Evidence that
a
-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 A6e.,Wenatchee,WA 98801, USA Received 5 June 1999; accepted 16 January 2000
Abstract
The effect of ethylene regulation ona-farnesene biosynthesis in preclimateric ‘Delicious’ and ‘Granny Smith’ apples was studied using an ethylene inducer and inhibitor,a-farnesene biosynthesis precursors, and protein transcription and translation inhibitors.a-Farnesene was not detectable when internal ethylene concentrations were less than 1ml l−1. Correlations between internal ethylene anda-farnesene production fit the exponential growth equation and were
significant in ‘Delicious’ (y=e0.17; r2=0.68) and ‘Granny Smith’ (y=e0.18; r2=0.83). When applied at harvest,
aminoethoxyvinylglycine (AVG) at 200 mg l−1 inhibited both internal ethylene accumulation and a-farnesene
production, whereas ethephon at 200 mg l−1accelerated both. Adding ethephon to AVG-treated fruit after 18 days
at 20°C induced internal ethylene accumulation anda-farnesene production. Ethephon induceda-farnesene produc-tion in discs from preclimacteric fruit peel as well as AVG-treated fruit peel, buta-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, a-farnesene biosynthesis was induced by 50 mM 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 ofa-farnesene by MAL or FPP. © Published by Elsevier Science B.V.
Keywords:a-Farnesene biosynthesis; Ethylene; Gene expression; Apples
www.elsevier.com/locate/postharvbio
1. Introduction
Biosynthesis of a-farnesene in apple fruit is developmentally regulated. It is present in newly forming fruit (Sutherland et al., 1977) and,
al-though 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 a-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 This research was supported by Washington State Fruit
Tree Research Commission.
* Corresponding author. Tel.: +1-509-6642280; fax: + 1-509-6642287.
E-mail address:[email protected] (Z. Ju)
glycine (AVG), a potent inhibitor of ACC syn-thase, inhibits both ethylene anda-farnesene pro-duction (Ju and Bramlage, 2000). It is not clear, however, whether the ethylene related increase in a-farnesene biosynthesis during fruit ripening is caused by gene expression, by enzyme activation, or by other mechanisms. Since a-farnesene accu-mulation is closely related to scald development in apples (Ingle and D’Souza, 1989), a better under-standing of its regulation will help in the design of new strategies to control this storage disorder.
Previous reports have shown that a-farnesene biosynthesis in apples involves several steps start-ing with acetyl CoA, with hydroxymethylglutaric acid (HMG), mevalonic acid, and farnesyl py-rophosphate (FPP) as important intermediate pre-cursors (Rupasinghe et al., 1998; Ju and Curry, 2000). By feeding fruit tissue different substrate precursors and/or different inhibitors, such as cy-cloheximide (CHI, protein synthesis inhibitor), actinomycin D (Act D, transcription inhibitor), AVG (ethylene synthesis inhibitor), or silver ni-trate (ethylene action inhibitor), we can ascertain the step or steps at which ethylene regulatesa -far-nesene biosynthesis. In this paper, we examine the relationship between ethylene production and a -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 hibitor, a protein transcription or translation in-hibitor, and three precursors intermediate in the biosynthesis of a-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 anda-farnesene production. Internal ethylene was measured in each of 40 fruit every day for 5 days,
after which fruit were divided into groups accord-ing to their internal ethylene concentration. When internal ethylene was below 2 ml l−1, fruit were grouped within 0.1 ml l−1 intervals, and when internal ethylene was above 2 ml l−1, fruit were grouped within 2ml l−1intervals. Fruit within the
same interval were used for a-farnesene
measurement.
AVG and ethephon were used to assess the effects of ethylene inhibition and induction of a-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 evi-denced by undetectable internal ethylene and a -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 a-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 indi-vidual fruit in each replication using gas chro-matography. 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 N2 carrier, H2,
and air were 30, 30, and 300 ml min−1,
respectively.
2.3. a-Farnesene measurement
a-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
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 al-lowed to desorb for 3 min in the injector with a constant temperature of 250°C. The oven temper-ature was increased from 35 to 250°C at a rate of 50°C min−1and held for 4 min. Helium was used
as carrier gas and the head pressure was main-tained 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. a-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 contain-ing 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 pre-cursor for a-farnesene biosynthesis. AVG and ethephon were used at a concentration of 200 mg l−1
, cycloheximide (CHI, protein synthesis in-hibitor) 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 a-farnesene evaporation, 2 ml of air was drawn out of the test tube, after which a 100 mm polydimethylsiloxane (PDMS) probe was introduced into the tube and allowed to adsorb volatiles for 30 min at 20°C. a-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 a-farnesene mea-surement.
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 a-farnesene were analyzed by the exponential growth equation using SigmaPlot (SPSS, Chicago, IL).
3. Results
3.1. Relationship between ethylene and
a-farnesene synthesis in‘Delicious’ and ‘Granny Smith’ apples
In both ‘Delicious’ and ‘Granny Smith’ apples, the correlations between internal ethylene and a-farnesene production fit a typical exponential growth equation and were significant (Fig. 1). a-Farnesene was not detectable before internal ethylene reached 1 ml l−1, but was detected in all fruit with internal ethylene greater than 1 ml l−1. ‘Granny Smith’ apples produced less ethylene and a-farnesene than ‘Delicious’.
3.2. Effects of AVG and ethephon treatments on a-farnesene and ethylene synthesis
-far-nesene increased early and then remained con-stant after reaching the maximum. AVG treat-ment at harvest reduced ethylene to B0.5ml l−1, anda-farnesene to less than detectable levels dur-ing the 30 days of storage. Ethephon treatment at harvest increased both internal ethylene anda -far-nesene production. When ethephon was applied to AVG-treated fruit at day 18, internal ethylene concentration increased and a-farnesene biosyn-thesis was induced.
3.3. Effects of precursor feeding on ethylene and a-farnesene production in control and
AVG-treated fruit peel
Similar results were obtained using discs from ‘Granny Smith’ fruit at harvest (before the climac-teric 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. a-Farnesene was not detected in these discs. MAL and FPP in
Fig. 1. Relationship between internal ethylene accumulation anda-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 anda-farnesene was
measured in grouped fruit with similar internal ethylene concentrations. Correlations between internal ethylene anda-farnesene were
Fig. 2. Effects of AVG and ethephon treatment on internal ethylene concentration anda-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−1ethephon was applied to AVG-treated fruit. Bars represent S.D. of the means.
duced a-farnesene synthesis, but HMG did not. Precursor feeding did not affect ethylene produc-tion. Adding CHI or Act D to the incubation solution did not affect precursor-induced a -far-nesene 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 sil6er
ion on ethylene and a-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 (B0.05 ml l−1) and undetectable a -far-nesene (Fig. 4). Adding 200 mg l−1 of ethephon
to the incubation solution for 48 h increased ethylene production and induced a-farnesene biosynthesis. When added to the ethephon treat-ment, CHI, Act D, and silver ion reduced ethylene production and totally inhibited a -far-nesene biosynthesis.
In discs from fruit treated with AVG alone, ethylene and a-farnesene were undetectable after 20 days of storage at 0°C. When added to these discs, ethephon increased ethylene production and a-farnesene biosynthesis, whereas the addition of CHI, Act D, and silver ion totally inhibiteda -far-nesene synthesis and reduced ethylene production by 50% compared with the ethephon treatment.
Fig. 3. Effects of precursor feeding on ethylene anda-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−1of AVG
at harvest and stored at 0°C for 20 days. HMG, hydroxymethylglutaric acid (50mM); MAL, mevalonic acid lactone (50mM); FPP,
farnesyl pyrophosphate (50mM). Bars represent S.D. of the means.
Fig. 4. Effects of AVG, ethephon, cycloheximide, actinomycin D, and silver ion on ethylene and a-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−1of 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,
4. Discussion
Our results show that ethylene is involved in regulating a-farnesene biosynthesis during fruit ripening in apple. When internal ethylene was below 1 ml l−1, a-farnesene was not detected in fruit peel (Fig. 1). The correlations of internal ethylene witha-farnesene production fit the expo-nential growth equation and were highly signifi-cant. Ethephon at 200 mg l−1 increased internal
ethylene concentration anda-farnesene biosynthe-sis, 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 a-farnesene production were in-duced. Ethephon did not induce a-farnesene syn-thesis in the presence of silver nitrate (Fig. 4), an inhibitor of ethylene function (Veen, 1987). There-fore, initiation ofa-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 a-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 ml l−1, MAL and FPP induced a-farnesene biosynthesis, but HMG did not (Fig. 3), indicating a-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 a-farnesene biosynthesis. In discs from preclimacteric fruit, ethephon induced a-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 a-farnesene biosynthesis. Silver ion also counteracted ethephon induced a -far-nesene production (Fig. 4), demonstrating that a-farnesene biosynthesis is a result of the normal action of ethylene.
Although HMGR appears to regulate a -far-nesene biosynthesis during fruit ripening in ap-ples, 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 Bram-lage, unpublished data). Although a non-meval-onate isoprenoid pathway has been reported in plastids (Eisenreich et al., 1996; Lange et al., 1998; Rodriguez-Concepcion and Gruissem, 1999), it appears that a-farnesene is synthesized entirely from mevalonate in apple fruit during ripening, since Lovastatin, a specific HMGR inhibitor, in-hibited a-farnesene biosynthesis (Ju and Curry, 2000). Furthermore, although HMG did not in-duce a-farnesene biosynthesis in preclimacteric fruit, it increased a-farnesene production in cli-macteric fruit (Ju and Curry, unpublished data). It is curious then, that preclimacteric fruit do not produce a-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 differ-ent HMGR isoforms. It has been reported that the HMGR gene is present in multiple forms, with two inArabidopsis(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 biosyn-thesis (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 concomi-tant accumulation of lycopene (Gillaspy et al.,
1993; Rodriguez-Concepcion and Gruissem,
1999). Thus, there is a possibility thata-farnesene and other end products of isoprenoid synthesis in apples are regulated by different HMGR genes or isoenzymes. Clearly, identifying and characteriz-ing these HMGR genes may lead to a new ap-proach in controlling scald in apples and warrants further investigation.
Acknowledgements
References
Bach, T.J., Wettstein, A., Boronat, A., Enjuto, M., Gruissem, W., Narita, J.O., 1991. Properties and molecular cloning of plant HMG-CoA reductase. In: Patterson, G.W., Nes, W.D. (Eds.), Physiology and Biochemistry of Sterols. American Oil Chemists Society, Washington, DC, pp. 29 – 49.
Barden, C.L., Bramlage, W.J., 1994a. Accumulation of antiox-idants in apple peel as related to postharvest factors and superficial scald susceptibility of the fruit. J. Am. Soc. Hort. Sci. 119, 264 – 269.
Barden, C.L., Bramlage, W.J., 1994b. Relationships of antiox-idants in apple peel to changes in a-farnesene and
conju-gated trienes during storage, and to superficial scald development after storage. Postharvest Biol. Technol. 4, 23 – 33.
Choi, D., Ward, B.L., Bostock, R.M., 1992. Differential in-duction and suppression of potato 3-hydroxy-3-methylglu-taryl Coenzyme A reductase genes in response to Phytophthora infestansand to its elicitor arachidonic acid. Plant Cell 4, 1333 – 1344.
Du, Z., Bramlage, W.J., 1994. Roles of ethylene in the devel-opment of superficial scald in ‘Cortland’ apples. J. Am. Soc. Hort. Sci. 119, 516 – 523.
Eisenreich, W., Menhard, B., Hylands, P.J., Zenk, M.H., Bacher, A., 1996. Studies on the biosynthesis of taxol: the taxane carbon skeleton is not of mevalonoid origin. Proc. Natl. Acad. Sci. USA 93, 6431 – 6436.
Enjuto, M., Balcells, L., Campos, M., Caelles, C., Arro, M., Boronat, A., 1994. Arabidopsis thalianacontains two dif-ferentially expressed 3-hydroxy-3-methylglutaryl-CoA re-ductase genes, which encode microsomal forms of the enzyme. Proc. Natl. Acad. Sci. USA 91, 927 – 931. Gillaspy, G., Ben-David, H., Gruissem, W., 1993. Fruits: a
developmental perspective. Plant Cell 5, 1439 – 1451. Huelin, F.E., Murray, K.E., 1966. Farnesene in the natural
coating of apples. Nature (Lond.) 210, 1260 – 1261. Huelin, F.E., Coggiola, I.M., 1970. Superficial scald, a
func-tional disorder of stored apples. V. Oxidation of a
-far-nesene and its inhibition by diphenylamine. J. Sci. Food Agric. 21, 44 – 48.
Ingle, M., D’Souza, M.C., 1989. Physiology and control of superficial scald of apples: A review. HortScience 24, 28 – 31.
Ju, Z., Bramlage, W.J., 2000. Cuticular phenolics and scald development in ‘Delicious’ apples. J. Am. Soc. Hort. Sci., in press.
Ju, Z., Curry, A., 2000. Lovastatin inhibits (-farnesene synthe-sis without affecting ethylene production during fruit ripening in ‘Golden Supreme’ apples. J. Am. Soc. Hort. Sci. 125, 105 – 110.
Lange, B.M., Wildung, M.R., McCaskill, D., Croteau, R., 1998. A family of transketolase that directs isoprenoid biosynthesis via a mevalonate-independent pathway. Proc. Natl. Acad. Sci. USA 95, 2100 – 2104.
Meigh, D.F., Filmer, A.A.E., 1969. Natural skin coating of the apple and its influence on scald in storage. III. Far-nesene. J. Sci. Food Agric. 18, 307 – 313.
Murray, K.E., Huelin, F.E., Davenport, J.B., 1964. Occur-rence of a-farnesene in the natural coating of apples. Nature (Lond.) 204, 80.
Narita, J.O., Gruissem, W., 1989. Tomato hydroxy-methylglu-taryl-CoA reductase is required early in fruit development but not during fruit ripening. Plant Cell 1, 181 – 190. Rodriguez-Concepcion, M., Gruissem, W., 1999. Arachidonic
acid alters tomato HMG expression and fruit growth and induces 3-hydroxy-3-methylglutaryl Coenzyme A reduc-tase-independent lycopene accumulation. Plant Physiol. 119, 41 – 48.
Rupasinghe, H.P.V., Paliyath, G., Murr, D.P., 1998. Biosyn-thesis of a-farnesene and its relation to superficial scald
development in ‘Delicious’ apples. J. Am. Soc. Hort. Sci. 123, 882 – 886.
Sutherland, O.R.W., Wearing, C.H., Hutchins, R.F.N., 1977. Production ofa-farnesene, an attractant and oviposition
stimulant for codling moth, by developing fruit of ten varieties of apple. J. Chem. Ecol. 3, 625 – 631.
Veen, H., 1987. Use of inhibitors of ethylene action. In: Reid, M.S. (Ed.), Manipulation of ethylene response in horticul-ture. Acta Hort. 201, 213 – 222.
Watkins, C.B., Barden, C.L., Bramlage, W.J., 1993. Relation-ships amonga-farnesene, conjugated trienes and ethylene
production with superficial scald development of apples. Acta Hort. 343, 155 – 160.
