Evidence that -farnesene biosynthesis during fruit ripening is mediated by ethylene regulated gene expression in apples

Postharvest Biology and Technology 19 (2000) 9 16 www.elsevier.com/locate/postharvbio Evidence that -farnesene biosynthesis during fruit ripening is mediated by ethylene regulated gene expression in apples Zhiguo Ju *, Eric A. Curry Tree Fruit Research Lab, USDA-ARS, 1104 N. Western A e., Wenatchee, WA 98801, USA Received 5 June 1999; accepted 16 January 2000 Abstract The effect of ethylene regulation on -farnesene biosynthesis in preclimateric Delicious and Granny Smith apples was studied using an ethylene inducer and inhibitor, -farnesene biosynthesis precursors, and protein transcription and translation inhibitors. -Farnesene was not detectable when internal ethylene concentrations were less than 1 l l 1. Correlations between internal ethylene and -farnesene production fit the exponential growth equation and were significant in Delicious (y=e 0.17 ; r 2 =0.68) and Granny Smith (y=e 0.18 ; r 2 =0.83). When applied at harvest, aminoethoxyvinylglycine (AVG) at 200 mg l 1 inhibited both internal ethylene accumulation and -farnesene production, whereas ethephon at 200 mg l 1 accelerated both. Adding ethephon to AVG-treated fruit after 18 days at 20 C induced internal ethylene accumulation and -farnesene production. Ethephon induced -farnesene production in discs from preclimacteric fruit peel as well as AVG-treated fruit peel, but -farnesene was undetectable when cycloheximide (CHI, 50 mm), actinomycin D (Act D, 50 mm), or silver nitrate (150 mg l 1 ) were added to the ethephon-treated discs. In preclimacteric fruit discs at harvest, with or without AVG treatment, -farnesene biosynthesis was induced by 50 M mevalonic acid lactone (MAL) or farnesyl pyrophosphate (FPP), but not by hydroxymethylglutaric acid (HMG). Adding CHI or Act D to these discs did not affect the induction of -farnesene by MAL or FPP. Published by Elsevier Science B.V. Keywords: -Farnesene biosynthesis; Ethylene; Gene expression; Apples 1. Introduction This research was supported by Washington State Fruit Tree Research Commission. * Corresponding author. Tel.: +1-509-6642280; fax: +1-509-6642287. E-mail address: ju@tfrl.ars.usda.gov (Z. Ju) Biosynthesis of -farnesene in apple fruit is developmentally regulated. It is present in newly forming fruit (Sutherland et al., 1977) and, although not detectable in preclimacteric fruit at harvest, increases rapidly during normal fruit ripening or during cold storage (Murray et al., 1964; Huelin and Murray, 1966; Meigh and Filmer, 1969; Huelin and Coggiola, 1970). This increase in -farnesene during fruit ripening or storage parallels the increase of internal ethylene (Watkins et al., 1993; Barden and Bramlage, 1994a,b; Du and Bramlage, 1994). Furthermore, preharvest application of aminoethoxyvinyl- 0925-5214/00/$ - see front matter Published by Elsevier Science B.V. PII: S0925-5214(00)00078-8

10 Z. Ju, E.A. Curry / Posthar est Biology and Technology 19 (2000) 9 16 glycine (AVG), a potent inhibitor of ACC synthase, inhibits both ethylene and -farnesene production (Ju and Bramlage, 2000). It is not clear, however, whether the ethylene related increase in -farnesene biosynthesis during fruit ripening is caused by gene expression, by enzyme activation, or by other mechanisms. Since -farnesene accumulation is closely related to scald development in apples (Ingle and D Souza, 1989), a better understanding of its regulation will help in the design of new strategies to control this storage disorder. Previous reports have shown that -farnesene biosynthesis in apples involves several steps starting with acetyl CoA, with hydroxymethylglutaric acid (HMG), mevalonic acid, and farnesyl pyrophosphate (FPP) as important intermediate precursors (Rupasinghe et al., 1998; Ju and Curry, 2000). By feeding fruit tissue different substrate precursors and/or different inhibitors, such as cycloheximide (CHI, protein synthesis inhibitor), actinomycin D (Act D, transcription inhibitor), AVG (ethylene synthesis inhibitor), or silver nitrate (ethylene action inhibitor), we can ascertain the step or steps at which ethylene regulates -farnesene biosynthesis. In this paper, we examine the relationship between ethylene production and farnesene biosynthesis in Delicious and Granny Smith apples at both the preclimacteric stage and during fruit ripening using an ethylene synthesis inducer or inhibitor, an ethylene function inhibitor, a protein transcription or translation inhibitor, and three precursors intermediate in the biosynthesis of -farnesene. 2. Materials and methods 2.1. Plant material Delicious and Granny Smith apples were harvested on September 23 and October 6, 1998, respectively. Immediately after harvest, 200 fruit from each cultivar were placed in cardboard trays and stored in the dark at 20 C for further use to assess the correlation between internal ethylene and -farnesene production. Internal ethylene was measured in each of 40 fruit every day for 5 days, after which fruit were divided into groups according to their internal ethylene concentration. When internal ethylene was below 2 l l 1, fruit were grouped within 0.1 l l 1 intervals, and when internal ethylene was above 2 l l 1, fruit were grouped within 2 l l 1 intervals. Fruit within the same interval were used for -farnesene measurement. AVG and ethephon were used to assess the effects of ethylene inhibition and induction of -farnesene production. Of the 320 fruit from each of the three replications in each cultivar, 160 were dipped at harvest in 200 mg l 1 of AVG (Retain, Abbott Laboratory, IL), 80 were dipped in 200 mg l 1 ethephon, and 80 dipped in water for 3 min. These fruit were preclimacteric as evidenced by undetectable internal ethylene and farnesene. Treated and control fruit were put in paper boxes. Half of them were kept at 0 C and half at 20 C. Half of the AVG-treated fruit held at 20 C were treated with 200 mg l 1 ethephon after 18 days and returned to storage at 20 C. Internal ethylene and -farnesene in fruit held at 20 C were measured every 6 days for up to 30 days. 2.2. Internal ethylene measurement Internal ethylene was measured from 10 individual fruit in each replication using gas chromatography. A glass column (610 mm 3.2 mm i.d.) packed with Porapak Q (90 100 mesh) was used. Oven, injector, and FID temperatures were 50, 50, and 200 C. Gas flows for N 2 carrier, H 2, and air were 30, 30, and 300 ml min 1, respectively. 2.3. -Farnesene measurement -Farnesene was measured by GS-MS with a Solid-Phase-Micro-Extraction (SPME) method as described previously (Ju and Curry, 2000). Five fruit from each replication were placed in a 4-l glass jar at 20 C. The jars were connected to a flow-through system with a flow rate of 50 ml min 1. After 2 h equilibration, a 100 m polydimethylsiloxane (PDMS) probe (Supelco, Bellefonte, PA) was introduced into each jar and

Z. Ju, E.A. Curry / Posthar est Biology and Technology 19 (2000) 9 16 11 allowed to adsorb volatiles for 10 min. The probe was immediately inserted into the injection port of a gas chromatograph (HP 5890, Hewlett Packard, San Fernando, CA). Adsorbed volatiles were allowed to desorb for 3 min in the injector with a constant temperature of 250 C. The oven temperature was increased from 35 to 250 C at a rate of 50 C min 1 and held for 4 min. Helium was used as carrier gas and the head pressure was maintained to give a constant flow rate of 1 ml min 1. Analysis was conducted using a HP wide bore column (30 m length 0.25 mm i.d., Hewlett Packard) with a splitless injection. Volatiles were identified by analysis of fragmentation profiles using a HP 5971 MS detector (Hewlett Packard) combined with confirmatory library matches. -Farnesene was quantified using the abundance of characteristic ion 93 and reported on a fresh weight basis as units kg 1 min 1.A reading of 1000 in abundance was defined as one unit. 2.4. Precursor feeding Thirty discs (3 mm thick) were taken from the peel of 10 fruit in each of the three replications of each treatment using a 2-cm diameter brass cork borer and immediately put into a 20-ml test tube containing 6 ml 1% (w/v) ascorbic acid. The discs were then transferred into clear test tubes containing a citrate buffer (0.2 M, ph 5.8) and one of the following: hydroxymethylglutaric acid (HMG), the substrate for hydroxyl-3-methylglutaryl CoA reductase (HMGR), mevalonic acid lactone (MAL), the immediate product of HMGR, and farnesyl pyrophosphate (FPP), the immediate precursor for -farnesene biosynthesis. AVG and ethephon were used at a concentration of 200 mg l 1, cycloheximide (CHI, protein synthesis inhibitor) and actinomycin D (Act D, transcription inhibitor) were used at a concentration of 50 mm, and silver nitrate (ethylene action inhibitor) at 150 mg l 1. The discs were incubated for 10 min and then transferred to Petri dishes and incubated for 48 h. A small piece of tissue paper was placed between the lid and the plate to ensure adequate oxygen. After incubation, the discs were again put into a clean test tube, which was sealed with a rubber septum cap. To accelerate -farnesene evaporation, 2 ml of air was drawn out of the test tube, after which a 100 m polydimethylsiloxane (PDMS) probe was introduced into the tube and allowed to adsorb volatiles for 30 min at 20 C. -Farnesene adsorbed by the probe was measured as described above. Ethylene production was measured using GC by taking a 0.5-ml air sample from the test tube after -farnesene measurement. 2.5. Statistics Data were analyzed by ANOVA procedures of SAS Statistical Software (SAS Institute, Cary, NC). Means were separated using Duncan s New Multiple Range Test at the 5% level. Correlations between internal ethylene and -farnesene were analyzed by the exponential growth equation using SigmaPlot (SPSS, Chicago, IL). 3. Results 3.1. Relationship between ethylene and -farnesene synthesis in Delicious and Granny Smith apples In both Delicious and Granny Smith apples, the correlations between internal ethylene and -farnesene production fit a typical exponential growth equation and were significant (Fig. 1). -Farnesene was not detectable before internal ethylene reached 1 l l 1, but was detected in all fruit with internal ethylene greater than 1 l l 1. Granny Smith apples produced less ethylene and -farnesene than Delicious. 3.2. Effects of AVG and ethephon treatments on -farnesene and ethylene synthesis The effects of AVG and ethephon on internal ethylene and -farnesene production in Delicious and Granny Smith were similar. Therefore, only data from Delicious are presented in Fig. 2. In control fruit, both internal ethylene and -far-

12 Z. Ju, E.A. Curry / Posthar est Biology and Technology 19 (2000) 9 16 nesene increased early and then remained constant after reaching the maximum. AVG treatment at harvest reduced ethylene to 0.5 l l 1, and -farnesene to less than detectable levels during the 30 days of storage. Ethephon treatment at harvest increased both internal ethylene and -farnesene production. When ethephon was applied to AVG-treated fruit at day 18, internal ethylene concentration increased and -farnesene biosynthesis was induced. 3.3. Effects of precursor feeding on ethylene and -farnesene production in control and AVG-treated fruit peel Similar results were obtained using discs from Granny Smith fruit at harvest (before the climacteric rise in ethylene) or fruit treated with AVG at harvest and stored at 0 C for 20 days, therefore, means are presented in Fig. 3. -Farnesene was not detected in these discs. MAL and FPP in Fig. 1. Relationship between internal ethylene accumulation and -farnesene production in Delicious and Granny Smith apples. Delicious and Granny Smith fruit were harvested on September 23, and October 6, 1998, respectively, and held at 20 C in the dark. Measurements were made within 5 days of harvest. Internal ethylene was measured in individual apples and -farnesene was measured in grouped fruit with similar internal ethylene concentrations. Correlations between internal ethylene and -farnesene were generated using the exponential growth equation. Data in the insert were from fruit containing 5 l l 1 internal ethylene.

Z. Ju, E.A. Curry / Posthar est Biology and Technology 19 (2000) 9 16 13 Fig. 2. Effects of AVG and ethephon treatment on internal ethylene concentration and -farnesene production in Delicious apples. Fruit were harvested on September 23, 1998. AVG (200 mg l 1 ) or ethephon (200 mg l 1 ) were applied at harvest. Arrow indicates the date that 200 mg l 1 ethephon was applied to AVG-treated fruit. Bars represent S.D. of the means. duced -farnesene synthesis, but HMG did not. Precursor feeding did not affect ethylene production. Adding CHI or Act D to the incubation solution did not affect precursor-induced -farnesene production in any of the treatments (data not shown). A similar response was found in Delicious fruit peel (data not shown). 3.4. Effects of ethephon, CHI, Act D, and sil er ion on ethylene and -farnesene synthesis in control and AVG-treated fruit peel Peel discs from Granny Smith apples kept for 10 days at 0 C contained very low levels of ethylene ( 0.05 l l 1 ) and undetectable -farnesene (Fig. 4). Adding 200 mg l 1 of ethephon to the incubation solution for 48 h increased ethylene production and induced -farnesene biosynthesis. When added to the ethephon treatment, CHI, Act D, and silver ion reduced ethylene production and totally inhibited -farnesene biosynthesis. In discs from fruit treated with AVG alone, ethylene and -farnesene were undetectable after 20 days of storage at 0 C. When added to these discs, ethephon increased ethylene production and -farnesene biosynthesis, whereas the addition of CHI, Act D, and silver ion totally inhibited -farnesene synthesis and reduced ethylene production by 50% compared with the ethephon treatment. Similar trends were obtained using fruit peel from Delicious apples (data not shown).

14 Z. Ju, E.A. Curry / Posthar est Biology and Technology 19 (2000) 9 16 Fig. 3. Effects of precursor feeding on ethylene and -farnesene production in AVG-treated and control fruit peel of Granny Smith apples. Fruit were harvested on October 6, 1998. Data are means from fruit at harvest and fruit treated with 200 mg l 1 of AVG at harvest and stored at 0 C for 20 days. HMG, hydroxymethylglutaric acid (50 M); MAL, mevalonic acid lactone (50 M); FPP, farnesyl pyrophosphate (50 M). Bars represent S.D. of the means. Fig. 4. Effects of AVG, ethephon, cycloheximide, actinomycin D, and silver ion on ethylene and -farnesene production in AVG-treated and control fruit of Granny Smith apples. Fruit were harvested on October 6, 1998. Control fruit were stored at 0 C for 10 days before use. AVG-treated fruit were treated with 200 mg l 1 of AVG at harvest and stored at 0 C for 20 days before use. CK, control; AVG, aminoethoxyvinylglycine (200 mg l 1 ); Eth, ethephon (200 mg l 1 ); CHI, cycloheximide (50 mm); Act D, actinomycin D (50 mm); Ag +, silver nitrate (150 mg l 1 ). Bars represent S.D. of the means.

Z. Ju, E.A. Curry / Posthar est Biology and Technology 19 (2000) 9 16 15 4. Discussion Our results show that ethylene is involved in regulating -farnesene biosynthesis during fruit ripening in apple. When internal ethylene was below 1 l l 1, -farnesene was not detected in fruit peel (Fig. 1). The correlations of internal ethylene with -farnesene production fit the exponential growth equation and were highly significant. Ethephon at 200 mg l 1 increased internal ethylene concentration and -farnesene biosynthesis, while AVG at 200 mg l 1 inhibited both (Fig. 2). When ethephon was applied to AVG-treated fruit after 18 days of storage at 20 C (Fig. 2) or to disks of AVG-treated fruit peel (Fig. 4), both ethylene and -farnesene production were induced. Ethephon did not induce -farnesene synthesis in the presence of silver nitrate (Fig. 4), an inhibitor of ethylene function (Veen, 1987). Therefore, initiation of -farnesene biosynthesis in fruit peel during fruit ripening needs both the presence and the normal functioning of ethylene. Our results also suggest that the induction of -farnesene biosynthesis by ethylene involves gene expression and de novo enzyme synthesis. In discs from fruit at harvest, or in AVG-treated fruit where ethylene was less than 0.05 l l 1, MAL and FPP induced -farnesene biosynthesis, but HMG did not (Fig. 3), indicating -farnesene biosynthesis in these fruit was limited by the step from HMG to mevalonic acid, and the enzyme catalyzing this step, HMG CoA reductase (HMGR), may be key in regulating -farnesene biosynthesis. In discs from preclimacteric fruit, ethephon induced -farnesene synthesis (Fig. 4). CHI, a protein synthesis inhibitor, and Act D, a transcription inhibitor, counteracted the induction effect of ethephon, indicating the involvement of both gene transcription and translation in ethylene induced -farnesene biosynthesis. Silver ion also counteracted ethephon induced -farnesene production (Fig. 4), demonstrating that -farnesene biosynthesis is a result of the normal action of ethylene. Although HMGR appears to regulate -farnesene biosynthesis during fruit ripening in apples, we cannot assume that preclimacteric fruit do not contain HMGR, since other end products from the isoprenoid pathway, such as ursolic acid, increase during fruit maturation (Ju and Bramlage, unpublished data). Although a non-mevalonate isoprenoid pathway has been reported in plastids (Eisenreich et al., 1996; Lange et al., 1998; Rodriguez-Concepcion and Gruissem, 1999), it appears that -farnesene is synthesized entirely from mevalonate in apple fruit during ripening, since Lovastatin, a specific HMGR inhibitor, inhibited -farnesene biosynthesis (Ju and Curry, 2000). Furthermore, although HMG did not induce -farnesene biosynthesis in preclimacteric fruit, it increased -farnesene production in climacteric fruit (Ju and Curry, unpublished data). It is curious then, that preclimacteric fruit do not produce -farnesene even though the necessary enzymes downstream of HMGR are present and functional. One explanation is that different end products from the same pathway may need different HMGR isoforms. It has been reported that the HMGR gene is present in multiple forms, with two in Arabidopsis (Enjuto et al., 1994), three in potato (Choi et al., 1992), and four in tomato (Bach et al., 1991). In tomato fruit, the HMGR1 gene is highly expressed during the cell division and expansion period in early fruit development when sterols are required for membrane biosynthesis (Narita and Gruissem, 1989), whereas the HMGR2 gene is not expressed in young fruit, but is activated during fruit maturation and highly expressed during fruit ripening, with the concomitant accumulation of lycopene (Gillaspy et al., 1993; Rodriguez-Concepcion and Gruissem, 1999). Thus, there is a possibility that -farnesene and other end products of isoprenoid synthesis in apples are regulated by different HMGR genes or isoenzymes. Clearly, identifying and characterizing these HMGR genes may lead to a new approach in controlling scald in apples and warrants further investigation. Acknowledgements The authors thank Carol Duplaga, Carol Pavelko, Doris Frederick, Dave Buchanan, and Dr Rodney Roberts from Tree Fruit Research Laboratory, USDA-ARS, Wenatchee, for their technical support.

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