genomic structure and sequence polymorphism of E,E-alpha farnesene synthase gene in Malus domestica; Borkh.; genomic structure and sequence polymorphism of E,E-alpha-farnesene synthase gene in Malus domestica; Borkh.
The deduced amino acid sequence of this enzyme shares the greatest degree of homology with a variety of monoterpene synthases, including alpha-(-)beta-pinene synthase, a gamma-terpinene synthase, and a (+)-limonene synthase from Citrus limon, which have 41-42% amino acid identity. A geraniol synthase from Cinnamomum tenuipilum and an isoprene synthase from Populus tremuloides have 42% identity to AFS1. The closest homology is with a linalool/nerolidol synthase from Fragaria ananassa (35% identity) and a (+)-delta-cadinene synthase from Gossypium hirsutum (33% identity).; Malus domestica Borkh., cultivar Law Rome
differences in the relative emissions of (E)-beta-ocimene and (E,E)-alpha-farnesene from accession Wassilewskija, a high-(E)-beta-ocimene emitter, and accession Columbia, a trace-(E)-beta-ocimene emitter, are attributed to allelic variation of closely related, tandem-duplicated terpene synthase genes, TPS02 and TPS03. The Wassilewskija genome contains a functional allele of TPS02 but not of TPS03, while the opposite is the case for Columbia. Recombinant proteins of the functional Wassilewskija TPS02 and Columbia TPS03 genes both show (E)-beta-ocimene and (E,E)-alpha-farnesene synthase activities. Differential subcellular compartmentalization of the two enzymes in plastids and the cytosol is responsible for the ecotype-specific differences in (E)-beta-ocimene/(E,E)-alpha-farnesene emission; differences in the relative emissions of (E)-beta-ocimene and (E,E)-alpha-farnesene from accession Wassilewskija, a high-(E)-beta-ocimene emitter, and accession Columbia, a trace-(E)-beta-ocimene emitter, are attributed to allelic variation of closely related, tandem-duplicated terpene synthase genes, TPS02 and TPS03. The Wassilewskija genome contains a functional allele of TPS02 but not of TPS03, while the opposite is the case for Columbia. Recombinant proteins of the functional Wassilewskija TPS02 and Columbia TPS03 genes both show (E)-beta-ocimene and (E,E)-alpha-farnesene synthase activities. Differential subcellular compartmentalization of the two enzymes in plastids and the cytosol is responsible for the ecotype-specific differences in (E)-beta-ocimene/(E,E)-alpha-farnesene emission
when farnesyl diphosphate, synthesised from 96% (E,E)-farnesol, is incubated with recombinant protein, (E,E)- and (Z,E)-alpha-farnesene are produced in a ratio of 96:4, respectively
AdAFS1 can function as a bifunctional enzyme possessing both sesquiterpene and monoterpene synthase activities. Exhibits significant monoterpene synthase activity, producing exclusively (E)-beta-ocimene. kcat/Km is about 5fold lower than kcat/Km for farnesyl diphosphate
the monoterpene synthase ((E)-beta-ocimene and beta-myrcene) and sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced
the monoterpene synthase ((E)-beta-ocimene and beta-myrcene) and sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced
the monoterpene synthase ((E)-beta-ocimene and beta-myrcene) and sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced
although (E,E)-farnesyl diphosphate is the preferred substrate, the enzyme accepts all four isomeric forms of the farnesyl diphosphate precursor. Both isomers of beta-farnesene are also synthesised by the enzyme presumably from a specific farnesene isomer. The enzyme also produces alpha-farnesene by a reaction involving coupling of geranyl diphosphate and isoprenyl diphosphate but at less than 1% of the rate with farnesyl diphosphate
the monoterpene synthase ((E)-beta-ocimene and beta-myrcene) and sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced
the monoterpene synthase ((E)-beta-ocimene and beta-myrcene) and sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced
the monoterpene synthase ((E)-beta-ocimene and beta-myrcene) and sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced
activity is dependent on K+, the potassium binding region is defined; MdAFS1 retains up to 12% of its activity in the absence of K+, enzyme contains K+ binding region, MdAFS1 exhibits a type II K+ response, MdAFS1 is not absolutely dependent upon M+ its unequivocal classification as type I or type II K+ activated, or that of any other terpene synthases, will not be possibl, then type I enzymes can exhibit type II kinetics and vice versa.
after treatment of fruits at harvest with a blocker of ethylene action, AFS1 mRNA declines sharply over the initial 4 weeks of cold storage, and falls to nearly undetectable levels by 8 weeks
S487K mutant has 35-45% of the wild-type activity with farnesyl diphosphate with no significant alterations in the alpha-farnesene isomer ratios produced and a small decrease in catalytic efficiency (wild-type and S487K respective kcat/Km values of 17.5 and 13.3 mM/s)
production of (E,E)-alpha-farnesene occurs in the epidermal (peel tissue) or adjacent hypodermal cell layers and the sesquiterpene can accumulate to high levels in the natural epicuticular coating of scald-susceptible apples during the first weeks of storage
expression of AdAFS1 is significantly higher in flowers than in leaf tissue. Within floral tissues, expression is highest in petals and stamens. AdAFS1 expression is low in sepals in both sexes and also in ‘Hayward’ pistils. AdAFS1 expression in ‘Hayward’ is co-ordinated with anthesis, with low levels of expression in unopened flower buds and high levels detected in open flowers. The temporal accumulation patterns of AdAFS1 mRNA is constitutive. No difference in expression between male and female flowers
CmTpsDul expression is specific to ’Dulce’ rind. CmTpsDul is not expressed in either the rind or flesh of ‘Noy Yizre’el’ melons at any developmental stage
CmTpsDul expression is specific to ’Dulce’ rind. CmTpsDul is not expressed in either the rind or flesh of ‘Noy Yizre’el’ melons at any developmental stage
expression of AdAFS1 is significantly higher in flowers than in leaf tissue. AdAFS1 expression is similar in leaves of both cultivars ‘Chieftain’ and ‘Hayward’
The encoded protein appears to lack approximately 20 amino acids that are present in the N-terminal chloroplast-targeting region of monoterpene synthases. Thus, the product of this TS gene is most likely localized in the cytosol.
After screening a cDNA library generated from the peel tissue mRNA, a full-length terpene synthase cDNA 1931 nucleotides long is isolated (hot phenol RNA extraction protocol is used). The 1728-bp open reading frame encodes the 576 amino acid protein. Expression of the apple gene in Escherichia coli.
mutated enzymes are generated using the QuickChange II site-directed mutagenesis kit. The PCR-based mutagenesis protocol is performed using pET-30a harbouring the MdAFS1 cDNAs as template. The single-site mutant enzymes overexpressed in Escherichia coli.
expression of AdAFS1 is significantly higher in flowers than in leaf tissue. Within floral tissues, expression is highest in petals and stamens. AdAFS1 expression is low in sepals in both sexes and also in ‘Hayward’ pistils. AdAFS1 expression in ‘Hayward’ is co-ordinated with anthesis, with low levels of expression in unopened flower buds and high levels detected in open flowers. The temporal accumulation patterns of AdAFS1 mRNA is constitutive. No difference in expression between male and female flowers. AdAFS1 expression is similar in leaves of both cultivars ‘Chieftain’ and ‘Hayward’
MdAFS1 transcript abundance increases rapidly in untreated Cortland and Law Rome fruit, reaching close to maximal values after 2 weeks of storage, whereas in Idared controls it increases through 4 weeks, declines slightly, and then increases to a maximum by 10 weeks. 1-Methylcyclopropene treatment initially suppresses MdAFS1 expression in all three cultivars. However, Cortland and Law Rome escape from this suppression after 10-15 weeks, with MdAFS1 transcript abundance increasing to maximal control levels by 15-20 weeks. In Cortland, but not in Law Rome, this delayed increase in expression occurrs sooner in green than in red tissue. MdAFS1 transcript levels increase gradually in 1-methylcyclopropene-treated Idared fruit, but throughout storage remains much lower than the maximum levels in untreated fruit
85% loss of sesquiterpene synthase activity compared with wild-type enzyme. Little change in K+-independent activity; 85% loss of sesquiterpene synthase activity compared with wild-type enzyme when K+ is present
exhibits marginally increased sesquiterpene synthase activities, and an approximate 2fold increase in monoterpene synthase activity compared with the wild-type enzyme; mutant exhibits marginally increased sesquiterpene synthase activities, and an approximate 2fold increase in monoterpene synthase activity compared with the wild-type enzyme
95% decrease in sesquiterpene synthase activity compared with wild-type enzyme. Little change in K+-independent activity; 95% decrease in sesquiterpene synthase activity compared with wild-type enzyme when K+ is present
mutant has 35–45% of the wild-type activity with farnesyl diphosphate, with no significant alterations in the alpha-farnesene isomer ratios produced and a small decrease in catalytic efficiency; The S487K mutant has 35-45% of the wild-type activity with farnesyl diphosphate, with no significant alterations in the alpha-farnesene isomer ratios produced and a small decrease in catalytic efficiency
mutant shows decreases in both sesqui- and monoterpene synthase activities compared with the WT enzyme, with mono-TPS activity being reduced more than sesquiterpene synthase activity. Sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced.; mutant shows decreases in both sesqui- and mono-terpene synthases activities, compared with the wild-type enzyme, with mono-terpene synthases activity being reduced more than sesqui-terpene synthases activity
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Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
myc-tagged and untagged AFS1 expressed protein in bacterial inclusion-body fractions is urea-denatured, purified and renatured prior to assay of enzymatic activity.
Because diphenylamine treatment leaves unwanted chemical residues on the fruit, restricts export markets, and creates environmental concerns, a long-range molecular genetic strategy for control of scald by reduction of (E,E)-alpha-farnesene synthesis in scald-susceptible apples is searched. The success of this strategy will rely on our ability to identify, clone, and characterize key genes involved in alpha-farnesene biosynthesis and its regulation by ethylene.
expression in Sacchaomyces cerevisiae leads to a titer of (-)-alpha-copaene of 9 mg/l at 48 h and around 7 mg/l in Eschrichia coli, and the titer of (+)-delta-cadinene is 6 mg/l in Saccharomyces cerevisiae and 3.5 mg/l in Escherichia coli
Huang, M.; Abel, C.; Sohrabi, R.; Petri, J.; Haupt, I.; Cosimano, J.; Gershenzon, J.; Tholl, D.
Variation of herbivore-induced volatile terpenes among Arabidopsis ecotypes depends on allelic differences and subcellular targeting of two terpene synthases, TPS02 and TPS03