纽荷尔脐橙CCS基因的克隆与分析

利用RT-PCR技术从纽荷尔脐橙果实cDNA中分离出辣椒红／辣椒玉红素合成酶（Capsanthin/Capsorubin synthase,CCS）基因（Ccs）全长。该片断长为1619bp,编码503个氨基酸。BLAST比较其氨基酸同源性发现,该序列所推导的氨基酸与已知的甜橙CCS完全一致,与胡萝卜和辣椒的CCS,以及马铃薯新黄质合成酶（neoxanthin synthase,NSY）有70％的同源性。此外,与番茄和柑橘等的番茄红素环化酶也有50%~70%的同源性。半定量RT-PCR分析表明,Ccs在纽荷尔脐橙果实成熟过程中呈先上升后下降的表达趋势,期间9月份的表达量最高。     

The tomato mutation nxd1 reveals a gene necessary for neoxanthin biosynthesis and demonstrates that violaxanthin is a sufficient precursor for abscisic acid biosynthesis

Carotenoid pigments are indispensable for plant life. They are synthesized within plastids where they provide essential functions in photosynthesis. Carotenoids serve as precursors for the synthesis of the strigolactone phytohormones, which are made from β‐carotene, and of abscisic acid (ABA), which is produced from certain xanthophylls. Despite the significant progress that has been made in our understanding of the carotenoid biosynthesis pathway, the synthesis of the xanthophyll neoxanthin has remained unknown. We report here on the isolation of a tomato (Solanum lycopersicum) mutant, neoxanthin‐deficient 1 (nxd1), which lacks neoxanthin, and on the cloning of a gene that is necessary for neoxanthin synthesis in both tomato and Arabidopsis. The locus nxd1 encodes a gene of unknown function that is conserved in all higher plants. The activity of NXD1 is essential but cannot solely support neoxanthin synthesis. Lack of neoxanthin does not significantly reduce the fitness of tomato plants in cultivated field conditions and does not impair the synthesis of ABA, suggesting that in tomato violaxanthin is a sufficient precursor for ABA production in vivo.     

A chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of the tomato white-flower locus

Carotenoids and their oxygenated derivatives xanthophylls play essential roles in the pigmentation of flowers and fruits. Wild-type tomato (Solanum lycopersicum) flowers are intensely yellow due to accumulation of the xanthophylls neoxanthin and violaxanthin. To study the regulation of xanthophyll biosynthesis, we analyzed the mutant white-flower (wf). It was found that the recessive wf phenotype is caused by mutations in a flower-specific beta-ring carotene hyroxylase gene (CrtR-b2). Two deletions and one exon-skipping mutation in different CrtR-b2 wf alleles abolish carotenoid biosynthesis in flowers but not leaves, where the homologous CrtR-b1 is constitutively expressed. A second beta-carotene hydroxylase enzyme as well as flower- and fruit-specific geranylgeranyl diphosphate synthase, phytoene synthase, and lycopene beta-cyclase together define a carotenoid biosynthesis pathway active in chromoplasts only, underscoring the crucial role of gene duplication in specialized plant metabolic pathways. We hypothesize that this pathway in tomato was initially selected during evolution to enhance flower coloration and only later recruited to enhance fruit pigmentation. The elimination of beta-carotene hydroxylation in wf petals results in an 80% reduction in total carotenoid concentration, possibly caused by the inability of petals to store high concentrations of carotenoids other than xanthophylls and by degradation of beta-carotene, which accumulates as a result of the wf mutation but is not due to altered expression of genes in the biosynthetic pathway.     

Determination of the stoichiometry and strength of binding of xanthophylls to the photosystem II light harvesting complexes

Xanthophylls have a crucial role in the structure and function of the light harvesting complexes of photosystem II (LHCII) in plants. The binding of xanthophylls to LHCII has been investigated, particularly with respect to the xanthophyll cycle carotenoids violaxanthin and zeaxanthin. It was found that most of the violaxanthin pool was loosely bound to the major complex and could be removed by mild detergent treatment. Gentle solubilization of photosystem II particles and thylakoids allowed the isolation of complexes, including a newly described oligomeric preparation, enriched in trimers, that retained all of the in vivo violaxanthin pool. It was estimated that each LHCII monomer can bind at least one violaxanthin. The extent to which different pigments can be removed from LHCII indicated that the relative strength of binding was chlorophyll b > neoxanthin > chlorophyll a > lutein > zeaxanthin > violaxanthin. The xanthophyll binding sites are of two types: internal sites binding lutein and peripheral sites binding neoxanthin and violaxanthin. In CP29, a minor LHCII, both a lutein site and the neoxanthin site can be occupied by violaxanthin. Upon activation of the violaxanthin de-epoxidase, the highest de-epoxidation state was found for the main LHCII component and the lowest for CP29, suggesting that only violaxanthin loosely bound to LHCII is available for de-epoxidation.     

Inhibition of zeaxanthin epoxidase activity by cadmium ions in higher plants

The effect of cadmium and zinc ions on violaxanthin cycle enzymes, violaxanthin de-epoxidase and zeaxanthin epoxidase, has been investigated on selected plant species, as well as in vitro. About 50% inhibition of zeaxanthin epoxidase by cadmium ions was found for duckweed (Lemna trisulca) and tomato (Lycopersicon esculentum) leaves but for apricot (Prunus armeniaca) leaves no cadmium inhibition of the epoxidation reaction was observed. The cadmium inhibition of zeaxanthin epoxidase in tomato was abolished by zinc ions. Zinc ions alone did not affect the activity of neither of the enzymes of the violaxanthin cycle. This suggests that mechanism of cadmium inactivation of the enzyme relies on cadmium interaction with a cysteine residue of the protein, important for the enzyme activity. The target cysteine in tomato epoxidase could be the cysteine residue present in the most conservative part of the molecule which is not present in the apricot enzyme sequence. Neither stimulation nor inhibition of violaxanthin de-epoxidase by cadmium ions both in vivo and in vitro studies was detected. It confirms the proposed mechanism of zeaxanthin epoxidation inhibition by cadmium ions because the cysteine residue in the conservative motif of violaxathin de-epoxidase is not present.     

Carotenoid specificity of light-harvesting complex II binding sites. Occurrence of 9-cis-violaxanthin in the neoxanthin-binding site in the parasitic angiosperm Cuscuta reflexa

The parasitic angiosperm Cuscuta reflexa has a highly unusual carotenoid composition in that it does not contain neoxanthin, an otherwise ubiquitous component of the major light-harvesting complex protein (LHCIIb) in all other higher plant species studied to date. Combined HPLC and mass spectrometric analysis has enabled us to detect in tissues of C. reflexa two new types of xanthophylls: lutein-5,6-epoxide and 9-cis-violaxanthin. We have isolated the LHCIIb complex from thylakoids and analyzed chlorophyll and carotenoid composition. The data show that the 9-cis-violaxanthin is present in amounts similar to that of neoxanthin in most plants. On the other hand, lutein-5,6-epoxide was found to be in substoichiometric quantities, suggesting a peripheral location similar to the loosely-associated all-trans-violaxanthin and also enabling suitable accessibility for the de-epoxidase (VDE). Absorption spectroscopy revealed close similarities of the excited state energies of neoxanthin and 9-cis-violaxanthin in vitro and in intact LHCIIb complex. Resonance Raman analysis clearly indicates a cis conformation of violaxanthin in the complex, confirming the pigment analysis data and proving that not only does violaxanthin replace neoxanthin as an intrinsic component of LHCIIb in C. reflexa but it also adopts the same 9-cis conformation of neoxanthin. These results suggest that the N1 binding site of LHCIIb preferentially binds 9-cis-5,6-epoxy carotenoids, which has implications for the features of this binding site and its role in the photosystem II antenna assembly and stability.     

Characterization and expression profile analysis of a new cDNA encoding taxadiene synthase from Taxus media

A full-length cDNA encoding taxadiene synthase (designated as TmTXS), which catalyzes the first committed step in the Taxol biosynthetic pathway, was isolated from young leaves of Taxus media by rapid amplification of cDNA ends (RACE). The full-length cDNA of TmTXS had a 2586 bp open reading frame (ORF) encoding a protein of 862 amino acid residues. The deduced protein had isoelectric point (pI) of 5.32 and a calculated molecular weight of about 98 kDa, similar to previously cloned diterpene cyclases from other Taxus species such as T. brevifolia and T. chinenisis. Sequence comparison analysis showed that TmTXS had high similarity with other members of terpene synthase family of plant origin. Tissue expression pattern analysis revealed that TmTXS expressed strongly in leaves, weak in stems and no expression could be detected in fruits. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in Taxol biosynthetic pathway in different tissues of Taxus plants. Phylogenetic tree analysis showed that TmTXS had closest relationship with taxadiene synthase from T. baccata followed by those from T. chinenisis and T. brevifolia. Expression profiles revealed by RT-PCR under different chemical elicitor treatments such as methyl jasmonate (MJ), silver nitrate (SN) and ammonium ceric sulphate (ACS) were also compared for the first time, and the results revealed that expression of TmTXS was all induced by the tested three treatments and the induction effect by MJ was the strongest, implying that TmTXS was high elicitor responsive.     

The Arabidopsis ABA-deficient mutant aba4 demonstrates that the major route for stress-induced ABA accumulation is via neoxanthin isomers

A novel abscisic acid (ABA)-deficient mutant, aba4, was identified in a screen for paclobutrazol-resistant germination. Compared with wild-type, the mutant showed reduced endogenous ABA levels in both dehydrated rosettes and seeds. Carotenoid composition analysis demonstrated that the defective locus affects neoxanthin synthesis. The ABA4 gene was identified by map-based cloning, and found to be a unique gene in the Arabidopsis genome. The predicted protein has four putative helical transmembrane domains and shows significant similarity to predicted proteins from tomato, rice and cyanobacteria. Constitutive expression of the ABA4 gene in Arabidopsis transgenic plants led to increased accumulation of trans-neoxanthin, indicating that the ABA4 protein has a direct role in neoxanthin synthesis. aba4 mutant phenotypes were mild compared with previously identified ABA-deficient mutants that exhibit vegetative tissue phenotypes. Indeed, ABA levels in seeds of aba4 mutants were higher than those of aba1 mutants. As aba1 mutants are also affected in a unique gene, this suggests that ABA can be produced in the aba4 mutant by an alternative pathway using violaxanthin as a substrate. It appears, therefore, that in Arabidopsis both violaxanthin and neoxanthin are in vivo substrates for 9-cis-epoxycarotenoid dioxygenases. Furthermore, significantly reduced levels of ABA were synthesized in the aba4 mutant on dehydration, demonstrating that ABA biosynthesis in response to stress must occur mainly via neoxanthin isomer precursors.     

Effect of hydroxylated carotenoid deficiency on ABA accumulation in Arabidopsis

Abscisic acid (ABA) is a sesquiterpene compound (C₁₅) derived from C₄₀ carotenoids. The immediate carotenoid precursors for ABA biosynthesis, 9- cis -violaxanthin and 9'- cis -neoxanthin, are produced from β -carotene by a series of hydroxylation, epoxidation, and isomerization reactions. Carotenoid hydroxylase deficient mutants contain severely reduced levels of violaxanthin and neoxanthin ( < 20% of wild type level) and provide a unique system to correlate carotenoid substrate availability and ABA production in photosynthetic tissues under non-stressed conditions. Quantitative measurements indicated that ABA levels in the carotenoid hydroxylase mutants are reduced nearly 50% compared to the wild type plants under non-stressed conditions. When drought-stressed, wild type plants showed up to a 17-fold increase in ABA levels, while ABA levels in the carotenoid hydroxylase mutants were only increased 6- to 7-fold (25% of wild type drought-stressed ABA levels). Expression of AtNCED3 ( Arabidopsis thaliana nine- cis -epoxycarotenoid dioxygenase 3, the rate-limiting activity for ABA biosynthesis) was induced in the carotenoid hydroxylase mutants, but to a lesser extent than the 40-fold increase in wild type plants. Therefore, the reduced ABA accumulation in response to drought-stress is at least partially due to the attenuated increase in AtNCED3 gene expression in the carotenoid hydroxylase mutants. The remaining violaxanthin and neoxanthin in the carotenoid hydroxylase mutants can not be converted into ABA, indicating that there is probably a separate pool of violaxanthin and neoxanthin that is not accessible to the cleavage enzymes, because it is sequestered in the light-harvesting complexes.     

Molecular cloning and characterization of the yew gene encoding squalene synthase from Taxus cuspidata

The enzyme squalene synthase (EC 2.5.1.21) catalyzes a reductive dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for the sterol and triterpene biosynthesis. A full-length cDNA encoding squalene synthase (designated as TcSqS) was isolated from Taxus cuspidata, a kind of important medicinal plants producing potent anti-cancer drug, taxol. The full-length cDNA of TcSqS was 1765 bp and contained a 1230 bp open reading frame (ORF) encoding a polypeptide of 409 amino acids. Bioinformatic analysis revealed that the deduced TcSqS protein had high similarity with other plant squalene synthases and a predicted crystal structure similar to other class I isoprenoid biosynthetic enzymes. Southern blot analysis revealed that there was one copy of TcSqS gene in the genome of T. cuspidata. Semiquantitative RT-PCR analysis and northern blotting analysis showed that TcSqS expressed constitutively in all tested tissues, with the highest expression in roots. The promoter region of TcSqS was also isolated by genomic walking and analysis showed that several cis-acting elements were present in the promoter region. The results of treatment experiments by different signaling components including methyl-jasmonate, salicylic acid and gibberellin revealed that the TcSqS expression level of treated cells had a prominent diversity to that of control, which was consistent with the prediction results of TcSqS promoter region in the PlantCARE database.     

Reduced levels of cadinane sesquiterpenoids in cotton plants expressing antisense (+)-delta-cadinene synthase

Cotton plants were transformed with an antisense construct of cdn1-Cl, a member of a complex gene family of delta-(+)cadinene (CDN) synthase. This synthase catalyzes the cyclization of (E,E)-farnesyl diphosphate to form CDN, and in cotton, it occupies the committed step in the biosynthesis of cadinane sesquiterpenoids and heliocides (sesterterpenoids). Southern analyses of the digestion of leaf DNA from R(o), T(o), and T(1) plants with Hind III, Pst I and Kpn I restriction enzymes show the integration of antisense cdn1-C1 cDNA driven by the CaMV 35S promoter into the cotton genome. Northern blots demonstrate the appearance of cdn synthase mRNA preceding CDN synthase activity and the formation of gossypol in developing cottonseed. T(2) cottonseed show a reduced CDN synthase activity and up to a 70% reduction in gossypol. In T(1) leaves the accumulated amounts of gossypol, hemigossypolone and heliocides are reduced 92.4, 83.3 and 68.4%, respectively. These data demonstrate that the integration of antisense cdn1-C1 cDNA into the cotton genome leads to a reduction of CDN synthase activity and negatively impacts on the biosynthesis of cadinane sesquiterpenoids and heliocides in cotton plants.     

Carotenoid binding sites in LHCIIb. Relative affinities towards major xanthophylls of higher plants.

The major light-harvesting complex of photosystem II can be reconstituted in vitro from its bacterially expressed apoprotein with chlorophylls a and b and neoxanthin, violaxanthin, lutein, or zeaxanthin as the only xanthophyll. Reconstitution of these one-carotenoid complexes requires low-stringency conditions during complex formation and isolation. Neoxanthin complexes (containing 30-50% of the all-trans isomer) disintegrate during electrophoresis, exhibit a largely reduced resistance against proteolytic attack; in addition, energy transfer from Chl b to Chl a is easily disrupted at elevated temperature. Complexes reconstituted in the presence of either zeaxanthin or lutein contain nearly two xanthophylls per 12 chlorophylls and are more resistant against trypsin. Lutein-LHCIIb also exhibits an intermediate maintenance of energy transfer at higher temperature. Violaxanthin complexes approach a xanthophyll/12 chlorophyll ratio of 3, similar to the ratio in recombinant LHCIIb containing all xanthophylls. On the other hand, violaxanthin-LHCIIb exhibits a low thermal stability like neoxanthin complexes, but an intermediate accessibility towards trypsin, similar to lutein-LHCIIb and zeaxanthin-LHCIIb. Binary competition experiments were performed with two xanthophylls at varying ratios in the reconstitution. Analysis of the xanthophyll contents in the reconstitution products yielded information about relative carotenoid affinities of three assumed binding sites. In lutein/neoxanthin competition experiments, two binding sites showed a strong preference (> 200-fold) for lutein, whereas the third binding site had a higher affinity (25-fold) to neoxanthin. Competition between lutein and violaxanthin gave a similar result, although the specificities were lower: two binding sites have a 36-fold preference for lutein and one has a fivefold preference for violaxanthin. The lowest selectivity was between lutein and zeaxanthin: two binding sites had a fivefold higher affinity for lutein and one has a threefold higher affinity to zeaxanthin.     

LEFPS1, a tomato farnesyl pyrophosphate gene highly expressed during early fruit development

Farnesyl pyrophosphate synthase (FPS) catalyzes the synthesis of farnesyl pyrophosphate, a key intermediate in sterol and sesquiterpene biosynthesis. Using a polymerase chain reaction-based approach, we have characterized LeFPS1, a tomato (Lycoperscion esculentum cv Wva 106) fruit cDNA, which encodes a functional FPS. We demonstrate that tomato FPSs are encoded by a small multigenic family with genes located on chromosomes 10 and 12. Consistent with farnesyl pyrophosphate requirement in sterol biosynthesis, FPS genes are ubiquitously expressed in tomato plants. Using an LeFPS1 specific probe, we show that the corresponding gene can account for most of FPS mRNA in most plant organs, but not during young seedling development, indicating a differential regulation of FPS genes in tomato. FPS gene expression is also under strict developmental control: FPS mRNA was mainly abundant in young organs and decreased as organs matured with the exception of fruits that presented a biphasic accumulation pattern. In this latter case in situ hybridization studies have shown that FPS mRNA is similarly abundant in all tissues of young fruit. Taken together our results suggest that several FPS isoforms are involved in tomato farnesyl pyrophosphate metabolism and that FPS genes are mostly expressed in relation to cell division and enlargement.     

Radiolabeling of a wound-inducible pyridoxal phosphate-utilizing enzyme: evidence for its identification as ACC synthase

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase, a pyridoxal phosphate-utilizing enzyme, catalyzes the conversion of S-adenosylmethionine to ACC, the rate-limiting step in the biosynthesis of the plant hormone ethylene. We report the partial purification (400-fold) of ACC synthase from wounded pink tomato pericarp. Further purification results in a decrease in specific activity apparently due to the instability of the enzyme. Radiolabeling of a pyridoxal phosphate-utilizing protein in the ACC synthase-enriched fraction was achieved by reduction using tritiated sodium borohydride. Evidence that this radiolabeled protein is ACC synthase is presented.