Α-farnesene Synthesis Research Articles

Histone deacetylase CsHDA6 mediates the regulated formation of the anti-insect metabolite α-farnesene in tea (Camellia sinensis)

α-Farnesene accumulated in tea plants following infestations by most insects, and mechanical wounding is the common factor. However, the specific mechanism underlying the wounding-regulated accumulation of α-farnesene in tea plants remains unclear. In this study, we observed that histone deacetylase inhibitor treatment induced the accumulation of α-farnesene. The histone deacetylase CsHDA6 interacted directly with CsMYC2, which was an important transcription factor in the jasmonic acid (JA) pathway, and co-regulated the expression of the key α-farnesene synthesis gene CsAFS. Wounding caused by insect infestation affected CsHDA6 production at the transcript and protein levels, while also inhibited the binding of CsHDA6 to the CsAFS promoter. The resulting increased acetylation of histones H3/H4 in CsAFS enhanced the expression of CsAFS and the accumulation of α-farnesene. In conclusion, our study demonstrated the effect of histone acetylation on the production of tea plant HIPVs and revealed the importance of the CsHDA6-CsMYC2 transcriptional regulatory module.

Dual Regulation of Cytoplasm and Peroxisomes for Improved Α-Farnesene Production in Recombinant Pichia pastoris.

Microbial production of α-farnesene from renewable raw materials is a feasible alternative to traditional petroleum craft. Recently, the research on improving α-farnesene production in Pichia pastoris mainly focused on cytoplasmic engineering, while comprehensive engineering of multiple subcellular compartments is rarely reported. Here, we first sought to confirm that the isopentenol utilization pathway (IUP) could act as a two-step shortcut for IPP synthesis in P. pastoris peroxisomes. In addition, we proposed dual regulation of cytoplasm and peroxisomes to boost α-farnesene synthesis in P. pastoris X33, thus the resultant strain produced 2.18 ± 0.04 g/L, which was 1.3 times and 2.1 times than that of the strain only with peroxisomal or cytoplasmic engineering, respectively. The α-farnesene production achieved 2.56 ± 0.04 g/L in shake flasks after carbon source cofeeding, which was the highest reported production in worldwide literatures to the best of my knowledge. Therefore, we propose these strategies as efficient approaches to enhancing α-farnesene production in P. pastoris, which might bring new ideas for the biosynthesis of high-value compounds.

De novo transcriptome assembly of transgenic tobacco (Nicotiana tabacum NC89) with early senescence characteristic.

The online version contains supplementary material available at 10.1007/s12298-021-00953-z.

Engineering the oleaginous yeast Yarrowia lipolytica for production of \u03b1-farnesene

BackgroundYarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production.ResultsWe constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica.ConclusionsYarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.

Heterologous production of α-farnesene in metabolically engineered strains of Yarrowia lipolytica

Herein, we studied the heterologous production of α-farnesene, a valuable sesquiterpene with various biotechnological applications, by metabolic engineering of Yarrowia lipolytica. Different overexpression vectors harboring combinations of tHMG1, IDI, ERG20 and codon-optimized α-farnesene synthase (OptFS) genes were constructed and integrated into the genome of Y. lipolytica Po1h. The engineered strain produced 57.08±1.43mg/L of α-farnesene corresponding to 20.8-fold increase over the initial production of 2.75±0.29mg/L in the YPD medium in shake flasks. Bioreactor scale-up in PM medium led to α-farnesene concentration of 259.98±2.15mg/L with α-farnesene to biomass ratio of 33.98±1.51mg/g, which was a 94.5-fold increase over the initial production. This first report on α-farnesene synthesis in Y. lipolytica lays a foundation for future research on production of sesquitepenes in Y. lipolytica and other closest yeast species and will potentially contribute in its industrial production.

Storage Temperature, Controlled Atmosphere, and 1-Methylcyclopropene Effects on α-Farnesene, Conjugated Trienols, and Peroxidation in Relation with Superficial Scald, Pithy Brown Core, and Fruit Quality of ‘d’Anjou’ Pears during Long-term Storage

Alternatives to ethoxyquin (Etq) are needed for controlling superficial scald of ‘Anjou’ european pears (Pyrus communis) during long-term storage. The current commercial standard storage conditions [Etq + −1 °C + controlled atmosphere (CA) with 1.5 kPa O2] reduced scald occurrence compared with control fruit (−1 °C + CA) during 6–8 months storage. At 1 °C in air, 1-methylcyclopropene (1-MCP) fumigation at 0.15 µL·L−1 at harvest was more efficient on reducing scald than Etq but did not prevent scald during 6–8 months storage. The 1-MCP-treated fruit at 1 °C in air developed their ripening capacity at 20 °C following 6–8 months storage but had deceased shipping ability (softening and yellowing of fruit). Although Etq inhibition of scald was associated with the inhibition of α-farnesene oxidation to conjugated trienols (CTols); 1-MCP reduced α-farnesene synthesis and thereby the availability of substrate to oxidize to CTols. CA storage at 1.5 kPa O2 totally prevented scald and retarded the loss of shipping ability without affecting the ripening capacity of 1-MCP-treated fruit at 1 °C through further decreases in the syntheses of ethylene, α-farnesene and CTols during 6–8 months storage. In addition, 1-MCP prevented a CA-induced disorder, pithy brown core (PBC), in ‘Anjou’ pears possibly through enhancing an oxidative/reductive metabolic balance during extended storage. In conclusion, the combinations of 1 °C + 1-MCP + CA is a potential commercial alternative to Etq for scald control while allowing the 1-MCP-treated ‘Anjou’ pears to recover ripening capacity during the shelf life period after 6–8 months storage.

Metabolic engineering of Escherichia coli for α-farnesene production

Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.

Superficial scald susceptibility and α-farnesene metabolism in ‘Bartlett’ pears grown in California and Washington

‘Bartlett’ pears grown in northern California (CA) consistently show development of the physiological storage disorder superficial scald, particularly after prolonged storage of 4–5 months in air. In contrast, fruit of this cultivar grown in central Washington (WA) are typically less susceptible to scald, exhibiting mild or no symptoms. Conjugated triene (CT) oxidation products of the sesquiterpene α-farnesene are thought to play a key role in scald induction in apples and pears. This study compared accumulation of α-farnesene and its CT products in peel tissue of CA- and WA-grown ‘Bartlett’ pears during air storage at −1 °C in relation to scald development after transfer to 20 °C. Pears were harvested from commercial orchards in 2006 and 2007 and stored under nearly identical conditions for up to 24 weeks. Peel tissue samples taken at harvest and at 2–4-week interval during storage were analyzed by HPLC to determine concentrations of α-farnesene and CTs. Measurements of flesh firmness, respiration, and ethylene production were also made at harvest and/or from 1 to 8 d after removal from storage to 20 °C. WA fruit from the second harvest in 2006 developed light superficial scald after 20 weeks of cold storage plus 5 d at 20 °C; all others were scald-free. By contrast, all CA fruit from both seasons showed light scald after 12–14 weeks, and moderate scald after 20–24 weeks, plus shelf life. Correspondingly, α-farnesene and CTs accumulated more rapidly and on average reached about twofold higher concentrations in CA compared with WA fruit over the first 8–12 weeks of storage. CA fruit also had an earlier rise in ethylene production, higher respiratory rates, and lower flesh firmness at harvest than WA fruit. These suggest advanced maturity, which may have contributed to the increased rates of α-farnesene synthesis and oxidation, and higher incidence of scald.

Combination of 1-Methylcyclopropene and Ethoxyquin to Control Superficial Scald of ‘Anjou’ Pears

Superficial scald of ‘Anjou’ pears (Pyrus communis) usually develops after cold storage of ≥3 months. Ethoxyquin has been used to control scald commercially. However, only a small amount of fruit can be treated within 7 days after harvest as recommended, and sometimes ethoxyquin causes phytotoxicity. Application of 1-methylcyclopropene (1-MCP) showed excellent scald control potential, with rapid and mass treatment feasible. However, fruit may lose normal ripening ability at a dosage of 1-MCP as low as 30 nL·L−1, whereas a dosage of ≤20 nL·L−1 is not enough to control scald. In this investigation, ‘Anjou’ pears treated with 25 nL·L−1 1-MCP immediately after harvest were stored at −1 °C for up to 5 months. After 1, 7, 30, or 60 days of cold storage, part of the fruit were treated with 1000 μL·L−1 ethoxyquin and the remainder was left untreated as nonethoxyquin controls. The incidence of superficial scald, the concentrations of α-farnesene and its conjugated triene (CT) oxidation products, and the ripening ability of fruit were measured after 3-, 4-, and 5-month storages. All fruit ripened properly within 7 days of shelf life at 20 °C regardless of treatment. 1-MCP treatment at harvest or ethoxyquin alone applied within 7 days adequately controlled scald for only 3 months. By contrast, 1-MCP + ethoxyquin controlled scald for 5 months, regardless of when ethoxyquin was applied from 1 to 60 days after the start of cold storage. Thus, a combination of 25 nL·L−1 1-MCP, which is easily applied and does not influence ripening ability, and a delayed application (up to 60 days) of 1000 μL·L−1 ethoxyquin, which is a low dosage that does not cause phytotoxicity on fruit, controlled scald sufficiently. Scald is linked with accumulation of CT oxidation products of α-farnesene. 1-MCP and ethoxyquin inhibited accumulation of CT in fruit peel by different mechanisms. 1-MCP inhibited the production by reducing α-farnesene synthesis and the oxidation to CT, whereas ethoxyquin worked by inhibiting the latter.

Combination of 1-Methylcyclopropene and Ethoxyquin to Control Superficial Scald of ‘Anjou’ Pears

Superficial scald of ‘Anjou’ pears (Pyrus communis) usually develops after cold storage of ≥3 months. Ethoxyquin has been used to control scald commercially. However, only a small amount of fruit can be treated within 7 days after harvest as recommended, and sometimes ethoxyquin causes phytotoxicity. Application of 1-methylcyclopropene (1-MCP) showed excellent scald control potential, with rapid and mass treatment feasible. However, fruit may lose normal ripening ability at a dosage of 1-MCP as low as 30 nL·L−1, whereas a dosage of ≤20 nL·L−1 is not enough to control scald. In this investigation, ‘Anjou’ pears treated with 25 nL·L−1 1-MCP immediately after harvest were stored at −1 °C for up to 5 months. After 1, 7, 30, or 60 days of cold storage, part of the fruit were treated with 1000 μL·L−1 ethoxyquin and the remainder was left untreated as nonethoxyquin controls. The incidence of superficial scald, the concentrations of α-farnesene and its conjugated triene (CT) oxidation products, and the ripening ability of fruit were measured after 3-, 4-, and 5-month storages. All fruit ripened properly within 7 days of shelf life at 20 °C regardless of treatment. 1-MCP treatment at harvest or ethoxyquin alone applied within 7 days adequately controlled scald for only 3 months. By contrast, 1-MCP + ethoxyquin controlled scald for 5 months, regardless of when ethoxyquin was applied from 1 to 60 days after the start of cold storage. Thus, a combination of 25 nL·L−1 1-MCP, which is easily applied and does not influence ripening ability, and a delayed application (up to 60 days) of 1000 μL·L−1 ethoxyquin, which is a low dosage that does not cause phytotoxicity on fruit, controlled scald sufficiently. Scald is linked with accumulation of CT oxidation products of α-farnesene. 1-MCP and ethoxyquin inhibited accumulation of CT in fruit peel by different mechanisms. 1-MCP inhibited the production by reducing α-farnesene synthesis and the oxidation to CT, whereas ethoxyquin worked by inhibiting the latter.

Ethylene and α-Farnesene Metabolism in Green and Red Skin of Three Apple Cultivars in Response to 1-Methylcyclopropene (1-MCP) Treatment

Relationships among alpha-farnesene synthesis and oxidation, ethylene production and perception, antioxidative enzyme activities, and superficial scald development in fruit of three commercial apple cultivars were investigated at the biochemical and gene transcriptional levels. Scald-susceptible Cortland and Law Rome and scald-resistant Idared apples were untreated or treated with the ethylene action inhibitor 1-methylcyclopropene (1-MCP) and stored for up to 25 weeks at 0.5 degrees C. Separate blushed (red) and unblushed (green) peel tissue samples were taken at harvest and after 2, 4, 6, 10, 15, 20, and 25 weeks of storage. Large increases in peel tissue concentrations of alpha-farnesene and its conjugated trienol (CTol) oxidation products occurred in untreated Cortland and Law Rome and were about 4-9-fold greater than those in Idared. In both Cortland and Law Rome, accumulation of CTols in green peel was nearly twice that in red peel. 1-MCP treatment delayed and attenuated alpha-farnesene and CTol accumulation in each cultivar. Activities of peroxidase (POX) and catalase (CAT) were lower in red peel than in green peel, with the exception of CAT in Law Rome, whereas no effects of 1-MCP on enzyme activities were detected except for Cortland. In control fruit, internal ethylene concentrations (IECs) increased during the first 4-6 weeks to reach highest levels in Cortland, intermediate levels in Law Rome, and low levels in Idared. In 1-MCP-treated fruit, IECs increased gradually to modest levels by 25 weeks in Cortland and Law Rome but were almost nil in Idared. Expression patterns of the alpha-farnesene synthase gene MdAFS1, the ethylene receptor gene MdERS1, and the ethylene biosynthetic genes MdACS1 and MdACO1 were generally in accord with the patterns of alpha-farnesene and ethylene production. In particular, MdAFS1 and MdACS1 showed similar patterns of expression in each cultivar. Among the controls, transcript levels increased more rapidly in Cortland and Law Rome than in Idared during the first few weeks of storage. In 1-MCP-treated fruit, transcript abundance in Cortland and Law Rome rose to untreated control levels after 10-15 weeks but remained low in Idared. Scald symptoms were restricted to unblushed skin, and the incidence in controls after 25 weeks was nearly 100% in Cortland and Law Rome compared with 1% in Idared. 1-MCP treatment reduced scald incidence to 14, 3, and 0% in Cortland, Law Rome, and Idared, respectively. Overall, the results support the proposed role of CTols in scald induction and indicate that alpha-farnesene synthesis is tightly regulated by ethylene. However, gene transcription alone does not account for the big differences in ethylene and alpha-farnesene production in Cortland, Law Rome, and Idared apples.

Unusual features of a recombinant apple α-farnesene synthase

A recombinant α-farnesene synthase from apple ( Malus × domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K + and all four isomers of α-farnesene, as well as β-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, ( Z)- and ( E)-β-ocimene and β-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for α-farnesene synthesis from FDP. Addition of K + reduced monoterpene synthase activity. The enzyme also produced α-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.

(29) MCP Treatment Makes Delayed Application of Ethoxyquin Effective in Control of Superficial Scald of `d'Anjou' Pears

Superficial scald of `d'Anjou' pears usually develops after storage of 2 months or longer. MCP application controls scald; however, fruit lose their ripening capacity if the dosage is &gt;30 ppb, but lower dosages cannot control scald substantially. In this study, fruit treated with or without 25 ppb MCP within 2 d after harvest were stored at –1 °C for up to 5 months. After 1, 7, 30, or 70 d of storage, part of the fruit were pulled from storage and treated with 1000 ppm ethoxyquin line spray and immediately returned to the storage, left untreated as non-ethoxyquin control. Incidence of superficial scald along with the concentrations of α-farnesene and conjugated trienes (CTs), and the ripening capacity of fruit were investigated after 3, 4, and 5 months storage. All fruit ripened properly within 7 d of conditioning at room temperature regardless of treatments. Both of MCP or ethoxyquin-1d (ethoxyquin was applied after 1 day storage at –1 °C) alone controlled scald for only 3 months; however, MCP + ethoxyquin controlled scald for 5 months, whenever ethoxyquin was applied between 1 to 70 d after storage. Thus, with 25 ppb MCP treatment, which is simple, with rapid and mass treatment available, a delayed application (up to 70 d) of ethoxyquin becomes effective to control scald. Furthermore, the later application of ethoxyquin within 70 d after MCP treatment, the less incidence of scald was observed. Scald is caused by the CTs oxidation products of α-farnesene. MCP and ethoxyquin inhibited accumulation of CTs of fruit peel by different mechanisms. MCP inhibited the production by influencing ethylene production through control of α-farnesene synthesis; however, ethoxyquin worked by inhibiting the oxidation of α-farnesene to the CTs.

Expression of α-farnesene synthase gene AFS1 in relation to levels of α-farnesene and conjugated trienols in peel tissue of scald-susceptible ‘Law Rome’ and scald-resistant ‘Idared’ apple fruit

Abstract Nontreated control and 1-MCP-treated fruit of the scald-susceptible ‘Law Rome’ and scald-resistant ‘Idared’ cultivars were compared with respect to scald incidence, internal ethylene concentration (IEC), α-farnesene metabolism, and expression of the gene encoding α-farnesene synthase ( AFS1 ), the final, rate-limiting enzyme in the α-farnesene biosynthetic pathway. The incidence of scald in nontreated ‘Law Rome’ apples after 20 weeks at 0.5 °C plus 1 week at 20 °C averaged 86%; 1-MCP treatment reduced increases in IEC and reduced the incidence of scald to less than 1%. Fruit of ‘Idared’ showed no scald regardless of the treatment. 1-MCP also inhibited α-farnesene production, suggesting that ethylene induces transcription of key genes involved in α-farnesene biosynthesis. In both ‘Law Rome’ and ‘Idared’, a sharp increase in AFS1 mRNA during the first 4–8 weeks of storage preceded a proportional rise in α-farnesene and a subsequent increase in CTols. However, maximum levels of AFS1 transcript, α-farnesene, and CTols were, respectively, 2.5-, 4-, and 33-fold greater in ‘Law Rome’ than in ‘Idared’ apples. 1-MCP suppressed the increases in AFS1 transcript and α-farnesene early in storage, although AFS1 expression and α-farnesene synthesis had recovered in treated ‘Law Rome’ fruit after 20 weeks, consistent with increasing IEC in these fruit.

Cloning and bacterial expression of a 3-hydroxy-3-methylglutaryl-CoA reductase cDNA ( HMG1) from peel tissue of apple fruit

As part of an effort to elucidate the mechanism of superficial scald in apple fruit, and ultimately to devise a molecular genetic strategy for control of this storage disorder, we have begun to clone and characterize gene products that regulate production of α-farnesene in peel tissue. Oxidation products of this sesquiterpene are thought to induce necrosis of cell layers just beneath the fruit skin, leading to development of scald symptoms. Here we report the cloning and bacterial expression of a cDNA encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the initial, rate-limiting enzyme in sesquiterpene biosynthesis via the mevalonic acid pathway. RT-PCR cloning based on the conserved catalytic domains of plant HMGRs yielded two different cDNA fragments from peel tissue mRNA, designated HMG1 and HMG3. The 3′ untranslated regions of the two cDNAs, obtained using 3′ RACE, were used to screen an apple peel cDNA library. A full-length transcript of HMG1 was obtained (2334 bp), with an open reading frame of 1827 bp, encoding a protein 608 amino acids in length. A partial HMG3 clone was isolated (1192 bp), composed of a 1143-bp coding region and a 49-bp 3′ untranslated region. RNA-gel blots of HMG1 showed a high level of expression in peel tissue at harvest and after 4 and 8 weeks of storage at 0.5 °C, even when tissue responsiveness to ethylene was blocked by prestorage treatment of fruit with 1-methylcyclopropene, which suppresses α-farnesene synthesis. Hence, the rapid rise in α-farnesene production that occurs in the initial weeks of storage cannot be attributed to increased expression of HMG1. E. coli cells transformed with the HMG1 transcript showed over 10-fold higher HMGR activity than those with the control vector. Bacterial expression was confirmed by immunoblots of HMG1 protein fused to a C-terminal myc tag, which showed a single immunoreactive band with a molecular mass of ∼69 kDa.

Cloning of hmg1 and hmg2 cDNAs encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase and their expression and activity in relation to α-farnesene synthesis in apple

In plants, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyses the synthesis of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A. It has been reported to be the rate-limiting enzyme in sesquiterpene and triterpene biosynthesis and is encoded by a small gene family. The accumulation of α-farnesene in the skin tissue of apple fruit during storage appears to be predominantly through the classical mevalonate pathway and not through the novel glyceraldehyde-3-phosphate/pyruvate pathway independent of HMGR action. The content of α-farnesene in the skin tissue increased during the first 8 weeks of storage at 0 °C in air, and started declining after 12 weeks. In contrast, HMGR activity in the total membrane and soluble fractions, was the highest at the time of harvest, but decreased during the first 8 weeks in storage and remained stable thereafter. The potent ethylene action inhibitor 1-methylcyclopropene inhibited α-farnesene evolution and HMGR activity by 97 and 30 %, respectively. As a first step in studying the molecular mechanism of apple HMGR regulation, we have isolated and cloned a full-length cDNA ( hmg1) as well as a fragment ( hmg2), using apple skin mRNA and RT-PCR in the presence of degenerate oligonucleotides designed against conserved regions of plant HMGR genes. Genomic Southern analysis using probes designed for the 3’-end of the two cDNA clones confirmed the presence of at least two HMGR genes in apple. The cDNA for hmg1 has an open reading frame of 1 767 nucleotides. Analysis of the nucleotide sequence revealed that the cDNA encodes a polypeptide of 589 residues with a relative molecular mass of 62.7 kDa. The hydropathy profile of the putative polypeptide indicated the presence of two highly hydrophobic domains near the amino terminus. Northern blot analysis confirmed that both hmg1 and hmg2 transcripts possessed a size of 2.4 kb. The two genes are differentially expressed during low temperature storage and in response to C 2H 4, with hmg1 being expressed constitutively and hmg2 being relatively more sensitive to developmental stimuli and ethylene.

083 Is HMGR the Key Regulator of α-Farnesene Biosynthesis of Apple?

We tested the hypothesis that conversion of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG CoA) to mevalonate (MVA) catalyzed by HMG CoA reductase (HMGR) is the rate limiting step for α-farnesene biosynthesis of apples. In higher plants, isopentenyl pyrophosphate (IPP) is derived via two pathways: 1) the classical mevalonate pathway, and 2) the novel glyceraldehyde-3-phosphate (GAP)/pyruvate pathway independent of HMGR action. When apple skin discs were incubated with MVA, or GAP and pyruvate, MVA increased α-farnesene levels in the skin but not GAP and pyruvate. Treating apple fruits with Lovastatin (1000 ppm), a competitive inhibitor of HMGR, inhibited α-farnesene accumulation in the skin by 20% to 50% during storage. Content of α-farnesene in the skin increased during the first 2 to 4 months in storage, and then decreased. In contrast, HMGR activity, as determined by the conversion of [4-3H]HMG CoA to MVA in the total membrane and soluble fraction, was the highest at the time of harvest and gradually decreased during 5 months of storage in air at 0 °C. The potent ethylene action inhibitor 1-MCP inhibited ethylene production and α-farnesene evolution by 99% and 97%, respectively. The effect of 1-MCP on in vitro activity of HMGR was marginal (≈30% inhibition). 1-MCP inhibited respiratory CO2 evolution by 50%, which suggests also that inhibition by 1-MCP of α-farnesene synthesis in apple could be regulated by the acetyl CoA pool. In plants, HMGR is encoded by a small gene family and differentially expressed. As the first step of studying the molecular mechanism of HMGR regulation, we have isolated a 444-bp fragment of apple hmgr gene using apple skin mRNA and degenerate oligonucleotides designed against conserved regions of plant hmgr genes.

DPA treatment alters α-farnesene metabolism in peel of ‘Empire’ apples stored in air or 1.5% O2 atmosphere

Treatment with diphenylamine (DPA), sometimes in combination with storage under low (≤1.5%) O2 atmosphere, is the major commercial means of limiting superficial scald in apple fruit. Synthesis and oxidation of the sesquiterpene α-farnesene are thought to be directly involved in induction of this storage disorder. Control of scald by DPA has been ascribed to its ability to block both in vitro and in vivo oxidation of α-farnesene to conjugated trienes (CTs), but a number of reports have indicated that DPA has multiple effects, including reduction of α-farnesene synthesis, ethylene production, and respiration. The time course and levels of α-farnesene and CT accumulation in peel tissue were compared in ‘Empire’ apples that were either DPA-treated or untreated and stored for up to 28 weeks at 0°C in air or under 1.5% O2 atmosphere. Also, it was found that the HPLC-UV method previously developed to quantify α-farnesene and CTs could be used to simultaneously measure the concentration of DPA residue. In air-stored fruit, DPA treatment protracted rather than diminished α-farnesene synthesis; a similar maximum level of α-farnesene was reached at 15 and 28 weeks in untreated and DPA-treated fruit, respectively. DPA treatment delayed the onset of CT production by ∼5 weeks and reduced CT accumulation more than 2.5-fold. The low O2 atmosphere was overall more effective than DPA treatment in reducing synthesis and oxidation of α-farnesene. In combination, however, DPA and low O2 had a synergistic effect, resulting in a ninefold reduction in α-farnesene and virtual elimination of CT production over 28 weeks. In both air and 1.5% O2, DPA residue in the peel tissue declined rapidly during the first 15 weeks of storage and more gradually thereafter, with an overall drop from ∼11 to 1.1 μg g−1.

Lovastatin Inhibits α-Farnesene Synthesis without Affecting Ethylene Production during Fruit Ripening in `Golden Supreme' Apples

Lovastatin is a specific hydroxymethylglutaryl coenzyme-A reductase inhibitor in animals and as such, is a potent cholesterol lowering pharmaceutical for human use. Because it has also been shown to inhibit α-farnesene in certain plants, we investigated its effects on ethylene and α-farnesene biosynthesis, volatile production, and fruit color during ripening in `Golden Supreme' apples [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. Immediately after harvest, fruit were dipped in Lovastatin solution for 2 min, allowed to dry, and stored in the dark at 20 °C for 30 days. Internal ethylene at harvest was low (&lt; 0.1 mL·L-1) and α-farnesene was undetectable. Both internal ethylene and α-farnesene increased in nontreated fruit during 30 days storage. Prestorage Lovastatin treatment did not affect ethylene synthesis, but at 1.25 or 2.5 mmol·L-1 nearly eliminated α-farnesene production. At 0.25 mmol·L-1, Lovastatin delayed the increase in α-farnesene production about 12 days and reduced total α-farnesene production by the end of storage compared with controls. When applied to nontreated preclimacteric fruit, ethephon at 1.4 mmol·L-1 increased both internal ethylene concentration and α-farnesene production. In Lovastatin-treated preclimacteric fruit, however, ethephon increased internal ethylene concentration without promoting α-farnesene synthesis. In another trial, after 30 days storage at 0 °C, fruit were treated with 1.25 mmol·L-1 Lovastatin and stored at 20 °C with air circulation for 20 days. These fruit accumulated similar amounts of ethylene as nontreated controls, but α-farnesene production decreased rapidly and was not detectable after 5 days. Treating with ethephon at 1.4 mmol·L-1 increased α-farnesene production in control fruit but not in Lovastatin-treated fruit. Lovastatin treatment did not affect the change in fruit color. Chemical names used: [1S-[1α (R °), 3α, 7β, 8β (2S °, 4S °), 8ab]]-1,2,3,7,8,8α-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthaienyl 2-methylbutanoate (Lovastatin); 2-chloroethylphosphonic acid (ethephon).

532 Lovastatin Inhibits α-Farnesene Synthesis without Affecting Ethylene Production during Fruit Ripening in Apples

At harvest, internal ethylene was &gt;0.5 mL·L–1 and α-farnesene concentrations were below detectable levels in `Golden Supreme', `Delicious', and `Granny Smith' apples. After 30 days of storage at 20 °C, control fruit produced high levels of internal ethylene and α-farnesene. Lovastatin at 100 to 1000 mL·L–1 did not affect ethylene synthesis, but significantly inhibited α-farnesene production. In `Golden Delicious', ethephon treatment increased ethylene synthesis in lovastatin-treated fruit but did not stimulate α-farnesene production. In lovastatin-treated fruit peel of `Delicious' and `Granny Smith', mevalonate (MAL) and farnesyl pyrophosphate (FPP) induced α-farnesene production, but hydroxymethylglutaric acid (HMG) did not. The induction of α-farnesene synthesis by MAL and FPP was concentration-dependent. Precursor feeding did not affect ethylene production in fruit peel. When high level of α-farnesene was detected in fruit peel (5 mm thick), it was not found in outer (adjacent to peel) and inner cortex (mid of flesh) tissues. Adding HMG, MAL, and FPP induced α-farnesene biosynthesis in both cortex tissues. When lovastatin was added to the feeding solution, MAL and FPP induced α-farnesene production but HMG did not.