Isolation and characterisation of carotenoid biosynthetic genes from Vitis vinifera

[摘要] Plants are constantly exposed to adverse environmental conditions including variations inlight intensity and the availability of water resources. These abiotic factors are expected toworsen as the changing global climate places additional daily and seasonal demands onplant growth and productivity. As plants are incapable of avoiding stress they havedeveloped a number of mechanisms to manage and adapt to the unfavourable conditions.Carotenoids represent one of these mechanisms; with the xanthophylls (oxygenatedcarotenes) playing an essential role in photoprotection following exposure to excess lightenergy. They are also precursors to the plant hormone abscisic acid (ABA) which plays aknown role in stomatal regulation and thus drought tolerance. Carotenoids have beenidentified as potential targets for genetic manipulation to meet the existing nutritionaldemands (particularly vitamin A) and to enable plants to survive the climatic variationspredicted. Thorough investigations into the regulation and functioning of each carotenoidbiosynthetic gene in vivo as well as the roles of their encoded proteins are prerequisite.Within the Grapevine Biotechnology Programme, a number of isoprenoid biosynthetic geneshave been isolated from Vitis vinifera L. cv. Pinotage. From this vast resource two geneswere chosen; namely a lycopene b-cyclase (b-LCY) and 9-cis epoxycarotenoid dioxygenase(NCED) for detailed in planta analyses to address knowledge gaps in our currentunderstanding of carotenoid biosynthesis in general, its regulation and the roles of the twotarget genes in these processes. Currently, the role of b-LCY within the chloroplasts is notwell known. Although the relationship between NCED overexpression, ABA levels, reducedstomatal conductance and increased tolerance to water stress has been well-established,comprehensive physiological analysis of the resulting mutants during conditions of bothwater availability and shortage is not well documented. To assess their in planta role,functional copies of both genes were isolated from Vitis vinifera (cv. Pinotage), characterisedand independently transformed into the genome of the model plant, Arabidopsis thaliana, inthe sense orientation under a constitutive promoter.In order to investigate these pertinent scientific questions and thus to evaluate thephysiological role of each gene in vivo, a number of technologies were developed and/oradopted. These included a high-performance liquid chromatography method for profiling themajor plant pigments in leaf tissue, a combination vapour phase extraction and electronimpact-gas chromatography/mass spectrometry method for the phytohormone profiling aswell as various physiological analyses including the use of chlorophyll a fluorescence toassess the photosynthetic and non-photochemical quenching (NPQ) capacities of the plants.Overexpression of grapevine b-LCY (Vvb-LCY) decreased lutein levels due to preferentialpartitioning of lycopene into the b-branch. This decrease was not met by an increase ineither b-carotene or the xanthophyll cycle pigments implying that Vvb-LCY is not able toregulate the flow of carbon through the pathway and provides additional evidence to thefluidity of this pathway whereby pigment levels are continually balanced. The decreasedlutein levels observed under low light (LL) did not compromise the plants' ability to induceand maintain NPQ over a wide actinic light range. Vvb-LCY transgenics also had lower neoxanthin levels (and specifically the cis-isomer) under both LL and following exposure tohigh light (HL), which could be correlated to an increase in malondialdehyde. Although notcorroborated, a novel and unexpected finding was an essential role for neoxanthin, andpotentially lutein, in preventing or at least reducing lipid peroxidation under HL stress. Thelower neoxanthin amounts may be due to silencing of the Arabidopsis b-LCY by theVvb-LCY, as the former may function as a NSY paralog as NSY is not encoded for in theArabidopsis genome. Clearly, this study has confirmed that Vvb-LCY partitions the carbonflux between the a- and b-branches, however, the catalytic action of this enzyme isdependent on the amount of substrate available and is thus not a regulatory step directingthe flux within the pathway. Enzyme kinetic and detailed transcriptional analyses wouldconfirm the above findings.Overexpression of grapevine NCED1 (VvNCED1) increased ABA concentrations, delayedseed germination and resulted in a slight to severe reduction in the overall plant growth rate.NCED cleaves the 9-cis xanthophylls regulating ABA synthesis. However, contrary toexpectations, constitutive levels of this regulatory enzyme did not deplete the total andindividual chlorophylls and carotenoids in well-watered plants. Instead the VvNCED1transgenics simply exhibited a lower chloroplastic pigment complement with no concomitanteffects on their photosynthetic capacity. Of particular interest, well-watered plantsoverexpressing the VvNCED1 gene had an increased NPQ capacity of which the thermalenergy dissipation component (qE) was the most significant. It has been speculated that thisNPQ is associated with the phenotype conferred by VvNCED1 overexpression and occursindependently of the xanthophyll cycle, and specifically zeaxanthin. This study confirmedthat VvNCED1 functions during drought tolerance via ABA regulation of stomatalconductance. A detailed study was done to understand the plants' response during waterdeficit. Typically, decreases in total and individual carotenoids and the maximum efficiencyof photochemistry (Fv/Fm) as well as the relative water and soil moisture content wererecorded. No changes were recorded in salicylic acid (SA) levels, while indole acetic acid(IAA) was positively correlated to ABA or vice versa. In contrast, the physiology of VvNCED1overexpressing lines was largely unaffected, indicating that a reduced stomatal conductanceprotects the plants against water stress.This study has resulted in the isolation and characterisation of a carotenoid biosynthetic gene(b-LCY) and an abscisic acid synthesising gene (NCED). Significant advancements in ourexisting knowledge of the in planta role of both genes have been achieved. We have alsoreaffirmed that strict regulatory control and fluidity exists within the carotenoid biosyntheticpathway whereby individual pigment levels are constantly brought back into balance despiteconstitutive expression of one of the pathway gene members. These analyses providevaluable baseline information about individual genes which can be extended upon with otheromic technologies in order to comprehend the full complexity involved in carotenogenesis.

[发布机构] Stellenbosch University