapevine in Arabidopsis and to analyse the transgenic population with regards to pigment content and levels as well as certain photosynthetic parameters.The transgenic lines were compared with wild type Arabidopsis (untransformed) plants and two xanthophyll cycle mutants under non-limiting conditions as well as a stress condition, specifically a high light treatment to induce possible photodamage and photoinhibition.Transgenic Arabidopsis lines over-expressing the two V. vinifera xanthophyll cycle genes, β-carotene hydroxylase (VvBCH) and zeaxanthin epoxidase (VvZEP), were established following Agrobacterium transformation.In addition to the untransformed wild type, two NPQ mutants, npq1 (lacking violaxanthin de-epoxidase) and npq2 (lacking zeaxanthin epoxidase), were used as controls throughout this study.The transgenic lines were propagated to a homozygous T3-generation, where stable integration and expression of the transgenes were confirmed in only 16% and 12% for VvBCH and VvZEP lines, respectively.No phenotypical differences could be observed for the transgenic lines compared to the wild type, but the npq2 mutant showed a stunted and 'wilty' phenotype, as was previously described.To evaluate the pigment composition of the transgenic lines a reliable and reproducible method was needed to analyse carotenoids from leafy material.To this end a new high-performance liquid chromatography (HPLC) method was developed for the quantitative profiling of eight major carotenoids and chlorophyll a and b.Emphasis was placed on baseline separation of the xanthophyll pigments, lutein and zeaxanthin as well as the cis-and trans-forms of violaxanthin and neoxanthin.The method effectively distinguished Arabidopsis wild type plantlets from the two NPQ mutant lines (npq1 and 2) and could possibly find application for green leafy tissue samples in general.The carotenoid content of the NPQ mutants were in accordance with previous reports.The lack of zeaxanthin epoxidase activity in the npq2 mutant resulted in the accumulation of zeaxanthin under both low and high light conditions.This high level of zeaxanthin was found to cause an initial rapid induction of NPQ at low to moderate light intensities, but this difference disappeared at high light, where zeaxanthin formation induced considerable NPQ in the wild type.Similarly, the npq1 mutant was unable to de-epoxidise violaxanthin to zeaxanthin under high light conditions, which resulted in severe inhibition of NPQ induction.Furthermore, these mutant plantlets were shown to be more susceptible to photoinhibition compared to that of the wild type.The over-expression of VvBCH resulted in a marked increase in the xanthophyll cycle pool pigments (violaxanthin, antheraxanthin and zeaxanthin) and reduced β-carotene levels under both low and high light conditions compared to that of the wild type, indicating elevated β-carotene hydroxylase activity possibly due to over-expression of the VvBCH gene.Similar to the induction of NPQ in the npq2 mutant, the increased levels of zeaxanthin in the VvBCH lines did not offer any additional photoprotection.This would suggest that the heightened zeaxanthin levels observed for the VvBCH lines do not necessarily enhance photoprotection, however it may protect the thylakoid membrane against lipid peroxidation as has been shown previously.The VvZEP lines however, showed reduce levels of zeaxanthin in high light conditions to that of the wild type, probably due to the competing epoxidation and de-epoxidation reactions of the xanthophyll cycle.This reduction in zeaxanthin synthesis in the VvZEP lines resulted in significant reduced NPQ induction compared to that of the wild type, a phenomenon also observed for the npq1 mutant.Similar to the npq1 mutant, these lines displayed significantly increased photoinhibition, which may be due to photodamage of the reaction centers if one considers the lowered photosystem II photochemistry efficiency and reaction center openness of these lines compared to the wild type.This may suggest that even small reductions in zeaxanthin amounts can result in an increase in photoinhibition, under high light conditions.This study and its results provide fundamental information regarding two grapevine-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 in mind.The results highlighted the complex regulation of this pathway, necessitating attention to flux control, simultaneous manipulation of several pathway genes, and the measurement of other compounds derived from this pathway when evaluating the possible applications of the carotenoid pathway of plants. OPSOMMINGPlante het, deur middel van evolusie, sisteme ontwikkel om effektief die son se stralingsenergie te absorbeer en vir koolhidraatproduksie te gebruik.Alhoewel hierdie sisteme die ligenergie vasvang, is hul sensitief vir 'n oormaat lig.Om hierdie sisteme te beskerm, het plante meganismes ontwikkel om die oormaat ligenergie veilig te verwyder en enige skade te herstel.Hierdie meganismes sluit die volgende in: die beweging van blare, selle en chloroplaste; die verandering van die antennagrootte vir ligvaslegging deur geenregulering en proteïendegradering; die verwydering van oormatige ligenegie deur 'n alternatiewe elektrontransportweg en deur die omskakeling van ligenergie na hitte.Van al hierdie meganismes is die hitteomskakeling die doeltreffendste.Dit betrek die pigmente van die xanthofilsiklus en kan verder ook gemeet word as nie-fotochemiese blussing van chlorofil-fluoresensie ("non-photochemical quenching", NPQ) Die xantofilsiklus is vir die eerste keer in 1962 opgemerk toe vasgestel is dat hierdie siklus deur lig beheer word.Sedertdien is verskeie Arabidopsis-en Chlamydomonas-mutante geïsoleer wat in hul xantofilsiklus beïnvloed is.Hierdie mutante het die belangrike fisiologiese funksies van die xanthofilsiklus in die beskerming van die fotosintesesisteme en die ensieme wat hierby betrokke is, uitgelig.Die belangrikheid van die xanthofilsiklus in fotobeskerming is dus 'n belowende teiken vir genetiese manupilering om plante meer weerstandbiedend teen ligstreskondisies te maak.Verskeie gene van die karotenoïedbiosintesepad in wingerd (Vitis vinifera L.) is in ons laboratorium geïsoleer.Die hoofdoel van hierdie studie was om twee xantofilsiklusgene van wingerd in Arabidopsis oor uit te druk en om die karotenoïedinhoud en -vlakke, asook sekere fotosintese parameters van die transgeniese lyne, te bestudeer.Hierdie transgeniese lyne is vergelyk met die wildetipe (ongetransformeerde Arabidopsis) en twee NPQ-mutante tydens ongestresde en gestresde toestande, spesifiek onder hoë ligstreskondisie om moontlike fotobeskerming en fotobeskadiging te induseer.Transgeniese Arabidopsis-lyne wat die β-karoteenhidroksilase-(VvBCH) en seaxantin-epoksidase-(VvZEP) gene oorruitdruk, is na Agrobacterium-transformasie gegenereer.Die ongetransformeerde wildetipe en twee NPQ-mutante, npq1 (defektief in violxantin-deëpoksidase) en npq2 (seaxantin-epoksidase), is as kontroles gedurende hierdie studie gebruik.Die transgeniese lyne is gepropageer deur die T3-generasie en die stabiele integrasie, en transkripsie van die transgene bevestig in net 16 en 12% vir VvBCH en VvZEP onderskeidelik.Geen fenotipe verskille is vir hierdie lyne opgemerk nie, behalwe die npq2-mutant wat 'n dwergagtige en verlepte voorkoms gehad het, wat ook ooreenkom met vorige bevindinge.Om die karotenoïedpigment-inhoud in die transgeniese lyne te evalueer was 'n akkurate en herhaalbare metode nodig om karotenoïedpigmente in blaarmateriaal te ontleed.'n Nuwe hoëdoeltreffendheid-vloeistofchromatografie (HDVC) -metode is ontwikkel om kwantifiseerbare profiele vir die hoofkarotenoïede en chlorofil a en b te lewer.Die ontwikkeling van die nuwe metode was veral van belang vir die sorgvuldige skeiding van die xantofilpigmente, seaxantin en luteïen, sowel as die sisen trans-vorme van violaxantin en neoxantin.Hierdie metode het dit moontlik gemaak om die wilde