Isolation and characterisation of carotenoid biosynthetic genes from Vitis vinifera
Plants are constantly exposed to adverse environmental conditions including variations in light intensity and the availability of water resources. These abiotic factors are expected to worsen as the changing global climate places additional daily and seasonal demands on plant growth and productivity. As plants are incapable of avoiding stress they have developed a number of mechanisms to manage and adapt to the unfavourable conditions. Carotenoids represent one of these mechanisms; with the xanthophylls (oxygenated carotenes) playing an essential role in photoprotection following exposure to excess light energy. They are also precursors to the plant hormone abscisic acid (ABA) which plays a known role in stomatal regulation and thus drought tolerance. Carotenoids have been identified as potential targets for genetic manipulation to meet the existing nutritional demands (particularly vitamin A) and to enable plants to survive the climatic variations predicted. Thorough investigations into the regulation and functioning of each carotenoid biosynthetic gene in vivo as well as the roles of their encoded proteins are prerequisite. Within the Grapevine Biotechnology Programme, a number of isoprenoid biosynthetic genes have been isolated from Vitis vinifera L. cv. Pinotage. From this vast resource two genes were 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 current understanding of carotenoid biosynthesis in general, its regulation and the roles of the two target genes in these processes. Currently, the role of b-LCY within the chloroplasts is not well known. Although the relationship between NCED overexpression, ABA levels, reduced stomatal conductance and increased tolerance to water stress has been well-established, comprehensive physiological analysis of the resulting mutants during conditions of both water 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), characterised and independently transformed into the genome of the model plant, Arabidopsis thaliana, in the sense orientation under a constitutive promoter. In order to investigate these pertinent scientific questions and thus to evaluate the physiological role of each gene in vivo, a number of technologies were developed and/or adopted. These included a high-performance liquid chromatography method for profiling the major plant pigments in leaf tissue, a combination vapour phase extraction and electron impact-gas chromatography/mass spectrometry method for the phytohormone profiling as well as various physiological analyses including the use of chlorophyll a fluorescence to assess the photosynthetic and non-photochemical quenching (NPQ) capacities of the plants. Overexpression of grapevine b-LCY (Vvb-LCY) decreased lutein levels due to preferential partitioning of lycopene into the b-branch. This decrease was not met by an increase in either b-carotene or the xanthophyll cycle pigments implying that Vvb-LCY is not able to regulate the flow of carbon through the pathway and provides additional evidence to the fluidity of this pathway whereby pigment levels are continually balanced. The decreased lutein levels observed under low light (LL) did not compromise the plants’ ability to induce and 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 to high light (HL), which could be correlated to an increase in malondialdehyde. Although not corroborated, a novel and unexpected finding was an essential role for neoxanthin, and potentially lutein, in preventing or at least reducing lipid peroxidation under HL stress. The lower neoxanthin amounts may be due to silencing of the Arabidopsis b-LCY by the Vvb-LCY, as the former may function as a NSY paralog as NSY is not encoded for in the Arabidopsis genome. Clearly, this study has confirmed that Vvb-LCY partitions the carbon flux between the a- and b-branches, however, the catalytic action of this enzyme is dependent on the amount of substrate available and is thus not a regulatory step directing the flux within the pathway. Enzyme kinetic and detailed transcriptional analyses would confirm the above findings. Overexpression of grapevine NCED1 (VvNCED1) increased ABA concentrations, delayed seed 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 to expectations, constitutive levels of this regulatory enzyme did not deplete the total and individual chlorophylls and carotenoids in well-watered plants. Instead the VvNCED1 transgenics simply exhibited a lower chloroplastic pigment complement with no concomitant effects on their photosynthetic capacity. Of particular interest, well-watered plants overexpressing the VvNCED1 gene had an increased NPQ capacity of which the thermal energy dissipation component (qE) was the most significant. It has been speculated that this NPQ is associated with the phenotype conferred by VvNCED1 overexpression and occurs independently of the xanthophyll cycle, and specifically zeaxanthin. This study confirmed that VvNCED1 functions during drought tolerance via ABA regulation of stomatal conductance. A detailed study was done to understand the plants’ response during water deficit. Typically, decreases in total and individual carotenoids and the maximum efficiency of photochemistry (Fv/Fm) as well as the relative water and soil moisture content were recorded. 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 VvNCED1 overexpressing lines was largely unaffected, indicating that a reduced stomatal conductance protects 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 our existing knowledge of the in planta role of both genes have been achieved. We have also reaffirmed that strict regulatory control and fluidity exists within the carotenoid biosynthetic pathway whereby individual pigment levels are constantly brought back into balance despite constitutive expression of one of the pathway gene members. These analyses provide valuable baseline information about individual genes which can be extended upon with other omic technologies in order to comprehend the full complexity involved in carotenogenesis.