Molecular analyses of candidate carotenoid biosynthetic genes in Vitis vinifera L.

[摘要] ENGLISH ABSTRACT:Plants cannot avoid stress and must therefore be capable of rapidly responding toextreme environmental changes. An inability to control and regulate thephotosynthetic process during stress conditions will lead to the formation of highlyreactive oxygen species that concomitantly causes photo-oxidative damage to thepigments and proteins of the photosynthetic apparatus. Since light is the primarysource of energy for the photosynthetic process, it is clear that plants arecontinuously required to balance the light energy absorbed for the photochemicalreactions against photoprotection in a dynamic way in order to survive. Carotenoidsare precursors of abscisic acid, but more importantly structural components of thephotosynthetic apparatus. During photosynthesis carotenoids function as accessorylight-harvesting pigments, and also fulfil a photoprotective function by quenching thereactive molecules formed during conditions that saturate the photosynthetic process.Due to the importance of carotenoids to plant fitness and human health (asVitamin A precursors) this study has attempted to isolate and characterise genes thatare directly, or indirectly involved in carotenoid biosynthesis in Vitis vinifera. In totaleleven full-Iength- and eight partial genes have been isolated, cloned andsequenced. These genes can be grouped into the following pathways: (i) the 1-deoxy-D-xylulose 5-phosphate (DOXP)/2-C-methyl-D-erythritol 4-phosphate (MEP)pathway (i.e. the plastidic isopentenyl diphosphate biosynthetic pathway); (ii) themevalonate pathway (i.e. the cytosolic/mitochondrial IPP biosynthetic pathway); (iii)the carotenoid biosynthetic pathway; (iv) the abscisic acid biosynthetic pathway (as adegradation product of carotenoids); and general isoprenoid biosynthetic pathways(as precursors of carotenoids).The full-length genes (i.e. from the putative ATG to the STOP codon) of DOXPsynthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), IPPisomerase (IPI), 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), phytoenesynthase (PSY), Iycopene ~-cyclase (LBCY), ~-carotene hydroxylase (BCH),zeaxanthin epoxidase (lEP), 9-cis-epoxy carotenoid dioxygenase (NCED), farnesyldiphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) havebeen isolated from cDNA. In addition, the full-length genomic copy and putativepromoters of DXS, PSY, LBCY, BCH, NCED and lEP have also been isolated fromgenomic DNA by the construction and screening of sub-genomic libraries.Alignments of the genomic copies of these genes to the corresponding cDNAsequences have provided useful information regarding the genomic organisation ofthese genes, including the intron-exon junction sites in V. vinifera. The copy numberof the DXS, PSY, LBCY, BCH, NCED and lEP encoding genes in the Vitis genomehave been determined. DXS, PSY, BCH and lEP are single copy genes, whereasLBCY and NCED have two and three copies, respectively.The transcriptional activity of the putative promoters of six of the isolated genes(i.e. DXS, PSY, LBCY, BCH, lEP and NCED) were tested with a transient reportergene assay. None of the putative promoters tested showed any transcriptionalactivity of the reporter gene. The transcription of these genes, has however beenshown using northern blot analysis and/or RT-PCR. Preliminary expression profilesfor PSY, LBCY, BCH, and lEP were determined in different plant organs and theexpression of these genes was generally higher in photosynthetically active tissues.The expression of these genes following different treatments (abscisic acid, NaCI andwounding) was also assayed. The functionality of five of the isolated full-lengthgenes (IPI, GGPS, PSY, LBCY and BCH) has been shown in a bacterial colourcomplementation assay.In silica analysis of the predicted protein sequences of all eleven isolatedgenes revealed that they are conserved and share a high degree of homology to thecorresponding proteins in other plant species. The sequences were further analysedfor conserved domains in the protein sequences, and these proteins typicallydemonstrated similar domain profiles to homologues in other species (plant, bacteriaand algae). The predicted protein sequences were further analysed for transitpeptides, the presence of which would provide evidence for the sub-cellularlocalisation of the mature peptides. Since these genes are involved in biosyntheticpathways that are active in discrete organelles, the sub-cellular localisation of most ofthese proteins is known. The carotenoid biosynthetic genes (PSY, LBCY, BCH andZEP), the abscisic acid biosynthetic gene, NCED, as well as the DOXP/MEP pathwaygenes (DXS, lytB and IPI) were all localised to the chloroplast. The mevalonatepathway gene, HMGS, was localised to both the cytosol and the mitochondria, andthe general isoprenoid precursor genes, FPS and GGPS, were localised to thecytosol and the chloroplast, respectively. All these results are in agreement with thelocalisation of the respective pathways.In order to increase our understanding of carotenoid biosynthesis and functionsin plants, we constitutively overexpressed one of the isolated genes (BCH) in themodel plant, Nicotiana tabacum. Plants expressing the BCH gene in the senseorientation maintained a healthy photosynthetic rate under stress conditions thattypically caused photoinhibition and photodamage in the untransformed controlplants. This result was inferred using chlorophyll fluorescence and confirmed usingCO2 assimilation rates and stomatal conductance. Chlorophyll fluorescencemeasurements indicated that the photo protective non-photochemical quenchingability of the BCH-expressing plants increased, enabling the plants to maintainphotosynthesis under conditions that elicited a stress response in the untransformedcontrol plants. An integral photosynthetic protein component, the D1 protein, wasspecifically protected by the additional zeaxanthin in the BCH sense plants. Plantsexpressing an antisense BCH proved the converse, i.e. lower levels of BCH resultedin decreased zeaxanthin levels and made the transgenic plants more susceptible tohigh-light induced stress. These results have shown the crucial role of carotenoids(specifically the xanthophylls) in the photoprotective mechanism in plants. Theincreased photoprotection provided by the BCH expressing plants suggests that thescenario in plants is not optimal and can be improved. Any improvement in thephotoprotective ability of a plant will affect both the fitness and productivity of theplant as a whole and will therefore find application in a number of crop plants on aglobal scale. This study has resulted in the successful isolation and characterisationof genes involved in the direct, or indirect, carotenoid biosynthetic pathways. Thefurther study and manipulation of these genes in model plants will provide usefulinsights into the physiological role of specific carotenoids in photosynthesis and inplants as a whole.

[发布机构] Stellenbosch University