BIOCHEMICAL AND MOLECULAFL CHARACTEIUZATION OF a-FARNESENE BIOSYNTHESIS IN RELATION TO SUPERFICIAL SCALD
DEVELOPMENT IN APPLE (Malus x domestka Borkh.)
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
H. P. VASANTHA RUPASINGHE
In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
January, 200 1
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BIOCHEMICAL AND MOLECULAR CHGIRACTERIZATION OF a-FARNESENEBIOSYNTHESIS IN RELATION TO SUPERFICLAL SCALD DEVELOPMENT IN APPLE (Malus x domestica Borkh.)
H.P. Vasantha Rupasinghe Advisors: University of Guelph, 200 1 Dr. Demis P. Murr Dr. Gopi Paliyath
Metabolism of a-famesene, a sesquiterpene that accurnulates in apple skin during cold storage, has been implicated in the development of superficial scald in pome fniits.
Biosynthesis of a-famesene occurs through the classical mevalonate pathway, and is formed directly fiom trans,trans-farnesyl pyrophosphate. This step is catalyzed by trans,~rans-a-faniesene synthase, an enzyme located mainly in hypodermaI and epidennaI cells of apple hit. The enzyme was purified seventy-fold fiom the cytosolic fraction, where activity was highest among sub-cellular fractions. The enzyme required a divalent rnetal (M~"or ~n'3for activity and exhibited allosteric kinetics: S(o.jlfor FPP was 84 j~rnol-~-'.The Hill coefficient (nH)indicated that the native protein was a trimer.
Activity of a-famesene synthase was not evident in apple skin at harvest, but was induced by Iow temperature storage and preceded the accumulation of a-famesene. In contrat, high activity of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) was present in the skin at harvest, but declined during the first 8 weeks in storage and then remained unchanged. The inhibition of ethylene (CzH4) action in apple fruit by 1- methylcyclopropene (1-MCP) reveaIed that C2& was required for a-farnesene synthesis and the development of superficial scald. However, activity of a-fmesene synthase \vas
not af5ected by Cz&. Since fiuit respiration was suppressed significantiy by 1-MCP, the
reghation of a-famesene biosynthesis by Cz& may be through control of glycolysis;
e-g., acetyl CoA availability limits isoprene synthesis and HMGR activity.
A full-length (hmgl) and a fragment (hmg2) of two cDNA clones comprising the
HMGR gene farnily were isolated f?om apple skin. The transcription product of hmgl
cDNA has an open reading fiame of 1767 nucleotides and encodes a protein of 589
polypeptide residues of 62.7 kD. The presence of two highiy hydrophobic domains near the amino terminus, a unique feature of plant HMGR genes, was recognized. The two genes were expressed differentially in response to developmental stimuli; hrngl being expressed relatively constitutively, and hmgZ being highly sensitive to low temperatures and Cr& The synthesis of a-farnesene possibly occurs through a complex of sequential metabolic enzymes or "metabolon" located in the cytosol/ER boundary, where hmg2 also may be involved. To rny wzye Pri-vanka. rny son Kalana. and rny dazcghter Viraji who received less of my attention while this thesis was heing prepared 1 wish to express my gratitude and appreciation to my CO-advisorsDrs. Demis
Murr and Gopi Paliyath for providing me the opportunity to study the postharvest biotechnology of fiuit. Appreciation also goes to Drs. Kckey Yada and Geza Hrazdena
(Corne11 University) for serving as members of my advisory cornmittee and for their guidance and encouragement during the doctoral study.
1 gratehlly acknowledge Dr. Daryl Rowan (HortResearch, NZ), Dr, Barry
McGIasson (University of Western Sydney, Australia), and Dr. Albert Purvis (University of Georgia) for sharing their experience. 1 thank Drs. Gordon Lange and Rodney Memll
(Department of Chernistry and Biochernistry) and Dr. Brian Allen (Department of
Mathematics and Statistics) of University of Guelph for advice in their areas of expertise.
Special thanks to Dr. John Proctor for reviewing the manuscripts written for publication fiom this thesis.
1 am very thankful to Kurt Almquist for his support given during the gene cloning and expression studies. 1 appreciate the technical assistance of Dr. Valerie Robinson, Dr.
Sandy Smith, and Robert Harrison for studies on 31~-~~~,DSC and freeze fracture, respectively, although the results of these works were not included in the thesis. 1 aIso owe many thanks to the laboratories of Drs. Barry Shelp and Judy Strommer for allowing me to use their facilities. 1 also thank Brad Cooney (Laboratory Services) for the timely semice provided on primer synthesis and DNA sequencing.
1 wish to thank Dr. John Cline (Department of Plant Agriculture, Simcoe) and
Ken Wilson (OMAFRA, Thornbury) for their help in collecting apple hit which were used in this study. 1 must also thank Len Wiley for his help harvesting and maintaining the storage of apple, and Ramany Paliyath, Valsala Kailidumbil, Bernie Watts, and
Rodger Tschanz for technical help.
1 gratefully acknowledge the frnancid assistance provided for this research by the
Ontario Apple Marketing Commission; the Natural Sciences and Engineering Research
Council of Canada (NSERC); the Ontario Ministry of Agriculture, Food and Rural
Af%airs (OMAFRA): Agriculture and Agi-Food Canada (AAFC); and the CanAdapt
Program of the Agriculturai Adaptation Council (AAC). TABLE OF CONTENTS
CHAPTER 1 General Introduction
Superficial scald Factors determining the susceptibility to scald development Control of superficial scald 2 -3.1 Diphenylamine @PA) 1-3-2 DPA is under scrutiny 1.3 -3 Attempts to find a DPA alternative 1 -3-4 Manipulation of ethylene 1-3 -5 Controlled atmosphere storage 1.3 -6 Heat treatments 1-3 -7 Prediction Biochemical theories of scaid development 1-4.1 ceFarnesene theory Biosynthesis of isoprenoids 1-51 HMGR as the key regulatory enzyme 1-5.2 GAP/pyruvate pathway 1-5.3 Sesquiterpene cyclases and synthases Oxidative stress and membrane degradation 1 -6.1 Lipid peroxidation 1.6.2 Antioxidants and antioxidant enzymes
1.6.3 Alternative oxidase and stress Research hypothesis and objectives Outline of the thesis
CHAPTER IX Biosynthesis of a-Farnesene and its Relation to Superficial Scaid Development in 'Delicious' Apples
3.1 Abstract 2.2 Introduction 2.3 Materials and Methods 2.4 Results and Discussion 2.5 Literature Cited CHAPTER III Sesquiterpene a-Famesene Synthase: Partial Purification, Characterization, and Activity in Relation to Superficial Scald Deveiopment in Apples 3-1 Abstract 3 -2' Introduction 3-3 Materials and Methods 3 -4 Results 3 -5 Discussion 3 -6 Literature Cited
CHAPTER IV Regulation of a-Famesene Synthesis by Ethylene
Study 1 Suppression of a-Famesene Synthesis in 'Delicious' Ap ple by Arninoethoxyvinylglycine (AVG) and 1-Methylcyclopropene (1-MCP)
4A. 1 Abstract 4A.2 Introduction 4A.3 Materials and Methods 4A.4 Results and Discussion 4A.5 Literature Cited
Study 2 Inhibitory Effect of 1-MCP on Ripening and Superficial Scald Deveiopment in 'Mclntosh' and 'Delicious' Apples
4B. 1 Abstract 4B.2 Introduction 4B.3 Materiais and Methods 4B.4 Results and Discussion 4B.5 Literature Cited
CHAPTER V Cloning and Expression of hmgl and /.mg2 cDNA Clones Encoding 3-Hydroxy-3-Methyiglutaryl Coenzyme A Reductase (HMGR) and its Activity in Relation to cc-Farnesene Synthesis in Apple
5.1 Abstract 5 -2 Introduction 5.3 Materials and Methods 5.4 Results and Discussion 5 -5 Literature Cited
CHAPTER VI GENERAL DISCUSSION 179
LITERATURE CITED IN GENERAL INTRODUCTION AND GENERAL DISCUSSION LIST OF TABLES
CHAPTER II
2.1 Incorporation of tram,trans-[l --'~]-farnes~l pyrophosphate into a- 47 famesene and farnesol by different tissue segments of 'Delicious' apple fruit.
CHAPTER III
3.1 Distribution of a-farnesene synthase activity arnong different sub- cellular fiactions of skin tissue of 'Deiicious' apple hit.
Partial purification of a-famesene synthase from skin tissue of 78 -Delicious' apple bit.
Content of hexane-extractable a-faniesene, and its catabolites in the 85 skin tissue of 'Delicious' apple in relation to severity of scaid, DPA and CA treatment.
CHAPTER IV
production and in vivo a-famesene synthase activity of 'Delicious' apples following treatment with AVG and 1-MCP.
Sumary of the statistical analysis of Fi,ve 4.2.1.
Flesh fimess of 'McIntosh' and 'Delicious' apples treated with 1pL.L' 1-MCP a day afier harvest at 20 OC for 18 h or not treated and stored 60 days (removal 1) and 120 days (removal 2) in air at O - 1 OC and 90 - 95 % relative humidity.
cc-Farnesene and conjugated triene alcohol (CTOL) contents in skin tissue of 'Mclntosh' and 'Delicious' apples treated with 1-MCP a day after harvest and stored in air at O - 1 OC with 90 - 95 % relative humidity for 120 days.
Incidence of superficial scald in 'McIntosh' and 'Delicious' apples treated with 1-MCP and stored 120 days in air at O - 1 OC with 90 - 93 % relative humidity.
Correlation (rZ) of mean Cz& production at removal tirne 1 with six postharvest measurements in 'Mclntosh' and 'Delicious' apples treated or untreated with 1-MCP. 4.2.6 Response of 'Mchtosh' apples to 25 @*L" 1-MCP in CA storage (3 % O2 + 4.5 % CO2 at 3 OC with 90-95 % relative humidity). Apples were removed fkom CA storage after 120 days and kept two weeks at 20 OC. Estimation of parameters were done as descnbed for air-stored apples.
CHAPTER V
5.1 Metabolic responses of apples treated with the Cr& action inhibitor. 1- methylcyclopropene (1-MCP, 1 Ci~-~'l).Each variate represents the mean value of response measured triplicate at 1, 5, and 11 day at 20 OC following removal fiom storage for 60 days at O OC in air.
a-Famesene content in the skin of apples treated with or without Lovastatin (200 rng-~-'),a specific HMGR inhibitor at harvest and stored at O OC storage in air. Data represent mean of 3 replicates I: SD.
Incorporation of radiolabel from precursors of isopentenyl diphosphate (IPP) into a-fâmesene in isolated skin tissue of Lovastatin-treated (200 rn@~-')apples. Data represent the mean of 3 replicates 2 SD. LIST OF FIGURES
CHAPTER 1
1.1 A proposed mechanism for the auto-oxidation of a-farnesene [denved fiorn Anet (1 969)].
1.2 Pathways of isoprenoid biosynthesis.
CHAPTER II
2.1 Proposed pathway and mechanism of a-famesene biosynthesis in apple 50 skin tissue.
2.2 cc-Farnesene content of scald-free and scaiding 'Delicious' apple skin 52 tissue as estimated by HPLC separation of hexane-exnacted components
3.3 Incorporation of radioactivity into a-famesene (A) and farnesol (B) 53 from tran~.rrans-[1-~~]-~~~in scalding (a) and scdd-free (U) 'Delicious' apple skin tissue during incubation.
CHAPTER III
3-1 Effects of MnClz (0)and MgCl? (a) on a-famesene synthase activity. 73
3 -3 Influence of incubation temperature on a-famesene synthase activity. 74 Enzyme was extracted frorn skin tissue of apples stored in CA for five months.
Partial purification of a-famesene synthase by ion eschange 76 chromatography on DEAE Sephacel (A) and gel perrneation chromatography on Sephacryl (B).
Effect of increasing farnesyl phyrophosphate (FPP) concentration on 80 initial velocity of a-farnesene synthesis.
a-Farnesene content (A), in vivo cc-farnesene synthase activity (B), and conjugated triene alcohol (CTOL) content (C) of the skin of 'Delicious' (*)and 'Empire' (O) apples during air storage at O OC.
vii a-Farnesene content (A),in vivo a-farnesene synthase activity (B), and conjugated triene alcohol (CTOL) content (C) of the skin of scald- susceptible (Delicious, McIntosh, Cortland, Idared, Rome Beauty). moderately scald-susceptible (Fuji), and scald-resistant (Empire. Gala, Mutsu, Jonagold, Northern Spy) cultivars of apple.
CHAPTER IV
4.1.1 Changes in hexane-extractable a-faniesene content in the skin and head- space a-farnesene of 'Delicious' appIes treated with AVG (O), 1-MCP (r)or left untreated (a) after removd from air storage at O OC in air for 8 weeks.
4.2.1 Cl& production (A,B), total volatile (C,D) and a-fmesene (E,F) emitted by 1-MCP-treated or untreated 'McIntosh' and 'Delicious' apples during 13 and 15 days, respectively, at 20 OC upon removal from 60 days in air at O - 1 OC with 90 - 95 % relative humidity.
CHAPTER V
cc-Famesene content (top) and in vivo HMGR activity in the membrane fraction (a), and the soluble fraction (O) (bottom) of the skin of 'Delicious' apples during storage at O OC in air.
Combined nucleotide sequence and the predicted arnino acid sequence of the encoded product of the cDNA clone correspondin; to hmgl from M. x dornestica cv. Delicious.
Alignrnent of deduced amino acid sequence of M. x domesticu hmgl with hmg 1 sequences of A. thaliana, C. aczcminara, G. hi?-sztrum,and H. brasiliensis.
Hydropathy index plot of the predicted amino acid residues of apple hmgl (1), A. thdiann hmgl (II), Hbrasiliemis hmgl (III), and C. acuminata hmg3 (IV).
Schematic representation of the domain structure of apple HMGR isoform encoded by hmgl.
Nucleotide sequence of the fragment of hmg2 from M. x dornesricu cv. Delicious, and the predicted amino acid sequence of the encoded product. 5.5 Genomic Southem blot hybridization analysis to verie two HMGR 158 genes.
5.6 Northern blot-hybridization of apple total RNA (IO pg per lane) probed 159 with hmgl and hmg2 specific probes to show the size(s) of transcnpts.
5.7 Expression of apple hmgl and hmg2 during the Cmonth storage at 0°C 161 in air.
5.8 Effect of the C2& action inhibitor 1-methylcycfopropene (1-MCP) on 162 the expression of apple hmgl and hmg2.
5.9 A simplified biosynthetic pathway for plant isoprenoids. PP, 165 pyrophosphate; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase- LIST OF APPENDICES
Appendix 5-1 Design of degenerate oligonucleotides for apple HMGR. CHAPTER 1
General Introduction
1.1 Superficial Scald
Superficial scald (storage or common scald) is a postharvest physiological disorder that affects several cultivars of apple (-Malus xdomestica Borkh.) during or afier prolonged low-temperature (O to 5 OC) storage in air or under controlled atrnosphere.
Scaid is manifested as an uneven browning or bronzing of the skin, aIong with the development of skin wrinkling and pitting with increasing severity (Bain, 1956; Bain and
Mercer, 1963; Ingle and DySouza, 1989; Wilkinson and Fidler, 1973). Scald reduces the organoIeptic quaiity and market value of apples, and those fruits are used only for processing. The currently adapted method of controlling scald, drenching apples with a synthetic chemicai diphenylamine (DPA) before storage, is under scrutiny because of health concerns. Thus. understanding the biochemical mechanism of superficial scald development and developing alternatives to control scald is highly warranted.
Histological examinations of affected hits revealed that the developrnent of scald is due to the progressive browning of the hypodermal cells (Bain, 1956). In slightly scalded tissue, the darnage is confined to the outer four to five plastid-bearing ce11 layers of the hypodermis, but does not penetrate beneath the hypodermal ce11 layer. Epidermal cells are not affected unless the disorder is very severe. In severely scalded tissue. the af3ected cells of the hypodermis collapse in a radial direction, foming sunken regions. In severely scalded tissue. cells of the outer cortex also become distorted, but the shape of the epidermal celis is not affected (Bain, 1956). Subsequently, Bain and Mercer (1963) observed the formation of additionai electron-dense materials in close association with the normal constituents of the vacuoles of hypodennal cells in scald developing tissue.
Beelectron-dense materials increase and accumulate in the tonoplast, as scald syrnptoms become more severe (Bain and Mercer, 1963). The breakdown of the tonoplast. allowing oxidattive browning due to @oly)phenoloxidase action, was proposed to be the ultimate symptom of the deterioration process. In 'Granny Smith' apples. the progression of scaId intensity is accompanied by a gradual decrease of ail types of oligomeric polyphenoIs
(Piretti et al., 1994? 1996). Ju and Bratnlage (2000) found a negative correlation between
Free phenolic concentration in fruit cuticle and scald development of apples. The content of total cimamic acid derivatives such as caffeic acid and chlorogenic acid, were significantly higher in scald-fiee tissue than scalded tissue on the same apple (Abdallah et al., 1997). However. total catechin derivatives (Abdallah et al.. 1997) and flavonoIs
(Abdallah et al., 1997; Piretti, 1996) were not correlated with scald deveIopment. Proton magnetic resonance imaging (MN) and volume selective spectroscopy (VOSY) studies show an increase in mobility of water in tissues with preliminary symptoms of scald. due to an increase in the ratio of free water to bound water (Paliyath et al., 1997). As well, nuclear magnetic resonance (NMR) imaging studies have shown that skin winkling of severely-scaIded tissues is due to significantly lower water content than that of the scald- fiee tissue (Golding et al., 1997).
The precise biochsrnical cause for the induction of superficial scald is still not clear. However, it is widely believed that scald devefopment involves an accumulation of a-famesene, an acyclic sesquiterpene hydrocarbon (CisH24; [3 E,BE]-3,7,Il-trimethyl-
1,3,6,1 O-dodecatetraene) present in the skin of apple fruit (Huelin and Murray, 1 966), and its oxidation to conjugated trienes (CT) (Huelin and Coggiola, 1970a). ïhere is no direct proof for this hypothesis, but a large body of evidence supporting this theory has accumulated in the literature which postdates the severity of scaid is influenced by the extent of a-famesene oxidation (Chen et al., 1990; Gallerani and Pratella, 1991 Huelin and Coggiola, 1970; Meir and Bramlage, 1988). On the other hand, methods which control scald also reduce cc-faniesene oxidation. The following sections give an ovewiew of the curent understanding of this physiological disorder.
1.2 Factors Determining the Susceptibility to Scald Development
Studies on superficial scaid date back to 1903 with emphasis on determining the factors iduencing scald deveioprnent and preventing its appearance (Bain, 1956; Brooks et al., 1919). Susceptibiliv to scaid is determined by several factors such as cultivar differences, environmenta1 conditions that app1es are exposed to during growth and development, stage of maturity at harvest, and storage atmosphere (Ingle and D'Souza,
1989). Apple cultivars such as Delicious, McIntosh, Cortland, Granny Smith, Idared.
Rome Beauty, Brarnley's Seedling and Fuji are susceptible to this disorder, whereas
Empire, Gala, Mutsu, Jonagold, and Spy are relatively resistant to scald development
(Emongor et al., 2994; Meigh, 1970; Meir and Bramlage, 1988). Scald susceptibility is influenced greatly by climatic variables, and thus, varies markedly among seasons
(reviewed by Emongor et al., 1994). In general, hot and dry conditions during fruit maturation promote the occurrence of scald, while moist, cool weather retards it (Fidler,
1956; Wilkinson and Fidler, 1973). Apples that are harvested later in the season receive prolonged exposure to low temperature and as a result, develop less scald during storage
(Barden and Bramlage, 1994a). The accumulated number of hours below IO OC to which apples are exposed during growth and development seems to reduce the incidence of
scald (Fidler, 1956; Merritt et al-, 196 1; Morris et 21-, 1963). In contrast, Lougheed et al.
(1992) reported little relationship between the number of hours below 10 OC and scald
severity on 'Mchtosh' and 'Delicious' apples. Imbalance of plant nutrients dso seerns to
influence scald incidence. Scald development is inversely proportional to calcium and
phosphorus levels in the hit, while nitrogen and potassium levels are positively
correlated with scald development (Emongor et al., 1994). FidIer (1956) postulated that
large apples which might result fiom high nitrogen nutrition could Iead to lower fruit
calcium by dilution. and are more susceptible to scald deveIopment than srnaII apples.
Micro nutrients like copper and cobalt increase scald, while bariurn and strontium reduce
the disorder (Wills and Scott, 1974).
PhysiologicaI maturity at harvest greatly influences the deveIoprnent of scald.
Less mature apples are highly vulnerable to scald development (Albrigo and Childers,
1970; Anet, 1972; Barden and Bramlage, 1994a; Chen et al., 1985; Fidler, 1973; Huelin and Murray, 1966; Whitaker et al., 1997), but scald develops on iùlly mature fruits as well (Emongor et al., 1994). Blanpied et al. (1991) observed a significant negative correlation (F-0.3) between starch index and scald index. Whitaker et al. (1997) found that less mature appies produce higher levels of a-farnesene and its presumed scald- causing oxidation product conjugated triene alcohol (CTOL) than more mature apples.
However, Barden and Bramlage (1994a) reported elevated levels of antioxidants, oc- tocopherol and carotenoids, and inhibition of lipid oxidation as harvest date of 'Cortland' and 'Delicious' apples progressed. Antioxidant concentration at harvest was inversely related to accumulated CT at the end of storage, and to scald development (Barden and Bramlage, 1994b). In contrast, over-mature 'Mchtosh' (Smock, 196 1) and -Golden
Delicious ' (Hardenburg and Siegelman, 195 7) developed scald more severely than early-
picked apples in controlled atmosphere (CA) and polyethylene liners. respectively.
Col~ectively,preharvest factors such as cultivar, rootstocks, weather, nutrients, plant
growth regulators, harvest rnaturity, hitsize, etc. are reported to influence incidence and
severity of scald by modifjmg fniit physiology. However, most of the literanire seems to
contradict one another and the precise mechanism of scdd development is not clear.
1.3 Control of Superficial Scald:
1.3.1 Diphenylamine (DPA)
Attempts to control scald have been reported hmthe early twentieth century (see
Brooks et al., 19 19); however, a successfd chemical procedure utilizing the synthetic
chemical diphenylarnine (DPA; N-phenyl-benzenearnine) and ethoxyquin (6-ethoxy-
2,2,4-trimethyl- 1.2-dihydroquinoline) has been in practice only since 1960 (Wilkinson
and Fidler, 1973). The etiology of control of superficial scald in apples was reviewed by
Brooks et al. (1 9 19), Fidler (1 954), Smock (1 96 1), Hardenburg (1965), Meigh (1969),
IngIe and D'Souza (1989) and recently, Wang and Dilley (1999). The pioneering research work done by Brooks and CO-workersobserved that scald is caused by the accumulation of toxic volatile esters or similar products of the apple and these could be carried away by ventilation or absorbed by fats or oils (Brooks et al,, 1919). Consequently, wrapping individual apples in oii-impregnated paper (Brooks et al.. 1919, 1923; Huelin and k~ett,1958; Meigh, 1967) or spreading oil-impregnated strips uniformly throughout the bins (Goldenberg et al., 1979) were used commercially to reduce scald for several decades. HueIin and Coggiola (1968) found that during storage, a-farnesene present in the apple 'coating' moved to the oiled-wraps uitil wraps contained more than twice as
much a-famesene as the fit. However, oil-impregnated papers were ineffective in
controlling severe scald, A milestone in scald research occurred when Smock (1955)
found that this disorder could be controlled completely by applying DPA to fnrit before
storage. Extensive trials done in a wide range of conditions in the USA (Smock, 1957),
Australia (Hall et al., 1961) and Europe (Staden, 1960) attested that a pre-storage dip of
apples in 21000 p~.~-lDPA was extremely effective in controlling scald. DPA was then
rapidly adapted as the standard commercial control of scald in the world. Ethoxyquin (6-
ethoxy-2,2,4-trimethy 1- 1,2-dihydroquinoline) (Srnock, 1957) and DPA hydrochloride
(Smock, 1961) have similar scald-reducing properties, but are less effective than DPA.
Huelin (1964) found that DPA-related compounds dibenzylamine, dicyclohexylarnine
and N-benzy laniline also were effective against scald. As well, wrapping apples with
ethoxyquin-impregnated papers reduced scald (Porritt and Meheriuk, 1970).
1.3.2 DPA is Under Scrutiny
Early investigations on DPA residue in treated apples reported that DPA is
metabolized rapidly during the first 30 weeks of storage, with 93% loss of DPA originally present (Huelin, 1 968). Distribution of DPA in apple fruit is restricted rnainly to the cuticular wax and epidemis to a depth of 1 mm (Hanekom et al. 1976; Huelin,
1968; Johnson et al., 1%O), and only about 0.1 mg.kg" DPA was detected in the flesh of
DPA-treated apples (Hall et al., 1961). Johnson et al. (1980) found that an initial DPA residue concentration of 2.3 mgkg-' was sufficient to give complete control of scald.
Fruit residue tolerance levels for DPA and ethoxyquin enforced by the Environmental
Protection Agency (EPA) are 10 and 3 mg.kg", respectively (Ingle and D'Souza, 1989). DPA determination methods reported earlier were based on spectrophotometric
procedures and codd have resulted in providing incorrect results. Based on recent
methods utilizing gas chrornatographic (GC) with either electron-capture or nitrogen-
sensitive thennionic detectors. the residue levels in skin of apples treated wîth 2000 PL.
L-~DPA were 3 -62 and 0.29 pgmcm-2 at O and 118 days, respectively, during storage
(Allen and HalI, 1980). These results are over 5-fold higher than previous estimations.
Recently, Kim-Kang and Robinson (1998) investigated the fate of DPA in stored apples
using radiolabelled-DPA. Interestingly, total radioactivity remained constant over the 40- week storage penod. The majority of the terminal residue, which was confïned largely to the peel, consisted of unmetabolized DPA. The major DPA metabolite in apple was
identified as a glucose conjugate of 4-hydroxy-DPA (4-OH-DPA), with additional metabolites characterized as glycosyl conjugates of 2-OH-DPA, 3-OH-DPA, or dihydroxy-DPA along with their nonconjugated forrns (Kim-Kang and Robinson, 1998).
Detailed clinical studies describing the effects of biological degradatives of DPA were not available until the writing of the thesis.
Because of the grotiring global concems on health and food safety. the use of chernicals such as DPA on fresh produce has corne under close scrutiny. Use of DPA on apples has already been banned in Germany, Holland, and Thailand. and is anticipated to occur in North Amerka and other parts of the wor1d- DPA has been reported to be a mitochondrial cytochrome oxidase inhibitor (Abood and Gerard. 1953). Recently, DPA has been classified as a possible teratogen and mutagen that targets the kidney and liver
(EPA's Registry of Toxic Effects of Chernical Substances No. .J.J7800000; MSDS, Sigma-
Aldrich). 1.3.3 Attempts to Find a DPA Alternative
It wodd be impractical to wrap apples in oiled paper because of the high cost of
wrapping involved with buik handling. Alternatively, Scott et al- (1995a) applied
different vegetable oils (sunflower, canola, castor, palm, and peanut) to 'Granny Smith'
apples and observed a reduction of scdd. Injection of natural monoterpenes (geraniol.
nerol, citronelloI, lindool, a-terpineol, menthol, menthone, and pulegone) into 'Granny
Smith' apples dso reduced the incidence of scald (Wills et al.. 1977). Phorone (2.6-
dimethylhepta-2.5-dien-4-one). an olefinic ketone, was found to reduce scald in 'Granny
Smith' (Scott et al., 1980), but little is known about the biological activity and toxicity of
this chemical. Because of the known antioxidant properties of DPA and ethoxyquin, a
wide range of other synthetic and natural antioxidants have been trsted on apple to
control scald. A postharvest dip of the food compatible antiosidant butylated
hydroxytduene (BHT; 2,6-Bis(1,l-dimethylethy1)-4-methylphenol)) reduced scald
(Gough et al., 1973, WiIls and Scott, 1977). However, BHT was required in extremely high amounts to produce an effect equivalent to that caused by DPA. and it induced skin spotting and exhibited an unpleasant taint (Little et al.. 1980). Anet and Coggiola ( 1974) exarnined a wide range of antioxidants and found that amine type antioxidants (NAr'- diphenyl-p-phenylenediarnine, NN'-di-s-butyl-p-phenylenediamine, N-isopropyl-Ar'- phenyl-p-phenylenediamine) were more effective than phenolic antioxidants (cc- tocopherol, t-butyl-4-methoxyphenol) to control scald. Other antioxidants such as ascorbyl palmitate and n-propyl gallate fomulated with Semperfresh (a sucrose ester- based fniit coating) (Bauchot et al., 1995a; Bauchot and John, 1996), citnc acid and ascorbic acid (Chellew and Little, 1995; Manseka and Vasilakakis. 1993) have shown lirnited control and variable results against scald. Semperfkesh alone reduced scald appreciably when apples were stored in CA (Chellew and Little, 1995). Reduction of scdd observed with antioxidants plus Semperfkesh is due partially to altered intemal gas atmosphere by the Sernpefiesh coating (Bauchot et al.. 1995b).
interestingly, when ethanol, which also can act as an antioxidant, is applied to apples as single dips (Chellew and Little, 1995). as an injection (Wills et al., 1977; Scott et al,, 1995), or as a vapor (Scott et al., 199%; Ghahramani and Scott, 1998a;
Ghahramani et al.. 1999), scald incidence was reduced significantly in 'Granny Smith' apples. Ethanol at the rate of 0.35 - 5.5 g=kg'reduced a-farnesene and CT contents in apple skin and completely controlled scald (Ghahramani and Scott, 1998a).
Subsequently. it was found that ethanol is not a specific inhibitor of superficial scald. as some other alcohols (propan- 1 -01, butan- 1-01, pentan- 1-01. hexan- l -oi) also reduced scald when evaporated at low temperatures (Ghahramani et al., 1999). Exposure of 'Delicious' apples to ethanol vapors for 24 h resulted in more than a 3-fold increase in the sum of concentration of aromatic ethyl esters, inciuding ethyl-2-methylbutyrate. which is the
'character impact compound' of apples (Berger and Drawert. 1984). Hotvever. ethanol taste was detected in apples treated with high levels of ethanol vapor (Scott et al., 1995b), and development of off-flavors also could occur due to prolonged exposure of apples to ethanol vapor. Commercialization of exogenous application of ethano l on apple has several barriers: it is heavily taxed in rnany countries, prohibited on food in some countries (Ghahramani and Scott, 1998b)' and there could be significant consumer resistance against use of ethanol on fresh produce. As yet, no alternative antioxidant or chemical for the control of scald on apples appears as promising as DPA. 1.3.4 Manipulation of Ethylene
In general, advanced maturity at harvest results in a lowered incidence of scald
(Emongor et al., 1994). Ethylene (Cz&) is involved in accelerating fruit ripening. hence, several researchers studied the effect of preharvest application of CIE&-generating compound khloroethyl phosphonic acid (EthephonTM)on scdd incidence. The response to ethephon was variable, it either reduced (Couey and Williams, 1973; Padifield, 1977;
Cw,1994; Lune et al., 1989a; Watkins et al., 1982) or prornoted (Windus and Shutak.
1977) scald incidence. Du and Bradage (1994a) exarnined the effect of ethephon on accumulation of a-famesene and CT in fiuit peel. Ethephon induced rapid and delayed effects on hit, the former being stimulation of C2% produciion, a-faniesene, and CT accumulation in fruit peel, which could increase scald development. The delayed effects included a disproportionately higher accumulation of CT258 than of CT28I during prolonged cold storage, which was associated with reduced scaId development (Du and
Brarnlage, 1994a). Du and Bramlage (1 994a) postulated that C2H4 seerns to be critically involved in scald development through a stimulation of the Ievel of a-farnesene and CT in hit peeI. Accumulation of a-faniesene in hit peel corresponded with the rate of
C2& production by the fniit (Meigh and Filmer, 1969 and Watkins et al., 1993). In contrast, pre-harvest application of succinic acid-3.2-dimethylhydrazide (Daminozide or
Alar) reduced CzHl production and delayed ripening of apples, but reduced scald development (Blanpied et al., 1967; Williams et al., 1964; Windus and Shutak, 1977).
However, Alar is already banned from use on apple. Preharvest sprays of other plant growth regulators such as gibberellins (G&+7), napthaleneacetic acid, abscisic acid, no control over scald (Ingle and D'Souza, 1989). Glutathione, ethylene dibromide, and dirnethyIsuIfoxide either increased scald or showed no control over scald (IngIe and
D'Souza, 1989). DPA treatment dso reduces C2& production by the fiuit in addition to suppressing a-faxnesene oxidation (Du and Bramlage, 1994a; Lurie et al., 1989b), indicating its effect is not solely as an antioxidant. Recently, several investigators have shown that removal of C& fiom the storage atmosphere using potassium perrnan, anat te
(PurafilM) can be beneficial in reducing the incidence of scald (Knee and Hatifieid. 198 1:
Little et al., 1985; Liu, 1986). Vice versa, 'Bramley's Seedling' apple treated with CzH3 in a flowing strearn caused an accumulation of a-famesene and earlier onset of scald
(Knee and Hatfield, 1981). The accumulation of a-farnesene in fruit peel closely paralleled the rate of C2& production by apple (Meigh and Filmer. 1969; Watkins et al.,
1993). In contrast, post-storage ethephon treatment of 'Cortland' apples increased the cc- famesene and CT content, but did not affect superficial scald (Watkins et al., 1993).
Dover (1985) found that even extremely low levels of C2& (0.05 p~m~-')in storage of
'Bramley's Seedling' apples did not result in reliable scald control. C7H4 scrubbing had no effect on scald in fiuit held under ultra low oxygen (ULO; 0.7 % or 1.5 % Oz) (Fica,
1991; Lau, 1990). Tt is evident that factors other than C2H4 concentration (e-g. gas composition) in storage can determine the rate of development of superficial scald.
Overdl, the roIe of CzH4 in scald development is not yet clear and the accumulated literature is conflicting.
1.3.5 Controlled Atmosphere Storage
Extensive studies have been conducted to deheate the effects of controlled atrnosphere (CA) storage on superficial scald development. Standard controlled atrnosphere (SCA, 2.5 - 5% COz and 2.5 - 4.5% Oz) alone reduced or delayed but did not
11 prevented scaid incidence (Ben-Arie et al., 1993; Blanpied and Smock, 1962: Fica. 199 1 :
Patterson and Workman, 1962). However, storage of DPA-treated apples in SCA storage
provided additional control of scald. Recently, with the growing desire to find alternative
non-chernical control strategies to DPA, several workers investigated the feasibility of
further reduction in the level of aunosphenc Oz (ULO: ultra low oxygen) (Gallerani et al..
1992; Ghahrarnani and Scott, 1998b; Graell et al., 1997; Lau, 1985, 1990: Lidster et al.,
198 1; Truter et d,1994; Wang and Dilley, 1999), initial low O2 stress (ILOS) (Lidster et
al., 1985, 1987; Truter et al., 1994), ILOS followed by ULO (Little et al. 1983) or
elevated CO1 (Ben-Arie et al.. 1993) in storage. Though successfül control of scald is
reported with ULO and ILOS techniques, under conditions such as in hot and dry seasons
where apples tend to develop high incidence of scald, DPA treatment, at least at a
reduced dose. was necessary for effective scald control (Graell et al.. 1997). Conversely.
prolonged storage of apples under stress levels of low-Or andor high-COz atmospheres
caused deleterious effects such as accumulation of ethanol and acetaldehyde. due to the
induction of anaerobic respiration, along with the development of offiflavors. increased
incidence of flesh browning, failure to npen after removal to air. and development of
low-O2 andor high-CO2 injury (Ke et al.. 1991; Lidster et al.. 1985; Little and Peggie,
1987; Nichols and Patterson, 1987; Patterson and Nichols. 198 8).
1.3.6 Heat Treatments
Hardenburg (1967) found that hot water dips of 30 ro 60 s duration at 54 OC inhibited scald on 'Stayman' and 'Delicious' apples, but were less effective on 'Rome
Beauty'. Treatment temperature and holding time are estremely sensitive variables and inappropriate treatment can result in either no scald control or skin injury (Ingle and D'Souza, 1989). Lurie et ai. (1991) reported that pre-storage heat ueatment of 38 OC for 4 days applied to 'Granny Smith' apples inhibited the accumulation of a-famesene and CT
in apple cuticle during storage, and controlled scald to the same degree as DPA-dipped apples (Lurie et al., 1991). Similar results were reported when apples were kept at 46 or
42 OC for 12 or 24 h, respectively, before storage (Klein and Lurie, 1992). However. the prestorage heat treatment was effective in preventing scald only for the first 3 months of storage, as scald development began thereafter (Klein and Lurie, 1992; Lurie et al..
199 1). Heat-treated apples fiequently had a yellowed skin upon removal from storage
(Klein and Lurie, 1992). Oipping heated apples in 1.5 % Ca& before storage synergisticaily reduced the severity of superficial scald, in addition to improving the general keeping quality (Klein and Lurie, 1994). Lurie et al. (1995) observed that prestorage heat treatrnent lowered membrane microviscosity and Ieakage and increased phospholipid content in membranes of heated apples than in unheated apples. Watkins et al. (1995) observed an interruption of O OC storage with a single warming period at 10 or
20 OC (intermittent warming) reduced scald development in 'Granny Smith' apples. The greatest reduction of scald occurred when apples were warmed for 3 to 5 days at 20 OC after 1 to 4 weeks at O OC (Watkins et al., 1995). However, loss of fmit quality, mainly yellowing and softening, due to advanced ripening is the risk associated with warming.
1.3.7 Prediction as a Scald Control Strategy
Having little progress with investigating alternative non-chernical control methods for apple scald, a few attempts have been made to develop theoretical models and prediction strategies to recognize the occurrence of scald in apples. By using the prediction stratea. apples that show a propensity for developing scald could be rnarketed
imrnediately. ïhose that are not susceptible could be subjected to long-term CA storage-
Considering environmental variables such as total cumulative hours the apples are
exposed to preharvest temperatures below a threshold temperature (IO OC) during
maturation/ripening, and its correlation with susceptibility to scald, attempts have been
made to predict the occurrence of scald (Blanpied et al., 1991; Bramiage and Barden.
1989; Little and Taylor, 198 1; Memt et al., 1961). Barden and Bramlage (1 994) reported
high correlation (r2=0.76 and r2=0.69 for 'Cortland' and 'Delicious', respectively) between
the cumulative preharvest hours below 10 OCto which fruit were exposed and the incidence
of scald. In another study, superficial scald decreased significantly on -Granny Smith' apples exposed to <10 OC for 100 h, and it was negligible when duration was extended :O
120-160 h (Thomai et al., 1998). A survey done at severai North American locations
showed that scald incidence on 5tarkrimson Delicious' apples was controlled by delaying harvest until fruit had received 90 hours below 10 OC. or when starch index was
5.3 or higher (Blanpied et al., 1991). However, preharvest prediction of scald based on cumulative orchard temperature seems to Vary by Iocality. season. and several other factors and appears unreliable (Lougheed et al.. 1992).
Interestingly, antioxidant content (ODzoovalue of hexane extract of apple peel) and activity in apple peel at harvest increased as a fùnction of hours below 10 OC before harvest (Thomai et al-, 1998). A significant high correlation benveen scald incidence and peel antioxidant content at harvest was reported for 'Cortland' (Bramlage and Meir, 1989) and 'Granny Smith' (2=0.913, p=O.Oj) (Thomai et al., 1998) apples. It appears that estimation of antioxidants at harvest could be a usehl predictor of resistance/susceptibility to superficial scald. However, absorbance at 200 nrn is not a
reliable parameter for estimating antioxidants, since many other components including
monorneric to oligomeric carbohydrates absorb at this wavelength.
Recently, usefulness of chlorophyll fluorescence as a nondestructive-predictor of
superficiai scald has been evaluated. A rapid decline in chlorophyll fluorescence
rneasurements [photochernical efficiency (Fv/Fm; where Fv=variable fluorescence=Fm-
Fo, Fm=rnaximum fluorescence, Fo--minimal fluorescence) and half-time for rise in Fv
(TIR)]preceded superficial scald development in appIes during storage (Song et al., 1997;
DeEll et al.. 1996: Beaudry et al., 1995). Mir et al. (1998) observed that FvEm and Fm
values declined in scald susceptible Tortland' and 'DeIicious' apples during storage, but the values remaincd stable in scald-resistant 'Empire7 apples. However, DeEll (1 996) observed a decline in FdFm ratio in 'Delicious' apples at a rate similar to that in apples treated with DPA or the C2Ha antagonist 1-MCP. Changes in chlorophyll fluorescence also paralleled or increased during fruit senescence, and. therefore. chIorophyl1 fluorescence does not appear to be related directly to superficial scald susceptibility per se (DeEl1 et al., 1996; Mir et al., 1998). At present, there are no precise and accurate means of predicting the occurrence of superficial scald.
1.4 Biochemical Theories of Scald Development
The earliest research work on understanding the cause of apple scald was reported at the begiming of the twentieth century (see Brooks et al., 19 19). Since oiled wraps and facilitated ventilation reduced scald, initially it was thought that scald is caused by toxic volatile organic metabolites released by apples (Brook et al., 19 19). Huelin and Kemett
(1958) investigated the effects of nineteen volatile products on scald development, but found artificial induction occurred only with butyric and caproic acids and butyl and 15 hexyl acetates at excessive concentrations in oil-wrapped 'Granny Smith' apples. The oil-
wrap was more effective in controlling scald than good ventilation (Huelin and Kennett,
1958), suggesting that control of scald may be due to "inhibitors" released to apple by oil
(Huelin, 1964).
1.4.1 'Alpha-Farnesene Theory'
1.4.1.1 Isolation of a-Farnesene
Foundation to the 'cc-faniesene theory' of scald development was laid first by
Murray and CO-workers in 1964. IdentiQing famesene fiorn a petroleum extraction of
cuticle w~xand oil fiaction of 'Granny Smith' apples, Murray et al. (1964) suggested that
farnesene has a role in the development of superficial scald. Later, Huelin and Murray
(1966) identified the a isomer as the authentic farnesene in the natural coating of apple
and this observation was confinned by others (Murray, 1969; Anet, 1970; Naves, 1966).
Subsequently. in two separate studies conducted by Murray (1969) and Anet (1970). the predominent isoform of a-farnesene was found to be trans(E),trans(E)-cc-hesene. along with a small proportion of the cis(Z),trans(E) isomer. The ratio of tl-ansfmns- to cis,trarzs-isomer in 'Granny Smith' apples was 300:l (Anet, 1970). Higher levels of a- faniesene found in earlier-picked apples which are more liable to scald, and relatively more cc-farnesene found in scald-susceptible 'Granny Smith' than in scald-resistant
'Crofton' apples (Huelin and Murray, 1966) were some of the reasons to claim a relation between a-farnesene and scald.
Initially, a-farnesene was suggested as the primary scald-causing agent, but Anet and
Coggiola (1974) found no correlation between a-farnesene leveIs in the waxy skin
16 coating of apples and scdd development. Instead. it was suggested that a-famesene was
oxidized to a group of conjugated trienes (CT; compounds with a series of 3 conjugated double bonds) and this oxidation was inhibited by DPA, both in hexane solution and in the naturai coating of apples during storage. Such circurnstantial evidence led to the suggestion that scald may be caused by the oxidation products of cc-farnesene viz. CT
(Anet. 1969; Huelin and Murray, 1966; Huelin and Coggiola, 1970b). A mechanism for the auto-oxidation of a-farnesene was proposed, where a-famesene cm be attacked by a free radical in two ways: (i) by abstraction of a hydrogen or (ii) by addition of an osygen radical to one of the unsaturated carbon bonds (Anet, 1969). The loss of a hydrogen ion fiom cc-farnesene initiates a fiee radical chain reaction yielding inter molecular free radicals, terminating in the conjugated triene peroxides (Figure 1.1) (Anet. 1969). The rapid auto-osidation of a-farnesene in viiro in Oz at 1 OC resulted in the formation of mainly two groups of conjugated triene hydroperoxides, namely (E,E)-1.5-dimethyl- 1 -(4- methylpent-3-eny1)hepta-2,4,6-trienyl hydroperoxide, with some of the (E.2) isomer. and a mixture of the el yrhl-O and rhreo (E,E)-3-(1 -hydroperoxy- 1 -meth+ 1-(4-methylhesa-
1.3,s-trieny1)tetramethylene peroxide (Anet, 1969). Afier reduction of the hydroperoside group of the conjugated triene hydroperoxides by sodium borohydride maBH4), two corresponding conjugated triene alcohols with their isomers were isolated and characterized (Anet. 1969). Spicer et al. (1993) produced a library of over twenty potemtial a-famesene oxidation products under varying conditions consisting of allylic alcohols, epoxides, bisepoxides, and diols. Recently. Ro~vanet al. (1 995) and Whitaker et al. (1997) unequivocally demonstrated that conjugated triene alcohol, 2.6,10- trimethyldodeca-2,7E.9E11 1-tetraen-6-01 (CTOL), is the major component of CT present
17 Figure 1.1 A proposed mechanism for the auto-oxidation of a-famesene [derived from Anet. (1969)l. A. a-famesene, B. free radical, C. peroxy radical, D. conjugated triene hydroperoxide 1: (E, E)-l,5-dimethyl-1-(4-methylpent-3-enyl)hepta-2,4,6-t~lhydroperoxide, E. conjugated triene hydroperoxide II: erythro and threo (E,E)-4-(1 -hydroperoxy- 1-methyI- 1-(4-methyihesa- 1,3,5-trieny1)tetramethylene peroxide, F. conjugated triene aIcohoI 1: 2,6,1 O-trimethyldodeca- 3,7E,9E,ll-tetraen-6-01 (CTOL), G. conjugated triene alcohol II: erythro and threo (E,E)-4(2- hydroxy- 1-methyl- 1-(4-methylhexa- 1,3 .j-trienyl)tetrameù1yIene peroxide. 18 in the apple cuticle. accounting for 88-95 % of the total with its 7E,9Z isomer constituting most of the remainder.
Conjugated trienes possess 2 characteristic UV absorbance maximum at 269 nrn. with subsidiary peaks at 258 and 281 nrn. Since the reports of Anet (1969) and Huelin and Coggiola (1970a,b), most of the earIy studies exclusively relied on spectrophotometric measurement of CT to quanti@ it in cmde hexane extracts of stored apples. However, a discrepancy whether absorption maxima (lm,)at 258, 269 and 281 correspond to different compounds or common absorption characteristics of these species is extensively discussed in the literature. The hypotheticd species of CT were designated on the 3 absorbance maxima as CT258, CT269, and CT28 1. Du and Brarnlage (1993) suggested that the levels of CT28 1 did not correlate with scald development, since apples stored at 20 OC developed substantial concentrations of CT28 1, but did not deveIop scald.
It was show that the ratio of absorbance at 258 nrn to absorbance at 281 nm
(CT258:CTX 1) of apple hexane extracts kvas more closely related to scald development.
High ratios (a)were associated with scald resistance (little or no scald development). while low ratios (4) were associated with significant scald development (Du and
Brarnlage. 1993). Huelin and Coggiola (1 968) observed a peak at about 253 nm in hesane extracts of Xrofton' and late-harvested 'Granny Smith' apples. in whicli scald was slight or absent. Recently, Whitaker (1998) found that the compound(s) responsible for the absorbance peak at 258 nm is a member of the family of phenolic fatty acid esters. which can lirnit the oxidation of a-famesene and thus prevent scald. However. Rao et al. (1998) obsewed no relationship between CT258:CTZ8 1 ratio and scald susceptibility of 'White
Angel' x 'Rome Beauty' apple selections. Lately, to overcome the problems associated with quantiwng CT by interfering compounds of crude extracts, a high-pressure liquid
chromatography (HPLC) technique was introduced (Gallerani and Pratella, 199 1; Rowan
et al.. 1995: Whitaker et al., 1997). Gallerani and PrateIla (199 1) isolated thirteen
compounds that absorbed at 268 nm with one of them having the most correlation with the onset of scaid (Gallerani and Pratella, 1991). Rowan et al. (1995) estimated CT by normal and reversed phase HPLC, which were only 12-35 % of the CT concentration estirnated by UV spectroscopy, indicating that previous analyses of appIe skin washes have seriously overestimated the triene concentrations.
1.4.4.3 Volatile Oxidation Products
The predominant volatile compound identified fiom oxidation of cc-farnesene was
6-methyl-5-hepten-2-one (MHO, rnethyl heptenone) (Anet, 1972b; Filmer and Meish,
1969). Anet (1972b) hypothesized that methyl heptenone is denved from an alkoxy radical of a-famesene. This radicaI can arise by homolytic fission of a hydroperoxide or dialkylperoxide formed fiom the auto-oxidation of cc-farnesene (Anet, 197%). Since the arnount of the ketone(s) forrned was comparatively small, free radicals generated during the decomposition of peroxides and hydroperoxides derived by auto-osidation of cc- famesene were suggested to be more responsible for scald development (Anet. 1973b).
Spicer et al. (1993) also found that this distinctively smelling ketone, MHO, was the major product of the allylic oxidation of cc-famesene, resuIting from cleavage of the Cg'
C7bond, Recently. Wee and Beaudry (1997) found that MHO is inversely correlated with cc-farnesene accumulation during storage. MHO is also a highly Iipophilic cornpound as judged by its high partition coefficient into Iipophilic polymers or solvents (Song et al..
1997b). In support of this theory. Mir et al. (1999) observed that MHO released from isolated fruit peel fiom DPA-treated 'Coaland' -apples was 8000-fold lower than that
fkom untreated apples. Despite the notion that MHO could be involved in scald development (Wee and Beaudry, 1997; Mir et al., 1999): its direct impact in scald development or toxicity is not clear.
1.4.4.4 Evidence Supporting the a- Farnesene Theory
This long standing hypothesis of the mechanism of scald development claims that
CT attack ce11 membranes, causing membrane perturbation and cellular darnage, leading to superficiai scald (Anet, 1969; Anet and Coggiola, 1974; Du and Brarnlage, 1993;
Huelin and Coggiola, 1970qb). The theory has become dominant by the build-up of consistently reported circusmstantial and correlative data as descnbed below.
During cold-storage, a-farnesene levels increased on the surface of apples to a
maximum during the first two months and then declined (Huelin and CoggioIa,
1968. 197Ob; Meigh and Filmer, 1969), while oxidation products of a-famesene.
CT, progressively accumuIated with storage time with a strong correlation to
severity of scald (Chen et al., 1990; Gallerani and Pratella, 199 1; Ghahrarnani and
Scott, 1W8a; Huelin and Coggiola, 1WOb; Meir and Bramlage. 1988);
DPA which controls scald. suppressed the oxidation of a-fmesene both in pure
solution and in the natural coating of apple (Anet and Coggiola, 1973: Huelin and
Coggiola, 1970a,b,c; Johnson et al., 1980);
More a-fmesene was found in earlier-picked apples, which are more liable to
scald (Anet, 1 972: Whitaker et al., 1997);
Oiled wraps which reduce scald, absorbed more than half of the a-farnesene that
accumulated dunng storage (Meigh, 1967; Goldenberg et al., 1979);
2 I (v) Application of previously oxidized a-famesene withiin marked circles on -Granny
Smith' apples induced scdd syrnptoms (Huelin and Coggiola, 1970b);
(vi) Recently identified predominant CT species, CTOL, in the apple peel correlated
closely with scald-susceptibility and occurrence (Whitaker et al.. 1997) and also
possessed W absorption maximas at 258, 269. and 281 nrn. characteristic of CT
previously observed by Anet (1969) and Spicer et al. (1993). Bioassays done
using CTOL showed that it is capable of producing scald-like symptoms on stored
apples (Brimble et al., 1994); and
(vii) Ethanol when applied as a dip, an injection. or as vapor controls scald and reduces
CT levels (Chellew and Little, 1995; Wills et al.? 1977: Scott et al.. 1995b;
Ghahrmani and Scott, 1998a; Ghahramani et al., 1999).
1.5. Biosynthesis of Isoprenoids
Murray (1969) and Anet (1970) were the first to identifi the predominant form of farnesene present in apple skin as rrans.trans-a-famesene, accompanied by a small proportion of the cis.trans isomer. Based on the abundance of the triterpene (Cjo) acids
(mainly ursolic and oleanoic) in the natural coating of apples. Murray ( 1969) su,,oaested that a-farnesene might originate from a farnesyl or a nerolidyl intermediate in the biosynthesis of these Cjo acids. a-Farnesene could not be prepared by dehydration of famesol or nerolidol with acid catalysts (Murray. 1969; Naves. 1966). Recently, Salin et al. (1995) reported that stmcturally similar tram-p-farnesene in maritime pine (Pinzls pinaster Ait.) needles was biosynthesized from rrans.truns-farnesyl pyrophosphate (FPP) of the isoprenoid pathway. The isoprenoid pathway provides the largest class of secondary metabolic products (over 22,000) found in higher plants, and include fiagrance components, sterols? carotenoids, tocopherols, hormones (gibberellins, abscisic acid, brassinosteroids. certain cytokinins), phytyl side chah of chlorophyll, phylloquinone' plastoquinone. ubiquinone, cytochrome a and phytoalexins (Figure 12) (Bach, 1995; McGarvey and Croteau. 1995).
Isoprenoids are composed of C5 isoprene units and are classified as hemi-(Cj), mono-
(CIO)and sesquiterpenes (Cij) as well as di- (Czo), tri- (Cjo) and tetra-terpenes (&). and also polyterpenes. Initially, a single pathway was found for the formation of the Cs monomer. isopentenyl diphosphate (IPP) (for recent reviews see Gray. 1987; Chappell.
1995a,b, Bach. 1995; McGarvey and Croteau, 1995). In this classic isoprenoid pathway- the precursor of isoprenoids, mevalonate, is synthesized by the condensation of three acetyl-CoA units via aceto-acetyl-CoA and 3-hydroxy-3-methyl-glutaryl-CoA(HMG-
CoA). These first two reactions are catalyzed by a single enzyme. utilizing ~e"and quinone as cofactors (Bach et., 1991; Weber and Bach, 1994), but have not been studied extensively in plants. HMG-CoA is converted to Cg mevalonate in an irreversible reaction catalyzed by HMG-CoA reductase (HMGR; EC 1.1.1.34). This enzyme catalyzes two reduction steps, each requiring NADPH. Mevalonate is sequentially phosphorylated by two separate soluble kinases, mevalonate kinase and phospho mevalonate kinase, to form 5-pyrophosphornevalonate. Formation of the "active CS isoprene unit", isopentenyl diphosphate (IPP) is then catalyzed by pyrophosphomevalonate decarboxylase (McGarvey and Croteau, 1 995). Rohmer GIUCOS~-1-P Pathway 4 Gly ceraldehy de-3 -P 1-Deoxy-D-xylulose-5-P wthase @on) I
Don redm toisomerase Acetyl CoA
HMG CoA 2-C-MethyI-D-erythri toI-4-P MVA 4 HMGR Pathway Mevalonate (IvfVA) J- C, kopentenyl-pp (IPP) - Dimethylallyl-PP(DMAPP)
Monoterpenes 4 C 10 Gerany LPP (GPP)
Sesqui terpenes 4 Side chains of cytochromes, ubiquinone C,, Farnesyl-PP (FPP) Iosoprenoid polymers - Squal ene, Phytosterol , Tri terpenoids Abscisic acid 4 C,, Geranylgerany 1- PP (GGPP) Diterpenes / GA Side chains of chlorophylls, tocopherols, and phylloquinone Phytoene, Carotenoids
Fig. 1.3 Pathways of isoprenoid biosynthesis. IPP dong with dimethylallyl pyrophosphate (DMAPP), an interconvertible
isomer of LPP, represent the '-activater monomer building blocks for al1 isoprenoids
(Chappell. 1995a.b). The first isomerization enzyme. IPP isomerase. requires a divalent
metai ion and operates through an unusud carbocationic mechanism (Lutzow and Beyer.
1988; McGarvey and Croteau, 1995). Isoprene, the simplest of the isoprenoids- is
synthesized directly fiom DMAPP by the enzyme isoprene synthase, eliminating the
diphosphate unit. Condensation of DMAPP with IPP in a head-tail fashion by various
prenyltransferases generates prenyl diphosphates of different chain lengths. The Cm
compound geranyl pyrophosphate (GPP) is catalyzed by GPP synthase. Addition of a
second IPP unit to GPP generates the Ciscompound FPP by FPP synthase; and addition
of a third IPP generates geranylgeranyl pyrophosphate (GGPP) by GGPP synthase; and
so on (Chappell, 1995a:b; McGarvey and Croteau, 1995). The family of enzymes
responsible for the conversion of GPP, FPP, and GGPP to the monoterpene.
sesquiterpene, and diterpene classes. respectively, are referred to as monoterpene.
sesquiterpene, and diterpene synthases or cyclases, and represent reactions committing carbon fiom the central isoprenoid pathway to the end products (Chappell, 1995a,b).
In higher plants at least three distinct semiautonomous subceiluIar cornpartments exist that synthesize isoprenoids: cytopIasrn/ER (sesquiterpenes and triterpenes e.g sterol), plastids (rnonoterpenes and diterpenes e-g. chlorophyll, carotenoids, prenylquinones) and mitochondria (ubiquinones). It is generally accepted that at least the final biosynthetic steps are bond to these compartments. The biosynthesis of particular mono- and diterpenes is generally attributed to the plastidic cornpanment, even if other subsequent biosynthetic steps and accumulation of the final isoprenoid may take place in compartments (Lichtenthaler et al., 1997b). 25 1.5.1 HMGR as the Key Regdatory Enzyme -
HMGR. a highly conserved enzyme in eukaryotes, catalyzes the rate limiring step
of IPP biosynthesis in anirnals and most of the isoprenoid biosynthesis in plants
(Chappe11 et al., 1991). In higher plants HMGR is encoded by a multigene farnily
(Lichtenthaler et al.. Z997b) which are distinguishable fiom each other by the sequence
differences at the 3 ' untranslated regions of the cDNAs (McCaskilI and Croteau. 1997).
HMGR is encoded by at least two distinctive genes in Arabidopsis, three in Hevea. at
least three in tomato, twelve or more in potato, and even larger multiple gene farnilies in
maize and pea (McCaskill and Croteau, 1997; Stermer et al., 1994). In tornato. hmgl is
highly expressed during early stages of fi-uit development, when sterol biosynthesis is required for membrane biogenesis during cell division and expansion, whereas hmg2 expression is not detectable in young hit, but is activated during hit maturation and ripening (Rodriguez-Concepcion and Gruissem, 1999). The presence of multiple genes is consistent with the hypothesis that different isoforms of HMGR are involved in separate subcellular pathways to produce specific isoprenoid end-products (Stermer et al.. 1994:
Rodriguez-Concepcion and Gruissem, 1999). Plant HMGR activity responds in vivo to a variety of developmental and environmental signals, such as ce11 division, light, and pathogen infection (Stermer et al.. 1994). Plants regulate HMGR activity at the level of mRNA by differential induction of HMGR gene farnily members, and post- translationally by enzyme modification (Stermer et al., 1994). HMGR utilizes NADPH and requires a reduced thiol group (dithiothreitol) for optimal activity (Broker and
Russell. 1975). HMGR is also regulated by a protein kinase cascade in which phosphorylation inactivates the enzyme (McCaskill and Croteau, 1997). Calcium, calmodulin, and proteolytic degradation may also have a role in regulation of plant
HMGR (Stenner et al., 1994).
HMGR activity was very low in purified choloropIast preparations, and plastidic
isoprenoid formation was not inhïbited by mevinolin, a highly specific inhibitor of
HMGR (Bach and Lichtenthaler. 1983; Rodriguez-Concepcion and Gruissem. 1999).
which raised doubts about the participation of HMGR and rnevalonate in plastidic
isoprenoid formation (Lichtenthaler et al., 1997b). Although HMGR has been considered as a primary control point for isoprenoid biosynthesis (Chappell et al., 1991), it does not appear to be the rate-Iimiting step of ail isoprenoids (Vogeli and Chappell. 1988:
Lichtenthaler et al,. I997b).
1.5.2 GAPIPyruvate Pathway
In addition to the classical mevalonate pathway which leads to the biosynthesis of isoprenoid compounds, recent studies indicate the presence of a mevalonate-independent
(non-mevalonate or Rohmer) pathway which was found first in eubacteria and green alga by Rohmer and collaborators (Rohmer et al., 1993). In this novel pathway pyruvate and glyceraldehyde-3-phosphate (GAP) are precursors of IPP, but not acetyl-CoA and mevalonic acid (Lichtenthaler et al., 1997a; Lange and Croteau, 1999). The pathway is believed now to be responsibie for the formation of al1 plastid-derived isoprenoid compounds in plants. including carotenoids, plastoquinones. the prenyl side chains of chlorophyll. (Lichtenthaler et al., 1997b; Kreunvieser et al., 1999; Rodriguez-
Concepcion and Gruissern, 1999), as well as rnonoterpenes (Lange et al.. 1998) and diterpenes (Eisenreich et al.. 1996). The first step in the pathway involves a tranketolase- type condensation reaction of pyruvate and GAP to yield l-deosy-D-xylulose-5- phosphate (DOXP). Genes encoding the enzyme which catalyzes this reaction, DOXP synthase, have been cloned fiom peppermint (mentha x piperita) (Lange et al.. 1998) and
pepper (Bouvier et al,, 1998). The second step is considered to involve an intrarnoIecular
rearrangement and subsequent reduction of DOXP to yield 2-C-methyl-D-erythrïto1-4-
phosphate. This step is catalyzed by DOXP reductoisomerase that has been isolated fiom
Arabidopsis thaliana and peppermint (Lange and Croteau, 1999). Because al1 plastidic
isoprenoids studied so far are formed via this new pathway, it is assumed that isoprene
synthesized in the chloroplasts is also produced via this metabolic route (Kreuzwieser et
al., 1999). Existence of the mevdonate-independent pathway provides an aItemative
route for the biosynthesis of isoprenoids and, therefore, HMGR may not be the only rate
Lirniting enzyme for isoprenoid biosynthesis. Thus, in higher plant, there esist two distinct and biochernically different IPP biosynthetic pathways: (1) the classical cytoplasmic acetatehevalonate pathway and (2) the novel, alternative GAP/pyruvate pathway apparently lirnited to the plaçtidic cornpartment.
1.5.3 Sesquiterpene Cyclases and Synthases
Sesquiterpenes, isoprenoid Cl, compounds, comprise a Iarge and diverse group of natural products. Sesquiterpenes originate fiom the acyclic precursor trans.rr-ans-FPP. and it is possible in theory to derive approximately 200 different sesquiterpene structural families (Zook et al., 1992) which include mainly phytoalexins. toxins or antibiotics. FPP can be diverted to sesquiterpene biosynthesis by unique enzymes known as sesquiterpene cyclases or synthases (Cane, 198 1; Cane, 1985; Croteau and Cane, 1985). Sesquiterpene cyclases have been punfied and characterized from hngi (Cane and Pargellis, 1987;
Hohn and VanMiddlesworth, 1986; Hohn and Plattner, 1989) and several higher plants that include sage (Salvia officinaZis L.) (Croteau and Gundy, 1984; Dehal and Croteau, l988), patchouli (Pogostemon cablin Benth.) (Croteau et al., 1987). tobacco (Nicoticzna
tabacurn L.) (Vogeli et al-, 1990), calamondin (Citrofortzcnella mitis) (Belingheri et al.,
1992), potato (Solarium tzrberosum L.) (Zook et al., l992), maritime pine (Pinus pinaster
Ait.) (Salin et al., 1993, and Cotton (Gossypium hirsutum L.) (Davis et al.. 1996). The enzymatic cyclization of FPP is initiated by ionization of the diphosphate ester to generate an allylic carbocation (McCaskill and Croteau, 1997). Al1 of the sesquiterpene cyclases that have been characterized have a cofactor requirement for divalent metal ions
(Croteau and Gundy. 1984; Croteau and Cane, 1985). M~'+is preferred to ~n'' in most of the identified sesquiterpene cyclases Pelingheri et al., 1992; Dehal and Croteau, 1988:
Salin et al.. 1995: Vogeli et al.. 1990). The role of the cation is presumed to be the neutralization of the negative charge of the pyrophosphate moiety, thus assisting in the ionization of the substrate to produce the allylic cation (Chayet et al., 1984). trans-p-
Farnesene synthase catalyzes the biosynthesis of the sesquiterpene olefin rrans-p- farnesene from FPP and has been purified to apparent homogeneity in maritime pine needles (Dehal and Croteau, 1988; Salin et al., 1995). This soluble enzyme is possibly a homodimer with 45-kDa subunits and its properties are similar to those of the sesquiterpene cyclases. Bernard-Dagan et al. (1 982) observed that the microsomal pellet of maritime pine needle extracts fomed mainly cyclic sesquiterpenes while supernatants synthesized more acyclic rrans-p-farnesene. Evidence also suggests that the biosynthesis of the cyclic Ci olefins is associated with endoplasmic reticulum membranes (Belingheri et al., 1988; Gleizes et al., 1980). 1.6 Oxidative Stress and Membrane Degradation
Two types of evidence suggest that superficial scald is induced or stimulated by an oxidative process. First, the scald-preventing compounds such as DPA are known to act as antioxidants in other systems. In addition, apple peel antioxidant activity estimated at harvest negatively correlated more strongly with scdd development than either cc- farnesene or CT level. Second, it is accepted widely that a reduction of oxygen concentration in the storage atmosphere, with or without an increase in carbon dioxide concentration, reduces the incidence of scald.
1.6.1 Lipid Peroxidation
Many storage-related disorders appear to have an etiology in membrane dysfunction (Poovaiah, 1986). Also, the first site of damage during the development of chilling injury is thought to be the membranes (Lyons et al., 1979). Lipid peroxidation occurs during senescence and low-temperature strcss (Du and BramIage, 1995). and is known to generate a variety of highly reactive oxygen species (ROS) known to damage membrane lipids (Turrens, 1997), including singlet oxygen ('OZ), the alkoxy radical
(RO'), and the peroxy radical (ROO'). On the other hand, a number of ROS' especially hydroxyl radical (OH') and superoxide radical (O;-) are able to initiate lipid perosidation reactions (Shewfelt and Purvis, 1995). Formation of lipid hydroperoxides (LOOK) also can be induced enzymatically by lipoxygenase (LOX; EC 1.13.1 1.12) (Lynch and
Thompson, 1984). However, provision of fiee fatty acid substrate, specially linoleic acid and linolenic acid. through the action of lipolytic acyl hydrolase is essential, since LOX cmnot utilize esterified fatty acids as substrate. Also. investigations have been conducted-to determine if lipid peroxidation has any relation to scald development (Du and Brarnlage, 1995; Feys et d., 1980). Activiw of
LOX in the peeI of 'Schone van Boskoop' appIe increases rapidly during storage. suggesting that LOX-mediated oxidation/hydroperoxidation of polyunsaturated fatty acids may be involved in the mechanism cif scald induction (Feys et al., 1980).
Conversely, Du and Bramlage (1995) found no marked differences in lipid peroxidation products (thisbarbituric acid-reactive substances), total peroxides or peroxidase activity in
'Cortland' apple tissue with different levels of scald syrnptoms.
1.6.2 Antioxidants and Antioxidant Enzymes
Antiosidants present in the apple skin were implicated as a factor controlling free radicds generated during the decomposition of peroxides and hydroperoxides by a- faniesene auto-oxidation (HueIin and Coggiola, 1970b; Anet, 1972; Anet and Coggiola.
1974; Anet, 1974). Among a wide range of antioxidants (amine, phenolic and sulphur- containing compounds) found to inhibit the auto-oxidation of a-farnesene in vitro, only amine-type antioxidants inhibited the auto-oxidation of a-farnesene in vivo (Anet and
Coggiola, 1974). Anet (1 974) separated eleven antioxidants fiom the cuticule of appIe. a, y, and 6-tocopherol being the only ones identified. and fûrther postulated that scald did not occur during storage if the hydrophobic antioxidant content remained adequate to prevent or limit a-fmesene auto-oxidation. Subsequently, circumstantiaI evidence suggested an inverse relationship between antioxidant potential of apple and development of scald (Barden and BrarnIage, 1994b,c; Gallerani et al., 1990; Meir and Bramlage.
1988). Meir and Bramlage (1 988) found that a-farnesene content was not affected by harvest date, but CT and scald incidence was decreased by later harvest. It has been
3 1 reported that antioxidants such as a-tocopherol (Meir and Brarnlage, 1988), anthocyanins
(Ju et al.. 1996), ascorbic acid and flavanols (Albrigo and Childers. 2970) increased in
apples with Iate harvesting. Gallerani et al. (1 990) found that total antioxidant capacity of
'Granny Smith' apple was significantly lower in scald-developing tissues compared with
scald-fkee tissue. For instance, a-tocopherol Ievel in scald-developing tissues was 3-fold
less (Gallerani et al.. 1990).
Furthemore, suppression of scald by holding apples in CA or ultra low 0, storage
(DeElI, 1996; Ghahramani and Scott, 1998; Lau, 1985. 1990; Lidster et al., 198 1: Little
and Peggie. 1987; Patterson and Workman. 1962) suggested that oxygen and ROS were
involved in CT formation and scald development. In general, effective enzymatic
antioxidant systems can protect tissues fiom deleterious and degradative reactions by
removing ROS (Rao et al.. 1996). Superoxide dismutase (SOD: superoxide:superoxide
oxioreductase: EC 1.15.1.1 ) is a ubiquitous enzyme reported to have enhanced activity in
plants that are resistant to low-temperature stresses (HalliweIl. 1985). However, Du and
Bramlage (1994b, 1995) found no relation of total SOD activity and the relative activities
of its different metalloenzymes to scald susceptible (-Cortland' and 'Delicious') and
resistant ('Empire') cultivars. As well. storase temperature of O OC vs 20 OC and DPA treatment scarcely affected SOD activity (Du and Bramlage. 1994b).
1.6.3 Alternative Oxidase and Stress
The alternative pathway of mitochondrial respiration diverges from the cytochrome pathway in the inner mitochondrial membrane at the ubiquinone pool and tramfers electrons directly to a second quinol osidase. the alternative osidase (AOX).
The AOX apparently reduces molecular oxygen to water. Unlike cytochrorne osidase, its electron transport from ubiquinone to water does not contribute to a transmembrane
potential (nonphosphorylating) and thus wastes two of the three energy coupling sites
(ATP synthesis) that are part of the cytochrorne pathway. However, the phosphorylating
potential fiom NADH dehydrogenase (Complex I) is retained, thus allowing some energy
production (Vaderberghe and McIntosh, 1997). AOX exists in the inner mitochondrial
membrane as a homodimer that is encoded by a nuclear gene(s) Aoxl. AI1 alternative
oxidase genes sequenced to date encode a highly similar protein. The partitioning of
respiratory electrons to the alternative pathway or AOX activity is regulated through
several factors: (i) regulation of AOX protein level through changes in gene expression,
(ii) level of reduced ubiquinol, the substrate, (iii) active reduced form of intermolecular disulfide bond of AOX protein, (iv) presence of activators such as a-keto acids. particularly pyruvate (Vaderberghe and McIntosh, 1997).
Apart from a physiological role in heat production of thermogenic floral or,oans to volatilize compounds during pollination (Meeuse, 1975). several other functions of the alternative pathway in plants have been suggested. Any metabolic condition that leads to accumulation of either reduced ubiquinone, mitochondrial NADPH. or pyruvate has the potential to increase electron flow to the alternative pathway (Vanlerberghe et al.. 1995.
1997). Such conditions may arise when there is an imbalance between upstream respiratory carbon metabolism and downstrearn electron transport due to changes in supply of, or demand for, carbon, reducing power, and ATP. When the cytochrome pathway is saturated or limited, the alternative pathway serves a regulatory function as an
"overflow" for excess electrons (Lambers, 1982). The most general function of alternative oxidase may be to balance carbon metabolism and electron transport, allowing synthesis of carbon skeletons by tricarboxylic acid cycle and eliminating excess electron flow to molecular oxygen that may result in the formation of ROS.
Recent studies support a protective role of AOX against oxidative stress in preventing the generation of ROS.by avoiding overreduction of the electron transport chah (Purvis and Shewfelt. 1993). When highly thermolabile cytochrorne oxidase is iniibited or ATP turnover is limited, electrons can leak from the mobiIe electron carrier
(ubiquinone) ro molecular oxygen and prodüce ROS, i.e. superoxide. ROS can be produced at two sites in the mitocliondria, first at the flavoprotein region of the interna1
NADH dehydrogenase, and second at ubiquinone (Rich and Boimer, 1978). The alternative pathway is induced in plant tissues exposed to low ternperatures. Cold- resistant cultivars and tissues generally develop a greater potential for electron flux through the alternat ive pathway than do cold-sensitive cultivars and tissues
(Vanlerberghe and McIntosh. 1992). Chilling stress Ied to lower cytochrorne osidase activity and protein Ievels in corn seedlings transferred to 14°C (Prasad et al., 1994).
Conditions that induce AOX expression, including chilling (Vanlerberghe and McIntosh.
1992): pathogen aitack (Lemon, et al. 1997), aginç (Hiser and McIntosh. 1990) and inhibition of cytochrome pathway. also cause an increase in cellular ROS formation. suggesting AOX may serve a more general function in al1 plant species by Iimiting rnitochondrial ROS formation. CoIlectively, it is apparent from the Iiterature that the alternative pathway may be able to maintair. a Iiigher percentage of its relative activity at low temperatures than the cytochrome pathway. As well. AOX rsliibi~sa rnechanism by which plant cells can protect themselves froin oxidative stress. 1.7 Research Hypothesis and Objectives
At present, superficial scald development in apples is controlled cornmercially by
postharvest treatrnent with the synthetic antioxidant diphenylamine (DPA). Because of
increasing concern over the potential undesirable effects of DPA residues or metabolites
to the consumer, development of alternative control measures are being sought. Thus.
understanding the precise physiological and biochemical mechanism of scald
development is critical.
From the accumulated literature, it is evident that the development of superficial
scald in apples can be viewed as a two-stage event where the induction events are
separate fiom symptom development. Huelin and Murray (1966) postulated that the
mechanism of superficial scald induction is centered around the accumulation and
degradation of a specific secondary metabolite, a-farnesene (an acyclic sesquiterpene). in the skin of apples during cold-storage. Since then, evidence showing that a-famesene
catabolites are the primary cause of scald development has accurnulated, and this hypothesis has dominated scald research. The major support for this "a-famesene hypothesis" is based on the tight correlation between the severity of scald and the arnount of a-farnesene oxidation products present in apple skin. It has been reported consistently that higher levels of putative scald-causing a-famesene catabolites (CT) existed in severely scald-developed tissue compared with scald-free tissue. Moreover. treatments with agents such as DPA and ethanol that control or suppress scald development also reduced the oxidation of a-farnesene to CT. However, it is clear that most of the evidence supporting this hypothesis is based on circumstantial evidence or correIative data.
Experiments that had attempted to show direct effect of a-famesene or CT on apple fruit
35 physiology produced inconclusive results, potentially due to the problems related to the hydrophobie nature of u-farnesene and instability of the intermediates, conjugated triene hydroperoxides, which are presumed to be the highly toxic CT species.
In this research, 1 propose to examine the validity of the a-farnesene hypothesis of scald development using biochemical and molecular biological means to study the regulation of a-farnesene biosynthesis in apple fitand its physioIogica1 role in scaId development. If a-famesene is directly involved in scald developmenh approaches to limit a-famesene biosynthesis could be developed to prevent scald formation in apple.
Overall, this thesis tests the hypothesis that a-famesene metabolism is directly involved in superficial scald development.
From the Iiterature, it is evident that the biosynthesis of a-farnesene and its regulation have not been investigated in relation to scald development in apple. Thus, the specific objectives of this research are:
1. To elücidate and characterize the biosynthetic pathway of a-farnesene in
apple hitin relation to superficial scald developrnent;
2. To understand how low temperature storage regulates the two key enzymes (a
-famesene synthase and HMGR) of a-farnesene biosynthesis;
3. To study the influence of Czl& on activity of cc-fmesene synthase and
HMGR, accumulation of a-famesene, and scald development;
4. To study the specificity of H-MGR genes in relation to a-famesene
biosynthesis.
5. Clone the gene(s) encoding the key regdatory enzyme(s) of a-farnesene
biosynthesis. 36 1.8 Outline of the thesis
a-Famesene belongs to the sesquiterpenes or Cij isoprenoid compounds.
Generally, plant sesquiterpenes originate kmthe acyclic precursor trans.trans-FPP.
Hence, initial experiments were conducted feeduig 14 C or 'H-labelled FPP to apple bit tissues to investigate the hunediate precursor of a-famesene. Radio-labelled early precursors of the isoprenoid pathway also were studied for incorporation of radioactivity into a-famesene. The capacity of a-famesene biosynthesis in different fruit tissues. scald-fiee and scald-developing skin tissues, was examined. The results of the above experiments are presented in chapter II.
From the above study, tram-irons-a-faniesene synthase was recognized as the terminal enzyme of a-faniesene biosynthesis that catalyzes the conversion of FPP to a- famesene. The enzyme is an ideal target for regulation of a-farnesene synthesis since it will not interfere with the metabolic flow of other isoprenoids. Since the gene(s) encoding a-farnesene synthase has not been cloned from any living organisms. one of the cloning strategies is the preparation of cDNA Iibraries with mRNA isolated from tissue e~chedfor u-famesene synthase mRNA, then screening the library with antibodies prepared against purified a-famesene synthase protein, or with ~Iigonucleotideprobes designed from the arnino acid sequence of the purified cyclase protein. With this ultimate goal, purification of a-fmesene synthase to homogenity \vas attempted using skin tissues of apple fruit. The enzyme was characterized using partially purified protein fractions. Changes in the enzyme activity in apple hitskin along with accumulation of cc
-famesene and its putative scald-causing catabolite, conjugated triene alcohol, were snidied in relation ro (i) low temperature storage and (ii) inherent susceptibility of eleven
selected cultivars to develop superficial scald. These data are discussed in chapter III.
From the above studies, it was apparent that cc-farnesene biosynthesis in apple
was initiated exclusively afier harvest and induced during the early phase of low
temperature storage. This finding raised several questions in regard to the regulation of a
-famesene biosynthesis. Mainly, what is the pnmary signal(s) inducing a-famesene
accumulation and what step in the a-famesene pathway perceives the signal(s). Most of
the findings fiorn Chapter III suggested that following harvest hit exposure to low
temperature during storage was the cause of this metabolic shifi in the isoprenoid
pathway. However, it is known also that endogenous C2& levels in apple fhit increase
during storage in parallel to the levels of a-famesene. Thus, CzHj may have a role in a-
famesene biosynthesis and scald development. In study l of chapter IV, the hypothesis that a-famesene synthase is induced by Cz& is tested. In study 2 of the sarne chapter. the role of C2& in a-famesene rnetabolism and scald development is discussed in detail.
Lack of a relationship between a-famesene levels and a-famesene synthase activity in response to C2i& inhibitors indicated that regulation of u-faniesene levels by
C2H4 in apple fruit is not at this terminal enzyme, but could occur at an upstream step of the isoprenoid pathway. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase
(HMGR) is considered to be a primary control point for most isoprenoid biosynthesis in plants. In Chapter V, changes in total HMGR activity were studied (i) during storage and
(ii) in apples treated with 1-MCP, an C2H4 action inhibitor. Also tested was the hypothesis that HMGR is the rate lirniting step of a-famesene biosynthesis in apples. The impact of inhibition of HMGR by ~ovastatin~~,a cornpetitive inhibitor of HMGR on a-
38 famesene accumulation in the skin of apple was studied. Experiments were also
performed to distinguish between two pathways which could Iead to formation of
isopentenyl pyrophosphate (IPP), namely (1) the classical mevdonate pathway, and (2)
the novel glyceraldehyde-3-phosphate (GAP)/pyruvate pathway independent of HMGR
action. HMGR is encoded by a multipIe gene family, and different isoforms are believed
to be involved in separate subcellular pathways to produce specific isoprenoid end-
products (Stermer et al., 1994; Rodnguez-Concepcion and Gruissem, 1999). A PCR-
facilitated method was used to clone and sequence the full-length of a HMGR gene
(hrngl) from skin of apple fruit. A £kagrnent of another HMGR gene (hmg2) was identified relying on a 3' untranslated region which is shown to be highly divergent arnong the members of the HMGR gene family (McCaskill and Croteau, 1997).
Expression of these two genes was studied in relation to accumulation of a-farnesene in the skin of apple. These results are described in Chapter V. CWTER11
Biosynthesis of a-Famesene and its Relation to Superficial ScaId Development
in 'Delicious' Apples
Published in J. Amer. Soc. Hort. Sci. 123:882-886 (1998)
ABSTRACT
a-Farnesene is an acyclic sesquiterpene hydrocarbon which is a constituent of the volatile components and the surface wax of apples (Malrrs xdornestica Borkh).
Although oxidation products of a-farnesene have been implicated in the development of superficial scald in apples, the relation between a-farnesene biosynthesis and scald development is not well understood. Iiz vivo labeling studies using isolated tissue segments shorved that a-farnesene is derived from fratzs,frans-
[1,2-"C or b3~]-farnesylpyrophosphate (FPP) mostly in the skin rather than cortex tissue. Among other Iabeled products, farnesol was over a hundred-fold higher cornpared to a-farneseae. However, HPLC analysis of hexane-ertractable components from apple skin revealed farnesol is not a predominant natural constituent of apple slcin tissue. In addition, trnns,trans-[l-'HI-farnesol was not converted to a-farnesene by apple skin tissue. Our results indicate that biosynthesis of a-farnesene in apple fruit tissue.occurs through the isoprenoid pathway, and the conversion of FPP to a-farnesene is catalyzed by a single sesquiterpene synthase enzyme, trans,trans-a-farnesene synthase, rather than via farnesol as an intermediate. A cornparison of a-famesene biosynthesis between scald-developing and scald-free regions of the same apple showed that incorporation of radiolabel into a-farnesene from ir~ns,trans-[l-~~]-~~~was nearly 3-fold Iower in scald- developing skin tissue than in scald-free skin tissue.
INTRODUCTION
Superficial scald is a serious storage disorder of several apple cultivars, characterized by brown or bronze discoloration of the skin (Bain and Mercer, 1963;
Meigh, 1969). a-Famesene, an acyclic sesquiterpene hydrocarbon (CljH24; [3 E76EJ-
3,7,11 -trimethyl- 1,; ,6, Wodecatetraene) which is a constituent of the surface wax of apples, has long been associated with the occurrence of this physiologicaI disorder
(Huelin and Coggiola, 1970a,b). Oxidation products of a-famesene. including hydroperoxides or conjugated trienes, are presumed to be the potentiaI causative factors for the development of scald (Anet, 1972). Conjugated trienes progressively accumulate on the surface of apples during storage. The concentration of trienes, as measured by UV spectroscopy, correlates more closely with the occurrence and severity of superficial scald than does the concentration of a-faniesene (Chen et al., 1990; Gallerani and Pratella,
1991; Hueiin and Coggiola, 1970b; Meir and BramIage, 1988). In the naturaI coating of apple, the a isomer of faniesene was isolated and identified as the predominant form
(Anet, 1970; Hueiin and Murray, 1966; Murray et al ., 1 964; Naves, 1 966). Subsequently,
Murray (1969) confirmed that trans,trans-a-farnesene fiom apples is predominant over the cÏs,trans isomer in a 300:l ratio. Based on the analyses of the triterpene (Cjo) acids
(mainly ursolic and oleanoic) in the natural coating of apples, Murray (1969) suggested
4 1 that a-farnesene might originate fiom a farnesyl or a nerolidyl intermediate. Recently.
Salin et al. (1995) reported that pans-B-farnesene in maritime pine (Phuspinaster Ait.) needles was biosynthesized from ~ms,trans-faniesyi pyrophosphate (FPP). As well. there are many reports on the biosyntthesis of sesquiterpene hydrocarbons fiom n-ans,tram-FPP (see Croteau and Cane, 1985).
Presently, superficiai scald is controlled commercially by posthmest treatment of appfes with the antioxidant diphenylamine (DPA) (Anet and Coggiola, 1974; Huelin and
Coggiola, 1970-b). Because of increasing concem over potential undesirable effects of
DPA residues or metabolites to the consumer, development of alternative non-chemical control rneasures are increasingly being sought (Ingle and D'Souza, 1989; Lurie et al.,
1991). A better understanding of the biochemicai basis of scald development will help in the design of non-chemical control strategies such as a preharvest prediction method.
Hence, the present study \vas undertaken to understand the biochemical rnechanisrn of u- farnesene biosynthesis in 'Delicious' apples which develop severe scald during storage.
MATERIALS AND METHODS
Plant material
'Delicious' apples were harvested at optimum maturity during the first week of
October 1996. frorn a commercial grower in Meaford, Ontario and were transported to
Guelph. Apples were cooled to O "C within 8 h of harvest and kept at O OC in air or in controlled atmosphere (CA) of 3 kPa O2 and 2.5 kPa CO2. Chernical reagents
~ram,hanr-[1-3~-farnesyi pyrophospate (7.4 x 10 I4 E3cpmol-'), rrarzs, tms-l1.2-
14Cl-famesyl pyrophospate (2.0 x 1 O' mol-'), tram,trarzs-[I -3~-farne~~l (2.1 x 1
~cprnol-'),[~-~~-rnevalonate (2.2 x 10" mol‘'), and [2-'"1-acetic acid sodium salt
(2.0 x 10" ~~.rnol-l)were purchased from Amencan Radiolabeled Chernicals (ARC)
Inc., St. Louis, Mo. A standard mixture of a, P isomers of farnesene was obtahed from
TCI, Tokyo; standards of farnesol and fmesyl acetate were fiom Aldrich Chem. Co..
Milwaukee, Wis.; and NADP and ATP were from Boehringer Mannheim Canada Inc..
Montreal, Que. Al1 other reagents were from Sigma-Aldrich Canada Ltd., Oakville, Ont.
Fruit fksue prepara fiorz
Apples stored in CA for five months (if not othenvise mentioned) were used in these experiments. Fruit were brought to room temperature the day before being used in the experiment. Fruit showing initial signs of scald on only one side were selected for these experiments such that they could be cut (axial plane) into scalding and scald-free halves. Under these experimental conditions. we observed for this specific cultivar, when left at room temperature, develops scald only on one side. This provided the added advantage of eliminating experimental errors due to physiological/biochemical differences arnong fmit. Cylindncd pieces of tissue were removed from the equatorial regions of scalding or scald-free halves using a cork borer (1 cm diameter), and divided into skin (2 mm thick; 1041T12 mg fiesh mass), and outer (closer to the skin) and inner
(closer to the core) cortex segments (5 mm thick; 273+-17mg fresh mass). Cefi-free extract preparation
Outer regions of appIe fruit tissue (0.5-0.75 cm) were removed and cut into 0.5 cm3 pieces and hornogenized using a polytron@ homogenizer (Brinkmann Instments.
Westbury, N.Y., mode1 PT 1O/3S) for 1 min in sodium phosphate buffer (0.1 mol-L-', pH
6.5) containing 5 mol.^" sucrose, 50 rnmol-L-' ascorbic acid' 2 rnmol-~-'EDTA, 1 mmol.~" DTT, 1 mmoL~-'PMSF and 50 g.~-lpolyvinylpyrrolidone. The homogenate was filtered throiigh four layers of cheesecloth, the filtrate was centrifiged at 730 x g for
10 min to remove starch and debris, and the supernatant was used as the cell-free estract.
Protein content of the extract was determined by the method of Bradford ( 1976).
In vivo incorpornfion of radiolabelledprecrtrsors
Fruit tissue segments, prepared as described above. were incubated in 4.5 mL amber glass-vials containing 950 pL of 0.1 rnol.~-' phosphate buffer (pH 7.2). The vials were seded tightly using screw caps with teflon-silicon septa. Air trapped in the vial was removed using a needle and tubing comected to a vacuum pump and flushed with pure
NZ (99.995 kPa). The reaction was started by adding 50 pL of radiolabelled substrate
(equivalent to approx. 25 prnol substrate; 1.85 x 10' Bq of radiolabelled precursor in 1 rnL final volume of 0.1 rno1.L-' phosphate buffer and 1 ~L~L-'Triton X-100) to the reaction vial containing tissue segments. The tissue segments were subjected to vacuum infiltration to facilitate the uptalce of the radiolabelled substrate and filled again with pure
Nz to minimize the possible oxidative breakdown of the product, cc-famesene. After 1 h of incubation with gentle shaking, the reaction was terminated by adding 0.1 mL of 1 rnoLL-' KOH and 1 mL hexane followed by vigorous mixing. A randomized complete-
44 block design with three fniit (blocks) was used for dl experiments. For time-course
cornparison of a-famesene synthesis in scalding vs scald-free tissue, each block consisted
of scdding and scald-fiee tissues each with four reaction times. Two experirnents were
conducted independently and data were pooled for statistical analysis (SAS release 6.12,
SAS Institute, Cary, N.C.)-
Separarion and analysis of radiolabelled produc fs
One-half milliliter of hexane extract was transferred to a test tube which contained a mixture of famesene, farnesol and fmesyI acetate standards, and evaporated to near dryness under N2. The remaining residue was redissoIved in 50 pL of hexane and 25 pL aliquots were spotted on a thin layer chrornatography plate (silica gel G, 60 s 1O-'' m, 20 x 20 cm, Whatrnan Inc., Clifton, N.J.). The plates were completely developed with hexane:ethyl ether (4: 1, v/v) and dned under N2. a-Famesene (RJ = 0.639), farnesyl acetate (RI = 0.505) and farnesol (RI= 0.203) were visualized in the presence of iodine vapor. The regions of the plate corresponding to authentic standards were scraped, mised with 5 mL of scintillation cocktail (~colume~,ICN, Costa Mesa, Ca). and their radioactivity determined by liquid scintillation counting (Becban LS6800, Mississauga.
Ont.). Controls, of no tissue segment and boiled tissue segment, were used to exclude the possible formation of compounds by nonenzymaric reaction(s). Nonenzymatic formation of a-farnesene and farnesyl esters was negligible. The 8% of total radioactivity that accumulated in the farnesol fraction was due to nonenzymatic conversion of the substrate. Anaiyskof a-farnesenecontenturingHPLC -
Two segments of fruit skin tissue, prepared as described above, were immersed in
2 mL of hexane. and kept for 24 h in a closed 4.5 mL glass-vial. Hexane-soluble a-
farnesene content was analyzed by HPLC by measuring absorbance at 233 nrn after
separation on a ~ova-Pak@Ci* column (3 -9 x 150 mm, 60 x 10-Io m, Waters Co. Milford.
Mass.). Acetonitrile (100 %) was used as the mobile phase, at a flow rate of 0.75 mL.minL
1 - Faniesol was eluted at 2.3 min, and farnesene at 2.8 min. Two experùnents were
conducted independently and data were pooled for statistical analysis (SAS release 6.12,
SAS Institute, Cary, N.C.).
RESULTS AM) DISCUSSION
Tissue locnlization ami a-farnesene biosynth esis
Experiments using isolated tissue segments showed that incorporation of radiolabel frorn tram.tram-[l -3~]-famesyl pyrophosphate (FPP) into a-famesene was
limited to the skin tissue when compared to the conex tissues (Table 2.1). Similar results were also obtained while using trans,frans-[l,2-'?]-~~~as substrate (data not presented). This provides direct evidence for the conversion of FPP into a-farnesene by apple skin tissue. Scald symptoms are confined to the hypodermal and epidermal ce11 layers of the miit (Fidler, 1950). Histological investigations by Bain (1956) and Bain and
Mercer (1963) also revealed that the browning of cells was limited to the epidermal layer and the first five or six layers of the hypodermis. Hence, it can be speculated that al1 biochemical changes which cause scald deveiopment, including the biosynthesis of Table 2.1. Incorporation of mans.tram-[ l -3HJ-faniesy~pyrophosphate into a-famesene
and farnesol by different tissue segments of 'DeliciousS apple fniit. Data are the means of
niplicate analyses.
Storage time in air or CA' at O OC
Tissue 3 weeks in air 30 weeks in CA 28 weeks in CA segment FNEy FOL" FNE FOL FNE FOL
Radioacriviîy on afiesh mass basis @pg-'eh-])
Skin 26 4810 73 3075 40 3046
Outer cortex O 4904 O 1943 O 1643
Imer cortex O 5280 O 2092 O 1719
'CA = Controlled Atrnosphere storage of 3.0 kPa O2 and 2.5 kPa CO2 at O OC
'FNE = a-famesene. Standard Error of Mean for FNE = 14
"FOL = farnesol, Standard Error of Mean for FOL= 130 a-famesene, could be localized in the few outer ce11 Iayers of apple fruit. At harvest. cc-
famesene formation fiom [1-'HI-FPP was very low (6 ~~.~-'*h-').but increased after 3
and 20 weeks in air and CA, respectively, and then declined afier 28 weeks in CA storage
(Table 2.1).
Arnong the other labeled products detected in the hexane extract. farnesol was
predominant but there was also a trace amount of farnesyl acetate. The formation of
farnesol from [ 1-3~j-~~~ was over one-hundred-fold higher than a-famesene formation
in Mt three weeks after harvest. Famesol formation in the skin, and in the outer and
imer cortex tissue segments was similar (Table 2.1). Formation of farnesol from FPP by
the action of phosphatases has been reported in other plant tissue systems (Croteau and
Karp, 1979; DehaI and Croteau, 1988; Vogeli and Chappel, 1988) and could account for
the large amounts of farnesol relative to a-famesene formed frorn FPP in apple tissue.
Thus, the possibility exists that faniesol may be an intermediate in the formation of u-
farnesene from FPP.
Trans,rrans-farnesol is the only possible isomer that could be hedfrom
trans,trans-FPP. and it is the only stereoisomer present in many essential oils (Naves.
1966). However. rrans. tram-[1-'HI-famesol was not convened to a-fmesene either in
isoIated tissue segments or in a cell-free system, although loss of radioactivity from
labeled farnesol was observed both in vivo and in vitro. In a cell-free system, labeled farnesol decreased with time in the presence of increasing M~'' concentration without formation of a-farnesene (data not presented), suggesting that farnesol may be metabolized into other product(s). The capability of phosphorylating farnesol in the presence of M~~'and cytidine triphosphate (CTP) to give the corresponding mono- (FP)
48 and diphosphate (FPP) through the action of farnesol (pyro)phosphokinase has been
reported in the 100.000 x g pellet fraction of Botryococcus braztnii Kützing B race (houe
et &, 1995). However, a-faniesene cannot be formed readily by dehydration of the
sesquiterpene alcohols. faniesol or-neroiidol (Murray, 1969). As well, dehydration of
farnesol cm yield on1y the tram-p-farnesene isomer (Brieger et al., 1969;Naves, 1966).
Labeled precursors of the isoprenoid pathway, [2-14~]-aceticacid and [5-'~]-
mevdonate, were incorporated into a-farnesene in trace arnounts in skin tissue segments
but not in cortex tissues. Ln both these experiments, farnesol formation was not higher
than that of a-fmesene (data not presented). However, incubation of [2-"CI-acetic acid
with the ceIl-fiee extract in the presence of 1 mmol.L-' coenzymeA, 5 rnrn01~~-~ATP, 10
mmol.~-~MgC12, 1 mmol.~" NADPH, 5 mmol.L-' ascorbate, and 1 rnmol.~-' Dm,or
incubation of [5-3~]-mevalonatein the presence of 50 pmol.~-l mevastatin (inhibitor of
HMGR), 5 rnmol*~"ATP. 10 rnrnol.~-~MgCI2, 1 mrnol.~" NADPH. 5 mmol.~-' ascorbate, and 1 mmol-L-' DTT did not lead to the formation of a-famesene. The incorporation of ['"J-acetate and [3~]-mevalonateinto plant sesquiterpenes has been reported (Cane, 198 1). The poor incorporation in the present study, however. may be due to the mobilization of the labeled precursors into several other pathways.
Our results revealed that a-farnesene biosynthesis in apple fruit skin is mainly through a direct conversion of FPP to a-farnesene rather than via faniesol as an intermediate. cc-Farnesene formation from FPP could directIy occur through a deprotonation reaction of an allylic carbocation (McGarvey and Croteau. 1993, catalyzed by a sesquiterpene synthase enzyme, trans, frans-cc-farnesene synthase (Fig. 2.1). Acetylw CoA [2-14C]Acetic acid 9 HMG CoA Mevastatin -. HMGR Mevalonate [5-3wMevaIonate
GPP
Farnesyl Pyrophosphate (FPP) [I-3H]FPP Rz3H [ 1.2-1J~~~~c=14c
O I m -p-O-p-0- 1 I O - aTftzrn eserz e syntlt me farneso 1 py ro p hosp ho ki nase
'allyIic carbocation'
[ 1- jH]famesoI R=jH
Figure 2.1. Proposed pathway and mechanism of a-famesene biosynthesis in apple skin tissue. HMG Co4 3-hydroxy-3-methylglutaryl-CoA;HMGR, HMG CoA reductase; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate. Isolation of n-ans,trans-a-farnesene has aiso been-reported in pears (Pyrus cornmunis L.)
(Murray, l969), hops (KzimuZus Zupdus L. var. Cordifolius Maxim.) (Naya and Kotalce,
1970), dwarfCavendish bananas (Musa accuminata Colla) (Wills et al., 1973, 'Tahitian' lime (Citrus Zatzj5olia Tanaka), 'Emperor' mandarin (Citrus reticulata Blanco), 'Marsh' grapehit (Citrus paradisi Macf), and 'Valencia' orange (Citrzn sinensis L Osbeck)
(Moshonas and Shaw, 1980; Yuen et al., 1993). This is the first report illustrating the biosynthesis of tram,tram-a-farnesene.
a-Farnesene metabolism itz scaidit~gand scald-free tissue.
To investigate the possible relationship between a-farnesene biosynthesis and superficial scald development, a-famesene content of the bit skin and incorporation of radiolabel into a-famesene &om [1 -3~-~~~were analyzed using skin tissues frorn scald- devefoping and scald-fiee regions of the same apple. Interestingly, a-famesene content. as estimated by HPLC separation and quantification by monitoring absorbance at 233 m. was nearly three times higher in scald-fiee skin tissue compared with scalding skin tissue
(Fig. 2.2). Famesol was not detected as a predominant constituent arnong the hexane- extractable components of scalding or scald-fkee tissue. Previous studies have shown that a-farnesene emanates fiom the fruit surface, and hexane-extractable a-famesene content of the skin is higher in scald-fkee appie tissues than scalding tissues (Paliyath et al., 1997;
Urhiting et al., 1997).
Labeled a-famesene and farnesol production fiom [1-'HI-FPP increased as a function of tirne (Fig. 2.3A,B). a-Famesene formation fiom [ I -'HI-FPP was three-fold scald-free scalding
Figure 2.2. a-Famesene content of scald-free and scalding 'Delicious' apple skin tissue as estimated by HPLC separation of heuane-extracted components. Each bar represenis the mean of 6 repkates. and the error bar indicates the standard error of the mean. I I I I O 20 40 60 90 Reaction Time (min)
Figure 2.3. Incorporation of radioactivity into a-famesene (A) and famesol (B) from Pans, ~~~~S-[I-'H]-FPPin scald-free (i)and scaldùig (0) 'Delicious' apple skin tissue during incubation Two experiments each with three fruits were conducted independently and data were pooled for the statistical analysis. greater in scald-fiee skin tissue (Fig. 2.3A). showing parallelisrn to the natural a-
famsene levels in the skin (Fig. 2.2). Farnesol formation fiom [1-'4-FPP was not
si&ficantly different (p10.05) between scalding and scald-free tissues (Fig. 2.3B). These results indicate that a-famesene formation is independent of famesol formation despite scald incidence. Accumulation of lower levels of cc-famesene and reduced cc-farnesene biosynthesis in scald-developing tissue suggest that cc-farnesene catabolism and/or a- farnesene biosynthesis are af5ected by scald development. The apparent fate of labeled a- farnesene and potential differences between the scald-developing and scald-free tissues are under investigation. The fact that hexane-extractable cc-farnesene content progressively increases during the first two months of storage and declines thereafter is well documented (Huelin and Murray, 1966; Meigh, 1969; Meigh and Filmer. 1969; Meir and Brarnlage, 1988). Therefore, it would be expected that a-famesene synthase attains maximal activity during the first 2-3 months in storage and then declines, afier which time the initial syrnptoms of scald are seen. If so. this could explain the present results of changes in a-famesene synthase activity during storage. In addition, HPLC provides a more precise estimation of a-farnesene level than spectrophotometric estimation of the crude skin extract' since spectrophotometric methods couid over-/under-estimate u- fxnesene content as a result of interference of other surface components in scald- developing apples. Whiting et al. (1 997) observed over 1 1 separate components of apple skin that absorb at 233 nm. Thus. this could be the reason for widely recognized poor correlation between concentration of a-farnesene content (measured by spectrophotometry) and the extent and seventy of superficial scald reported by several Iaboratories (Chen et al., 1990; Gallerani and Pratella, 199 1; Huelin and Coggiola 1970b;
Meir and Bramlage. 1988).
In smq,our results indicate that a-famesene formation in apple tissue is
through the isoprenoid pathway via FPP, and is localized predominantiy in epidermal and
hypodermal ce11 layers of fruit. Conversion of FPP to a-farnesene is catalyzed by a single
sesquiterpene synthase, trans,trans-a-famesene synthase, rather than via farnesol as an
intermediate. Reduced biosynthesis of a-faniesene fiom [1-3~]-~~~,and Iower content of
accumulated a-farnesene in the skin of scald-developing tissue, is due either to enhanced
catabolism of a-farnesene and/or to inhibition of specific enzyrne(s) of the cc-farnesene
biosynthetic pathway, or may simply reflect enhancement of cellular damage as scald
foms.
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'Valencia' orange. J. Sci, Food Agr. 6735-339. CHAPTER III
Sesquiterpene a-Farnesene Synthase: Partial Puriftcation, Characterization, and
Activity in Relation to Superficial Scald Development in Apples
Published in J. Amer. Soc. Hort. Sci. 125111-119 (2000)
ABSTRACT
To decipher the relation between a-farnesene metabolism and the development of superficial scald in apples, trans,trans-a-farnesene synthase, the enzyme that catalyzes the conversion of farnesyl pyrophosphate to a-farnesene, \vas partially purified from skin tissue of 'Delicious' apples (Malits xdomestica Borkh.) and characterized. The total and specific activities of the enzyme were higher in the cytosolic fraction than in membrane fractions. a-Farnesene synthase \vas purified
70-fold from the cytosolic fraction by ion exchange chromatography and gel permeation, and the native molecular weight was estimated to be 108,000. The enzyme had optimal activity at a pH of 5.6 and absolutely required a divalent metal ion such as M~'+or ~n'+for activity. It exhibited allosteric kinetics, S(Od)for farnesyl pyrophosphate being 84+18 pnol.~"',and a Hill coefficient (nFr) of 2.9, indicating the number of subunits to be two or three. Enzyme activity was highest between 10 and 20 OC, while 50 % of the maximal activity was retained at O OC, and decreased during further storage. In vivo a-farnesene synthase activity was minimal at hawest, then increased rapidly during 16 weeks storage in air at O OC, and decreased during further storage. Activity of a-famesene synthase, a-farnesene content, and the conjugated triene alcohol (the putative scald-causing oxidation product of a-farnesene) content in the skin tissue were not correlated to the inherent nature of scald-susceptibility or resistance in 11 apple cultivars tested.
INTRODUCTION
Superficial scald is a senous postharvest physiological disorder affecting several cultivars of apple (Malus xdomestica Borkh.). The disorder is characterized by an uneven browning or bronzing of the skin, associated with death of the hypodemal cells, along with the development of skin wrinkling and pitting with increasing severity (Bain and
Mercer, 1963). Susceptibility to scald is determined by several factors such as cultivar diEerences, environmental conditions that apples are exposed to during growth and development, stage of maturity at harvest, and storage atmosphere (Ingle and D'Souza.
1989). Fruit of the apple cultivars Delicious, McIntosh Cortland, Granny Smith, Idared.
Rome Beauty. and Fuji are susceptible to this disorder, whereas those of cultivars such as
Empire, Gala, Mutsu, lonagold, and Northem Spy are resistant to scald development
(Emongor et al., 1994).
a-Famesene, an acyclic sesquiterpene hydrocarbon (C jH24; [3 EY6E]-3,7,1 1- trimethyl- 1.3.6.1 O-dodecatetraene) which is a constituent of the skin ce11 layers, has been associated with the occurrence of scald. Oxidation products of a-farnesene that include intermediate hydroperoxides and conjugated trienes, have been suggested to perturb membrane lipids, causing disruption of endodermal cells and thereby leading to the
'bronzed' appearance on the skin of scalded apples (Anet, 1972; Huelin and Coggiola,
61 1WOa,b). Conjugated trienes, as rneasured by ultraviolet (UV) spectroscopy at 258, 269.
and 281 nm, progressively accumulate on the surface of apples during storage and closely
correlate with the occurrence and severity of superficial scald (Chen et al.. 1990:
Gallerani and Pratella, 199 1; Ghahrarnani and Scott- 1998; Huelin and Coggiola 1970b:
Meir and Bramlage, 1988). Most of the early studies have relied on direct UV absorbante
measurements of crude extracts of the skin for quantification of a-famesene and
conjugated trienes. Concentrations of trienes measured by HPLC were only 1245% of
the uiene concentration estimated by W spectroscopy (Rowan et al., 1995), indicating
that previous analyses of apple skin washes may have seriously overestimated the triene
concentrations. Rowan et al. (1995) also identified a conjugated triene alcohol, (CTOL;
2,6,1 O-trimethyldodeca-7,7E.9E, 1 1-tetraen-6-01)? as the major component of conjugated
trienes in the apple cuticle, accounting for 88-95% of the total, and its 7E.92 isomer
constituting most of the remainder. CTOL 2lso possessed W absorption maxima at 258.
269, and 281 nrn. characteristic of conjugated trienes (Spicer et al.. 1993). CTOL is
capable of producing scald-like syrnptoms on stored apples in bioassays (Brimble et al..
1994; Rowan. Personai Communication).
a-Famesene is synthesized in apple skin tissue by the direct conversion of trans,trans-famesyl pyrophosphate (FPP) (Rupasinghe et al., 1998). The step is catalyzed by a single sesquiterpene synthase enzyme, trans,rrans-a-farnesene synthase.
Interestingly' in vivo trans.trans-a-famesene synthase activity was nearly three times lower in scald-developing skin tissue when compared with scald-free skin tissue
(Rupasinghe et aI., 1998). In addition, hexane-extractable cc-farnesene content of skin tissue measured by HPLC (Rupasinghe et al., 1998; Whiting et al., 1997), and a-
62 farnesene evolution into the head-space (Paliyath et al., 1997; Whiting et al., 1997) were
2- to 3-fold greater in scald-fiee sides than scald-developing sides of apples. These obse~ationsraised the question whether differences in a-farnesene Ievels between scald- developing and scald-fiee tissues are due to differences in biosynthesis of a-faniesene or its catabolism. In general, sesquiterpene biosynthesis is catalyzed by sesquiterpene cyclases or synthases (Cane, 198 1; Croteau and Cane, 1985). Sesquiterpene synthases have been purified and characterizcd fiom several higher plants that include sage (Salvia oflcinalis L.) (Croteau and Gundy, 1984; Dehd and Croteau, 1988), patchouli
(Pogostemon cablin Benth.) (Croteau et al., 1987), tobacco (Nicotiana tabacum L.)
(Vogeli et al., 1WO), calamondin (Citrofortunella miris) (Belinghen et al., 1992). potato
(Solarium tuberosum L.) (Zook et al., 1992), maritime pine (Pinzrs pinaster Ait.) (Salin et al., 1995), and Cotton (Gossypiurn hirsutum L.) (Davis et al., 1996).
A synthetic antioxidant, diphenylarnine (DPA), inhibits oxidation of u-farnesene to conjugated trienes (Chen et al., 1990; Huelin and Coggiola, 1970a,b; Smock. 1955) and is used commercially to prevent the development of scald (Ingle and D'Souza. 1989).
However, future use of DPA is under scrutiny because of its possible undesirable biological degradation products (Kim-Kang et al., 1998). The development of alternative control strategies requires a better understanding of the role of a-famesene and its oxidation products in the development of superficial scald. In particular. the ability to markedly down regulate or inhibit a-fârnesene biosynthesis could be used to demonstrate unequivocally the physiological role of a-farnesene in scald development and help develop alternative strategies to control superficial scald. Thus, the objective of the present study was to characterize rrans, tram-a-farnesene synthase and examine the
63 relation between a-famesene metabolism and susceptibility to superficial scaid
developrnent in apples.
MATERIALS AND METHODS
Plant materiaf
'Delicious' apples were harvested at optimum maturity during the first week of
Oct. 1996. from a commercial grower in Meaford, Ontario. Apples were cooled to O OC within 8 h of hanrest and stored at O OC in air or in controlled-atmosphere (CA) of 3 kPa
Oz and 2.5 kPa COz. These apples were used for extraction, purification and characterization of a-farnesene synthase. For monitoring a-famesene synthase activity, and a-famesene and CTOL contents of the skin of 'Delicious' and 'Empire7 apples during storage, and comparing the activity and content arnong different cultivars, apples were harvested at commercial rnatunty from the Horticultural Research Station, Simcoe.
Ont. between 14 Sept. and 1 8 Oct. 1997. Apples were stored as above.
Substrates and Ch emical reagents
Radiolabelled tram.tram-[ l -'~]farnes~lpyrophospate (FPP) (7.4 x 1 O ''
~~.rnol-',1 pCi = 0.037 mega Bq) was purchased fiom Arnerican Radiolabelled
Chemicals (ARC) Inc., St. Louis, Mo. A standard mixture of a and P isorners of farnesene was obtained from TCI, Tokyo; standards of farnesol and fmesyl acetate were from Aldrich Chem. Co., Milwaukee, Wis.; and NAD, NADH, NADP, NADPH, ATP, and ADP were from Boehringer Mannheim. Canada Inc, Montreal, Que. Synthetic CTOL
(2,6,1 O-trimethyldodeca-2,7E,9E,1 1-tetram-6-01} was a gifi from D.D. Rowan, 64 Horticulture and Food Research Institute Ltd., New Zedand. Al1 other chernicals were
obtained fiom Sigma-Aldrich Canada Ltd., Oakville, Ont.
In vitro enzyme assay
a-Famesene synthase activity was determined by incubating the enzyme extract
(200 pg of protein, unless otherwise mentioned) in a final volume of 1 mL reaction
mixture containing 0.1 rnol.~-' MES buffter (pH 56), 10 m.tnol~~-~MgCl2 and 0.15
mmole~-~MnClr. Assays were carried out at 20 OC in 4.5 rnL amber glass-vials. The
reaction was started by the addition of 50 pL of radiolabelled substrate prepared as
follows. Ten microliters of authentic 11-'HIFPP (ARC) was transferred to a test tube and
adjusted to 1 rnL final volume by adding 990 PL of 0.1 mol.^" MES buffer CpH 5-61 and
shaken vigorously. Fifty microliters of this solution contained approximately 25 pmol of
radiolabelled FPP. Afier 20 min of incubation wïth gentle shaking, the reaction \vas
terminated by adding 0.1 mL of 1 rnol.~-' KOH and 1 mL of rt-hesane followed by
vigorous mixing. Control experiments were performed with boiled enzyme in whicli
nonenzyrnatic formation of or-fmesene was negligible.
The influence of several cofactors (NAD. NADH, hTADP. NADPH, ATP, and
ADP) were tested at 20 and 50 rnmol.~" and the antioxidants (ascorbic acid and cc-
tocopherol) at 100 to 50 rnrnolm~-'.To create anaerobic conditions reaction vials were
flushed three tirnes of 1 min each with pure Nz before adding the substrate. To assess the possibility of blocking farnesol formation corn FPP, three pyrophosphatase inhibitors
(ammonium vanadate, ammonium molybdate, and sodium fluoride) were tested independently at concentrations of 1 to 10 mrnol.~-'. The experiments of enzyme activity 65 as a function of substrate concentration and calculation of S(oa were conducted using
substrate containing hot and cold FPP in a ratio of 990 Bq : 1 nmol of cold FPP (1 3,164 dphnmol-' ).
In vivo enqyme assay
Cylindrical pieces of tissue were removed fiom the equatorial regions of fruit
using a cork borer (1 cm diameter), and the skin tissues (I to 2 mm thick; 105112 mg
fiesh weight) were excised using a surgical blade- Two tissue disks were incubated in 4.5
mL amber giass-vials containing 950 pL of 0.1 rnol.~-~MES buffer @H 5.6). The vials
were sealed tightly using screw caps with teflon-silicon septa. The reaction was started by adding 50 pL of radiolabelled substrate (equivalent to -25 pmol substrate) to the reaction vid containing skin tissues. The skin tissues were subjected to vacuum infiltration to facilitate the uptake of the radiolabelled substrate. Afier 1 h of incubation with gentle shaking, the reaction was terminated by adding 0.1 mL of 1 rnol.~-' KOH and 1 mL of n- hexane followed by vigorous mixing.
Separation and anaiysis of radiolabelied prodlict
A one-half milliliter aliquot of the n-hexane extract was transferred to a test tube which contained 20 pL of 0.05% mixture of fmesene isorners and evaporated to dryness under Nz. The residue remaining was redissolved in 50 pL of n-hexane and a 25 pL aliquot was spotted on a thin layer chromaiography plate (TLC; silica gel G, 20 x 30 cm,
LKSD, Whatman Inc., Clifton, N.J.). The plates were developed completely with hexane:ethyl ether (4: 1, v/v) and dried under N2. a-Farnesene (Rf= 0.66) was visualized
66 in the presence of iodine vapor. The region of the plate corresponding to authentic
standard was scraped, mixed with 5 mL of scintillation cocktail (Ecolume, ICN, Costa
Mesa, Calf,), and radioactivity determined by liquid scintillation counting (Beckman
LS6800, Beckman Canada Inc., Mississauga, Ont.). The assay product separated by TLC
and corresponding to farnesene was eluted and compared with the elution of authentic
farnesene isolated by HPLC, which was again confïrrned to be exclusively a-farnesene
by GC-MS analysis as descnbed by Paliyath et al. (1997).
Enzyme extraction and prtri_fcation
Since in vivo synthesis of a-farnesene was found mainly in the skin rather than
cortex tissue of apple hit (Rupasinghe et al., 1998), skin tissue was used for enzyme
preparation. About 200 g of outer cortical tissue (0.25 to 0.5 cm depth) including the
cuticle was removed and cut into 0.25 to 0.5 cm3 pieces and homogenized using a
Polytron homogenizer (Brinkmm Instruments, Westbury, N.Y.. mode1 PT 10/35) for 1 min in 150 mL of 0.1 mmol-~-'sodium phosphate buffer (pH 6.5) containing 0.25 rno1.L-
sucrose, 50 mmol-L" ascorbic acid sodium salt, 2 mrnole~-'EDTA? 1 mrnol-L" DTT,
10 rnmol-L-' MgCL, 1 mmole~-' PMSF and 50 g-~-'polyvinylpyrrolidone. The homogenate was filtered through four layers of cheesecloth, the filtrate was centrifuged at
750 gn for 10 min to remove starch and debris, and the supemarant (crude extract) was used as the enzyme source. The crude extract was centrifuged at 10.000 x g for 20 min.
The resultant pellet was resuspended in 0.1 ~oI-L-'sodium phosphate buffer (pH 6.5) containing 0.25 mol-L" sucrose, 1 mmole~-'MgC12 and I rnrnol-L'I DTT (buffer A) and comprised the chloroplast~mitochondrial membrane fraction (Edward and Gardestrom,
67 1987). The supernatant was centrifuged again at-105,000 x g for 60 min to yield the
microsornal membrane pellet and cytosol. The pellet O btained afier ultracentrifugation
was resuspended as mentioned above to provide the microsornal fraction. For membrane
solubilization studies, a total membrane £?action was obtained by centrifuging crude
extract at 105,000 x g for 60 min and resuspending the resulting pellets as mentioned
earlier. One hundred micrograms of total membrane was incubated with either 0.5 to 3
mmol.~-' CHAPS (3-[-3-cholarnidopropyl] di-rnethylammonio- 1-propanesulphonate) or
0.0 1 to O. 1 % Triton X-100 for 60 min at 4 OCbefore the enzyme assay-
The cytosolic Fraction was loaded on a DEAE Sephacel colurnn (1.6 x 23 cm)
previously equilibrated with buffer A at a flow rate of 0.5 mlemin-'. The colurnn was
washed with 75 mL of the sarne buffer to remove unbound proteins. The bound proteins
were eluted with 5 mL volumes of 0.1 mole^' to 0.6 rnol.~-[NaCl in 0.05 mol.^-' steps
(0.1, 0.15, 0.2 .. . mole^'). a-Famesene synthase was estirnated in each fraction and the
most active fractions were pooled and used for gel permeation chromatography. Al1 steps
were carried out at 4 OC. A Waters AP-1 colurnn (10 x 500 mm, Waters Ltd.,
Mississauga, Ont.) packed with Sephacryl S300-HR equilibrated with 0.0 1 rnol.~'~Tris
buffer (pH 6.5) was used for size exclusion chromatography at a flow rate of 0.7 rnlamin-
' using a HPLC systern (Waters 626 LC system). a-Famesene synthase activity was determined in 1.4 mL fiactions. Protein concentrations were determined according to the method of Bradford (1 976) using bovine serum albumin as the standard. Estimation of relative molecular weiglzt
The Sephacryl column was calibrated with a Sigma MW-GF-200 kit that
consisted of the following standard markers; horse heart cytochrome C (12.400), bovine
erythrocytes carbonic anhydrase (29,000), bovine semm albumin (66,000), yeast alcohol
dehydrogenase (150,000), and sweet potato P-amylase (200,000). The void volume (V,)
was determined with Blue Dextrm (2.0001000) using 0.0 1 rnola~-'Tris buffer (pH 6.5).
The elution profile was monitored by the absorbance at 280 nm.
AnaCysis of a-famesene, CTOL, met/tyf heptenone (1MHO), nnd methyl hepten ol
(MHOL) contents in the skin iising HPLC
Two segments of fmit skin tissue, prepared as described for the iiz vivo enzyme
assay, were immersed in 2 mL of n-hexane and lcept for 24 h at 4 OC in a closed 4.5 mL
amber glass-vial. Hexane-soluble a-farnesene and its catabolites were analyzed by HPLC
using a Nova-Pak Ci* coIurnn (3.9 x 150 mm). The absorbance maxima used for a-
famesene and CTOL were 233 and 269 nrn. respectively. MHO (6-methyl-j-hepten-2-
one) and MHOL (6-methyl-5-hepten-2-01) were monitored at 2 10 nrn. the absorbance
maximum determined for these compounds. Acetonitnle (100 %) was used as the mobile
phase, at a flow rate of 0.75 mlmin-'. Elution rimes for famesene. CTOL. MHO, and
MHOL were 3.1, 2.34, 1-48 and 2.14 min, respectively. Quantification of each metabolite
was performed using a standard curve prepared with the corresponding authentic compound. StatMcal anaCysis
Determinations of the effects of pH, buffers. metal ions, heterotrophic effectors.
and temperature on a-famesene synthase activity were conducted in trïplicate and
statistically analyzed using a completely randomized experimental design (CRD).
Analyses of in vivo a-famesene synthase activity, a-famesene (skin and head-space) and
CTOL content in the skin during storage of different apple cultivars were performed also
using CRD with three replicates. Time of rernoval fiom storage and cultivar were
assigned as the main factors of the factorial treatment combination. Replicates were
apples that were obtained randomly from 3 boxes (a box contained 80 to 120 apples) of each specific cultivar stored at O OC in air. Al1 statistical analyses were done using SAS release 6.12 (SAS Inst., Inc., Cary, N.C.).
RESULTS
a-Farnesene synthesis during in vitro reaction
a-Farnesene synthase activity was low in the various preparations analyzed.
During the time-course studies of a-famesene formation, the rate of reaction was neariy linear up to 20 min and reached a plateau at 30 min. Therefore. in further experiments the incubation period was restncted to 20 min to obtain an optimal level of a-famesene among the reaction products. In general, the crude extract from CA-stored apples converted 0.1% to 1% of [1-'H]FPP to a-famesene. However. nearly 20 % of radiolabel frorn [1-3~]~~~was converted to farnesol. possibly due to the action of pyrophosphatases
(Croteau and Karp, 1979; Dehal and Croteau, 1988). Addition of pyrophosphatase inhibitors such as ammonium vanadate, ammonium molybdate, or sodium fluoride (1 to 70 10 mmo1.~-') dunng enzyme assays (Croteau and Karp, 1979) substantially blocked
farneso1 formation. but also adversely aEected a-famesene synthase act ivity . Conducting
the enzyme assay under anaerobic conditions or in the presence of antioxidants (ascorbic
acid and a-tocopherol) either marginally improved or inhibited a-famesene synthase
activity (data not presented). This suggests that a-fmesene formed is not spontaneously
oxidized to other products and lost during separation and andysis.
Buffer and pH.
The effect of pH on a-fmesene synthase activity was determined using 0.1
mol.^-' acetate and sodium phosphate buffers (pH range fiom 4 to 8). Enzyme activity
increased rapidly from pH 5.2, reached a maximum at pH 5.6 and declined thereafter
showing a typical bell-shaped activity protile. Therefore. al1 subsequent assays were conducted at pH 5.6. Similar properties have been reponed for most other sesquiterpene synthases with pH optima ranging from 6 to 7 (Croteau and Cane, 1985; Croteau et al..
1987; Dehd and Croteau, 1988; Salin et al., 1995; Vogeli et al.. 1990; Zook et al.. 1992).
The activity of a-fmesene synthase was cornpared in four different buffer systems at pH
5.6 (acetate. MES (2-IN-morpholino]ethanesulfonic acid). Tris-HCI. and sodium phosphate) each at 50 and 100 rnmol.~-'. The ionic strength of the buffer systerns had no significant effect (p=O.O5) on a-famesene synthase activity, but the highest enzyme activity was obtained in MES @Ka at 25 OG6.1) and hence this buffer was used for subsequent enzyme assays. Cofactor requirement - Meta1 ions
The only cofactor required for a-famesene synthase activity was a divalent metal ion, specifically M~~+or bAn2+. The highest enzyme activity with ~n"was at 0.5 mmol.~-', and at h4n2+ concentrations above 0.5 mmol.~-' activity decreased (Fig. 3.1). a-Farneçene synthase activity increased with increasing concentration of M~'' up to 20 mrno1.~-' and further increases in M~'' concentration did not promote enzyme activity
(Fig. 3.1). A relatively high activity was observed at 0.25 rnrnole~-' MnClz and 10 mrnol.~-~MgCl? in combination.
Temperature
a-Farnesene synthase activity increased from O to 10 OC, reached a plateau between 10 and 20 OC, and declined rapidly above 20 OC (Fig. 3.2). Enzyme activity was lowered only by 50% at O OC cornpared with the activity at the optimum temperature range (10 to 20 OC) (Fig. 3.2). This observation supports Our notion that a-farnesene biosynthesis actively occurs in apples stored at O to 4 OC.
Isolation and partial purification of a-farnesene synthase
The highest total activity and specific activity of a-fmesene synthase were found in the crude extract and the highest proportion of activity was confined to the cytosol rather than the membrane fraction (Table 3.1). Enzyme activity was the lowest in the microsomal fraction, with intermediate levels in the chtoroplast/mitochondrial fraction
(Table 3.1). The first step of purification by ion-exchange chromatography on DEAE a-farnesene synthase activity (Bqlmg proteinlh) a-farnesene synthase activity (Bqlmg proteinlh) Table 3.1. Distribution of a-famesene synthase activity among different sub-ceIlular fiactions of skin tissue of 'Delicious' apple miit. Apples were stored for five months in
CA at 3.0 kPa O2 and 2.5 kPa CO3. The data presented are representative of three independent experiments showing similar resufts.
Cellular Total protein Total activity Specific activity fraction (mg) (~cph-') (~cprn~-'-h-')
Cnide extract 64.4 3 1,045 482
Cnide extract without debris
Chloroplast +
Mitochondria
Microsome
Cytosol 18.8 8,3 17 332 + Protein A Radioactivity
Fraction nurnber
Figure 3.3. Partial purification of a-famesene synthase by ion esc hange chromatography on DEAE Sephacel (A) and gel permeation chromatography on Sephacryl (B). The conditions are as described in Materials and Methods. Sephacel resulted in a 20-fold purification (Table 3.2. Fig. 3.3A). cc-Farnesene synthase
was eIuted from îhe column with 0.35 rno1.~-' NaCl indicating that the enzyme has a
relatively large negative charge. Subsequent gel penneation chromatography on Sephacryl
further increased the purity of the enzyme preparation (70-fold) (Table 3.2, Fig. 3.3B).
Poor recovery from this step together with the instability of partially purified enzyme
restricted further purification of a-faniesene synthase to homogeneity. The rnost active
enzyme preparations were obtained fiom apples removed from CA storage during the
period of March to June and these apples had to be kept at O OC in air for an additional 1
to 2 weeks to attain maximal activity- The enzyme activity remained stable for a
maximum period of two to three rnonths only if stored as a crude extract at -80 OC.
Incubation of the total membrane fiaction (105,000 x g pellet) with 0.5 to 5 rnrnol.~-~
CHAPS (3-[-3-cholamidopropyl] di-methylammonio- l -propanesuIphonate) or 0.0 1 to O. 1
% Triton X-100 did not enhance cc-faniesene synthase activity in the solubilized fraction,
but inhibited the activity with increasing concentration (data not presented).
Apparent molecular weight of a-farnesene synthase
The molecular weight. of a-farnesene synthase was estimated using standard
molecular weight markers ranging fkom 12,000 to 200,000 (Sigma MW-GF-200 kit) and
size exclusion column chromatography. Using a calibration curve of V,N, against log molecular weight and determining the V,N, of eluted cc-farnesene synthase activity peak, an apparent molecular weight of 108,000 was estimated. Table 3.2. Partial purification of a-faniesene sfithase from skin tissue of 'DeIicious7
apple hit. The cytosol was subject to anion-exchange chromatography on DEAE-
sephacel. Active fractions were pooled and subjected to gel filtration using a Sephacryl
s300-HRCO~W.
purification Total Total Specific Recovery Purification step protein activity activity factor
(mg> (~q*h'9 (~~*m~-'*h-') (%)
cytos01 9 1-20 1 1,476 126 100 --
DEAE-Sephacel 2.1 O 5,3 16 253 1 46 20
Sephacryl 0.27 2,3 85 8,83 3 21 70 Enzyme kinetics
The partially purified a-famesene synthase obtained after DEAE-Sephacel-
chromatography exhibited a sigrnoidal pattern of enzyme kinetics (Fig. 3.4). Substrate
concentration at half maximal velocity, S(o-j>was 84k18 pole~-l for FPP as determined
from the results of three independent estmations. A slope (Hill coefficient. nH)of î.9kO.j
was obtained frorn Hill plots of [og FPP concentration against log [v/(V,,-Y)] (the insct
of Fig. 3.4). These results suggest that a-famesene synthase is an allosteric enzyme
comprising two or three subunits. a-Famesene synthase activity was not influenced by
cofactors such as NAD. NADH. NADP. NADPH, ATP. and ADP (data not presented).
In vivo a-farnesene synthase activity and a-farnesene and CTOL content during storage of different cultivars
a-Farnesene was not detectable in the skin tissue of scald-susceptible .Delicious' or scald-resistant 'Empire' fmit before attaining physiological maturity or ar harvest (Fig.
XA). a-Famesene content increased rapidly in both cultivars from 4 to 10 weeks afier harvest during storage in air at O OC (Fig 3.5A). Ten weeks afier harvest. u-farnesene content was nearly 3-fold higher in 'Delicious' apples than that in 'Empire'. and then declined rapidly (Fig. 3.3A). By contrast, a-famesene content in -Empire' apples increased gradually during storage and showed a marginal decline afier 23 weeks of storage (Fig. ;.SA). u-Famesene synthase activity was similar in -Delicious' and
'Empire' apples (Fig. 3-33). The changes in enzyme activity during storage nearly paralelled changes in a-famesene content in the skin. except during the latter part of storage where the decline in 0 O 0000 "Y w t-amo_ 8 [FPP] (prnol-L-')
FPP concentration (pmol=~-l)
Figure 3.4. Effect of increasing famesyl phyrophosphate (FPP) concentration on initial velocity of a-famesene synthesis. The enzyme source was a 20-fold purified cytosolic extract from CA-stored apple skin tissue. The assay was conducted in the presence of 100 mrnol.~-' MES buffer at pH 5.6, containing 10 rnmoi.~-' MgC12 and 0.23 mmol-L" MnC12-The substrate contained both "hot" and "cold" FPP in a ratio of 990 Bq : 1 mol. The curve is representative of three separate experirnents showing similar results. The inset shows determination of S(o-jland number of sub units (nH) of native a-farnesene synthase protein using Hill plot. -5 O 5 10 15 20 25 30 35 Weeks after harvest
Figure 3.5. cr-Famesene content (A). in vivo a-farnesene synthase activity (B). and conjugated triene alcohoI (CTOL) content (C) of the skin of -Delicious' (e) and 'Empire' (O) apples during air storage at O OC. A11 pararneters are expressed on a fresh weight basis and each data point is a mean of three separate sets of replicates. a-farnesene content was more rapid in 'Delicious' apples than the decline in enzyme
activiv (Fig. 3.5A,B). CTOL, the putative scald-causing oxidative product of a-
famesene, was very low in the skin of 'Delicious' and negligible in 'Empire' up to 26
weeks in storage (Fig. 3.5C). CTOL content increased sharply in 'Empire' at week 18,
but decreased thereafter (Fig. 3.X). In 'Delicious', maximal levels of CTOL were
observed at week 25, afier which the CTOL leveI declined (Fig- 333. Interestingly, the
cudative arnount of CTOL proàuced in storage by 'Delicious' and 'Empire' was
sirnilar, regardless of their inherent susceptibility to scald.
The relation between a-famesene synthase activity and a-farnesene levels was
explored Merby analysis in a wide variety of apple cultivars with differing degrees of
scald-susceptibility. The susceptibility of the cultivars was confirmed by storing them in
air for 4 to 6 months at O OC and evaluating for scald. In addition to -Delicious' and
'Empire', four scald-susceptibIe (Mclntosh, Cortland, Idared, and Rome Beauty), one moderately scald-susceptible (Fuji), and four scald-resistant f Gala, Mutsu, Jonagold, and
Northern Spy) cultivars of apple were evaluated. Data were acquired at three selected storage intervals during which the a-famesene and CTOL contents or a-farnesene synthase activity reached their maxima. 'Delicious', Tortland'. -Rome Beauty'. -Fuji', and 'Mutsu' showed a distinctively higher amount of accumulated a-farnesene in the skin compared with the other six cdtivars (Fig. 3.6A). Interestingly. scald-resistant 'Mutsu' had the highest amount of a-farnesene (Fig. 3.6A). As observed previously (Fig. 3.5). a- famesene synthase activity of different cultivars was not proportional to the corresponding a-farnesene content (Fig. 3.6A, B). For example, the enzyme activities of a-Farnesene Synthase CTOL Content Activity a-Farnesene Content 'Jonagold' and 'Northern Spy' fruit were the highest, yet the accumulated a-farnesene was relatively low (Fig. 3.6A, B). Those cultivars that produced high arnounts of CTOL were 'Delicious', 'CortlandT, 'Empire', 'Mchtosh', and 'Rome Beauty' (Fig. 3.6C).
However, scald-susceptible 'Idared' had relatively low amounts of CTOL while scald- resistant 'Empire' and 'Mutsu' had relatively hi& amounts of CTOL (Fig. 3.6C). Linear regression analysis of a-farnesene content, CTOL content. and percentage scald development gave an r value of 0.23 between a-fmesene and percentage scafd, and
0.56 between CTOL and percentage scald. a-Famesene, CTOL, MHO, and MHOL content in relation to superficial scald development.
In agreement with some of our previous studies (Paliyath et al., 1997; Rupasinghe et al., 1998; Whiting et al., 1997), a-farnesene content was higher in scald-free tissue and decreased with increasing propensity to scdd (Table 3.3). Accumulated CTOL content of scald-developing and severely-scalded tissue was 50% and 30% higher. respectively. compared with scald-free tissue (Table 3.3). Diphenylamine (DPA) treatment did not influence a-famesene content, but decreased CTOL content by 90% (Table 3.3). CA storage suppressed a-farnesene and CTOL contents by 58% and 66%, respectively, compared with air storage at O OC. Content of MHO did not have any relation to the severity of scald, but MHOL content was 60% and 20% higher in scald-developing and severely-scalded tissue, respectively (Table 3 -3). Table 3 -3- Content of hexane-extractable a-famesene, and its catabolites in the skin tissue
of 'Delicious' apple in relation to severity of scald, DPA and CA treatment. Apples were
stored at O OC in air for 17 cveeks. The metabolites were extracted in n-hexane by
immersing 20 skïns fiom equatoriai region of ten apples for 24 h. Analysis was done as
described in the materials and methods. Data are representation of 2 experiments showing
similar results,
Scald Metabolite
severity a-Farnesene CTOL MHO MHOL
Scald-free apple 53 1 168 57 35
Healthy tissue of 508 scald-developing apple
Scald-developing tissue
DPA-treated, scald-fiee
ND, not determined
8 5 DISCUSSION
The current theory on the mechanism of superficial scald development in apple is
ceitered around the biosynthesis and degradation of the sesquiterpene a-famesene
present in the skùi tissue. However; recent work conducted in our laboratory consistently
revealed that a-famesene content and evolution in scald-free apples are tiiree-times
higher than in scald-developing apples (Paliyath et al., 1997; Rupasinghe et al.. 1998:
Whiting et al.. 1997). In the present study, we have attempted to answer some of the basic
questions arising from the above observations as to whether i) the differences in a-
faniesene levels in the skin tissues denved from scald-free and scald-developing apples
are due to their differences in a-farnesene biosynthetic capacity or degradation, and ii)
the inherent levels of a-famesene and conjugated trienes of different apple cultivars have
any reIation to scald susceptibility. We have also examined the characteristics of a-
faniesene synthase, which converts farnesyl pyrophosphate to a-famesene (Rupasinghe et
al., 1998). Most of the early studies on scald have relied on direct UV absorbance rneasurements of crude hexane extracts of the cuticular or surface region at absorption maxima of 232 nm and 269 nm for quantification of a-faniesene and conjugated trienes, respectively. We have reexarnined the level of a-famesene and its putative metabolites such as CTOL (Rowan et al., 1995) in reIation to the development of superficial scald using more accurate analytical tools such as HPLC, GCISPME, and GC-MS.
The highest u-farnesene synthase activity was in the cytosolic fraction, though fractionation adversely affected recovery. Biosynthesis of sesquiterpenes in other plant tissues is associated with two specialized subcellular compartments, the c~tosoVendoplasmicreticdurn (ER) boundary (Belingheri et al., 19%; Gleizes et al..
1980) and plastids (Bernard-Dagan et al., 1982). in leaves of Pinz~~pinasrer Ait.' the
38,000 x g supernatant was involved mainly in the biosynthesis of the acyciic sesquiterpene pans-B-famesene, whereas cyctic sesquiterpene hydrocarbons were synthesized by the microsomal pellet (ER) (Bernard-Dagan et al., 1982). However. the presence of high Ievels of a-fimesene synthase activity in the supernatant does not imply that it is a cytosolic enzyme. In general, sesquiterpene synthases are rather hydrophobie and possess relatively bw pl values (Croteau and Cane, 1985). Bernard-Dagan et al.
(1982) observed that most enzymes involved in sesquiterpene biosynthesis could be solubilized easily during fiactionation and that the enzyme activity is associated presurnably with a membrane cornpartment. Therefore, it is likely that a-farnesene synthase could be bound weakly to plastid or ER membranes and dissociated during the homogenization process. To test this assurnption, the 105,000 x g pellet (totaI membrane) was incubated with different detergents to observe possible enhancement of cc-faniesene synthase activity in the solubilized fraction. However, the enzyme activity was inhibited with CHAPS or Triton X-100. This implies that either the enzyme is not membrane- associated or the detergents adversely affected enzyme activity. However, based on high activity in the cytosolic fraction, together with a low pH optimum (5.6), it could be suggested that cc-farnesene synthase is localized in the cytosol, loosely bound to the ER or in other vesicular compartments.
In common wi th O ther enzymes of sesquiterpene biosynthesis, a-farnesene synthase required divalent metal ions for its activity, with relatively more affrnity for
MI? than M~~+(Belingheri et al., 1992; Cane, 1981; Dehal and Croteau, 1988; Salin et
87 al., 1995; Vogeli et ai., 1990). The presence of-a divalent metal ion was an absolute
requirement for this enzyme, since only a trace level of enzyme activity was detected in
the absence of metal ions in the reaction mixture. A divalent metd ion is the only
CO factor required for most kno wn sesquiterpene synthases (Croteau and Gundy, 19 84;
Croteau and Cane, 1985). Half maximal stimulation of a-faniesene synthase occurred at a
concentration of 2 to 2.5 rnrnol@~-'M~~~ which ais0 reflects the physiological cytosolic
levels of M~"(Hepler and Wayne, 1985). The divalent metal ion binds to the
pyrophosphate moiety of the allylic CO-substrate, so as to neutralize/shield negative
charges of the pyrophosphate moiety and to make it a better leaving group in the
ionization step (Chayet et al., 1984; McGarvey and Croteau, 1995). Ionization of the
allylic pyrophosphate (FPP) leads to formation of a charge-stabilized allylic carbocation
(Chayet et al.. 1984; McCaskill and Croteau, 1997; McGarvey and ~roteaÜ,1995) as an
intermediate of sesquiterpene formation.
Unlike B-famesene synthase (Salin et al., 1995), a-famesene synthase in apples
exhibits typical sigmoidal enzyme kinetics with increasing FPP in the reaction mixture.
The estirnated Sco5,value was 84+18 pnolm~-'(rnean+SD) and the Hill coefficient (nH)
was 2.9, suggesting the number of subunits of a-famesene synthase to be two or three.
None of the dinucleotide heterotropic effectors tested influenced enzyme activity. The native rnolecular weight estimated for a-farnesene synthase after gel filtration was
108,000 and was similar to that estimated for tram-p-farnesene synthase in needles of
Pinus pinaster Ait. (Salin et al., 1995). SDS-PAGE of purified tram-p-famesene synthase shows a single band at 45,000 (Salin et al., 1995), supporting the potential dimeric or multirneric nature of functionally active faniesene synthase in apples.
88 The decline in cc-faniesene content after attaining maximal levels during storage
was more rapid than that of a-farnesene synthase activity, and was more evident in scald-
susceptible 'Delicious' than scald-resistant 'Empire'. The decline in a-farnesene content
and increase in CTOL is relatively stoichiometric, the increase in concentration of CTOL
nearly reflecting the decline in tissue a-faniesene content. The decline in a-farnesene
synthase activity could reflect a depletion of substrates and cofactors involved in the
isoprenoid pathway. The increase in CTOL could result fiom increased peroxidation, an
inherent feature of advancing senescence in hit tissues. Therefore. the present results
revealed that the decline of a-farnesene during prolonged cold storage was related to both
Iowered a-farnesene synthase activity as well as enhanced oxidation of a-famesene to
CTOL-like compounds. The syrnptorns of scald usually appeared during this period.
Cornparison of scald-susceptible and scald-resistant cultivars showed that a-
farnesene content in the skin of hit does not correlate with their inherent. relative scald-
susceptibility. Scald-resistant 'Mutsu' produced the highest levels of a-famesene.
whereas scald-susceptible 'Idared' and 'McIntosh' had relatively low amounts of a-
farnesene. To test the possibility whether varying rates of a-farnesene evolution observed
were due to differences in skin permeability, head-space a-farnesene content of the II
cultivars was rneasured 12 weeks after harvest and compared with that of the
corresponding a-famesene content in the skin. The saturated a-farnesene level in the
head-space was 50- to 100-fold less compared with the a-famesene content of skin (on a
fresh weight basis). As well, there was a significant high correlation (r- = 0.88) between a-farnesene content in the skin and a-faniesene level in the head-space (data not presentesl). Hence, it can be suggested that poor correlation between a-famesene content and a-famesene synthase activity (r = 0.05) is not due to differences in skin permeability among the cultivars. It is possible that the regulation of cc-faniesene biosynthesis in apple skin could be upstream of the isoprenoid pathway. The enzyme 3-hydroxy-3- methylglutaryl-Coenzyme A reductase (HMGR) is considered a primary control point for isoprenoid biosynthesis in plants and is regulated by a variety of developmental and environmental signals (McCaskill and Croteau, 1997; McGarvey and Croteau. 1995).
Therefore, a lack of correlation between cc-famesene synthase activity and cc-faniesene present in the skin could have resulted from differences in available FPP for this terminal enzyme of cc-faniesene biosynthesis. Cultivars which produce high arnounts of cc- farnesene appear to produce relatively more CTOL, except 'Fuji' and 'Empire'. However, prelirninary analysis on the endogenous levels of CTOL, the presurned scald-causing u- faniesene catabolite (Huelin and Coggiola. 1970a,b; Whitaker et al.. 1997), shotved relatively poor correlation (r = 0.56) with the scaid index of the eleven different apple cultivars tested. Similarly, Rao et al. (1 998) found a poor relationship between conjugated trienes (CT258, CT38 1, and the C258KT28 1 ratio) and scald susceptibility of 'White
Angel' x 'Rome Beauty' apple selections.
Analysis of cc-farnesene in relation to scald severity revealed a close relation between reduced cc-faniesene content and the seventy of scald, consistent with the observations from Our previous studies (Paliyath et al., 1997; Rupasinghe et al., 1998;
Whiting et ai., 1997). The earlier evidence supporting the role of conjugated triene as the primary scald-causing agent was its close correlation to the severity of scald, and suppression of conjugated triene accumulation by DPA treatment (Huelin and Coggiola, 90 1970a,b; Meigh and Filmer, 2969). In agreement with the above conclusion, we observed
a 90% reduction in CTOL content after DPA treatrnent. Storage of apples under low-
oxygen atmosphere (CA storage) is another means of suppressing development of scald
(Ingle and D'Souza, 1989). The reduction in both a-fmesene (58%) and CTOL (66%) in
CA-stored apples compared with apples stored in air indicates that low oxygen af3ects a-
farnesene oxidation as well as general a-famesene metabolism, implying a role for
oxygen in these metabolic processes- MHO is an in vitro (Anet, 1972; Filmer and Meigh,
1971) as weIl as in vivo (Mir et al, 1999; Mir and Beaudry, 1999) oxidative product of u-
faniesene. Recently, Mir et al. (1999) found that MHO released fkom DPA-treated
Tortland' hit peels was 8000-fold lower than fi-om peel sarnples of control hit and
suggested that MHO could somehow be involved in scald development. In contrast, our
analysis suggested that MHO content in the skin did not have any direct relation to the
severity of scald. However. similar to CTOL, MHOL was 60% higher in scald-developing
tissue, suggesting further reduction of the ketone to alcohol. MHOL has been observed to
be present in the head-space volatiles of 'Jonagold7 apples and was 3-fold higher in air-
stored apples than in CA-stored appIes (Girard and Lau, 1995). However, no MHO was detected in their analysis (Girard and Lau, 1995), suggesting that it may have been reduced to MHOL in this apple cultivar. These observations taken together with those of
Buttery and Ling (1993) that tomato (Lycopersicon esczrlentzrm Mill.), a fniit that does not develop scald, evolves large quantities of MHO as a volatile during ripening and that
MHO is a catabolite of p-carotene. another long chain isoprenoid compound, cast considerable doubt on the notion that MHO is involved directly in scald development. The absence of a-famesene in the skin of apples at harvest and the rapid increase
in a-famesene content and a-famesene synthase activiv during storage at O OC indicates
that developmental regulation of a-faniesene synthase activity and a-fâmesene
biosynthesis took place following detachment of mature fniit fiom the tree and/or
possible induction by low temperature stress. Similar to the present results, it has been
observed previously that hexane-extractable a-faniesene content progressively increases
during the first 2 rnonths of storage at O OC and declines thereafter (Huelin and Murray,
1966; Meigh and Filmer, 1969; Meir and Bramlage, 1988; Wtiitaker et al., 1997).
Interestingly. the level of a-farnesene in the skin of lime (Citnis iat*Zia Tanaka), mandarin (Cirrus reticulata Blanco), and grapefhit (Cirrus paradisi Macf), which do not develop scald, also increases during 4 to 6 weeks storage at O OC (Yuen et al., 1995). In our study, a-farnesene synthase was active at Iow temperatures and retained 50% of the activity in crude apple skin extract at O OC compared with its ma~imumactivity between
lO to 20 OC.
Cornparison of the a-farnesene content of 'Delicious' appIes stored at O OC and 5
OC revealed that cc-faniesene biosynthesis is 1.5- to 3-fold higher at O OC than at 5 OC at 8 and 10 weeks afier harvest (data not presented). Wills et al. (1975) found that a-famesene levels of 'Dwarf Cavendish' bananas (Musa amminata Colla) cm be induced by low temperature storage. As well, in 'Anjou' pears (Pyrus communis L.) that contain similar levels of a-famesene as in apples, a-famesene levels can be increased 6-fold by holding miit for 3 d at O OC compared with those stored at 10 OC (Rupasinghe, Paliyath and Mur, unpublished data). Recently. a-farnesene has been identified as a major volatile induced by insect herbivory in Cotton (Gossypium hirsuturn L.) (Paré and Tumlinson, 1997).
These results suggest the possibility that a-fesene is a low temperature or stress-
induced secondary metabolite. This would support the earlier notion by Watkins et al.
(1995), who designated scald as a typical chilling injury. Scald development has also
been reIated to the production of free radicals through a-farnesene catabolism. Under
chilling conditions, disruption of electron Qow in the mitochondria could lead to the generation of superoxide radicals (Puis et al., 1995). Thus, the generation of free radicals and active oxygen species couId serve as a potential pnmary cause(s) for the development of superficial scald. Antioxidants such as a-tocopheroI (Anet, 1974; Barden and BrarnIage, 1994; Gallerani et al., 1990; Meir and Bramlage, 1988), and ascorbic acid and flavanols (Albrigo and Childers, 1970; Barden and Brarnlage, 1994) are associated with resistance to scald development. Recently, Rao et al. (1998) found higher activities of the HIOZ-degrading enzymes. guaiacol-peroxidases and catalases in scald-resistant selections of 'White Angel' x 'Rome Beauty' apples. Therefore, in apples, scald susceptibility or resistance could depend primarily on the generation of active oxygen species andlor the ability of the natural antioxidant defense system to effectively scavenge free-radicals generated during postharvest storage.
In conclusion, Our results do not support the hypothesis that scald-susceptibility of apple cultivars is related to the arnount of a-fmesene produced and its oxidation to conjugated trienes and other metabolites. Instead they support the idea that conversion of a-farnesene to CTOL occurs potentially through a free radical-mediated oxidation as a secondary event of scald developrnent, and that the inherent ability of the hit tissue to counteract or cope with oxidative stress plays a significant role in determining scald
susceptibility or resistance in apples.
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3445. CHAPTER IV - St~dy1
Suppression of a-famesene synthesis in 'Delicious' apples by
aminoethoxyvinylglycine (AVG) and 1-methylcyclopropene (1-MCP)
Published in J. Plant PhysioI. Mol. Biol. 6:195-198 (2000)
ABSTRACT
'Delicious' apples (Malus xdomestica Borkh.) treated with either AVG or 1-
MCP and stored for 8 weeks at O OC exhibited suppressed ethylene (C&) production and a-farnesene content in the skin when compared with untreated apples. In vivo E,E-a-farnesene synthase activity in fruit treated with the C7H4 inhibitors was not correlated with the corresponding level of a-farnesene in the skin nor that emitted during head-space analysis.
INTRODUCTION
Endogenous C2& production during storage is closely relsted to the accumulation of cc-farnesene ([3E,6EJ-3,7.11 -trimethyl-1,3,6,10-dodecatetraene) in the skin and superficial scald development in apple. Accumulation of a-famesene in fruit skin increases progressively during the first 2 months of cold storage, reaches a maximal level after 2 to 3 months m-d seems to parallel endogenous Czbin appIe (Watkins et al., 1993;
Whitaker et al., 1997). RemovaI of CzHjfrom the storage atmosphere by permanganate
(Liu, 1986) or blocking C2& action using diazocyclopentadiene (DACP) (Gong and Tian,
1998) retards a-famesene accumulation in apple skin. The activity of E,E-cc-farnesene
101 synthase, which catalyses the conversion of famesyl pyrophosphate to a-famesene. is
very low at harvest and increases during the first 2 to 3 months in storage, coincident with
changes in skin a-famesene level (Rupasinghe et al., 2000). Based on this evidence, we
hypothesized that C& may be involved in the regdation of a-famesene biosynthesis at
E.E-a-famesene synthase. Thus. our objective was to evaluate the effects of Cz&
biosynthesis (AVG) and action (1-MCP) inhibitors on de novo a-famesene levels and
E. E-a-famesene synthase activity in 'Delicious' apple bit.
MATERIALS AND METHODS
Plant material and treatments
'Delicious' apples were harvested at optimum maturity (based on interna1 CfiJ
and starch test) during the first week of October 1997, fiom the Horticultural Experiment
Station, Simcoe, Ontario. Apples were transported to Guelph and were cooled to O OC
within 8 h of harvest. Apples which had been stored at O OC in air for 8 weeks were
removed to 20 OC and divided into 5 samples of 25 fniit each. One sample was dipped for
10 min in D.I. water containing 200 rng.L-' AVG (~e~ain~~;Abbon Laboratories, North
Chicago, Illinois. USA) and 0.5 ~LL"ABG 701 lTM (Abbott Laboratories, North
Chicago, Illinois, USA) as surfactant. A second sarnple was placed in a sealed glass-jar and exposed to 20 p~.~-'1-MCP (~th~l~loc~~;Bio Technologies for Horticulture Inc.,
South Carolina, USA) for 12 h. Three additional samples of apples, the first dipped for 10 min in D.I. water containing surfactant, another sealed in a glass-jar for 12 h. and the third placed on the lab bench. were used as untreated controls. Post-treatment C2H4production on these three different controls were the same. Al1 treatments were applied at 20 OC in the dark and apples were rnaintained under the sarne conditions for the rest of the monitoring period.
Analysis of C2)4 production
Three hits (388fl0 g) were sealed gas-tight in a 2 L glass-jar for 15 min- A 3- rnL gas sarnple \vas withdrawn using a syringe fiom the head-space and analyzed using a
Hewlett Packard 5880A gas chrornatograph equipped with a Poropak Q colurnn and flame ionization detector (FID).
Analysis of head-space a-farnesene using SPMWGC
Three hit(38850 g) were seaied gas-tight in a 2 L glass-jar for 15 min. A solid phase microextraction (SPME) probe (100 pm diarneter, polydimethyl siloxane coating) was exposed to the head-space for 15 min to allow hydrophobie volatile cornponents emanating fiom the apples to be absorbed on the probe. The absorbed volatiles were allowed to desorb for 5 min and were analyzed by a Hewlett Packard 5890 gas chromatograph equipped with a 30 m x 0.25 mm capillary colurnn (HP5; Supelco
Canada, Mississauga. Ontario, Canada). Oven temperature was linearly raised from 100 to 250 OC at a rate of IO OC per min. Eluted components were detected by an FID: a- farnesene eluted at 1 1.4 min. Analysis of skin a-farnesene content using HPLC
a-Farnesene levels were determined as described by Rupasinghe et al. (3000).
Cylindrical pieces of tissue were removed from the equatonal region of apple hits using a cork borer (1 cm dia.), and 2 skin tissues (2 mrn thick; 104k12 mg fiesh weight) were immersed in 2 mL of hexane, and kept for 24 h at 4 OC in a closed 4-5-mL glass vial. a-
Farnesene was analyzed by reversed-phase HPLC (Waters Chromatography. Milford,
Massachusetts, USA) by measuring absorbance at 233 nrn derseparation on a 3.9 s 150 mm CI8 column (Nova-Pak; Waters Chromatography, Milford, Massachusetts, USA),
Acetonitde (1 00 %) was used as the mobile phase, at a flow rate of 0.75 rn~~rnin-' .
Analysis of in vivo a-farnesene synthase activity
As described in Rupasinghe et al. (2000), two skin tissue segments. prepared as described above. were incubated in 4.5-mL amber glass viaIs containing 1-mL final volume of 0.1 rnol.~-' MES buffer (pH 5-6) and 25 pmol [l-'HIFPP (American
Radiolabeled Chernicals (ARC) ïnc., St. Louis, Montreal, Canada.). The tissue segments were vacuum infiltrated prior to 1 h of incubation with gentle shaking at 20 OC. The reaction was terminated by adding 0.1 mL of 1 rnol.~-'KOH and 1 mL hesane. One-half mL of hexane extract was transferred to a test tube which contained 20 pL of 0.05 % farnesene standard (TCI, Tokyo. Japan). and evaporated to dryness using a Stream of pure
Nz.The remaining residue was redissolved in 50 pL of hexane and a 25 PL aliquot was spotted on a thin layer chromatography plate (silica gel G. 60 s IO-'' m. 20 x 20 cm;
Whatman Inc., Clifion, New Jersey, USA)- The plate was completely developed with hexane:ethyl ether (4: 1. v/v) and dried under Nr. a-Farnesene (RI= 0.66) was visualized
1 O4 in the presence of iodine vapor. The region of the plate corresponding to authentic
standard was scraped. mixed with 5 mL of scintillation cocktail colur urne^. ICN. Costa
Mes* California, US&,e--. and radioactivity was determined by Iiquid scintillation counting
(Beckman LS6800, Mississagua, Ontario, Canada).
Statistical analysis
A completely randomized design with 3 replicates (hit or fniit discs) was used in the experiment to analysis each parameter. For the time course analyses of Cz& production and head-space a-famesene, the sarne set of apples was used throughout the experiment to minimize experimental error. These hyo parameters were analyzed considering time as the repeated measure factor. Andysis of a-farnesene content in the skin and a-famesene synthase activity were perfomed with treatments and time as a factorial combination. Statistical anaIysis was done using a software program (SAS release 6.12, SAS Institute, Cary, North Caroiina, USA).
RESUlLTS AND DISCUSSION
Despite Our knowledge on the progressive accumulation of the sesquiterpene hydrocarbon a-faniesene in apple skin tissue during storage and its relation to endogenous and extemal levels of Cz&, the mariner by which C2& might regulate a- farnesene synthesis is not clear. Previous snidies revealed that changes of DI vivo E,E-a- famesene synthase activity during storage closely parailel cc-farnesene content in the skin
(Rupasinghe et al., 2000) and endogenous Cz& production (Watkins et al., 1993). We attempted in this study to evaluate the influence of endogenous CzHl on de novo a-
1O5 famesene content and E, E-a-farnesene synthase activity by blocking Ct& perception in
apple fruit pnor to attaining the maximal levels of a-farnesene and cc-farnesene synthase
ac6vity during cold storage.
Upon removal from storage after 8 weeks at O OC in air. CI& production at 20 OC
of untreated apples (control) increased until day 6 of post-treatment (Table 4.1.1). C2& production of AVG- and 1-MCP-treated apples during the post-treatment penod kvas approxirnately 90% lower than untreated apples (Table 4.1.1). Similar to the inhibition of
C& production. AVG and 1-MCP treatments significantly reduced hexane-extractable a-famesene content in the skin (Fig. 4.1.1A) and a-famesene evolved fkom apples (Fig.
4.1.1 B). cc-Farnesene content of untreated, AVG- and 1-MCP-treated apples was similar on the first day afier treatment, but AVG- and 1-MCP-treated apples had 75% lower a- farnesene content than control apples on the sixth day afier treatment (Fig. 4.1.1A). a-
Farnesene evolution by apples was over 100-fold less than skin a-faniesene content, and was similar in apples frorn AVG and 1-MCP treatments (Fig. 4.1.1). Conversely, in vivo
E,E-a-farnesene synthase activity decreased upon removal from O OC to 20 OC and the activiq rernained similar among untreated, AVG- and 1-MCP-treated apples during the post-treatment period (Table 4.1.1).
Lack of a relationship between a-farnesene levels and a-farnesene synthase activity in response to C2H4 inhibitors indicates that regulation of cc-farnesene levels in apple fruit is not at this terminal enzyme but could occur at an upstream step of the isoprenoid pathway. Therefore, the previously observed changes in cc-farnesene ievels Table 4.1.1. C2& production and in vivo a-famesene synthase activity of 'Delicious' apples following treatment with AVG and 1-MCP. Apples removed fiom air storage at O
OC afeer 8 weeks were subjected to AVG and 1-MCP as described in Materials and
Methods.
Cfiproduction rate onj-esh weighr basi.9 (nLmg-'
Control
AVG
1-MCP 8 -5 1.O 2.1 SEM = 1 .O
a-Farnesene synthase activity on fresh weighr basiss (pmol. kg" 4')
Control
AVG
1-MCP 4.0 6.3 SEM = 0.7
'C2& production rate and in vivo a-famesene synthase activity of apples at rernoval from O OC before treatment. 350 A 300 - - O - AVG 1-MCP
250 O - 4
200 1 I - \
1 LSD,,,, = 0.18
O 2 4 6 8 10 12 Days after treatment
Figure 4.1.1. Changes in hexane-extractable a-farnesene content in the skin and head- space a-faniesene of 'Delicious' apples treated with AVG (O), 1-MCP (V) or lefi untreated (a) afier removal fiom air storage at O OC in air for 8 weeks. Data represents the mean of 3 replicates. during storage which paralleled changes of E, E-a-farnesene synthase activity could not be due to Cz&-induced E.E-a-famesene synthase activity. The enzyme 3-hydroxy-3- methylglutaryl-coenryme A reductase (HMGR) is considered as a primary control point for isoprenoid biosynthesis in plants, in particular, the gene hmgl of Xevea brasiliensis L. is inducible by Cl& (Chappell? 1995). Golding et al. (1998) observed that application of
1-MCP significantly inhibited C2& production as well as respiration in ripening banana
(Musa sp. Cavendish). Therefore, an apparent limitation in the availability of the isoprenoid pathway precursor, acetyl CoA, could be a reason for suppression of a- farnesene synthesis by the two C2& inhibitors, AVG and l-MCP. Alternatively, the possibility of Cz&-mediated partitionhg of isoprenoid precursors and intermediates into other pathways. and a-famesene catabolism also exist but remain unclear. Further research is needed to address the Czh-dependent regulatory rnechanisrn of a-famesene biosynthesis in apple. Interestingly, the observed profound inhibitory effects of AVG and
1-MCP on C2HJ production and action even afier 8 weeks of storage suggest new postharvest implications for the handling of climacteric fruits utiiizing 1-MCP.
LITERATURE CITED
Chappell, J. 1995. The biochemistry and molecular biology of isoprenoid metabolism.
Plant Physiol. 107: 1-6.
Golding, J.B., D. Shearer, S.G. Wyllie, and W.B. IvIcGlasson. 1998. Application of
1-MCP and propylene to identify ethylene-dependent ripening processes in
mature banana fniit. Postharvest Biol. Technol. 14:87-98. Gong, Y. and M.S. Tian. 1998. Inhibitory effect of diazocyclopentadiene on the
development of superficial scald in 'Granny Smith' apples. Plant Growth Reg.
26:117-21.
Liu, F .W. 1986. Responses of Daminozide-treated 'Delicious' and 'Idared7 apples to
simulated low-ethylene CA storage. J. Amer. Soc- Hort. Sci. 11 1:7 16-7 2 9,
Rupasinghe, H.P.V., G. Paliyath, and D.P. Murr. 2000. Sesquiterpene a-farnesene
synthase: partial purification, characterization, and activity in relation to
superficial sca1d development in apples- J. Amer. Soc, Hort. Sci. 135: 1 1 1-1 19.
Watkins, C.B., C.L. Barden, and W.J. Bramlage. 1993. Relationships among a-famesene,
conjugated trienes, and ethylene production with superficial scald development of
apples. Acta Hort. 343 :155-1 60.
Whitaker, B. D., T. Solornos, and D.J. Harrison. 1997. Quantification of or-farnesene and
its conjugated trieno1 oxidation products fiom apple peel by C 18-HPLC with UV
detection. J. Agric. Food Chem. 45:760-765. CHAPTER IV - Study 2
Inhibitory Effect of 1-MCP on Ftipening and Superfilcial Scald Development in
'McIntosh' and 'Delicious' Apples
Published in J. Hort. Sci. & Biotech. 79211-276 (2000)
ABSTRACT
'McIntoshYand 'Delicious' apple were treated with the cornpetitive ethylene
(C2H4) antagonist 1-methylcyclopropene (1-MCP), to evaluate its feasibility as a postharvest tool for use by the apple industry. The threshold concentration of 1-
MCP required to inhibit de novo CrHi production and action was 1 ~~LoL-'.1-MCP treatment completely inhibited C2Hi production in apples for 6 to 10 days at 20 OC following storage at O OC in air or controlled atmosphere for 60 or 120 days. I-MCP- treated apples were significantly firmer (13 to 20 N) than untreated apples following storage and post-storage handling for 7 or 14 days at 20 OC. Total soluble solids of apples was not affected by 1-MCP treatment. Inhibition of total volatiles and cc- farnesene emanated by apples by 1-MCP treatment was similar for CrHr production. Contents of cc-farnesene and its putative superficial scald causing catabolite, conjugated triene alcohol, in the skin were reduced 60 to 98 % by 21 p~m~'l1-MCP. Treatment with 1-MCP suppressed the incidence of superficial scald in 'McIntoshYand 'Delicious' apples by 30% and 90°h, respectively. INTRODUCTION
Ethylene (C2&) is involved in accelerating bit npening and senescence and is
also related to superficial scald development in apple (Malus xdornestica Borkh.). The
newest member of a gaseous cycloolefin family of C2& action inhibitors, 1-
Methylcyclopropene (1 -MCP), inhibits C2& action in ornamentals and certain fruits
(Sisler and Serek. 1997; 1999). Recent snidies indicate that 1-MCP can suppress CI%
production and thereby delay ripening of rnany climacte5c fi-uits such as pears (Pyuus
cornmunis L. cv. Passe-crassane) (Lelièvre et al., 1997b), banana (Musa sp.) (Wyllie et
al., 1998; Golding et al., 1998; Sisler and Serek, 1997), plums (Prunus salicina Lindl)
(Abdi et al., 1998) and tomato (Lycopersicon esculen~urnMill) (Nakatsuka et al., 1997;
Sisler and Serek, 1997). Cl& plays a critical role in the ripening of climacteric hit by triggering several ripening-related physiological changes (Lelièvre et al., 1997a). The burst of C2hproduction during ripening with transition fiom Systent 1 to Systern 2 C2& production (autocatalytic Ca) is concomitant with increased abundance of 1- aminocyclopropane- l -carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) transcripts in fmits such as apple (Ross et al., 1992). pear (Lelièvre et al.. 1997b), and tomato (Nakatsuka et al., 1998). Nakatsuka et al. (1997, 1998) further showed that 1-
MCP can inhibit the positive feedback regulation for the expression of both ACS and
AC0 genes during tornato fruit ripening.
Superficial scald, a serious storage disorder of certain apple cultivars, is believed to be caused by oxidation products of a-faniesene, a natural sesquiterpene hydrocarbon present in the fmit epidermis (Whitaker et al., 1997). The oxidation products of a- farnesene are referred to as conjugated trienes, the majority (~90%)of which are
112 comprised of two isomers of 2,6.1 O-trirnethyldodeca-2,7E,9E,1 1 -tenaen-6-01 (CTOL)
(Rowan et al., 1995). The endogenous C2& levels of apple fit(Meigh and Filmer,
1969; Watkins et al., 1993; Du and Bramlage, 1994; Whitaker et al., 1997) and the amount of Cz& accurnulated in storage atmospheres (Knee and Hatifield. 198 1; Little et al., 1985; Liu, 1986; Lau, 1990) influence the content of a-famesene in apple skin and superficial scald development. Diphenylarnine (DPA), a commercial anti-scald chemical, dso reduces Cz& production (Lurie et al., 1989; Du and Bramlage, 1994). Furthermore, the Czl& action inhibitor diazocyclopentadiene (DACP) delayed ripening and depressed a-famesene production and superf~cialscald in 'Granny Smith7 apple (Gong and Tian,
1998), emphasizing the role of C2& as the major limiting factor in the long-term storage of apples.
Therefore, there exists great potential to use 1-MCP, an extremely effective competitive inhibitor of C2& action, as a postharvest tool to extend the storage life of apples. The purpose of this research was to assess: (1) the effective concentration and influences of 1-MCP on C& production. fmit sofiening, and volatile production. and (2) the involvement of CZ& in a-famesene metabolism and development of superficial scald in 'McIntosh' and 'Delicious' apples stored in air or CA.
MATERIALS AND METHODS
Plant material and posti" arvest treatments
'McIntosh' and 'Delicious' apples (Malus xdornestica Borkh.) were harvested, respectively, on September 08 and October 01, 1998 fiom a commercial grower in
Meaford, Ontario [Harvest maturity indices of 'McIntosh' and 'Delicious', respectively:
113 fimess, 72 and 73.3 N; soluble solids, 11.8 and 10.3%; starch-iodine index (Corne11
chart) 3.7 and 3-21. Fmit were transported to Guelph and allowed to equilibrate to 20 OC
overnight. One day after harvest three boxes (replicates) of apples each containing 110
fruit were placed in sealed plastic containers (0.25 m3) and exposed to 0, 0.01, 0.1. 1.0.
10 or 100 ci^.^-' 1-MCP (EthylBlocTM,Biotechnologies for Horticulture, Inc., 75 1
Thunderbolt Dr., Waltersboro, SC 29488, USA) according to the manufacturer's
instruction at 20 OC for 18 h. Following treatment. the apples were placed in cold storage at O - 1 OC and 90 to 95% relative humidity (RH). Afier 60 and 120 days of storage. 60
fruit fiom each box were removed fiom each treatment for post-storage evaluation. In addition, one day after harvest six boxes of 'Mckosh' apples each containing 120 hit were stored separately in two sealed charnbers (900 L volume) under controlled atmosphere (CA; 3% Oz + 2.5% CO2 for the first 30 days, then 3% O? + 4.5% COI thereafier; 3 OC: RH 90 - 95%.). One charnber was esposed to 25 ,LLL.L-' 1-MCP one day after establishing CA conditions. Fruit were removed for analysis after 120 days in CA.
Upon removal from storage the following analyses were performed on fruit kept up to 3 weeks in air at 20 OC. Ethylene and volatile analyses were performed at 2- or 3- day intervals; firmness was assessed at 1-, 7- and 14-days; a-farnesene and CTOL in skin was analyzed immediately upon removal; scald incidence was assessed afier 7 days. at
20 OC.
Eight apples were sealed gas-tight in a 4 L glass-jar for 30 min. A 3-mL gas sample from the head-space was analyzed using a Hewlett Packard j880A gas
Il4 chromatograph (Hewlett Packard Co ., Avondale, Pemsylvania, USA) equipped with a
Poropa. Q column and flame ionization detector (FID). The lowest measurabIe amount of
Cz& in the head-space was about 0.0 1 ~L,.L-'.
Firmness
Flesh firmness of apples was determined at harvest and 1. 7 or 14 days post-
storage at 20 OC after removal from air or CA storage. Fimess was measured using an
Effegi pressure tester fitted with an 11 mm (diameter) tip on pared opposite sides (blush and green) of the apple after peel removai. The measmement on each treatrnent was done on three replicates each containing 10 fiuits. The juice expressed during firmness evduation was used to determine total solids content uing a hand-held refiactometer.
An alysis of lr ead-space volafile arz d a-farnesene rrsing SPME/GC
Hydropho bic volatile and a-farnesene Ievels were measured by the method descnbed by Paliyath et al. (1997). Eight apples were sealed gas-tight in a 4 L glass-jar for 15 min at which time a solid phase microextraction (SPME) probe (1 00 pm diameter, polydirnethyIsiloxane coating; Supelco Canada, Mississauga. Ontario. Canada) was exposed to the head-space for another 30 min to allow- hydrophobic volatile components emanating from the apples to be adsorbed. The adsorbed volatiIes were analyzed by a
HewIett Packard 5890 gas chromatograph (Hewlett Packard Co., Avondale,
Pemsylvania, USA) equipped with a HP5 30 m x 0.25 mm capillary column. The SPME probe was desorbed for 5 min, then the oven temperature was raised linearly from 55 to
250 OC at a rate of 10 OC per min and eluted components detected by FID. Famesene,
II5 which eluted at 15.4 min, and the total volatile content are expressed in UNts. where one
Unit of a-famesene is 10" x peak area and for total volatiles is the cumulative peak area
of the total profile x 10?
Anaiysk of a-furnesene adCTOL contena in t/te skin ushg HPLC
Cylindrical pieces of tissue were removed from the equatorial region of apple
using a cork borer (1 cm diarneter), and the skin (2 mm thick; 104+12 mg fresh weight)
was excised. A total of 6 skins per treatrnent, two skin tissues fiom each apple of 3
replicates, were irnmersed in 6 mL of hexane and kept for 24 h at 4 OC in a closed glass-
vial. Hexane-soluble a-famesene and CTOL contents were analyzed by reversed-phase
HPLC (Waters Chromatography. Milford, Mass.. USA) by measuring absorbance at 233
and 269 nm, respectively. Separation of the components was accomplished with a Nova-
Pak Cis column (3.9 x 150 mm, Waters Chromatography, Milford. Mass., USA). using
acetonitrile (100%) as the mobile phase at a flow rate of 0.75 rn~~rnin-'.Authentic cc-
farnesene was obtained from TCI, Tokyo and CTOL was a generous gifi of Dr. D.D.
Rowan, Horticulture and Food Research Institute Ltd., New Zealand.
Scald Nlcidence
Scald incidence (%) was assessed on 3 replicates of 25 fruit each which had been held 7 days at 20 OC after removal fiom storage. Scald was quantified on each hitusing a O to 3 scale, where O = no scald; 1 = slight injury with diffiise light brown: 2 = moderate injury with definite browning; and 3 = severe injury with dark brown-black discoloration.
Data presented are % hitaffected with scald (1, 2 and 3 ratings combined).
116 Sfaf Lstical unaiysk
The expenmental design for analyses of hexane-extractable a-famesene, CTOL or
and incidence of scald was a completely randornized design with a factorial arrangement
of cultivars and 1-MCP concentrations (2 x 6 factorial) with 3 replicates. Percentage
scald values were subjected to Arcsin square root percentage transformation for analysis
of variance.
For statistical analysis of head-space C25,total volatile and a-famesene, separare
quadratic regressions were fitted for each 1-MCP concentration (Y, = Pei + Pl, day + ,û3
day' + E, where i=I-MCP concentrations and j=days at 20 OC). The intercept, slope, and curvature of the quadratic regressions for 1-MCP concentrations were compared with the control (no 1-MCP) using an F-test. For flesh firmness analysis, cultivar, 1-MCP concentration. removal time and days at 20 OC were arranged (2' x 3 x 6 factorial) in a completely randomized design wirh 3 replicates. A replicate comprises the mean firmness value of 10 fmits which are randomly selected from a box of apples.
The cornparison of two treatment means of the CA storage study, was determined by a t-test. SAS release 6.12 was used for al1 statistical analysis.
RESULTS AND DISCUSSION
'McIntoshWand 'Delicious' apples that were treated with 1-MCP at I~L~L-'and greater and stored 60 days in air at O - 1 OC exhibited complete inhibition of C2H4 production for at least 6 days post-storage at 20 OC (Fig. 4.2.1A.B). With longer post- storage penods. Cz& production increased slightly. However. apples treated with t 1 p~.~-'1-MCP exhibited over 96% inhibition of C2H4 production (Fig. 4.2.lA,B).
Il7 Extending the storage period to 120 days resulted in inhibition of CzH4 production by
85% (data not presented), indicating the long-term effectiveness or binding-stability of 1-
MCP at low temperature.
The threshold concentration of 1-MCP required to inhibit Cr& production and
counteract C2& action in stored apples was estimated to be 1.O p~m~-'.Above 1.0 p~4~"
1-MCP, there was no Merinhibition of C2& production (Fig. 4.2.1A.B; Table 4.2.1).
Concentration of 1-MCP necessary for maximum response seems to vary from crop to
trop' and with maturity, duration and temperature of exposure. 1-MCP completely
protects carnations (Dianrhw caryophyllus L.) and banana against Cz& afker exposure to
a very low concentration of 0.5 ~L.L-[for 24 h at 24 OC (Sisler and Serek. 1997. 1999).
Seven ~LL-'1-MCP was required with tomato to give a similar response (Sisler and
Serek, 1997). In carnation, the effectiveness of LMCP is four-fold higher at room temperature (24 OC) than at 4 OC (Sisler and Serek, 1997). The comparatively higher amount of 1-MCP required to block C2H4 action in apples could be due either to poor penetration of 1-MCP through the epicuticular wax into the flesh or to the presence of a large pool of C2H4 receptors. In 1-MCP-treated apples, gradua1 recovery of C& production with prolonged cold storage or during holding at 20 OC suggests either the synthesis of new receptor proteins (Sisler et al., 1996; Sisler and Serek, 1999), metabolism of the 1-MCP receptor protein complex, or release of the bound 1-MCP from the receptor sites thus regaining Cz& sensitivity. Nakatsuka et al. (1997) showed the ability of 1-MCP to completely eliminate Cz& action and feedback regulation of endogenous C2H4 production at transcriptional Ievel in mature green tomato fruit. 'Delicious'
Figure 4.2.1. C& production (AB),total volatile (C,D) and oc-farnesene @,FI emitted by 1-MCP-treated or untreated 'Mchtosh' and 'Delicious' apples during 13 and 15 days respectively, at 20 OC upon removal from 60 days in air at O - 1 OC with 90 - 95 relative humidity. 1 Unit of total volatile or a-famesene = peak area x 10? Table 4.2.1 . Summary of the statisticd anaiysis of Figure 4.2.1.
Cultivar 1-MCP concentration (~L-L-')
'Mchtosh' 'Delicious'
Total volatile (unit.kg-' F
'Mchtosh' 'Delicious'
Head-space a-fanesene (unir .kg-' FW)
'Mchtosh' 'Delicious'
8 88 *+* , . Significantly different fiom the control at P 1 O.Ojl 0.0 1 or 0.00 1. respectively: NS: Not Significant at P I0.05. Concomitant with the inhibition of endogenous Cz& production, 'Mclntosh' and
'Delicious' apples treated with 21 p~e~-l1-MCP showed a significant delay in miit
sohening (Table 4.22). Flesh fmess of 'Mchtosh' and 'Delicious' apples at harvest
was similar (72k0.9 md 7332N; respectively), but fruit sofiening during storage was
very abrupt in conuol and, to a lesser extent. in 1-MCP-treated 'McIntosh' apples
compared to that in -Delicious' apples (Table 4-22). Two weeks after transfer Eom cold
storage to 20 OC? both 'Mchtosh' and 'Deiicious' apples treated with I~L~L-'1-MCP
were significantly fimer (13 to 20 N) compared with untreated apples. The level of
firmness retention by 1-MCP-treated hit after storage is quite phenomenal and
comrnercially significant, especialIy for 'Mchtosh' and other cultivars known to soften
excessively during storage. As well, it should be noted these hit were delayed 48 h
(equilibration and treatment time) afier harvest before cooling to O - 1 OC, yet fruit
firmness was drarnatically improved with 1-MCP treatment. Fruit softening is one of the
ripening processes that is most sensitive to C2H4 (Lelièvre et al.. 1997a). Abeles and
Takeda (1990) descnbed the enzymes responsible for softening in apples during ripening
to be rnainly exo- or endo-polygalacturonase rather than cellulase. Significant inhibition
of softening by 1-MCP in the present study substantiates that C2H4 is involved in augmenting the activity of sofiening-associated enzymes. In contrast, 1-MCP had no significant effect on total soluble solid content of apple (data not presented). Similar results were reported in 1-MCP-treated pulp of banana (Golding et al., 1998) and apple
(Blankenship and Unrath, 1998). Table 4-22Flesh firmness of -McIntosh3and 'Delicious' apples treated with 1 p~.~-l1 - MCP a day afier hanest at 20 OC for 18 h or not treated and stored 60 days (rernoval 1) and 120 days (removai 2) in air at O - I OC and 90 - 95 % relative hurnidity. The Firrnness of 'McIntosh' and *Delicious' apples at harvest was 72 and 73 N, respectively.
Cultivar Removal Days at O 0.0 1 O. 1 1 10 100 (days) 20 OC Fleshfiumness (N)
'Mchtosh'
'Delicious'
SEM = 1.02; n = 3. df = 144 Significance: c"'. Tm*.R"', Il"*,C x T"', C x R*". c x D', C x T x R"'. C x T x R x D"*, T x R', T x D"'. R x D"'
8 *** , Significant at P 1 0.05 or 0.00 1, respectiveiy, where C = cultivar' T = 1-MCP concentration, R = removal tirne, D = day at 20 OC Inhibition of the total volatile components and a-fmesene production of apples
by 1-MCP (21 ~LL")correlated highly wÏth its effect on endogenous Cz& production
(Figure 4-21, Table 4.2.5). Most of the volatiles of 'Delicious' and 'Mchtosh' apples are
fatty acid esters and a-famesene (Paliyath et al., 1997). Arnong them. famesene and
esters of hexanol and hexanoic acid are the most abundant (Paliyath et al., 1997). Most of
the Cs-derived volatiles that we detected fiom SPME/GC are among the '-character
impact compounds" contributing to aroma in apples reported by Berger (1 99 1). Recently,
it was shown that a-faniesene is biosynthesized through the isoprenoid pathway
(Rupasinghe et al., 1998). Regdation of respiration during ripening in climacteric fmits could be mediated through Cr&. Therefore, suppression of total volatile and a-faniesene synthesis by 1-MCP could be intervened, at least in part, through its inhibition of respiration and apparent limiting of acetyl-CoA, which is the cornrnon precursor of isoprenoid and fatty acid biosynthesis. In support of this notion, Song et al. (1997) observed that 1-MCP-treated apple tissue converted butanol and butyic acid to their corresponding C4-esters, suggesting an apparent limitation in the availability of precurson. Similarly, Fan et al. (1 998) found that production of ester volatiles by ripening apples was regulated by Cz& and inhibited by an CzH4 action inhibitor. the irradiation product of diazocyclopentadiene (DACP). Furthemore, reduction and delaying of volatile production and its association to respiration/ç02 evolution in response to 1 -MCP have been reported in 'Golden Delicious' apples (Song et al., 1997), banana (Golding et al.,
1998; Wyllie et al., 1998) and plums (Abdi et al., 1998).
Contents of hexane-extractable a-famesene and its major catabolite CTOL in the skin tissue were 2- to 3-fold lower in 'McIntoshYthan 'Delicious' apples at 120 days after
123 storage (Table 4-23). The inhibition of accumulation of a-famesene in the skin by 1-
MCP (1 p~.~-l)compared to untreated apples was more pronounced in 'Delicious' (97%) than in 'Mchtosh' (64%) apple (Table 4.2.3). Interestingly. the incidence of scald on 1-
MCP ($1 p~*~-')-treated 'McIntosh7 and 'Delicious' was, respectively, 30% and 90% less than untreated apples (Table 4-24).
Though the precise rnechanism of superficial scald development is stilI obscure. it is believed that scald is a result of darnage to hypodermal cells from a-faniesene oxidation products. mainly CTOL (Rowan et al.? 1995). A substantial amount of literature has accumulated supporting the conception that Cl& is critically involved in scald development through stimulation of the level of a-fmesene and conjugated trienes in fi-uit peel (Meigh and Filmer. 1969; ffiee and Hatifield, 198 1; Little et al.. 1985; Liu.
1986; Wadüns et al., 1993; Du and Bramlage, 1994). Recently, Gong and Tian (1998) reported that the Cr& action inhibitor DACP drarnatically reduced the accumulation of a-fmesene and conjugated triene, and inhibited the development of superficial scald in
'Granny Smith' apple. We also found a strong relationship between C2H4. u-famesene.
CTOL and scald in 'Delicious' apple but not in 'McIntosh' (Table 4-23). The poor correlation between a-farnesene and scald in 'McIntosh' may be due to an alternative mechanism for scald development not invoiving a-famesene metabolism. Rao et al.
(1998) suggested that superficial scald is also related to the cellular effciency in metabolizing active oxygen species. Iri future work, 1-MCP couid be used as a mode1 to investigate such CzHa-dependent biochemical events in relation to superficial scald development. Table 4.2.3. a-Famesene and conjugated triene alcohol (CTOL) contents in skin tissue of 'Mc1ntosh'- and 'Delicious' apples treated with 1-MCP a day after hanrest and stored in air at O - 1 OC with 90 - 95 % relative humidity for 120 days. Analyses were done immediately upon removai of the fniit from storage. Data are the means of triplicate analyses.
-- I -MCP concentration (p~.~-l)
'Delicious' 766 672 569 22 14 10
SEM = 9.6; n=3, df = 24 Significance c". T". C x T"
CTOL content (pg. g-fFW)
'Delicious' 3 17
SEM = 7.0; n=3, df = 24 Significance c"', T"', C x T"'
58 885 Significant at P ( 0.0 1 or 0.00 1, respectively, where C = cultivar and T = 1-MCP concentration Table 4.2.4. Incidence of superficial scaId in 'Mchtosh' and 'Delicious' apples treated with 1-MCP and stored 120 days in air at O - I OC with 90 - 95 % relative hurnidity. Scaid wai assessed afier fitwere removed from cold storage to 20 OC for 7 days. Data are the mean of 3 repIicates each containing 25 fruit.
Cultivar 1-MCP concentration ($.L-')
'~caldincidence (%fruit affecteu')
'Delicious' 91 89 78 29 32 14
SEM of transformed data = 3 -5;n=3. df = 24 Significance T"', C x T*"
'~rcsinsquare root percentage transformation with detransformed rneans presented. *** Significant at P 5 0.00 1, where C = cultivar and T = 1-MCP concentration Table 4-25. Correlation (r') of mean C2& production at removal time 1 with six postharvest measurements in 'Mchtosh' and 'Delicious' apples treated or untreated with 1-MCP.
Measurement 'Mcintosh' 'Delicious'
Correlation co-eflcienr (r2)
Flesh firmness 0.99 0.99
Total volatiles 0.94 0.99
Head-space cc-farnesene 0.9 1 0.99
a-Famesene in the skin 0.97 0.99
CTOL in the skin 0.87 0-94
Scald incidence 0.29 0.94 'Mchtosh' apples treated with 25 ~.L.L-' 1-MCP one day after placing in CA
storage at 3 OC showed similar effects to apples which had been treated with 1-MCP at 20
OC and placed into cold storage (Table 4.26). Flesh fimess of 1-MCP-treated
'Mchtosh' apples after CA storage was about 50% higher than that of untreated apples held in CA. However, 1-MCP had only a marginal effect on scald incidence of CA-stored
'Mchtosh' apples due in part to the reduction of scald by CA storage.
In conclusion, the novel Czi& antagonist 1-MCP profoundly blocked C2E& action and delayed Cz&-dependent ripening and superficial scald development in 'Mclntosh' and 'Delicious' apples. The significant reduction of sofiening in 'McIntosh' apples which has poor storability due to excessive sofiening, and a 90% reduction in scald incidence in
'Delicious' apples indicates great potential of incorporating 1 -MCP into long-term storage of apples. 1-MCP did not affect total soluble solids, but reduced total volatile evolution which could negatively affect apple aroma. Therefore. hrther research is needed to study the effect of 1-MCP on character impact volatiles and to develop procedures to recover, if necessary, the charactenstic apple aroma. Table 4.2.6. Response of 'McIntahYapples to 25 p~.~-L1-MCP in CA storage (3 % O2 + 4.5 % CO2 at 3 OC with 90-95 % relative humidity). Apples were removed fkom CA storage afier 120 days and kept Noweeks at 20 OC. Estimation of parameters were done as descnbed for air-stored apples.
Parameter Untreated 25 p~.~-l1-MCP significance control 1-MCP relative to control
Mean prodzrction over 13 days at 20 OC (dekg-' F ~.h-')
FZesh firmness (iV) at harvest I day after removal 7 days oflei- rernoval 14 days ajier- removal
Mean total volatik prodzrction over 13 days at 70 OC (rinit. kg-'~R?l
Mean œ-famesene evolrrtion over 13 days at 20 OC (mit. kf'~rti) cc-Farneseile conlent in the skin (pg. g-'FW
CTOL content in the skin (pg. g"f~
Scald incidence (% frzrit ofected)
** *** , , Significant at P 5 0.05, 0. 01 or 0.00 1, respectively; NS: No Abdi, N., E.B. McGlasson, P. Holford, M. Williams, and Y. Mizrahi. 1998. Responses of climacteric and suppressed-climactenc plums to treatment with propylene and 1- methylcyclopropene. Postharvest Biol. Technol. 1429-39. Abeles, F.B. and F. Takeda. 1990. CeIIulase activity and ethylene in ripening strawberry and apple fruits, Sci, Hort. 42269-275. Berger, R.G. 199 1. Fruits 1, p. 283-304. In: H. Maarse (eds.). Volatile compounds in foods and beverages. Marcel Dekker, New York. Blankenship. S. M. and C.R. Unrath. 1998. Ethylene inhibitor, 1-methylcyclopropene. delays apple softening. HortScience 33 :469 (Abstr.) Du, 2. and W.J. Bramlage. 1994. Role of ethylene in the development of superf~cialscald in Tortland' apples. J. Amer. Soc. Hort. Sci. 1 19516-523. Fan. X.. J.P. Mattheis. and D. Buchanan. 1998. Continuous requirement of etliylene for apple fmit volatile synthesis. J. Agi. Food Chem. 46: 1959- 1963. Golding, J.B., D. Shearer, S.G. Wyllie, and W.B. McGlasson. 1998. Application of 1- MCP and propylene to identify ethylene-dependent ripening processes in mature banana Fruit. Postharvest Biol. Technol. 14:87-98. Gong, Y. and M.S. Tian. 1998. Inhibitory effect of diazocyclopentadiene on the development of superficial scald in 'Granny Smith' apples. Plant Growth Reg. 26:117-121. Knee, M. and S.G.S. Hatfield. 198 1. Benefits of ethylene removal during apple storage. Am. Appl. Biol. 98: 157- 165. Lau. O.L. 1990. Effkacy of diphenylamine, ultra-low oxygen, and ethylene scrubbing on scald control in 'Delicious' apples. J. Amer. Soc. Hort. Sci. 115:959-961. Leliovre, J-M.. A. Latchi, B. Jones, M. Bouzayen. and J-C Pech. 1997a. Ethylene and fruit ripening. Physiol. Plant. 10 1:727-739. Leliovre, J-M.. L. Tichit?P. Dao, L. Fillion, Y-W. Nam, J-C. Pech, and A. Latchi. 1997b. Effects of chilling on the expression of ethylene biosynthetic genes in Passe- Crassane pear (Pyrus commzinis L.) fnuts. Plant Mol. BioI. 338474355. Little, CR.. H.J. Taylor? and F. McFarlane. 1985. Postharvest and storage factors affecting superficial scald and core flush of 'Granny Smith' apples. HortScience 20: 1080- 1082. Liu, F.W. 1986. Responses of Daminozide-treated 'Delicious' and 'Idared' apples to sirnulated low-ethylene CA storage. J. Amer. Soc. Hort. Sci. 1 1 1 :7 16-71 9. Lurie, S.. S. Meir. and R. Ben Arie. 1989. Preharvest ethephon sprays reduce superficial scald of 'Granny Smith' apples. HortScience 24: 104-1 06. Meigh, D.F. and A.A.E. Filmer. 1969. Natural skin coating of the apples and its influence on scald in storage. III. a-Famesene. J. Sci. Food Agi. 20: 139-143. Nakatsuka A.. S. Shiomi. Y. Kubo. and A. Inaba. 1997. Expression and interna1 feedback regulation of ACC synthase and ACC oxidase genes in ripening tomato hit. Plant Ce11 Physiol. 38: 1103-1 1 10. Nakatsuka, A., S. Murachi, H. Okunishi, S, Shiomi, R. Nakano, Y. Kubo. and A. Inaba. 1998. Differential expression and intemal feedback regdation of 1- aminocyclopropane- 1-carboxyiate synthase, 1-arninocyclopropane- 1 -carboxylate oxidase, and ethylene receptor genes in tomato bitduring development and npening Plant Physiol. 118: 13%- 1305. PaIiyath, Cr. MD. Whiting, M. Stasiak, D.P. Mun, and B.S. Clegg. 1997. Volatile production and fruit quality during development of superficial scaid in 'Red Delicious' apples. Food Res. Intl. 30:95-103. Rao, M.V.. C.B. Watkins, S.K. Brown, and N.F. Weeden. 1998. Active oxygen species metabolism in 'White Angel' x 'Rome Beauty' apple selections resistant and susceptible to superficial scald. J. Amer. Soc. Hort. Sci. 123299404. Ross, G.S., M.L. Knighton, and M. Lay-Yee. 1992. An ethylene-related cDNA from ripening apptes. Plant Mol. Biol. 19231-238. Rowan, D.D.. J.M. Allen, S. Fielder, J.A. Spicer, and M.A. Brimble. 1995. Identification of conjugated triene oxidation products of a-farnesene in apple skin. J. Agr. Food Chem. 43 2040-2045. Rupasinghe. H.P.V.. G. Paliyath, and D.P. Murr. 1998. Biosynthesis of a-farnesene and its relation to superficial scald development in 'Delicious' apples. J. Amer. Soc. Hort. Sci. 123:882-886. Sisler, E.C., E. Dupille, and M. Serek. 1996. Effect of 1-metl~ylcyclopropeneand methylenecycloprpane on ethylene binding and ethylene action on cut carnations. Plant Growth Reg. 18:79-86. Sisler, E.C. and M. Serek. 1997. Inhibitors of ethylene responses in plants at the receptor level: Recent developments- Physiol. Plant. 100577-582. Sisler, E.C. and M. Serek. 1999. Compounds controlling ethylene receptor. Bot. Bull Acad. Sinic. 40: 1-7. Song, J., M.S. Tian, D.R. Dilley, and R.M. Beaudry. 1997. Effect of 1-MCP on apple fniit ripening and volatile production. HortScience 32536 (abstr.) Watkins, C.B., C.L. Barden, and W.J. Bramlage. 1993. Relationships among a-famesrne. conjugated û3enes, and ethylene production with superficial scald development of apples. Acta Hort 343 :155-1 60. Whitaker. B.D., T. Solornos, and D.J. Harrison. 1997. Quantification of a-famesene and its conjugated trienol oxidation products from apple peel by C 18-HPLC with W detection. J. Agr. Food Chem. 45:760-765, Wyllie, S-G,J.B. Golding, W.B. McGlasson, and M. Williams. 1998. The relationship between ethylene and aroma volatiles production in ripening climacteric fmit. Developments in Food Science 4O:3 75-3 84. CHAPTER V Cloning and Expression of hmgl and kmg2 cDNA Clones Encoding 3-Hydroxy-3-MethylglutarylCoenzyme A Reductase (HMGR) and its Activity in Relation to a-Farnesene Synthesis in Apple Submitted to Plant Physiol. Biochem. (2000) ABSTRACT In plants, 3-hydroxy-3-rnethylglutaryl coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate (MVA) from 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA), is the rate limiting enzymes in sesquiterpene and triterpene biosynthesis, and is encoded by a small gene family. The accumulation of a-farnesene in the skin tissue of apple fruit during storage is predominantly through the classical mevalonate pathway, and not through the novel glyceraldehyde-3- phosphate (GAP)/pyruvate pathway independent of HLMGR action. The content of a-farnesene in the skin increased during the first 12 weeks in storage at O°C in air, and then decreased. In contrast, MMGR activity in the total membrane and soluble fractions kvas the highest at the time of harvest, but decreased during the first 8 weeks in storage, and remained stable thereafter. The potent ethylene (C2H4) action inhibitor 1-rnethylcyclopropene (1-MCP) inhibited a-farnesene evolution and HMGR activity by 97 and 30 %, respectively. As the first step in studying the molecular mechanism of apple HMGR regulation, we isolated and cIoned a full- lengh (hmgl) and a fragment (hmg2) of cDNA using apple skin mRNA and 134 degenerate oligonucleotides designed against conserved regions of plant HMGH genes. Genomic Southern analysis using probes designed for the 3'-end of the hvo cDNA clones confirmed the presence of at least two HMGR geoes in apple. The transcription product of hmgl cDNA has an open reading frame of 1767 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 protein indicates the presence of two highly hydrophobie domains near the amino terminus. Northern blot analysis confirmed that the both Iimgl and Img2 transcripts possessed a size of 2.4 kb. The two genes are differentially expressed during low temperature storage and in response to Cz&, with lzmgl being expressed constitutively and hm& being relatively more sensitive to developmental stimuli and c2E4- INTRODUCTION A highly conserved enzyme in eukaryotes, 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), catalyzes the conversion of 3-hydroxy-3-methylglutar).lcoenzyme A (I-IMG-CoA) to mevalonate (MVA), the rate limiting step of isopentenyl diphosphate (IE'P) biosynthesis (Chappe11 et al., 1995; Lichtenthaler et al., 1997). However, in addition to the mevalonate pathway, IPP is derived through a non-mevalonate pathway (Rohmer or glyceraldehyde-3-phosphate (GAP)/pymvate pathway) in certain isoprenoid pathways (Lange and Croteau, 1999; Lichtenthaler et al., 1997; Rohmer et al., 1993). In general, the classicd cytoplasmic mevalonate pathway provides IPP for sesquiterpenes and triterpenes (Lichtenthaler et al., 1997). The Rohmer pathway is believed to be responsible for the formation of al1 plastid-derived isoprenoid compounds in plants, including carotenoids. plastoquinones, the prenyl side chains of chlorophyll (Kreuzwieser et al., 1999; ~ichtenthaleret al., 1997; Rodriguez-Concepcion and Gruissem, 1999), as well as monoterpenes (Lange et al., Z 998) and diterpenes (Eisenreich et al.. 1996). In higher plants, HMGR is encoded by a multigene farnily in nuclear DNA (Godoy-Hernandez et al., 1 998; Lichtenthaier et al.. 199 7;Stermer et al,, 1 994). HMGR is encoded by at least two distinctive genes in Arabidopsis haliana (Caelles et al.. 1989; Monfar et al.. 1990). Cotton (Gos.ypittrn hirszltzrrn L.) (Loguercio et al.. 1999), and rice (Oïyzu sativcr) WeIson et al.. 1994); by three genes in rubber (Hevea brusiliensis) (Chye et al., 1992), tomato (Lycopersicon esculentum) (Weissenborn et al., 1995; Yang et al., 199 1). and potato (Solanzun tzrberoszrm) (Choi et al., 1992); and an even larger complex muItiple gene family in maize (Zea mays) and pea (Pisum sativzrm) (Weissenborn et aI.. 1994). KMGR isoforms are expressed differentially in response to a variety of developmental and environmentai stimuli. such as fmit development. phytohormones. endogenous protein factors. light. and infection (Stenner et ai.. 1994). In tomato. hmgl is highly expressed during early stages of fruit developrnent, when stero1 biosynthesis is required for membrane biogenesis during ce11 division and expansion (Narita and Gruissem, 1989); whereas hmg2 expression is not detectable in young fruit. but is activated during fmit maturation and ripening (Gillaspy et al.. 1993; Rodriguez- Concepcion and Gruissem, 1999). Hmgl mEWA of A. thaliana accumulates in al1 parts of the plant, while the presence of hmg2 mRNA is restricted to young seedlings, roots? and inflorescences (Enjuto et al.. 1994). Cotton hmg2 encodes the largest of a11 plant HIMGR enqmes described to date, and contains several functional specialization features that include a unique 42 amino acid sequence located in the region separating the arnino- terminal domain and carboxy-terminal catalytic domain, that is absent in hmgl (Loguercio et al- 1999). The presence of multiple HMGR genes in plants is consistent with the hypothesis that different isoforms of HMGR could be involved in separate subcellular pathways to produce specific isoprenoid end-products (Bach, 1993; Chappell et al.. 1995: Srere. 1987: Stermer et al.. 1994). It has been suggested that some of these isoforms may be involved in separate subcellular pathways for specific isoprenoid biosynthesis throu,oh metabolic channels, or 'metabolons' (Sterrner et al., 1994). A nurnber of investigators have reported a correlation between the induction of isoprenoid biosynthesis, particularly of sesquiterpenes. and HMGR enzyme activity (Chappell et al.. 1995). In potato. the expression of specific HMGR genes has been correlated with the accumulation of steroids or sesquiterpenes (Choi et al., 1992). Chye et al. (1992) observed that only hmgi \vas inducible by CrH4 among other HMGR genes. and also speculated that distinct isoprenoid pathways do occur for rubber biosynthesis in brasiliensis. In cotton. hn7g.2 has been be associated with the synthesis of specific sesquiterpenes in developing embryos (Lo,ouercio et al., 1999). These results also support the concept of metabolic channels. or arrays of isoenzymes. independently regulated and specifically dedicated to the production of particular isoprenoids (Chappell. 1995). The acyclic sesquiterpene a-farnesene (CIjHa; [3 E,6E]-3.7,11 -trimethyl- 1,3,6,10- dodecatetraene) accumulates in the skin of apple fruit exclusively after harvest during low temperature (0-1 OC) storage (Rupasinghe et al., 2000a). The estent of oxidation of a- farnesene to conjugated trienes has been shown to'be proportional to the development and severity of the postharvest physiological disorder superficial scald (Chen et al., 1990; Huelin and Coggiola 1970; Meir and Brarnlage, 1988). Biosynthesis of a-famesene in apple skin is highly regulated by temperature (Rupasinghe et al.. 2000a) and C2H4 (Gong and Tian. 1998; Rupasinghe et al., 2000b,c). However, biochemical and molecular mechanisms of modulation of HMGR activity in relation to a-famesene biosynthesis of apple has not reported reported. Identification of regulatory factors of HMGR and cloning of HMGR gene(s) that mat potentially be directed to a-famesene biosynthesis will provide important insight into the understanding and the role of a-fmesene in supeficial scaid development in apple. Therefore, as a first step towards understanding the regulation of a-famesene accumulation by HMGR, total N? vitro HMGR enzyme activity and expression of two novel cDNA clones, hmgl and hmg2, encoding HMGR were snidied in the skin tissue of apple in relation to low temperature storage and C2HJ action. MATERIALS AND METHODS Plant material and postharvest treatments 'Delicious' apples (Malus x domesrica Borkh.) were harvested on October 05. 1999. from the Horticul~ralResearch Station of the University of Guelph, Simcoe. Ontario [Harvest maturity indices: firmness, 73.3 N; soluble solids, 10.3 %; starch-iodine index (~omell'chart),3.21. Apples were transported to Guelph and were cooied to O OC within 8 h of harvest and stored at 0-1 OC in air with 90 to 95% relative humidity (RH). These apples were used for monitoring HMGR activity and expression during storage. For HMGR inhibition snidy, one sample of apples (120 fhits) was dipped for 20 min in D.I. water containing 200 rnge~-'Lovastatin (Merck Frosst) with 0.5 IILL-'ABG 701 lTM(Abbon Laboratories) as surfactant. A second sample of 120 fniit was dipped only in D.I. water with surfactant (control). To snidy the effect of Czl& inhibition. apples were allowed to equilibrate to 20 OC overnight after harvest and exposed to O or 600 nL-L- ' 1-MCP (EthylBiocTM,generously supplied by Dr. Harlow Warner, Rohm and Haas Inc.) for 18 h. Following treatrnent, the apples were placed in cold storage at O OC and 90 to 95% RH for 60d. For the precursor channeling study, either 0.25 Ki ~[5-~~]rnevalonicacid (20 ~immol-' ; ARC) or a mixture of 0.25 pCi [2-14~]pynivicacid (5 rn~i~mmol-' ; ARC) and 25 mM GAP (Sigma) dissolved in 10 mM MES buf3er (pH 5.6) were vacuum infiltrated (2 min) into isolated apple skin tissues (6 discs each 2 mm thickness and 1 cm diameter) and incubated at 20 OC for 90 min. a-Famesene was isolated by incubating the treated discs in hexane ovemight at 4 OC. Apples treated with Lovastatin (200 rngm~-')at harvest and stored for 10 weeks in storage at O OC in air were used for the experiment. To veri@ for the potential non-enzyrnatic.incorporationof radiolabel into a-famesene. controls with boiled apple skins were used. HMGR enzyme assay A modification of the method described by Chappe11 et al. (1995) was used. Approximately 200 g of outer cortical tissue (0.25 to 0.5 cm depth) including the cuticle was removed and cut into 0.25 to 0.5 cm3 pieces and homogenized using a Polytron (BrÏnkrnann Instruments, mode1 PT 10/35) for 1 mui in 150 mL of 0.1 mmo1-~-'MOPS buf6er (pH 7.5) containing 0.25 MOLL*'sucrose, 1 rnrnole~-~EDTA, 5 rnrnole~-~DTT. 5 mmol-~-'MgCI2, 1 mmol=~-'PMSF, 3 % BSA, and 50 ge~-' polyvinylpyrroIidone. The homogenate was filtered through four Iayers of cheesecloth, and the filtrate was centrifuged at 3000 g. for 10 min to remove starch and debris. The supematant was centrifuged at 105.000 g, for 45 min. and the resulting pellet was resuspended in 0.1 mole^-' sodium phosphate buf5er (pH 6.5) containing 75 rnrnole~-' DTT. This and comprised the total membrane fi-action (Edward and Gardestrom, 1987) and the supernatant was considered as the soluble fraction. Aliquots of total membrane or supematant equivalent to 100 pg of protein (Bradford. 1976) were incubated for 45 min at 3 0 OC in a final assay volume of 250 pL containing IO0 mrnol*~-~sodium phosphate buffer (pH 7.0), 3 rnmol.~-~NADPH. 10 mmoL~-'DTT, 20 prnolmL-' HMG-CoA, and 0.025 pCi DL-13-"CIHMG-COA (58 rn~i=mrnol-*; Arnersham). The assay was teminated by addition of 20 pL of 5 rnrno1.~-' mevalonate lactone and 50 pL of 6 N HC1 followed by vortexing. The mixture was incubated for an additional 30 min at room temperature to allow for any radiolabeled mevdonate formed to Iactonize. Afier addition of 100 pL of saturated potassium phosphate (pH 6.0) and 300 pL of ethylacetate, the sarnples were briefly vortexed and centrifuged, and an aliquot of the upper organic phase was used to deterrnine radioactivity by scintillation counting. The chernical nature of the product was conformed by analyzing the ethylacetate fraction on a thin layer chromatography plate, which was developed completely with chloroform:acetone (21, v/v), and measuring the radioactivity in the MVAL-containing zone (RF 0.6). The assay was performed in triplicate. 140 RNA isolation and cDNA construction Five grams of appIe skin tissue (0.5 to 1 mm thickness) was ground to a fine powder with a mortar and pestle in Iiquid Nz. The powder was added immediately to 15 mL of preheated (65 OC) lysis buffer (150 rnM Tris, 50 mM EDTA, 4% SDS. 2% PVP, and 1% (vh) P-rnercaptoedianol, adjusted to pH 7.5 with boric acid) and vortexed for 30 sec at room temperature. The suspension was homogenized with a Polytron at maximum speed for 20 sec. Cold absolute ethanol (0.25 volumes) and 5 M potassium acetate (0.1 volumes) then were added and mixed for a Mer 30 sec. One volume of chloroform:isoamyl alcohol (24:l) (Sigma) was added to the homogenate, mixed with vortex stirring and then centrifuged at 20,000 g for 10 min at 4 OC. The recovered aqueous phase was extracted twice with an equal volume of Tris (pH 8.0) equilibrated phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma). The RNA was selectively precipitated by adding LiClz to a final concentration of 3.2 rnoL~-'and incubating ovrrnight at -20 OC. RNA was pelleted by centrifugation at 10.000 g for 20 min at 4 OC. The pellet was resuspended in 2 mL of DEPC-treated water, and sodium acetate (final concentration of 0.3 mole^-') and 6 mL of cold absolute ethanol were added. Afier I h incubation at -20 OC. RNA was pelleted by centrifugation for 10 min at 12.000 g and the pellet was resuspended in 200 pL of RNase-free water and stored at -80 OC. mRNA was synthesized using a OligotexTMmRNA Spin-Column kit (Oligotes). The total RNA utilized for mRNA \vas extracted from the skin tissue of 'Delicious' apples stored 12 weeks in air at O OC. Synthesis of cDNA by reverse transcription of mRNA was performed using a SMARTTURACE cDNA amplification kit (CLONTECH). A 20 pl, aliquot of reaction mumire contauiing 1 pg of rnRNA, 1 pg Oligo (dT) and sterile distilled water was heated to 70 OC for 2 min and chiiled quickly on ice. Afier a bnef centrifugation of the tube, 4 pL of the first strand buffer (5x), 2 pL 0.1 molm~'DTT, and 1 pL 10 mmol~~"dNTP xnk (10 rnmo1.~-' each dATP, dGTP, dCTP, and dTTP at neutral pl!?) were added. The contents were mixed gently and RNase H Reverse transcriptase was added and incubated for 90 min at 42 OC. The reaction was inactivated by heating at 72 OC for 7 min afier adding 100 PL of tricine-EDTA buf5er. The cDNA was stored at -20 OC. Cloning of apple Izmgi and /.mg2 Conserved regions of the plant HMGR gene farnily were identified by multiple sequence alignment of the predicted arnino acid sequences of five selected HMGR genes (GI 167488, Catharanthtcs roseus; GI 169485, Solanltm tttberosttm; GI I 763234. Camprotheca acuminata; GI 1 9746, Nicatiana sylvesrris; and G I 20723 22, Oryzu sarii-n) using the BIock Maker program. Highly conserved regions were assigned to the consensus-degenerate hybrid oligonucleotide primer (CODEHOP) algorithm (Rose et al.. 1998) and the foI1owing pIant-specific HMGR-degenerative oligonucleotide sequences were generated; the fonvard primer, 5 ' -GCTTCTGTTATTTATCTGCTGGGATT(C/T) TT(C/T)GG(AIG/C/T)(NC/T)T-3 ' (ASVIYLLGFFGI) and the reverse primer being, 5 ' -AGCAACCAGACATCCTTCAGTAGT(A/G/C/T)GCCAT(NG/C)GG-' (PMAT TEGCLVA). The poIdymerasechain reaction (PCR) was carried out using an AdvantageTM cDNA PCR kit (CLONTECH) with apple cDNA as the template in the high fidelity buffer. The PCR reaction conditions were: 1 min at 94 OC, plus 35 cycles at 94 OC (30 142 sec). 68 OC (2.2 min) and a final extension step at 72 OC for 7 min. The resultant 349 bp PCR product (hmgl) was used to design fonvard and reverse HMGR-specific prirners for 3 '- and 5 ' -RACE, respectively (SMARFM RACE cDNA amplification kit' CLONTECH). These two fragments were then used to design primers (forward 5'- TGTCCTTTCCTCTTCTCTCCTCCGCC-3' and reverse 5 ' -CTTCAAGCTCAGA AGTTAGAGCCT'TTC AAGTTC-3 ' ) to obtain the a full length cDNA clone (hmgT).To obtain the £kgment of hg2 cDNA clone, additional degenerative oligonucleotide primers (forward) was designed to the 3 ' region: 5 ' -GCTCCACCGGTGACGCTA TGGG(A/G/CTT)TGAA-3 ' (CSTGDAMGMN), TCCCACTGCATCACCATGATGGA (A/G)GC-3 ' (SHCITMMEA) and 3 ' RACE were performed by the procedure described above. DNA sequences were detemined after subcloning the PCR product into E-coli using an AdvanTApe cloning kitTM (CLONTECH), and by the chain termination me thod (Sanger et al., 1977). The sequence of both strands was determined using synthetic oligonucleotide primers. DNA sequences were analyzed using the MBS on-line tools. Construction of DIG-UTP RNA probes Gene specific probes were prepared for hmgl (309 bp) and hm@ (385 bp) using PCR with oligonucleotide primers drsigned to carboxy-terminal coding region and the 3 '-untranslated region. The forward and reverse primer pairs are: 5' - CACGTGTCTGTCACCATGCCTTCAATT-3 ' and 5 ' -CTTCAAGCTCAGAAGTTAG AGCCTTTCAAGTTC-3 ' for hmgl, and 5 ' -CCTATCGACGGCAAGGACCTTCATG T-3 ' and 5 ' -CCCGAACCGTCGAGCTTACTTATTTCTCT-3 ' for hmg2. Antisense vanscripts of each probe were made in vitro in the presence of digoxigenin (DE)- 143 labelled-UTP using a MAXIscnptTMin vitro transcription Kit (Ambion) and DIG RNA labeling mix (Roche). Southern blot analysis using apple genomic DNA Genomic DNA was extracted using DNeasy Plant ~axi~kit (Qiagen) fiom buds and immature leaves of 'Delicious' apple to improve the yield of DNA. DNA was digested with 3 restriction endonucleases EcoRI, BumHI, and Pst1 (Fermentas), and the resultant fragments were separated by electrophoresis in 0.8% (wh) agarose gels. The gels were blotted onto a positively-charged ~n~ht~tar-plusTMmembrane (Arnbion) using ~outhern~ax~~Southern blotting kit (Ambion). The membranes were prehybridized in ultrahybTMhybridization buffer (Ambion) for 1 hr at 50 OC then hybridized with digoxigenin-labelled apple hmgl and hmg2 RNA probes at 50 OC for 16 hrs. The membrane was washed twice in 2x SSC, 0.1 % SDS for 5 min at room temperature (low stringency) and twice in 0.1 x SSC. 0.1 % SDS for 15 min at 50 OC (high stringency). Cherniluminescent detection was performed as described in the DIG Northern Starter kit (Roche). Expression analyses using Northern blots For northern blot analysis, 10 pg of total RNA frorn apple skin was denatured at 65 OC for 10 min in RNA sample loading buffer (Sigma). separated by electrophoresis in 1.0% (wh) formaldehyde agarose gels, and blotted ont0 ~ri~ht~tar-~lus~~membrane (Ambion) using bIorthernMaxTMnorthern blotting kit (Arnbion). The RNA bound to the membrane was cross-linked by baking at 80 OC for 15 min. The membranes were 1 44 prehybndized in ~ltrah~b~hybridization buffer (Arnbion) for 30 min at 68 OC then hybridized with digoxigenin-labelled apple hrngl and hmg2 RNA probes at 68 OC for 16 hrs. The membrane was washed twice in 2x SSC, 0.1 % SDS for 5 min at room temperature (low stringency) and twice in 0.1~SSC, 0.1 % SDS for 15 min at 68 OC (high stringency). Cherniluminescent detection were performed as descnbed in the DIG Northern Starter kit (Roche). RESULTS HMGR activity in apple skin during storage The content of a-farnesene in the skin of 'Delicious' apples increased during the frst 12 weeks in storage at O OC in air. and then decreased (Fig. 5.1). In contrast, HMGR activity? as determined by the conversion of [4-3~~~~CoA to MVA in the total membrane and soluble fraction, was highest at the rime of harvest and gradually decreased during the first 8 weeks, and then remained constant during the rest of the storage period (22 weeks). Incorporation of radiolabelled-IPP precursors To determine whether the glyceraldehyde-3-phosphate (GAP)/pyruvate pathway exists in the skin tissue of apple fruit to generate IPP durhg the time HMGR activity declined, ~[~-~~]rnevalonicacid or a mixture of [2-''~]~yruvicacid and GAP was incubated with isolated apple skin tissues. Incorporation of radiolabel into a-famesene was 17-fold higher with mevalonic acid compared with the mixture of pyruvic acid and Weeks in storage Fig. 5.1. a-Famesene content (top) and in vivo HMGR activity in the membrane fraction (a), and the soluble fraction (O) (bottom) of the skin of 'Delicious' apples during storage at O OC in air. All parameters are expressed on a fresh weight bais and each data point is the mean of three replicates. GAP (Table 5.1). As well, in a separate study when apple skin discs were incubated with unlabelled MVA, or a mixture of GAP and pyruvate, a-famesene levels analyzed by reverse-phase HPLC fiom hexane extract of the incubated skïn tissue was higher in MVA-treated skins compared with that of GAP and pyruvate-treated skins (data not presented). Inhibition of HMGR activity by Lovastatin When apple fmit were treated with Lovastatin (1000 ppm), a cornpetitive inhibitor of HMGR. a-famesene accumulation in the skin was suppressed by 23 to 54 % dunng storage (Table 5.2). Lack of a complete inhibition of a-farnesene synthesis by Lovastatin could be due to poor uptake and incorporation of the inhibitor into the cells of apple skin where HMGR is located. These results together imply that in apple fruit, the biosynthesis of a-farnesene occurs predominantly through the classical MVA pathway and not through the GAPlpyruvate pathway. Relation behveen CzHJand HMGR activity Apples treated with the C2& action inhibitor. 1-MCP (0.6 p~m~-l)afier harvest exhibited complete inhibition (100%) of Czl& production upon removal from storage (Table 5.3). a-Farnesene content in the skin and in the head-space volatiles of I-MCP- treated apples was inhibited by 72% and 100%, respectively. In ilifroactivity of HMGR in apple skin tissue was inhibited by 30% when C2& action was blocked by 1-MCP. In addition, 1-MCP inhibited respiratory CO2 evolution by 50% (Table 5-3) which suggests Table 5.1. Metabolic responses of apples treated with the C& action inhibitor. 1- methylcyclopropene (1-MCP, 1 ~L,.L-'). Each variate represents the mean value of response measured triplicate at 1, 5, and 1I day at 20 OC following removal from storage for $0 days at O OC in air. Metabolic response Untreated 1-MCP Inhibition Significance C25production (pL/kg FW/h) 114 0.49 - 100% **ic HMGR activity (nrnohg proteinh) 3 7.4 26.1 3 0% * a-Farnesene content in the skin (pg/g FW) 62 1 126 72% * a-Famesene evolution (unitkg FW) 7.23 0.0 1 -100% **+ CO2 production (mL/kg/h) 8.6 3.5 59% *** *, **, *** Signifrcant at P<0.05,0.01 or 0.001, respectively. Table 5.2. a-Famesene content in the skin of apples treated with or without Lovastatin (200 rng*~-'),a specific HMGR inhibitor at harvest and stored at O OC storage in air. Dara represent mean of 3 repkates t SD. Treatment Weeks at O OC storage Total a-Farnesene content in the skin (pg/g Fw Control 396 k 35 567 t 48 446 I 39 1409 Lovastatin 182 5 21 277 +, 31 338 t 26 797 Table 5.3. Incorporation of radiolabel fiom precursors of isopentenyl diphosphate (IPP) into a-farnesene in isolated skin tissue of Lovastatin-treated (200 rngrn~-')apples. Data represent the mean of 3 replicates 1 SD. Precursor Radioactivity incorporated into Ratio a-farnesene (dpm) ~[~-~~]rnevalonicacid 8154 t 790 17: 1 A mixture of [2-"CI 373 t 58 pyruvic acid and 25 mM glyceraldehyde-3- phosphate (GAP) that inhibition by 1-MCP of a-famesene synthesis in apple could also be through respiratory regulation, that provides substrate ace~lCoA for isoprenoid biosynthesis pathway. Isolation and analysis of apple cDNA for lzmgl and Izmg2 genes A 549 bp PCR product was obtained fiom the cDNA library made from mRNA isolated fiom the skin tissue of 'Delicious' apples which had been stored for 12 weeks at O OC in air, using degenerate primers designed and based on published higher plant HMGR sequences. The fragment was sequenced, its identity contiirmed as an HMGR gene fiagment, and it was used to design apple HMGR-specific prïmers for 5'- and 3 ' - RACE. Forward and reverse primers were designed to the resultant 5 '- and 3 '- fragments, respectively. to obtain a full-length cDNA (hmgl) with an open reading frame of 1767 bp (Fig. 5.7A). Apple hmgl rnRNA encodes a protein of 589 amino acids with an estimated molecular mas of 62.727 Da and an isoelectric point (pi) of 5.79. As a cornparison, the apparent molecular mass of HMGR protein of H. brasiliemis estimated by western biot analysis is 59 kDa (Chye et al.. 199 1). The deduced amino acid sequence of apple hmgl cDNA was aligned with that of other plant HMGR genes and showed very close sirnilarity (Fig. 5.2B). The predicted arnino acid sequence of apple hmgl shares 79% identity with hmgl gene of Campiofhecu acuminata (Burnett et al., 1993, 75% identity with hmgl of H. brasiliensis (Chye et al.. 1991), 80% identity with hmgl of G. hirsurum (Loguercio and Wilkins, 1998), and 71% identity with hmgl of A. fhaliana (Caelles et al., 1989). Among al1 identified plant HMGR genes, the N-terminal region differs greatiy both in length and amino acid 150 1 - ATGAAGGTGAAGGK;GK;GACCACGAGAACGACGITGGllGTC~CGGGGCCAAGGCCI:CC - 60 -MKVKVVDHENDVGVVGAKAS 61 - GACGCCcrCiCCGCTGCC~ACCTGA(=rAACGCCGTCIT~CACTCT~~~CC- 120 -DALPLPLYLTNAVFFTLFFS 121 - GTCL;T~A~CCTCC?TAffCGTTGGCGCGAGAAGATACCGACGCCGCC- 180 -VVYFLLTRWREKIRTSTPLH 181 - GTCGTTAACCITTCCGAGATCGTCGCGATACTCGCGTI'CGTCGCCTCCrrCATCTACITG - 24 0 -VVNLSEIVAILAFVASFIYL 24 1 - CTCGGA?TCTTCGGGATCGA~CGK;~CGffCATT~CCGCCCCAGCAATGAC~C- 3 0 0 -LGFFGIDFVQSLILRPSNDV 3 0 1 - TGGGCCGCCGACGATGACGAGGAGGAGCACGAGCGTAACGACGCCC - 3 60 -WAADDDEEEHERLILKDDAR 3 6 1 - AAA~CC~GGCCGGAcTCGACKiCAGCCCAATTCCCCAAA~GCCCCTGTK;~- 42 0 -KVPCGAGLDCSPIPQIAPVA 4 2 1 - GCI1(;CCGCCCCCAAAGCTCITGCACAG~ATAAAGAGGTAGTCCTCTCCACT - 4 8 0 -AAAPKAVAQKVFDKEVVLST 4 8 1 - CCCTGCGAT?3CACCGCCCAGCCGfiGACGGAGGAAGATGAGGAGGTGGTCAAGTCCGTG - 54 0 -PCDFTAQPLTEEDEEVVKçV 5 4 1 - GTGGCGGGAACCATCCCITCffACTCTCK;GAGTCAAAGCTKiGAGATK;CAGGAGGGCG - 6 0 0 -VAGTIPSYSLESKLGDCRRA 60 1 - GCGGCTATCAGGCGCGAGGCGCITCAGAGGATCACAGGAAAGTCKTGGGTGGTCTGCCA - 6 6 0 -AAIRREALQRITGKSLGGLP 661 - TTGGAGGGG;TCGAmACGAGTCAATITTGGGTG - 720 -LEGFDYESfLGQCCEMPVGY 721 - CfiCRGATTCCAGTfGGGATK;CTGGGCffCrrATGCTCGAZGCAGAGA~CCGTA- 78 0 -VQIPVGIAGPLMLDGREFSV 781 - CCAATGGCCACCACCGAAGGITGCTn;C;rn;CCAGCACCAACCGK;G~ChAAGffATC - 840 -PMATTEGCLVASTNRGFKAI 84 1 - AACTK;TCCGGCGGAGCCACCAGK;~CrC;AGAGATACAGCT- 9 0 0 -NLSGGATSVLLRDGMTRAPC 90 1 - GTGAGGITCAA(=rCTGCTAAGAGAGcrC;CCGAGTK;AAA~~ACTTGGAAGAACCCAAA - 9 6 0 -VRFNSAKRAAELKFYLEEPK 9 6 1 - AATTA%ACAC~GTCCACGGTTITCAACAGGTCAkGCAGATTCG~AGG~~ACA- 102 0 -NYDTLSTVFNRSSRFGRLQT 1O 21 - ATTAAGTGTGCCATK;ffGGGAAGAACrrGTACATGAGATTCACCTGCAGCACCGGTGAT- 1 O 8 O -1KCAIAGKNLYMRFTCSTGD 108 1 - GCTATCGGGATGAACATGGTCTCWGGTGIY;CAAAACGTCrn;GA?TTCCTCCAGAAC - Il4 0 -AMGMNMVSKGVQNVLDFLQN 1141 - GACTTCCCM;ACATGGATGn;ATfGGAA~CCGGCAACTACn;CTCTGACAAGAAGCCC - 1200 -DFPDMDVIGISGNYCSDKKP 120 1 - G~CAGM;AACTGGATPGAAGGCCGCGGCCGCGGCAAATCGGTGGTCrCTGAGGCKt"rGATCAAG - 126 0 -AAVNWIEGRGKSVVCEAVIK 12 6 1 - GGTGATGTGGTGCAGAAGGTGTTGAAAACC~ATGTGGCGTCCCTGTGCGAG~AACATG - 132 O -GDVVQKVLKTNVASLCELNM 13 21 - CITAAGAACCTTACTGGGTCX'GCAATGGCTGGAGCCCICC - 13 8 0 -LKNLTGSAMAGALGGFNAHA 13 9 1 - AGCAACATCGTCTCCGCCATCTACATCGCTACCGGCCA - 144 0 -SNIVSAIYIATGQDPAQNVE 14 4 1 - AGTTmCACTG~TTAC~ZATGGAAcc~T~TGA~A~ACtfi~c~CT- 15 0 0 -SSHCITMMEPINDGQDLHVS 1501 - GTCACCATGCCITCAATTGAGGTKGTACI'GTTGGT - 1560 -VTMPSIEVGTVGGGTQLASQ 156 1 - TCAGCITGTCK;AAC~CITGGAGK;AAGGC;TGCTAACAGGGAGGCACCAGGATCAAAT - 1620 -SACLNLLGVKGANREAPGSN 1621 - GCAAGA~GGCCA~CK;GTTCTGTTCTK;CIY;GRGAG~ffmCATG- 168 0 -ARLLATVVAGSVLAGELSLM 16 8 1 - T~GCTATCTCAGCK;GACAGCTK;TGAATAGTCACATGAAATACAACAGATCAAGCAAA - 174 0 -SAISAGQLVNSHMKYNRSSK 174 1 - GATGTCTCAGCTGTTGCATCCGCITAAqaactcqaaa~aa- 18 0 0 -DVSAVASAf 1801 - aagccgaaagcatggccggaacacgtctgaataafgccgacagaatcagtgttg - 1860 1861 - acctgqcacggagagaagaggatagggaaataatgaacagaacaaacatacag Fig. 5.2. (A) Combined nucleotide sequence and the predicted amino acid sequence of the encoded product of the cDNA clone corresponding to hmgl from M. x dornesrica cv. Deiicious. The amino acid sequence is shown in one-letter code below the corresponding codons. Lower case letters indicate untranslated regions and the asterisk (*) indicates the stop codon. Regions used to design the oligonucleotide primers to synthesize hmgl specific RNA probe are underlined. GenBank accession number is AF3 157 13. 10 6 0 70 8 O 90 LOO LOO 190 200 220 220 130 240 Fig. 5.2. (B) Alignrnent of deduced amino acid sequence of M. x dornestica hmgl with hmgl sequences of A. [haliana, (GI 123340; Caelles et al., 1989), C. acuminara (GI 28988 1 ; Burnett et al., 1993), G. hirszrtttrn (GI 2935298; Loguercio and Wilkins, 1998) and H.brasi1iensi.s (GI 18835; Chye et al., 1991). Dots denote spaces introduced to maximize alignment and black shaded amino acid stretches indicate regions identical among the five comparisons. sequence (Fig, 5.3B). The hydropathy profile of the protein, deduced by the algorithm of Kyte and Doolittle (1982), shows close sirnilarity to the typical structural features of other plant HMGR genes; e.g.. hmgl of A. thaliana (Learned and Fink, l989), H brasfiensis (Chye et al.? 199 1). and hmg3 of C. acurninata (Maldonado-Mendoza et al., 1997) (Fig. 5-3A). The presence of tsvo hydrophobie regions located at residues 22 to 46 and 67 to 86 (Fig. 5.3B), each of which is long enough to span a membrane bilayer, is conserved in other HMGR genes characterized in plants. HMGR is a membrane-bound protein (Bach, 1987: Gray, 1987), and it has been postulated previously that these N-terminal hydrophoic regions couId correspond to tram-membrane domains (Caelies et ai., 1989; Chye et al., 1992; Loguercio et al., 1999; Narita and Gruissem, 1989). Furthemore, the carboxy- temini of a11 plant HMGR genes are highly conserved and the catalytic site of the enzyme is located within this region (Chye et al., 1992). As a strategy to isolate other HMGR genes fiom apple hit, additional degenerative oligonuc leotides (forward) were designed to 3' region, and 3'-RACE were performed. A 565 bp fragment of cDNA clone (hmg2) was isolated (Fig. 5.4). and the coding region (303 bp) of hmg2 fragment showed 79% nucleotide sequence homology and 87% amino acid homology to previously identified apple hrngl. Amino acid position Fig. 5.3 (A) Hydropathy index plot of the predicted arnino acid residues of apple hmgl O, A. haliana hmgl (II), Hbrasiliensis hmgl Cm), and C. acuminata hmg3 (N).The average hydrophobicity of each arnino acid was calculated using the algorithm of Kyte and Doolittle (1982) over a window size of 9 residues, and was plotted as a function of amino acid position. Positive values indicate that free energy is required for transfer to water, hydrophobic regions. Bars I and 2 indicate probable two transmembrane domain (membrane-spanning) regions. Apple hmgl Fig. 5.3 (B) Schematic representation of the domain structure of apple HMGR isoform encoded by hmgl. Abbreviations: Hl and H2, highly conserved membrane-spanning sequences; LS, highly conserved Iurnenai sequence. Nurnbers inside the domains indicate the respective nurnber of arnino acid residues. 1 - GAGCCTATCGACGGCAAGGACCTll~TGTCTCAGTCACCATGCCTTCCATTGAGGTGGGA - 60 -EP~DGKDLKVSVTMPS~EVG 61 - ACAGTTGGAGGAGGAACCCAGCTTGCATCCCAAGCAGCCTGCTTGAACCT'GCTGGGTGTA - 120 -TVGGGTQLASQAACLNLLGV 121 - AAGGGTGCAAGCAGAGAGACTCGCCCGGTTCAAACTCTAGGAAATTGGC~C~TTGTAGCC- 180 -KGAsRDSPGSNSRKLATIVA 181 - GGTTCAGTGCTCGCCGGAGAGCTCTCGCTGATGTCAGCAATTGCAGCTGGACAGCTGGTC - 240 -GSVLAGELSLMSAIAAGQLV 241 - AATAGCCACATGAAilTACAACAGATGTCTCnAAACTTGCCGGCCCCCGC - 300 -NSHMKYNRSSKDVSKLAGPR 301 - TAAacgtggaaaggagagaatacaatgtcataaacacccttqgaacctttggtgtatgt - 360 - t 361 - saqaaataaqtaaqctcqacq4ttCg9qggtttaagaatgtccgaaccagtgtgtgata - 420 421 - attaaacttgagatgctgacacagtgatatttcatgcgtcaacaagtttgtcggtgcgcc - 480 481 - atgtgtttgtgacataaagccacgtgttgggagcaagagattccaagcatctggtttcg - 540 541 - gggtgtctgttttttaacatgtgta Fig. 5.4. Nucleotide sequence of the fkagrnent of hmg2 from M. x dornesticu cv. Delicious, and the predicted arnino acid sequence of the encoded product. The amino acid sequence is shown in one-letter code below the corresponding codons. Lower case letters indicate untranslated regions and the astensk (*) indicates the stop codon. Regions used to design the oligonucleotide primers to synthesize hmg-7 specific RNA probe are underlined. GenBank accession number is AF3 16 1 12. Southern blot analysis on apple genomic DNA To verifi that the cDNAs of &mg1 and hmg2 are derived from two different genes, genomic southem analyses were performed using the gene specific DIG-UTP RNA probes as described in the Materials and Methods. The hybndizatiom performed under high stringency conditions, where probes behave as specific for each gene, indicate that each probe identifies a different set of genomic fragments (Fig. 5.5). Under low- stringency hybridization conditions a mutually complementaxy pattern of bands was observed, indicating that both genomic sequences are closely related. The results confirm that cDNAs of apple hrngl and hm& resemble two independent genes, and there are at least two genes in the HMGR gene farnily of apple. Several well docurnented studies confiirm the presence of multiple copies of HMGR genes occurs in plants (Caelles et al.. 1989; Choi et al., 1992; Chye et al., L992; Godoy-Hernandez et al., 1998; Lichtenthaler et al., 1997; Loguercio et al., 1999; Manfar et al., 1990; Nelson et ai., 1994; Sterrner et al.. 1984; Weissenbom et al., 1995; Yang et al., 1991). The detection of HMGR activity in cytosolic and membrane fiactions (Fig. 5.1) also supports the presence of several genes encoding HMGR for separate isoprenaids in apple fruit. Northern blot analysis on apple total RNA The transcnpt size of hmgl and hrng2 was estirnated by Northern blot analysis. Under high stringency conditions where the probes behave as specific for each gene. both hmgl and hg2 genes showed a similar size of 2.4 kb (Fig. 5.6). Similady. htng transcnpts of H. brasiliensis (Chye et al., 1991, 1992) and A. !haliana (Caelles et al., 1989) showed a size of 2.4 kb. However, the relative abundance of hmgl transcript in the hmgl Fig. 5.5. Genomic Southem blot hybridization analysis to veriw two HMGR genes. Apple genomic DNA (10 pg per lane) was digested with BanzHI (lanes l), EcoRI (lanes 2), and Pst1 (lanes 3). Fragments were electrophoreticaily fractioned, bound on a positively charged membrane (T3rightstarTM,Ambion) and hybridized with DIG-labeled RNA probe representing either the hmgl cDNA clone or hmg2 cDNA clone. DNA molecular size markers are indicated at the right. Fig. 5.6 Northern blot-hybridization of apple total RNA (10 pg per lane) probed with hmgl (A) and hmg2 (B) specific probes to show the size(s) of tcanscripts. The probe used was the antisense RNA of pCRII cDNA insert. Mobility of molecular size rnarkers (bp) is indiateci. skin of apple miit was considerably higher than that of hmg2. Expression of Itmgl and hmg2 in relation to storage and CIIL, Expression of hmgl and hmg2 genes at harvest and at 4-week intervals during the 16-week storage period was performed (Fig. 5.7). The hmgl minscript levels were at a constant abundance during storage, but declined margindly afier 16-week of storage. In contrast, hmg2 showed relatively less abundance of transcript during storage and a peak of accumulation at 8-week after harvest. Therefore, it can be concluded that hmgl is expressed constitutively during storage, but accumulation of hmg2 rnRNA appears to be peak at the time hita-farnesene content and C2& production are high during storage. In our mode1 system, the C2& action inhibitor 1-MCP suppressed the expression of hmg2 completely, but that of hmgl, only partially (Fig. 5.8). DISCUSSION Characterization of HMGR activity in apples during storage HMGR activity was detectable in vitro in both membrane and soluble fractions isolated from skin tissue of apples dunng 5-month storage in air at O OC. Plant HMGR is membrane-bound (Gray, 1987; Bach, l987), and the presence of two transmembrane domains in HMGR protein suggests that the enzyme is associated with the endoplasmic reticulum (ER) (Chye et al., 1991; Enjuto et al., 1994). Recently, Campos and Boronat (2000) demonstrated that in A. thaliana these sequences function as interna1 signal sequences by specifically interacting with the signal recognition particle (SRP) and mediate the targeting of the protein to ER-derived membranes. Although it is evident that Fig. 5.7 Expression ofapple hmgl and hmg2 during 0,4, 8, 12, and 16 weeks of storage at 0°C in air. Total RNA was extracted fiom the skin tissue of apple fhits and stored at -70 OC until used for the Northern blot-hybridization. RNA sarnples were hybridized with the DIG RNA-labeled probe specific to hmgl and hmg2 cDNA clones. The 18s rRNA stained with ethidium bromide and photographed before blotting is shown below the blot. hmgl Fig. 5-8 Effect of the C2H4 action inhibitor 1-rnethylcyclopropene (1-MCP) on the expression of apple hmgl and hm@ (lane 1, untreated; Iane 2, 1-MCP-treated). Apples were exposed to O or 0.6 (L(L-1 1- methylcyclopropene (1-MCP, Rohm and Haas) after harvest for 18 h and placed in storage at O (C in air with 90 to 95% RH for 60d. HMGR is associared with microsomal membranes, the enzyme activity has been detected also in mitochondrial and plastid membranes (Brooker and Russell, 1975; Chappell et al., 1995). The presence of HMGR activity in the soluble fraction of apple skin tissue extracts suggests the soluble fiaction prepared as 105,000 g, supernatant potentially contains light weight fjcagments of cellular membranes formed during the homogenization process of extraction. Alternatively, HMGR protein rnay proteolytically release a cytosolic fragment containing the active site that is fidly hctional as the native enzyme- The enzyme responsible for the Ci isoprenoid intermediate faniesyI diphosphate (FPP), FPP synthase. is observed to be in the cytoplasmic compartrnent and its product FPP serves as a precursor for sesquiterpene and sterol biosynthesis in cytoplasrn/ER (Lichtenthaler, 1993). SimiIarly, the highest specific and total activity of trans,trans-a-farnesene synthase. which catalyzes the conversion of FPP into cc-farnesene, in apples was Iocated in the cytosolic fraction (Rupasinghe et al- (2000a). Therefore, it could be speculated that enzymes invohed in a-farnesene biosynthesis are localized in cytosol/ER boundary and could operate as a highly organized supramolecular cornplex. Plant HMGR is encoded by a multigene family (Godoy-Hernandez et al.. 1998: Lichtenthaler et aI., 1997; Stermer et al., 1994). The enzymes involved in the subsequent isoprenoid pathway, such as IPP isomerase, FPP synthase, and GGPP synthase are also encoded by small gene farnilies (Scolnik and Bartley, 1996). This evidence supports the notion that separate subcellular pathways for specific isoprenoid formation could exist through complexes of sequential rnetabolic erqmes or "metabolons" in plants (Chappell et al., 1995; Srere, 1987; Stermer et al., 1994). The close proximity of enzymes responsible for catalyzing consecutive steps of a metabolic pathway may be used to increase the metabolic flow by assunng the 163 channeling of the intermediate. ~ccurnulationof the stress metabolite a-famesene in the skin of apples is triggered by low temperature storage and reaches a peak during 4 to 12 weeks in storage (Rupasinghe et al., 2000a). In contrast, in vitro HMGR activity was the highest at the time of harvest and gradually decreased during the first 8 weeks of storage and then remained constant during the rernainder of the storage period. The lack of a correlation between HMGR activity and a-famesene accumulation raises the question as to whether isopentenyl diphosphate (PP) supply for a-farnesene biosynthesis occurs through the HMGR-independent Rohmer pathway or non-mevalonate pathway during the time when decline in HMGR activity was observed. The existence of the novel pathway for IPP formation fiom glyceraldehyde-3-phosphate (GAP) and pyruvate, where the HMGR reaction is unnecessary. has been well established in higher plants (Fig. 5.9) (Eisenreich et al., 1996; Kreuzwieser et al., 1999; Lange et al., 1998; Lichtenthaler et al., 1997b; Rodriguez-Concepcion and Gmissem, 1999). However, the present results indicate that incorporation of radiolabelled or unlabelled mevalonic acid into a-farnesene is favored over a mixture of GAP and pymvic acid (precursors of Rohmer pathway) in isolated apple skin tissues. Therefore, it is evident that the biosynthesis of a-faniesene occurs predominantly through the classical MVA pathway in apple fruit. This conclusion is supported ako by the observation that Lovastatin, a competitive inhibitor of HMGR, inhibits a-famesene accumulation significantly (by 25 to 54 %) in apple skin during storage. Recently, Ju and Curry (2000) also found that when Lovastatin is applied to apple miit tissue at high concentrations, a-famesene biosynthesis is suppresed to undetectable levels in 'Delicious' and 'Granny Smith' apples. On the other hand, the GAPIpymvate I 64 Glucose- I -P Glyceralde hyde-3 -P I -Deoxy-D- xylulose- 5-P J- (Dom Pyruvate I Classical / Rohmer Acetyl CoA Pathway I MVA JI Pathway HMG CoA [z]j+- Lovastatin MevaIonate (MVA) C Isopentenyl-pp (IPP) C-) D imethylall yl-PP (DMAPP) Morioterpenes - C Geranyl-PP (GPP) Sesquiterpenes C 15 Farnesyl-PP (FPP) a-Farnesene Diterpenes Cz0Geranylgeranyl-PP (GGPP) Fig. 5.9. A simplified biosynthetic pathway for plant isoprenoids. PP, pyrophosphate; HMGR, 3-hydroxy-3-rnethylglutarylcoenzyme A reductase. pathway is responsible mainly for the formation of plastid-denved isoprenoid compounds in plants, including carotenoids, plastoquinones, the prenyl side chains of chlorophyll, (Kreuzwieser et al., 1999; Lichtenthaler et ai., 1997; Rodriguez-Concepcion and Gniissem, 1999), rnonoterpenes (Lange et al., 1998) and diterpenes (Eisenreich et al., 1996). However,. the classical cytoplasmic mevalonate pathway is specialized to provide IPP for sesquiterpenes and triterpenes (Lichtenthaler et al., 1997). Together these results imply that in apple fniit, the biosynthesis of a-famesene occurs predominantly through the classical MVA pathway. Isolation of Itmgl and Iimg2 cDNA from apple and their expression during storage To Merstudy the regulation of HMGR activity in relation to the accumulation of a-famesene in apple fruit, a molecular approach was employed. With these objectives. we cloned a full length (hmgl) and a fragment (hmg2) of cDNAs of two HMGR genes from the skin of apple miit using a strategy utilizing sequence similarities among previously cloned plant HMGR genes. Al1 plant HMGR genes identified to date share some cornmon structural features (Maldonado-Mendoza, 1997). They are highly conserved in the carboxyl terminal region, highly divergent in the arnino terminal region, and possess two putative tram-membrane dornains. The presence of two tram-membrane domains in the arnino terminal region is a common feature of al1 the HMGR genes cloned to date from plants, but differ fiom the animal HMGR gene which has seven tram- membrane domains (Liscum et al., 1985). HMGR is a membrane-bound protein (Bach, 1987; Gray, 1987), and it has been previously postulated that these amino terminal hydrophoic regions could correspond to tram-membrane domains (Caelles et al., 1989; 166 Chye et al., 1992; Loguercio et al., 1999; Narita and Gruissem, 1989). The cataiytic site of the enzyme is located within the highly conserved carboxyl terminai region (Chye et al., 1992). We hypothesized that the induction of specific HMGR isoqme(s) involved in an independent isoprenoid pathway couId leads to the accumulation of cc-farnesene in apple skin during storage. Northem blot analysis revealed that hmgl and hmg2 of apple were expressed differentially in apples during cold storage. It is interesting to note that hmgl is constitutively expressed while hm@ showed the highest levels of transcnpt when the accumulation of cc-famesene in the skin increases during storage. On the other hand, the abundance of hmg,7 transcript is high at the time when endogenous Cz& production is high in appIes during storage. However, the abundance of hmg2 mRNA was relatively low compared with that of hmgl. The differential regulation of hmgl and hm& expression in apple is consistent with the theory that levels of the different HMGR isozyrnes in plants are modulated in response to specific developmental and stress signals (Stermer et al., 1994). However, expression of neither hmgl nor hmg2 resembles the in vitro changes of HMGR activity during storage. The poor correlation could be the resuit of one or a combination of factors: e-g., changes occurred in other unidentified HMGR isoforms, post-transcriptional events such as mRNA processing, transcript stability. nucleocytopIasmic transport, translation efficiency, ador protein modification and haIf- Iife. HMGR is regulated aIso by a protein kinase cascade in which phosphorylation inactivates the enzyme (McCaskill and Croteau, 1997). As an example, the catalytic domain of the HMGR enzyme fiom the hmgl gene of A. rhaliana expressed in E. coli was reversibly inactivated by a Brassica oleracea HMGR kinase in a cell-fiee system (Dale et 167 al., 1995). Under stress conditions, phosphorylation and inactivation of HMGR enzyme couid occur to conserve energy until the stress situation is resolved (Corton et al., 1994). As ' well, Calcium, calmodulin, and proteolytic degradation also may have a role in regdation of plant HMGR (Stermer et al., 1994). In tomato, however, bodi HMGR activity and mRNA levels are high in early stages of hit development, when rapid ce11 division occurs, as weII as in the subsequent early stages of cellular expansion (Narita and Gruissem, 1989). Narita and Gnlissem (1989) postulated that the final period of miit expansion and ripening is not dependent upon HMGR activity, but instead utilizes a pre- existing pool of pathway intermediates such as isoprene units. Ethylene stimulates HMGR activity and regulates hmg2 expression Our results indicate that C2&-mediated stimulation of a-farnesene biosynthesis is partly due to the induction of HMGR activity by C2H+ In apples, the Cr& action inhibitor 1-MCP suppressed the expression of hmg2 completely and hmgl partially. 1- MCP inhibited respiratory COz evolution by 50 %, suggesting that inhibition of a- farnesene synthesis in apple by l-MCP could be regulated also through the available acetyl CoA pool that is utilized by isoprenoid pathway. However. it is clear from the Iiterature that C* or other stimuli which induce C2H3 production cari influence differential expression of the isogenes of HMGR. In rubber, hmgl is induced by CzHl while hmg3 expression remains stable indicative of a house-keeping nature of the gene (Chye et al., 1992). Furthemore, hmgl is expressed predominantly in the laticifers, the cells specific to rubber biosynthesis (Chye et al., 1992), thus it is postulated that hmgl of rubber encodes the HMGR enzyme involved in rubber biosynthesis. In tomato, hmgl 168 expression is very high in early stages of fhït development but declines during ripening (Jelesko et ai., 1999; Narita and Gruissem, 1989) but hmg2 is highly expressed during ripening @odriguez-Concepcion and Gruissem, 1999). In C. acuminata, hmgl rnRNA increased in response to wounding but hmg2 and hg3 transcript levels remained unaffected (Maldonado-Mendoza et al., 1997). In potato, hmg2 rnEWA levels are elevated in response to wounding or fimgal elicitors suggesting that hmgZ is a defense-related gene or hmg2 is the major elicitor-induced isogene (Yang et al., 1991). Plants regulate HMGR activity at the level of mRNA by differential induction of HMGR gene farnily members, and post-translationally by enzyme modification (Stermer et al., 1994). In C. acuminata apices hmgl is expressed at high levels, hmg3 is moderateiy expressed, and hmg2 transcripts are absent (Maldonado-Mendoza et al., 1997). It is speculated that the expression of specific HMGR genes in specific organs and tissues in the plant could be used as a mechanism for regulating the supply of mevalonate to metabolic pathways localized in those places. We anticipate identification of regulatory factors of HMGR and cloning of HMGR gene(s) specifically directed to a-farnesene biosynthesis will provide important insight into understaridine the role of a-farnesene in plant metabolism and more especially in apple fruit. The Iack of correlation between the a-faniesene content in the skin of apple and changes in HMGR activity during storage perhaps suggests independent regdation of events in vivo. a-Famesene synthesis is very iow at the tirne of harvest but increased rapidIy during low temperature storage. However, in vivo activity of a-faesene synthase, the enzyme that catalyzes the conversion of FPP to a-famesene, was minimal at harvest, then increased rapidly in apple miit during the storage in paraIlel to a-farnesene 169 accumulation (Rupasinghe et al., 2000a). Therefore, a-famesene accumulation is potentially replates at the level of a-farnesene synthase. The high HMGR activity at the time of harvest reflects the increased requirement of isoprenoid pathway intermediate during steady state metabolism during growth and deveiopment. It can be speculated that when apple hitis detached fiom tree and stored at low temperature, total metabolism of apple decreased and requirement of isoprenoid intermediate declines. Under the new steady state conditions, even low MGRactivity (50-100 rnmolmg-'-h") may be enough to provide required intermediates for synthesis of a-famesene. On the other hand, it could be speculated that the pool of mevalonate or other intermediates of isoprenoid pathway that are channeled into a-farnesene biosynthesis are synthesized prirnarily during the hit development phase pnor to harvest. During dark and low temperature storage, a metabolic shifi of rnulti-branched isoprenoid pathways may result in the accumulation of a-farnesene in the skin of apple. Similady, accumulation of a polyisoprene, natural rubber (cis-l,4-polyisoprene) and rubber transferase activity are stimulated at low temperature (Ji et al., 1993). Et is not clear how the individual enzymedgenes of the rnulti-branched isoprenoid pathway in plants are compartmentalized or interact (Bach, 1995). There is increasing evidence that a cornmitment to a particular branch pathway entails synthesis of HMGR isoforms that are developmentalIy and/or spatially regulated in order to differentially modulate metabolic channels, even within the sarne ce11 (Chappell, 1995). Plants regdate HMGR activity at the level of mRNA by differential induction of HMGR gene farnily members, and post-translationally by enzyme modification (Stermer et al., 1994). As well, the molecular mechanisms controlling the HMGR enzyme activity are largely unknown. At present it is diffïcult to define the relative contribution of each 170 HMGR gene to the total HMGR activity. In future, examining the additional HMGR isoforms and inactivation of specific HMGR genes by antisense or CO-suppressionwill reveal the contribution of individual MGR isozymes in isoprenoid metabolism. Answering these questions will help to elucidate how this key regulatory enzyme of isoprenoid pathway operates at the biochemical and molecular level. Since HMGR has an important roIe in a diverse array of kit physiology, a better understanding of its regulation is necessary to enhance the postharvest quality of apple fit. LITERATURE CITED Bach, T.J. 2987. Synthesis and metabolism of mevalonic acid in plants. Plant Physiol. Biochem. 25:163-178. Bach, T.J. 1995. Some new aspects of isoprenoid biosynthesis in plants - A review. Lipids 30: 19 1-202. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72248-254. Brooker, .J .D. and D.W. Russell. 1975. 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Biochem. Eng. 55: 109-146. Meir, S. and W.J. Bramlage. 1988. Antioxidant activity in Cortland apple peel and susceptibility to superficial scald afier storage. J. Amer. Soc. Hort. Sci. 1 13:412- 418. Monk, M., C. Caelles, L. Balcells, A. Ferra, F.G. Hegardt, and A. Boronat. 1990. Molecular cloning and characterization of plant 3-hydroxy-3-methylglutaryl coenzyme A reductase. In: Tower, G.H.N. and H.A. Stafford (eds.): Biochemistry of the mevalonic acid pathway to terpenoids. Recent Advances in Phytochemistry 24:83-97. Plenum Press, London, NY Narita, J.O. and W. Gruissem. 1989. Tornato hydroxymethylglutaryl-CoAreductase is required early in hitdevelopment but not during ripening. The Plant Cell. 1: 18 1- 190. Nelson, A.J., P.W. Doemer, Q. Zhu, and C.J. Lamb. 1994. Isolation of a monocot 3- hydroxy-3-methylglutaryl coenzyme A reductase gene that is elicitor-inducible. Plant Mol. Biol. 25:401-412. Rodriguez-Concepcion, M. and W. Gruissem. 1999. Arachidonic acid alters tomato HMG expression and hitgrowth and induces 1-hydroxy-3-rnethyIg1utql coenzyme A reductase-independent lycopene accumulation. Plant Physiol. 1 19:44 1-48. Rose, T.M., E.R. Schultz, J.G. Henikoff, S. Pietrokovski, CM. McCallum, and S. Henikoff. 1998. Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res- 26: 1628- 1635. Rupasinghe, H.P.V., G. Paiiyath, D.P. Murr. 2000a. Sesquiterpene a-farnesene synthase: partial purification, characterization, and activity in relation to supeficial scald development in 'Delicious' apples. J. Amer. Soc. Hort. Sci. 125 (1): 1 1 1-1 19. Rupasinghe, H.P.V., D.P. Murr, G. Paliyath, and L. Skog. 2000b. hhibitory effect of 1- MCP on ripening and superficial scald development in 'Delicious' and 'Mclntosh' apples. J. Hort. Sci- & Bioteck 74 (3):271-276. Rupasinghe, H.P.V., D. P. Mun; G. Paliyath, and J. R. DeE11.2000~.Suppression of a- farnesene synthesis in 'delicious' apples by arninoethoxyvinylglycine (AVG) and 1-methylcyclopropene (1-MCP). J. Plant Phyiol. Mol. Biol. 6: 195- 198. Sanger, F., S. Nicklen. and A.R. Coulson. 1997. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA. 7454634467. Scolnik, P.A. and G.E. Bartley. 1996. A table of sorne cloned plant genes involved in isoprenoid biosynhesis. Plant Mol. Biol. Rep. 14505-3 19. Schwender, J., M. Seemann, H.K. Lichtenthaler, and M. Rohmer. 1996. Biosynthesis of isoprenoids (carotenoids, sterols, prenyl side-chains of chlorophylls and plastoquinone) via a novel pyruvate/glyceraIdehyde 3-phosphate non-mevalonate pathwayin the green alga Scenedesmus obliquus. Biochem. J. 3 16:73-80. Srere, P.A- 1987. Complexes of sequential metabolic enzymes. Annu. Rev. Biochem. 56:89-124. Stermer, B.A.. G.M. Bianchini, and K.L. Korth. 1994. Regulation of HMG-CoA reductase activity in plants. J. Lipid Res. 35: 1 133-1 140. ~eissenborn,D.L., C.J. Denbow, M. Laine, S.S. Lang, 2. Yang, X. Yu, and CLCramer. i 995. HMG-CoA reductase and terpenoid phytoalexins: molecular speciaIization within within a complex pathway. Physiol. Plant. 93:393-400. Yang, S., H. Park, G.H. Lacy, CL., and Cramer. 1991. Differentid activation of potato 3- hydroxy-3-methylglutaryl coenzyme A reductase genes by wounding and pathogen challenge. Plant Ce11 3:397-405. CWTERVI General Discussion Superficial scald, a major physiological disorder of certain apples, develops during prolonged low-temperature storage. The disorder is controlled effectively by drenching apples at harvest with a synthetic chemicd diphenylamine (DPA), but the continued use of DPA is uncertain due to possible undesirable heaith effects of DPA residues. Research on understanding the biochemical bais of the disorder began around the turn of the twentieth centq (Brooks et al., 1919). in 1964, Murray and CO-workers identified a highly abundant molecule, a-famesene, from a petroleurn extraction of the cuticle wax of 'Granny Smith' apples. It was suggested that the accumu~ationof cc- faniesene in the skin of apple during low-temperature storage played a major role in the development of superficial scald. Initidly, a-famesene was suggested as the primary scald-causing agent. Later, Anet (1 969) proposed a rnechanisrn involving free radical- induced auto-oxidation of a-faniesene that could cause the production of other scald- causing metabolites. Accordingly, an abstraction of a hydrogen or an addition of an oxygen radical to one of the unsaturated carbon bonds of a-famesene, initiates a free radical chain reaction yielding intermolecular free radicals and subsequent conjugated triene peroxides. Since then, a-faniesene catabolites, conjugated trienes, were considered as the scald causing metabolites. As a result, literature dernonstrating the circumstantial evidence to support the "a-famesene theory" of scald development has accumulated. Most of the previous studies focused pnmarily on isolation of conjugated trienes from the apple skin and to give them a functional role. Recently, Rowan et al. (1995) found that 179 conjugated triene dcohol, 2,6,1 O-trimethyldodeca-2,7E,9E,1 1-tetraen-6-01 (CTOL), one of the conjugated trienes that Anet (1969) proposed, was the major component (88-95 %) among a-famesene catabolites present in apple skin. In spite of the fullness of ckcumstantial and correlative data to support the notion that conjugated trienes have a close association with scald development, their ability to damage cellular membranes or organelles causing superficial scald has not been understood fully. In this research, the blocking of a-famesene biosynthesis de novo as a genetic proof (or disproof) for the validity of the 'a-farnesene rheory' of scald development was proposed. If a-farnesene is directly involved in scald development, this approach will elirninate completely scald development in apple cultivars which are susceptible to this disorder. There are no reports claiming any beneficial contribution of a-farnesene to postharvest fruit quality of apples. Interestingly, a-farnesene is the major volatile in apples and its major volatile breakdown products, 6-methyl-5-hepten-2-one (MHO) and 6-methyl-5-hepten-2-01 (MHOL), are rninor components of the hydrophobie volatile profile of apple. Ho wever, these compounds are not "character impact compounds" which contribute to the characteristic aroma of apples (Berger, 1984). From the literature, it was evident that the biosynthesis of a-farnesene and its regulation had not been investigated in relation to scald development in apple. Once the biosynthetic pathway and specific enzyme(s) involved are identified, appropriate biochemical or molecular approaches could be directed to block or bio-engineer a-famesene synthesis to suppress its accumulation in the skin of apples during storage. Cloning the gene(s) encoding the specific enzyrne(s), and construction of antisense transgenic plants could be used as one approach to block a-fmesene biosynthesis and study its eEect on scald development. if indeed a-famesene is involved in apple scald development. Biosynthetic pathway of a-famesene in apple fruit. cc-Famesene belongs to the group of sesquiterpenes or Ci isoprenoid compounds which originate from the acyclic precursor trans,trans-farnesyl pyrophosphate (FPP). With the goal of elucidating a-farnesene biosynthetic pathway in apple fmit, in vivo feeding experiments were carried out using frans, tram-[1,2- I4c or I -'a-FPPwith isolated apple fmit tissues to investigate the immediate precursor of a-famesene (Chapter II). Incorporation of radioactivity fiom FPP to a-fmesene was detected only in the skin tissue but not in the cortex tissues. Therefore, this study demonstrated that biosynthesis of cc-farnesene is confined to the metabolically active outer epidermal and hypodermal cell layers of hit. Arnong other labeled products, farnesol was over a hundred-fold higher compared to a-famesene. However, HPLC analysis of hexane-extractable components from apple skin revealed farnesol is not a predominant natural constituent of apple skin tissue. The present results indicate that the terminal step of a-famesene biosynthesis in apple fruit tissue occurs by direct conversion of rranxtt-nns-FPP to a- famesene. It was, therefore, proposed that formation of an unstable intermediate, allylic carbocation followed by a deprotonation catalyzed by a single sesquiterpene synthase enzyme, rrans,rrans-a-famesene sythase, is the biochemical mechanism of conversion of FPP into a-faniesene. In plants, the isoprenoid pathway provides over 22,000 secondary metabolic products . One of the mechanisms of differential regulation of this multi-branched 181 pathway is the presence of two independent channels to synthesize its universal precursor isopentenyl phyrophosphate (IPP). In addition to the classical mevalonate (MVA) pathway, the presence of a mevalonate-independent pathway (non-mevaIonate or Rohmer pathway) (Rohmer et ai., 1993) has been reported. In this novel pathway, pyruvate and glyceraldehyde-3-phosphate (GAP) are the precursors of IPP, but not acetyl-CoA or rnevaionic acid (Lichtenthaler et al-, 1997% b; Lange and Croteau, 1999). To distinguish between which pathway is channeled into a-famesene biosynthesis, R[S-'H]MVA or a mixture of [2-'4~]pyruvicacid and GAP was incubated with isolated apple skin tissues (Chapter V). Incorporation of radiolabel into a-famesene was 17-fold higher with MVA compared wiîh the precursors of the Rohmer pathway. The enzyme, 3-hydroxy-3- methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) which catalyzes the synthesis of MVA from 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) is the rate limiting step of IPP biosynthesis in classical MVA pathway (Chappell et ai., 1995a.b). When apple hit at harvest were treated with Lovastatin (200 mg-L"), a cornpetitive inhibitor of HMGR, a-faniesene accumulation in the skin was suppressed considerably (25 to 54 %) during storage. Lack of complete inhibition of a-farnesene accumulation by Lovastatin could be due to the poor incorporation of the inhibitor into the cells of apple skin where HMGR is located. Recendy, Ju and Curry (2000b) found that when Lovastatin is fed in high concentrations (1000 rng.~-') in an oil emulsion, a-farnesene emitted to the head-space is inhibited over 90 % during storage of bDelicious7 and 'Granny Smith' apples. Together these results imply that in apple fruit the biosynthesis of a-famesene occurs predominantly through the classical MVA pathway and not through the GAPlpyruvate pathway. Properties of trans,trans-a-farnesene synthase and its relation to superficial scald. The initiai studies (Chapter II) revealed that ms-trans-a-famesene synthase is the terminal enzyme of a-farnesene biosynthesis that catalyzes the conversion of FPP to a-famesene. It is apparent that the enzyme is an ideal target for regulation/blocking of a- farnesene synthesis without disrupting any other metabolic flow to the rest of the isoprenoid pathways. Gene(s) encoding a-fiunesene synthase have not as yet been cloned fiom plants, and any cloning strategy for a-farnesene synthase would require preparation of cDNA Iibraries with rnRNA isolated from tissue enriched for a-farnesene synthase, then screening the library with mtibodies prepared against purified a-famesene synthase protein. With this ultimate goal, purification of a-famesene synthase to homogeneity was attempted using skin tissues of apple hit(Chapter III). The total and specific activities of the enzyme were highest in the cytosolic fraction and cc-farnesene synthase was punfied 70-fold from the cytosolic fraction by ion exchange chromatography and size elution chrornatography. The poor recovery afier the gel permeation together with the instability of the partially punfied enzyme restricted further protein purification. However, the enzyme was characterized from the partially purified protein fractions and had sirnilar biochemical properties to other higher plant sesquiterpene synthases and cyclases. The resuits suggest that cc-farnesene synthase is an allosteric enzyme comprising two or three subunits. The in vifro enzyme activity \vas highest between 10 and 20 OC, while 50 % of the maximal activity was retained ôt O OC showing a cold-stable nature of the dimenc or trimek structure of the native protein. In vivo a-famesene synthase activity along with the content of accumulated a- farnesene and its major catabolite (conjugated triene alchohol, CTOL) was studied in 183 relation to (i) scald severïty, (ii) inherent susceptibility of cultivars to develop superfcial scald, and (iii) exposure to prolonged low temperature storage (Chapter m). A cornparison of the skin tissues firom scald-developing and scald-fiee regions of the sarne apple showed that both a-famesene content and a-famesene synthase activity were nearly 3-fold lower in scald-developing skin tissue than in scald-fiee skin tissue. This reflects either enhanced catabolism of a-farnesene into CTOL-like compounds and/or the metabolic dysfunction is simply secondary event of cellular darnage due to scald development. a-Famesene was not detectable in the skin tissue of scald-susceptible 'Delicious' and scald-resistant 'Empire' £i-uit before attaining physiological maturity or at hanrest, but increased rapidly in both cultivars during the first 4 to 10 weeks of storage in air at O OC, and then declined rapidly. Therefore, it is apparent that a-famesene biosynthesis in apple is initiated afier harvest and induced during the early stages of ripening. Yuen et al. (1995) found that the level of a-farnesene in the skin of Cirrus species which do not develop scald, also increases during storage at O OC. 1 suggest that a-farnesene is a stress metabolite (and a biomarker) of low temperature stress in certain hits including apple. The increase in CTOL could result fiom increased peroxidation. an inherent feature of advancing senescence in fiuit tissues (Shewfelt and Rosario, 2000). The decline in or-famesene during prolonged cold storage was related to both lowered a- faniesene synthase activity as well as enhanced oxidation of a-farnesene to CTOL-like compounds. The symptoms of scald usually appeared during this penod in storage. From the above studies, several basic questions were raised with regard to the regulation of a- famesene biosynthesis. Mainly, what is the primary signaI(s) to induce a-famesene accumulation in apple fruit and what step of the a-famesene pathway recognizes the 184 signal(s). It was previously mentioned that low temperature during storage was a possible cause of this metaboric shifi in the isoprenoid pathway (Chapter m). However, it is also known that endogenous Cz& levels in appIe fit increase during storage in close parallel to the levels of a-farnesene (Watkins et al., 1993; Whitaker et d., 1997). Thus, it is speculated that Cz& also may be involved in regulating a-farnesene biosynthesis and scaid development. Ethylene stimulates a-farnesene biosynthesis and influences scald deveIopment. 'Delicious' apples stored for 8 weeks at O OC and treated with either an ethylene (Cz&) production inhibi~or[arninoethoxyvinylglycine (AVG)] or Cz& action inhibitor [1 -methylcyclopropene ( 1 -MCP) ] had suppressed Cz& production and a-farnesene content in the skin when compared with untreated apples, indicating that c;& regulates a-farnesene biosynthesis (Study 1, Chapter IV). Previous literature on the influence of Cz& on scald developrnemt is highly controversial. However, the endogenous Cz& levels of apple fruit (Meigh and Filmer, 1969; Watkins et al., 1993: Du and Brarnlage, 1994a; Whitaker et al.. 1997) and the arnount of Cr& accumulated in storage atmospheres (Knee and Hatifield, 1981; Linle et al., 2985; Liu, 1986; Lau, 1990) has been shown to influence superficial scald development. The objective of Chapter IVA was to utilize 1- MCP treatrnent as a strong Cz& action inhibitor in apple fruit to test the hypotheses that Cz& stimulates scald development. Further investigations (Chapter IVB) revealed that the exposure of 'Deliclous' and 'Mclntosh' apples to 1-MCP (2lppm at room temperature for 18 h) at harvest has a strong ability to eliminate completely C2& action and feedback regulation mf endogenous C2& production. Inhibition of Cz& action by 1- MCP also resdted in cornpiete inhibition of a-fmesene emanated by apples- The contents of a-famesene and CTOL in the skin were reduced by 60 to 98 % der 1-MCP treatment- As well, 1-MCP treatment suppressed the incidence of superficial scald in 'Mchtosh' and 'Deiicious' apples by 30 % and 90 %, respectively, suggesting that CI& has a distinct influence in scald development. This study also tested whether 1-MCP could be used as a potential tool to replace DPA, together with its tremendous ability to retain fruit quality especially flesh firrnness during storage of apple (Chapter IVB). The suppression of Cl& production and action in apple, did not influence in vivo activity of a-farnesene synthase (Chapter NA). These results suggested that the regulation of a-famesene by Cz& could occur at an upstream step(s) of a-famesene synthase in the isoprenoid pathway. Further experiments (Chapter V) suggested that Cz& regulates a-famesene synthesis through two major mechanisms: (i) regulating gl ycolysis thereby controlling the avaiIability of acetyl CoA for isoprenoid synthesis, (ii) regulating 3-hydroxy-3-methylgIutary1-coenzymeA reductase (HMGR), which is considered to be the primary regdatory point of IPP biosynthesis in the classicaI MVA pathway. To understand the regulation of u-farnesene biosynthesis at the HMGR level changes in rotal HMGR activity in membrane and soluble fractions were studied during storage of 'Delicious' apples (Chapter V). Total HMGR activity was the highest at harvest, but declined during the first 8 weeks in storage and then remained unchanged. It is apparent that total HMGR activity is high when demand for total isoprenoid synthesis is high during hit growth and development, and then declines as fiuit metabolism is suppressed due to low temperatures during storage. This observation reiterates that the rapid increase of a-famesene synthase activity that preceded the accumulation of a- fmesene in the skin tissue is the key regdatory step controlling a-farnesene biosynthesis in stored apples. It is suggested that a-famesene synthase is a cold-inducible stress protein in apple hit which is largely responsible for the accumulation of a-fmesene during storage. Cloning of differentially expressed HMGR genes. In plants, HMGR is encoded by a multiple gene family, and different isoforms are believed to be involved in separate subcellular pathways to produce specific isoprenoid end-products (Chappe11 et al., 1995a,b; Stermer et al., 1994). 1 hypothesized that a- farnesene biosynthesis in apple skin occurs through a metabolic channel or "metabolon". Studying the expression of genes encoding HMGR will provide a better understanding of the regulation of HMGR at the rnolecular level in response to low temperature storage and Cz&. In addition to that, if a HMGR isoform specific for cc-farnesene biosynthesis is identified, a possibility of blocking the a-famesene biosynthesis pathway at the gene level would be achieved. Therefore. a PCR-facilitated approach was used to clone and sequence the füll Iength of a HMGR gene (hmgl) from the skin of apple fhit (Chapter 5). A fiagrnent of another HMGR gene (hmg2) was also cloned and sequenced relying on a 3' untranslated region which is shown to be highiy divergent among the members of the HMGR gene farnily (McCaskiIl and Croteau, 1997). Genomic Southern analysis using the probes designed to the 3'-end of the two cDNA clones confirmed these two novel cDNA clones represent two separate genes. The predicted arnino acid sequence of apple hmgl shares 70 to 80% identity with the rnost of the HMGR genes cloned from higher plants. The hydropathy profile of the protein indicated the presence of two highly hydrophobie domains near the amino terminus which is a unique feahlle of ail identified plant HMGR genes. These two domains are believed to be the two membrane spanning dobains associated with the endoplasmic reticulum. Northem blot andysis indicated that the size of both hmgl and hmg2 -transcripts also agreed with the size of other plant HMGR genes. The two apple HMGR genes are differentially expressed during low temperature storage and in response to C2&, hmgl being constitutively expressed and hmg2 being relatively more sensitive to stimuli such as low temperature and CzH+ The hmgl transcript levels were almost stable during storage up to 12 weeks, but become relatively less abundant der 16 weeks of storage. In contrast, hmg2 showed relatively less abundance of transcript during storage compared with hmgl and a peak of accurnu~ationat 8 weeks afier harvest. It is interesting to note that the highest levels of mRNA of hmg2 and cc-farnesene levels are temporally conelated in the skin of apple hit. Based on these results, 1 propose that hmg2 may be involved in a metabolic pathway specialized to synthesize a-famesene in hyperdermal and epidermal cells of apple hit. The regulation of hmgl and hmg2 expression in apple is consistent with the theory that the levels of different HMGR isozymes in plants are modulated in response to specific developmental and stress signals (Bach, 1995; Chapell, 1995a,b). Other enzymes in the subsequent steps of the isoprenoid pathway, such as IPP isomerase. FPP synthase, and GGPP synthase (Scolnik and Bartley. 1996), are also encoded by small gene families, but little is known about the expression of their genes. This evidence supports the notion that separate subcellular pathways for specific isoprenoid formation could exist through complexes of sequential rnetabolic enzymes or "rnetabolons" in plants (Chappell et al., 1995qb; Srere, 1987; Stermer et al., 1994). The "metabolon" concept was originated after the observation of sedirnents of tfie soluble Krebs tricarboxylic acid cycle enzymes with cross-linking reagents to form large complexes (Robinson and Srere, 1985). Now. a considerable body of evidence supports the idea that sequential enzymes within a metabolic pathway interact with each other to form these highly organized enzyme complexes (Velot et al., 1997). Organization of enzymes as "metabo1ons" offers several advantages: (i) very high catalytic rates can easily be attained with a restricted nurnber of intermediate molecules, (ii) metabolites can specifically be directed towards one or another pathway (channeling), and (iii) unstable intermedÏates are protected since their life-times become greatly reduced (Beeckrnans et al., 1993). In other words, the close proximity of enzymes responsible for catalyzing consecutive steps of a metabolic pathway may be used to increase the metabolic flow, by assuring the efficient channeling of the intermediates. Nevertheless, the in situ existence of these weak enzyme complexes has not been demonstrated because many of them are dissociated during isolation due to dilution effects. Recently, in a cornputer-modeIing study performed using the sequential Krebs TCA cycle enzymes fiom yeast rnitochondria, malate dehydrogenase (MDH), citrate synthase (CS), and aconitase (ACO), a substantial interacting surface areas have been observed (Veiot et al., 1997). Based on this evidence, 1 propose enzymes of a- farnesene biosynthesis are clustered and operate as a functional complex of enzymes possibly Iocated in the cytoso1ER boundary. The abundance of hmg2 transcript in paralle1 to the u-famesene accumulation suggests that hmg2 could possibly be the candidate for the enzyme complex compnsing the a-farnesene biosynthesis. Under those assurnptions, I presume that cloning of the full-length hmg2 and subsequent application of antisense mRNA technology may resdt in suppression of a-famesene biosynthesis in apple. This thesis indicates that a-faniesene synthase is the key regdatory enzyme leading to the accumulation of a-famesene during storage of apple, and thus the key step to target for inhibiting cc-farnesene synthesis leading to presumed scald causing catabolites. Most of the terpene cyclases were cloned using conventional cloning strategïes, preparïng cDNA libraries then screening the library with antibodies prepared against the purified cyclase protein or with specific oligonucleotide probes. However, my attempt to follow the above approach (Chapter III) was lirnited because of the low activity and instability of partially purified a-faniesene synthase protein. However, Chappe11 (1995a.b) described that evolution of plant cyclases may be from a single ancestral gene or may be a reflection of convergent evolution. The similarity in intron placement within some sesquiterpene cyclase genes suggest the possibility of a mechanism to conserve functional ciornains that are responsible for discrete partial steps of the overall cyclase reaction. Therefore, the similarities between sequences of identified plant cyclase genes would help in cloning a-famesene synthase gene. Obviously, further research is warranted which should focus on cloning of a-farnesene synthase gene(s) based on degenerative primers designed to the regions of plant sesquiterpene cyclase proteins that are absolutely conserved. Sequencing information of the recently cloned B- farnesene synthase gene fiom peppermint (Crock et al., 1997) will be an asset in following this approach. Multiple mechanisms in regulating scald development. It was observed that the major catabolite of a-farnesene, a conjugated triene alcohol (CTOL), was higher in scald-developing tissue compared with scald-fiee tissue. However, there was no strong correlation between the levels of accumulated CTOL in the skin during storage and the inherent susceptibility or resistance of any of the cultivars to scald development (Chapter III). This observation suggests that the conjugated triene alone is not the primary and sole deciding factor in scald susceptibility. I presurned that scald-resistant cultivars may possess efficient defensive mechanisms that can combat free radicals including conjugated trienes formed dwing storage. According to the rnechanism of auto-oxidation of a-farnesene proposed by Anet (1969), a-farnesene should be attached primarïly by a free radical to initiate the chain reaction to form toxic cc-famesene catabolites. Therefore, the theory itself suggests that the presence and abundance of free radicals or reactive oxygen species (ROS) is the prirnary cause of scald development. There is no direct evidence to prove that intemediate free radical molecules or terminal conjugated trienes resuIting from auto-oxidation of cc-farnesene are more deleterious than ROS to cause cellular darnage. Therefore, there exists the possibility that ROS cm cause cellular darnage leading to superficial scald development without any association with cc- farnesene. The Ievel of ROS present in plant tissues is controlled mainly by (i) their generation catalyzed by oxidases and by the leakage of electrons from single-electron reduced components in the electron transport chain directly to oxygen, and (ii) the control of ROS scavenging enzymes and lipid-solubIe and water-soluble antioxidants (Scandalios. 1993). Therefore, factors involved in oxidative stress could have direct influence on deciding the susceptibility of apple to scald development. Scald-resistant apples may possess inherently some chernical defense mechanisms against the build up of active oxygen species during low temperature storage. Prolonged low temperature stress can alter the equilibrium between production of ROS and eEciency of scavenging ROS in favor of increased levels of ROS (Prasad et al., 1994; Purvis and Shewfelt, 1993: Scandaiios, 1993). This balance between Phe formation and detoxification of ROS is critical to cell survival during low temperame storage (Zhang et al., 1995). There is evidence that suggests supefiicial scald is induced or stimulated by an oxidative process. First. the scald preventing compounds, DPA and ethoxyquin, are both known to act as antioxidants in other plant systems. Second, it is widely accepted that a reduction of oxygen concentration in the sto-rage atmosphere, with or without an increase in carbon dioxide concentration, reduces the incidence of scald. Third, antioxidcmt activity at harvest has been negatively conelated more with scald development than either a-faniesene or conjugated trienes (Bdage and Meir, 1989; Thomai et al., 1998). As well, scald development exhibits many characteristics simikir to typical chihg inj~iry (Watkins et al.. 1995). Recently, Rao et al. (1 998) found higher activities of the H202- degrading enzymes (peroxidases and catalases) in scald-resistant selections of 'White Angel' x 'Rome Beauty' apples. Sala (1998) found that chilling-tolerant cultivars of mandarin have more efficient antioxidant enzyme systems. Therefore, further research is necessary to explore the relative contribution of natural antioxidants and antioxidant enzymes present in apple towards scald susceptibility. Recent smdies reported in the litera~erevealed that the alternative respiratory path in mitochondria has a protective role against oxidative stress in preventing the generation of ROS (Purvis and Shewfelt, 1993; Wagner and Krab, 1995; Millar and Day. 1997). Factors that inhibit cytochrome oxidase, such as low temperature, stimulate expression of alternative oxidase (AOX) and its activity (Vanlerberghe and McIntosh, 1997). Abood and Gerard (1953) reported that DPA inhibits mitochondrid cytochrome oxidase. It could be presumed that higher electron partitioning to the alternative respiratory pathway in apple fruit could lead to resistance against development of superficial scald in apples. Therefore, in apples, scald susceptibility or resistance could depend primarily on the natural mechanisms which control the balance between generation and scavenging of ROS levels during storage. For many years conjugated trienes in apple peel were presumed to be onIy a mixture of hydroperoxides and epoxides. Conjugated trienes have been quantified based on their characteristic absorbance maxima at 258, 269, and 281 nrn. Initially, Du and Bramlage (1993) thought that differences in absorbance at 258 and 281 nm were attributable to different oxidation products or metabolites of a-famesene. Unexpectedly, a "conjugated triene" with relatively strong absorbance at 258 nrn negatively correlated with scald incidence (Du and Brarnlage, 1993). However, Whitaker (1 998) identified the compound responsible for observed high absorbance at 258 nm as a farnily of phenolic fatty-acid esters. The partially purified compound showed a second absorbance maximum at about 206 and two small shoulders at about 294 and 305, resembling the typical characteristics of isoflavonoids (Whitaker, 1998). Therefore, natural antioxidants such as flavonoids present in the apple skin may also have a role in scavenging ROS, thus deciding scald susceptibility. In an early study, it was found that exposure of appIes to gamma radiation reduced scald in 'McIntosh' apples (Phillips and Poapst, 1960). Massey et al. (1964) also reported that relatively low dosage of gamma radiation can markedly reduce the incidence of scald in Rome BeauS., Mchtosh, and Cortland apples. Radiation shifted the principal respiratory system of apples fkom the Embden-Meyerhof-Parnas glycolytic pathway to the pentose phosphate pathway (PPP) within 3 weeks afier radiation (Faust et al., 1967)- As well, chernical treatments or environmental conditions that reduce scaid aiso favor the PPP. thus it was postulated that the active PPP provides resistance to scald development in apples (Faust et al., 1967). Generation of NADPH through PPP could facilitate the efficiency of operation of the antioxidant enzyme defense system. Therefore, further research on operation of PPP during storage in relation to scald susceptibility appears warranted. In concIusion, this thesis provides noveI information on the characterization of a- farnesene metabolism at the biochemicai and molecular level, with the emphasis on its relation to superficial scald development in apple. Overall, the findings provide a strong foundation towards genetic or biochemical manipulation of the putative scald causing stress metabolite, a-farnesene. Preliminary analyses indicated that neither a-famesene nor its major catabolite, conjugated triene alcohol, is involved in scald development. However, further investigation is necessary to discover the detailed biochemical changes in the ce11 leading to the complex physiological breakdown sirnply called "superficial scald". Literature Cited in the General Introduction and General Discussion Abdallah, A.Y., M.I. Gil, W. Biasi, and E.J. Mitcham. 1997. 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MDSRRRSPTVTAKAAAGELP----~PHEGQNQQPSIPESDVLPLFLYWWF~LFF56 MDVRRRPVKPLYTSKDASAG----EPLKQQE---VSSPKASDALPLPLYLTNGLFFTME 53 MDVRRRSEKPAYPTKEFAAGEKPLKPHKQQQEQDNSLLIASDALPLPLYLTNGLFFTMFF 60 MDWPTKSLWAKTAAAG----EPLKPHHQN-HSSLKASDALPLPLnTNGVFFTLFF 55 SVMYnLTRWREKIRNATPLHVVTLSELAALASL SVIYLVSFFG DNQSLIYKFNNE 116 ------GEILAICGL SLIYLLSFFG WVQSWSNSDDE 35 SVMYET,LVRWREKIRNSIPLHWTLSELLAMVSL SVTYLLGFFG FVQSFVSRSNSD 113 SVMYYLLSRWREKIRNSTPLHWTFSELVAIASL SVIYLLGFFG EITQSNSRDNND 120 SVAYFLLHRWREKIRTSTPLHIVTVSELAALISL fSVIYLLGFFG DWQSFITRASHD 115 KPAPLITPQNSEEDEDIIKAWAGKIPSYSLESKLGDCKRQRITGKSLEGL 226 CAAPKKMP---EEDEEIVAEWAGKI?SWLETRLGDCR~GIRREAVRRTTGRERGL 127 KPSPIIMPALSEDDEEIIQSWQGKTPSYSLESKLGDCMRAQRTGKSLEGL 231 KPAPLVTPAASEEDEEIIKSWQGKMPSYSLESKLGDCKRAASIaKEALQRITGKSLEGL 236 DPVPVIAP-TSEEDEEIIKSWAGTTPSYSLESRLGNCKRAAAIRREALQRLTGKSLAGL 222 gi 1 167488 gi12072322 gil i69485 gi119746 gi 11763234 PLDGFDYESILGQCCEMAVGWQMAVGIAGPLLLDGREYL ILASGGANSVLLRDGMTRAPVVRFGTAKRAAELK~EDTQNFTISVVKSSRFAKLQ346 SVQCAIAGKNLYIRF SKGVQNVLEFLQTDYPDMDVLGISGNFCADKK 406 RVKCAVAGRNLYMR SKGVQNVLDYLQDDFPDMDVISISGNFCSDKK 307 GIQCAIAGKNLYIT SKGVQNVLDYLQSEYPDMDVIGISGNFCSDKK 4 1f RIQCAIAGKNLYMR SKGVQNVLDYLQNEYPDMDVIGISGNFCSDKK 416 GIHCALAGQNLYMR SKGVQNVLDFLQNDFSDMDVIGISGNFCSDKK 402 Fo~rd2 PAAVNWIEGRGKSWCEAIIKEEIVKTVLKTEV~LIELXLAGSAIAGLGGFNA466 SAAVNWIEGRGKSVVCEAVIKEEWKKVLKTNVQSLVELNVIKNLAGSAVAGALGGA 367 P~VNWIEGRGKSWCEAIIKEEWKKVLKTEV~VELNMKNLTGSGALGGA471 PAAVNWIEGRGKSWCEAIITEEVVKKVLKTEV~LVELNMLKNLTGSGALGG476 PAAVNWIEGRGKSWCESIIKEEVVRKVLKTNVASLVELNMLNLTGSGALG 462 Fomrd 3 QSACLNLLGVKGASKDSPGANSRLLATIVAGSVLAGELçLMSASAGQLVRSHYNRSS 586 QSACLDLLGVKGANRESPGSNARLLAAVVAGELSGLVQSMKYNRSS 487 QSACLNLLGVKG~NRDAPGSNARLLATIVAGSVLAGELSLSAISAGQLVKSHMKYNRSI 591 QSACLNLLGVKGANilEVPGSNARLLATIVAGSVLAGELSLMSAISAGQLVKS~YRST 596 QSRCLNLLGVKGASKESPGSNSRLLATIVAGSVLAGELSLMSAIGQLVKSHMKYtRSS 581 KDITNIASSQLESDS 601 RDKSKVAS------495 K'ISK------596 KDVTKASS------604 KDITKVSS------589 Appendix 5.1 The alignment of amino acid sequences of HMGR genes of Catharanihzrs roseiis (GI 1 67488); Solmzm tuberosum (GI 169485); Camptotheca acuminata (GI 1 763 2%); Nicotiana sylvestris (GI 19746): and Oryza safiva (GI 2072322) to show the consewed regions used to design degenerate oligonucIeotides.