Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Cloning of hmg1 and hmg2 cDNAs encoding 3-hydroxy-3methylglutaryl coenzyme A reductase and their expression and activity in relation to -farnesene synthesis in apple
Handunkutti P.V. Rupasinghea, Kurt C. Almquistb, Gopinadhan Paliyathb*, Dennis P. Murra
a b
Department of Plant Agriculture (Horticultural Science Division), University of Guelph, Guelph, Ontario N1G 2W1, Canada Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada
Received 9 April 2001; accepted 2 July 2001 Abstract In plants, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyses the synthesis of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A. It has been reported to be the rate-limiting enzyme in sesquiterpene and triterpene biosynthesis and is encoded by a small gene family. The accumulation of -farnesene in the skin tissue of apple fruit during storage appears to be predominantly through the classical mevalonate pathway and not through the novel glyceraldehyde-3phosphate/pyruvate pathway independent of HMGR action. The content of -farnesene in the skin tissue increased during the rst 8 weeks of storage at 0 C in air, and started declining after 12 weeks. In contrast, HMGR activity in the total membrane and soluble fractions, was the highest at the time of harvest, but decreased during the rst 8 weeks in storage and remained stable thereafter. The potent ethylene action inhibitor 1-methylcyclopropene inhibited -farnesene evolution and HMGR activity by 97 and 30 %, respectively. As a rst step in studying the molecular mechanism of apple HMGR regulation, we have isolated and cloned a full-length cDNA (hmg1) as well as a fragment (hmg2), using apple skin mRNA and RT-PCR in the presence of degenerate oligonucleotides designed against conserved regions of plant HMGR genes. Genomic Southern analysis using probes designed for the 3-end of the two cDNA clones conrmed the presence of at least two HMGR genes in apple. The cDNA for hmg1 has an open reading frame of 1 767 nucleotides. Analysis of the nucleotide sequence revealed that the cDNA encodes a polypeptide of 589 residues with a relative molecular mass of 62.7 kDa. The hydropathy prole of the putative polypeptide indicated the presence of two highly hydrophobic domains near the amino terminus. Northern blot analysis conrmed that both hmg1 and hmg2 transcripts possessed a size of 2.4 kb. The two genes are differentially expressed during low temperature storage and in response to C2H4, with hmg1 being expressed constitutively and hmg2 being relatively more sensitive to developmental stimuli and ethylene. 2001 ditions scientiques et mdicales Elsevier SAS -farnesene / apple / ethylene / HMGR / Malus domestica / 1-MCP / post-harvest storage DIG, digoxigenin / GAP, glyceraldehyde-3-phosphate / HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase / IPP, isopentenyl pyrophosphate / 1-MCP, 1-methylcyclopropene / MVA, mevalonate / RACE, rapid amplication of cDNA ends
1. INTRODUCTION
A highly conserved enzyme in eukaryotes, 3-hydroxy-3-methylglutaryl coenzyme A reductase
*Correspondence and reprints: fax +1 519 824 6631. E-mail address: gpaliyat@uoguelph.ca (G. Paliyath). Present address: Guelph Centre for Functional Foods, Laboratory Services, University of Guelph, 95 Stone Road West, Guelph, Ontario N1H 8J7, Canada. Present address: Department of Environmental Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
(HMGR), catalyses the conversion of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) to mevalonate (MVA), the proposed rate-limiting step of isopentenyl pyrophosphate (IPP) biosynthesis [6, 21, 24]. However, in addition to the mevalonate pathway, IPP is derived through a mevalonate-independent pathway (Rohmer pathway or glyceraldehyde-3-phosphate (GAP)/pyruvate pathway) for the biosynthesis of certain isoprenoids [13, 24] (gure 1). In general, the classical cytoplasmic mevalonate pathway provides IPP for sesquiterpene and triterpene biosynthesis [24]. The Rohmer pathway is believed to be responsible for
934
Figure 1. A simplied biosynthetic pathway for plant isoprenoids. PP, pyrophosphate; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase.
the formation of all plastid-derived isoprenoid compounds in plants, including carotenoids, plastoquinones, the prenyl side chain of chlorophyll, as well as monoterpenes and diterpenes [19, 21, 24]. In higher plants, HMGR is encoded by a multigene family in nuclear DNA [24, 39]. HMGR is encoded by at least two distinct genes in Arabidopsis thaliana [4], cotton (Gossypium hirsutum L.) [26], and rice (Oryza sativa) [31]; by three genes in rubber (Hevea brasiliensis) [11], tomato (Lycopersicon esculentum) [30], and potato (Solanum tuberosum) [9]; and an even larger complex multiple gene family in maize (Zea mays) and pea (Pisum sativum) [39]. HMGR isoforms are expressed differentially in response to a variety of developmental and environmental stimuli, such as fruit development, phytohormones, endogenous protein factors, light and pathogen infection [39]. In tomato, hmg1 is highly expressed during early stages of fruit development, when sterol biosynthesis is required for membrane biogenesis during cell division and expansion [30]. The expression of hmg2 is not detectable in young fruit, but is activated during fruit maturation and ripening [18, 32]. Hmg1 mRNA of A. thaliana accumulates in all parts of
the plant, while the presence of hmg2 mRNA is restricted to young seedlings, roots and inorescence [14]. Cotton hmg2 encodes the largest of all plant HMGR enzymes described to date and contains several functional specialization features that include a unique 42 amino acid sequence located in the region separating the amino-terminal domain and carboxyterminal catalytic domain, that is absent in hmg1 [26]. 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 specic isoprenoid end-products through metabolic channels, or metabolons [6, 39]. A number of investigators have reported a correlation between the induction of isoprenoid biosynthesis, particularly that of sesquiterpenes, and HMGR enzyme activity [6]. In potato, the expression of specic HMGR genes has been correlated with the accumulation of steroids or sesquiterpenes [9]. Chye et al. [11] observed that only hmg1 was inducible by C2H4 among HMGR genes, and also speculated that distinct isoprenoid pathways do occur for rubber biosynthesis in H. brasiliensis. In cotton, hmg2 has been associated with the synthesis of specic sesquiterpenes in developing embryos [26]. These results support the concept of metabolic channels, or arrays of isoenzymes that are independently-regulated and dedicated to the production of specic isoprenoids [6]. The acyclic sesquiterpene -farnesene (C15H24; [3E,6E]-3,7,11-trimethyl-1,3,6,10-dodecatetraene) accumulates in the skin of apple fruit exclusively after harvest and during prolonged low temperature (0 to 1 C) storage [37]. The extent of oxidation of -farnesene to conjugated trienes has been shown to be proportional to the development and severity of the post-harvest physiological disorder supercial scald [8, 16, 28]. Biosynthesis of -farnesene in apple skin is highly regulated by temperature [37] and C2H4 [34, 35]. However, biochemical and molecular mechanisms of modulation of HMGR activity in relation to -farnesene biosynthesis in apple has not been reported. Identication of regulatory factors of HMGR and cloning of HMGR gene(s) that may potentially be directed to -farnesene biosynthesis, will provide important insight into the understanding and the role of -farnesene in supercial scald development in apple fruit. Therefore, as a rst step towards understanding the regulation of -farnesene accumulation by HMGR, total in vitro HMGR enzyme activity and expression of two novel cDNA clones encoding HMGR in apple, hmg1 and hmg2, were studied in relation to low temperature storage and C2H4 action.
935
Figure 2. -Farnesene content (A) and in vitro HMGR activity in the membrane fraction and the soluble fraction (B), in Delicious apples during storage at 0 C in air. All parameters are expressed on a fresh weight basis and each data point is the mean of three replicates.
a separate study when apple skin discs were incubated with unlabelled MVA, or a mixture of GAP and pyruvate, -farnesene levels (HPLC analysis) were higher in MVA-treated skin tissue than that of GAP and pyruvate-treated skin tissue (data not presented).
Table I. Incorporation of radiolabel from precursors of isopentenyl pyrophosphate into -farnesene in isolated skin tissue of Lovastatin-treated (200 mgL1) apples. Six discs (1 cm in diameter and 23 mm in thickness) isolated from three apples were incubated in the presence of radiolabel. Data represent the mean of three such replicates. The -farnesene formed was extracted with hexane, concentrated by evaporation in a stream of nitrogen and subjected to separation using thin layer chromatography. Region corresponding to authentic -farnesene was scraped and mixed with scintillation uid to quantify the radiolabel. Precursor R[5-3H]-mevalonic acid A mixture of [2-14C]-pyruvic acid and 25 mM glyceraldehyde-3-phosphate Radioactivity incorporated into -farnesene (pmol) 741 0.01
936
Table II. -Farnesene content in the skin of apples treated with or without Lovastatin (200 mgL1), a specic HMGR inhibitor. Apples were treated soon after harvest and stored at 0 C in air for 8, 12 or 16 weeks. Data represent mean of three independent estimations SD. For each estimation, a total of six discs (1 cm in diameter and 23 mm in thickness) were excised from three apples and extracted with 3 mL hexane overnight. After clarication by ltration through a 0.22micron lters, an aliquot was subjected to HPLC separation using a Nova-Pak C18 column and acetonitrile as the mobile phase. -Farnesene was quantied using a standard curve. Treatment -Farnesene content in the skin (gg1 FW) Storage period (weeks) 8 Control Lovastatin 396 35 182 21 12 567 48 277 31 16 446 39 338 26 1 409 797 Total
was inhibited by 30 % when C2H4 action was blocked by 1-MCP. In addition, 1-MCP inhibited respiratory CO2 evolution by nearly 60 % (table III) which suggests that inhibition of -farnesene synthesis in apple by 1-MCP, could also be mediated through respiratory regulation, which provides C2 skeletons (acetyl CoA) for isoprenoid biosynthesis.
These results further imply that, in apple fruit, the biosynthesis of -farnesene occurs predominantly through the classical MVA pathway and not through the GAP/pyruvate pathway.
Table III. Metabolic responses of apples treated with the C2H4 action inhibitor, 1-methylcyclopropene (1-MCP, 600 nLL1). Each variate represents the mean value of response measured in triplicate after 5 d at 20 C following removal from storage for 60 d at 0 C in air. One unit of volatile -farnesene = peak area 106. *, **, *** Signicant at P < 0.05, 0.01 or 0.001, respectively. Metabolic response C2H4 production (Lh kg ) HMGR activity (pkatmg1 protein) -Farnesene content in the skin (gg1) -Farnesene evolution (unitkg1) CO2 production (mLh1kg1)
1 1
937
Figure 3. Combined nucleotide sequence and the predicted amino acid sequence of the encoded product of the cDNA corresponding to hmg1 from M. domestica cv. Delicious. 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 hmg1 specic RNA probe are underlined. GenBank accession number is AF315713.
938
Figure 4. Alignment and comparison of deduced amino acid sequence of M. domestica hmg1 with hmg1 sequences of A. thaliana (GI 123340; [4]), C. acuminata (GI 289881; [27]), G. hirsutum (GI 2935298; [26]) and H. brasiliensis (GI 18835; [11]). Dots denote spaces introduced to maximize alignment and black shaded amino acid stretches indicate regions identical among the ve comparisons.
tum [25] and 72 % homology with hmg1 of A. thaliana [4]. Among all the identied plant HMGR genes, the N-terminal region differs greatly both in length and amino acid sequence (gure 4).
The hydropathy prole of the protein, deduced by the algorithm of Kyte and Doolittle [20], shows close similarity to the typical structural features of other plant HMGR genes; e.g. hmg1 of A. thaliana [22], H.
939
brasiliensis [10], and hmg3 of C. acuminata [27] (gure 5A). The two hydrophobic regions located between residues 22 to 46 and 67 to 86 (gure 5B), each of which is long enough to span a membrane bilayer, are conserved in other HMGRs characterized in plants. HMGR is a membrane-bound enzyme [1, 15]. It has previously been postulated that these N-terminal hydrophobic regions could correspond to trans-membrane domains [4, 11, 26, 30]. Furthermore, the carboxyl-termini of all plant HMGRs are highly conserved and the catalytic site of the enzyme is located within this region [11]. HMGR belongs to a multigene family. As a strategy to isolate other HMGR genes from apple fruit, additional degenerate oligonucleotide primers (forward) were designed for the 3-end region, and 3-RACE performed. A 565-bp fragment of cDNA (hmg2) was isolated (gure 6), and the coding region (303 bp) of hmg2 fragment showed 79 % nucleotide sequence homology and 87 % amino acid homology to the apple hmg1.
Figure 5. Sequence and domain characteristics of HMGRs. A, Hydropathy index plot of the predicted amino acid residues. Apple hmg1 (I), A. thaliana hmg1 (II), H. brasiliensis hmg1 (III), and C. acuminata hmg3 (IV). The average hydrophobicity of each amino acid was calculated using the algorithm of Kyte and Doolittle [20] over a window size of nine residues, and was plotted as a function of amino acid position. Positive values indicate that free energy is required for transfer to water, characteristic of hydrophobic regions. H1 and H2 indicate two probable trans-membrane domain (membrane-spanning) regions. B, Schematic representation of the domain structure of apple HMGR isoform encoded by hmg1. Abbreviations: H1 and H2, highly conserved membrane-spanning sequences; LS, highly conserved lumenal sequence. Numbers inside the domains indicate the respective number of amino acid residues.
Figure 6. Nucleotide sequence of the fragment of hmg2 from M. domestica cv. Delicious, and the predicted amino acid sequence of the encoded product. The amino acid sequence is shown in oneletter 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 hmg2 specic RNA probe are underlined. GenBank accession number is AF316112.
940
Figure 7. Genomic Southern blot hybridization analysis to verify the number of HMGR genes in apple genome. Apple genomic DNA (10 g per lane) was digested with BamHI (lanes 1), EcoRI (lanes 2) and PstI (lanes 3). Fragments were electrophoretically separated, transferred to a positively charged membrane (BrightStar, Ambion) and hybridized with DIG-labelled RNA probe representing either the hmg1 or hmg2 cDNA. DNA molecular size markers are indicated on the right.
Figure 8. Northern blot-hybridization of apple total RNA (10 g per lane) probed with hmg1 (A) and hmg2 (B) specic DIG-labelled RNA probes to show the size(s) of transcripts. Mobility of molecular size markers (bp) is indicated on the right.
cDNAs are from two independent genes, and there are at least two genes in the HMGR gene family of apple. Several well-documented studies conrm the presence of multiple copies of HMGR genes in plants [4, 9, 11, 24, 26, 31, 39, 40]. The detection of HMGR activity in cytosolic and membrane fractions (gure 2) also suggests that these enzymes might have derived from different HMGR genes in apple fruit.
2.7. Northern blot analysis on apple total RNA The transcript size of hmg1 and hmg2 was estimated by northern blot analysis. RNA was isolated from the apple skin tissue and subjected to agarose gel electrophoresis. After blotting to nylon membranes, the RNA was allowed to hybridize with DIG-labelled hmg1 and hmg2 RNA probes. Under high stringency conditions where the probes behave as specic as possible for each gene, both hmg1 and hmg2 genes showed a similar size of 2.4 kb (gure 8). This is similar to the results obtained from H. brasiliensis [10, 11] and A. thaliana [4] where the transcript size was found to be of the order of 2.4 kb. The relative abundance of hmg1 transcript in the skin of apple fruit was considerably higher than that of hmg2. 2.8. Expression of hmg1 and hmg2 in relation to storage and C2H4 production To study the expression of hmg1 and hmg2 genes during post-harvest storage, RNA was isolated from
apple skin tissue at harvest and at 4-week intervals during the 16-week storage period and subjected to northern blot analysis (gure 9). Abundance of the hmg1 transcript was nearly constant during the rst 8 weeks of storage, and declined during further storage up to 16-weeks. In contrast, hmg2 showed a relatively lesser abundance of transcript during early periods of storage, with increasing abundance noticed at 4 weeks and a peak of accumulation at 8-week after harvest. Thus, it appears that hmg1 is expressed constitutively during storage of apples, whereas the accumulation of hmg2 mRNA, appears to peak at the time when fruit -farnesene content (gure 2) and C2H4 production (data not shown) are also high during storage. In our model system, the C2H4 action inhibitor 1-MCP suppressed the expression of hmg2 completely, but only partially that of hmg1 (gure 10).
3. DISCUSSION
The development of supercial scald in apples has been extensively studied, however, the explanations for the biochemical mechanisms of scald development are still hypothetical. The biosynthesis and degradation of the sesquiterpene -farnesene has drawn considerable attention from researchers. Recent studies from our laboratory have revealed several interesting aspects on the biosynthesis of -farnesene [3437]. -Farnesene is synthesized by the enzyme -farnesene
941
ow of substrates. Thus, these studies were performed in apple fruit to understand the potential regulation of isoprenoid pathway and -farnesene biosynthesis at the level of HMGR.
Figure 9. Expression of apple hmg1 and hmg2 during 0, 4, 8, 12, and 16 weeks of storage at 0 C in air. Total RNA was extracted from the skin tissue of apple fruits and stored at 70 C until used for the northern blot hybridization. RNA samples were hybridized with the DIG RNA-labelled probe specic to hmg1 and hmg2 cDNA. The 18S rRNA stained with ethidium bromide and photographed before blotting is shown below the blot.
Figure 10. Effect of the C2H4 action inhibitor 1-methylcyclopropene (1-MCP) on the expression of apple hmg1 and hmg2 (lane 1, untreated; lane 2, 1-MCP-treated). Apples were exposed to 0 or 600 nLL1 1-methylcyclopropene after harvest for a period of 18 h and placed in storage at 0 C in air with 90 to 95 % relative humidity for 60 d.
synthase, which catalyses the direct conversion of farnesyl pyrophosphate to -farnesene through an allylic carbocation intermediate which possesses a positive charge at its C2 position. An elimination of a proton from the C1 position and rearrangement would create double bonds at C1 and C3 positions leading to the formation of farnesene (3,7,11-trimethyl, 1,3,6,10dodecatetraene) [36]. -Farnesene synthase activity increased during storage of apples, almost in parallel with the accumulation of -farnesene in the fruit skin [37]. However, the existence of additional regulatory steps became apparent when studies revealed that inhibition of ethylene biosynthesis in apples by 1-MCP treatment reduced -farnesene accumulation but did not alter -farnesene synthase activity [34]. A potential regulatory site that could affect -farnesene biosynthesis is at the level of HMGR, through regulating the
942
seeds, wherein, the HMGR activity was the highest 1012 d after pollination, when the mitotic activity was the highest. The endosperm-localized HMGR activity declined by over 80 % during maturation. However, during the same period the level of abscisic acid, an isoprenoid product, increased [29]. The lack of a positive correlation between HMGR activity and -farnesene accumulation also raises the question regarding the origin of isopentenyl pyrophosphate supply for -farnesene biosynthesis, whether it occurs through the HMGR-dependent pathway or through the HMGR-independent Rohmer pathway during the period when the decline in HMGR activity was observed. The existence of this novel pathway for IPP formation from GAP and pyruvate, which bypasses the HMGR reaction, is well established in higher plants (gure 1) [13, 19, 21, 24, 32]. However, the present results indicate that incorporation of radiolabelled or unlabelled mevalonic acid into -farnesene is relatively favoured over a mixture of GAP and pyruvic acid in isolated apple skin tissues, even considering the differences in specic activity and potential internal dilution. Therefore, it is evident that the biosynthesis of -farnesene occurs predominantly through the classical MVA pathway in apple fruit. This conclusion is also supported by the observation that Lovastatin, a competitive inhibitor of HMGR [1], inhibits -farnesene accumulation signicantly (by 25 to 54 %) in apple skin during storage. Recently, Ju and Curry [18] found that when Lovastatin was applied to apple fruit tissue at high concentrations (1 gL1), -farnesene biosynthesis was suppressed to undetectable levels in Delicious and Granny Smith apples. The GAP/pyruvate pathway is responsible mainly for the formation of plastid-derived isoprenoid compounds in plants, including carotenoids, plastoquinones, the prenyl side chains of chlorophyll, [19, 24, 32], monoterpenes and diterpenes [13]. However, the classical cytoplasmic mevalonate pathway is specialized in providing IPP for sesquiterpenes and triterpenes [24]. Together, these results imply that in apple fruit, the biosynthesis of -farnesene occurs predominantly through the classical MVA pathway.
3.2. Isolation of hmg1 and hmg2 cDNAs from apple and their expression during storage
To further study the regulation of HMGR activity in relation to the accumulation of -farnesene in apple fruit, cDNAs of two HMGR genes, the rst having a full length (hmg1) and the second being a fragment (hmg2), were cloned. All plant HMGR genes identied to date share some common structural features [27].
They are highly conserved at the carboxyl-terminal region, highly divergent at the amino-terminal region, and possess sequences for two putative trans-membrane domains, which is a common feature of all the HMGR genes cloned to date from plants, but differ from the animal HMGR genes which possess sequences for seven trans-membrane domains. HMGR is a membranebound enzyme [1, 15], and it was previously postulated that these amino-terminal hydrophobic regions could correspond to trans-membrane domains [4, 11, 26, 30]. The catalytic site of the enzyme is located within the highly conserved carboxyl-terminal region [11]. From our study, it appears that the induction of specic HMGR isozyme(s) could be involved in the accumulation of -farnesene in apple skin during storage. Northern blot analysis revealed that hmg1 and hmg2 were differentially expressed in apples during cold storage. It is interesting to note that hmg1 is expressed constitutively while hmg2 showed the highest levels of transcript when the accumulation of -farnesene in the skin also attained a high level during storage [37]. In addition, the abundance of hmg2 transcript increased in parallel with endogenous C2H4 production. However, the abundance of hmg2 mRNA was relatively low compared with that of hmg1. The differential regulation of hmg1 and hmg2 expression in apple is consistent with the theory that levels of the different HMGR isozymes in plants, are modulated in response to specic developmental and stress signals [39]. These may involve posttranscriptional events such as mRNA processing, transcript stability, nucleo-cytoplasmic transport, translational efficiency, and/or protein modication and proteolysis. HMGR is also regulated by a protein kinase in which phosphorylation inactivates the enzyme. As an example, the catalytic domain of the HMGR enzyme from the hmg1 gene of A. thaliana expressed in E. coli, was reversibly inactivated by a Brassica oleracea HMGR kinase in a cell-free system [12]. Calcium, calmodulin and proteolytic degradation may also have a role in the regulation of plant HMGRs [39]. In tomato, both HMGR activity and mRNA levels are high at early stages of fruit development, when rapid cell division occurs, as well as during the early stages of cellular expansion [30]. Narita and Gruissem [30] postulated that the nal period of fruit expansion and ripening is not dependent upon HMGR activity, but instead utilizes a pre-existing pool of pathway intermediates such as isoprene units. Thus, several internal and external factors regulate the expression and activity of HMGR.
943
Ontario (harvest maturity indices: rmness, 73.3 N; soluble solids, 10.3 %; starch-iodine index (Cornell chart), 3.2). Apples were transported to Guelph and were cooled to 0 C within 8 h of harvest and stored at 0 to 1 C in air with 90 to 95 % relative humidity. These apples were used for monitoring HMGR activity and expression during storage.
944
3 % BSA, and 50 gL1 polyvinylpyrrolidone. The homogenate was ltered through four layers of cheesecloth, and the ltrate was centrifuged at 3 000 g for 10 min to remove starch and debris. The resulting supernatant was centrifuged at 105 000 g for 45 min and the membrane pellet was resuspended in 0.1 M sodium phosphate buffer (pH 6.5) containing 2.5 mM DTT. The supernatant containing soluble proteins was collected for further experiments. Aliquots of total membrane or supernatant, equivalent to 100 g protein were incubated for 45 min at 30 C in a nal assay volume of 250 L containing 100 mM sodium phosphate buffer (pH 7.0), 3 mM NADPH, 10 mM DTT, 20 M HMG-CoA, and 0.925 kBq DL-[3-14C]-HMGCoA (215 MBqmmol1; Amersham). The assay was terminated by the addition of 20 L 5 mM mevalonate lactone and 50 L 6 N HCl followed by vortex stirring. The mixture was incubated for an additional 30 min at room temperature to allow the radiolabelled mevalonate formed to lactonize. After addition of 100 L saturated potassium phosphate (pH 6.0) and 300 L ethyl acetate, the samples were briey vortexed and centrifuged, and an aliquot of the upper organic phase containing the mevalonate lactone, was used to quantify the radiolabel by liquid scintillation counting. The chemical nature of the product was conrmed by analysing the ethyl acetate fraction using thin layer chromatography along with authentic mevalonate lactone, in a solvent system containing chloroform and acetone (2/1, v/v), and measuring the radioactivity in the mevalonate lactone-containing zone (Rf = 0.6). The assay was performed in triplicate.
tively precipitated by adding LiCl2 to a nal concentration of 3.2 M and incubated overnight at 20 C. RNA was pelleted by centrifugation at 20 000 g for 20 min at 4 C. The pellet was resuspended in 2 mL DEPC-treated water, and sodium acetate (nal concentration of 0.3 M) and 6 mL cold absolute ethanol were added. After 1 h incubation at 20 C, RNA was pelleted by centrifugation for 10 min at 12 000 g and the pellet was resuspended in 200 L RNase-free water and stored at 80 C. The messenger RNA was isolated from the total RNA using an Oligotex mRNA Spin-Column kit (Oligotex). The total RNA extracted from the skin tissue of Delicious apples stored for 12 weeks in air at 0 C, was used. Synthesis of cDNA by reverse transcription of mRNA was performed using a SMART RACE cDNA amplication kit (Clontech). A 20-L aliquot of reaction mixture containing 1 g mRNA, 1 g oligo (dT) and sterile distilled water was heated to 70 C for 2 min and chilled quickly on ice. After a brief centrifugation of the tube, 4 L rst strand buffer (5), 2 L 0.1 M DTT, and 1 L 10 mM dNTP mix (10 mM each dATP, dGTP, dCTP, and dTTP at neutral pH) were added. The contents were mixed gently and RNase H reverse transcriptase was added and incubated for 90 min at 42 C. The reaction was inactivated by heating at 72 C for 7 min after adding 100 L tricine-EDTA buffer. The cDNA was precipitated with ethanol and stored at 20 C.
945
The PCR reaction conditions were: 1 min at 94 C, plus 35 cycles at 94 C (30 s), 68 C (2.2 min) for annealing and a nal extension step at 72 C for 7 min. The resulting 549-bp PCR product (hmg1) was used to design forward and reverse HMGR-specic primers for 3- and 5-RACE, respectively (SMART RACE cDNA amplication kit, Clontech). The two fragments obtained after 3- and 5- RACE were then used to design primers (forward 5-TGTCCTTTCCTCTTCTCTCCTCCGCC-3 and reverse 5-CTTCAAGCTCAGAAGTTAGAGCCTTTCAAGTTC-3) to obtain a full length cDNA clone (hmg1). To obtain the fragment of hmg2 cDNA clone, additional degenerate oligonucleotide primers (forward) was designed for the 3 region: 5-GCTCCACCGGTGACGCTATGGG(A/G/C/T)TGAA-3 corresponding to the amino acid sequence (CSTGDAMGMN), and 5-TCCCACTGCATCACCATGATGGA(A/G)GC-3 corresponding to the amino acid sequence (SHCITMMEA) to perform 3-RACE by the procedure described above. DNA sequences were determined after subcloning the PCR product into E. coli using an AdvanTAge cloning kit (Clontech), and by the chain termination method [38]. The sequence of both strands was determined using synthetic oligonucleotide primers. DNA sequences were analysed using the MBS on-line tools.
DNA was digested with three restriction endonucleases, EcoRI, BamHI, and PstI (Fermentas), and the resulting fragments were separated by electrophoresis in 0.8 % (w/v) agarose gels. The gels were blotted on to a positively-charged BrightStar-Plus membrane (Ambion) using SouthernMax Southern blotting kit (Ambion). The membranes were prehybridized in Ultrahyb hybridization buffer (Ambion) for 1 h at 50 C, and then hybridized with digoxigenin-labelled apple hmg1 and hmg2 RNA probes at 50 C for 16 h. The membrane was washed twice in 2 sodium chloride/sodium citrate (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 50 C (high stringency). Chemiluminescent detection was performed as described in the DIG Northern Starter kit (Roche).
Acknowledgments. This research was supported by grants from the Ontario Ministry of Agriculture, Food and Rural Affairs, and Agriculture and Agri-Food Canada. We would like to thank Dr Geza Hrazdina, Cornell Agricultural Experimental Station, Geneva, New York, and Drs Mike McLean and Owen Van Cauwenberge, Department of Plant Agriculture, for their expert advice and help. We are also grateful to Drs Judy Strommer and Barry Shelp, Department of Plant Agriculture, for allowing us to use their lab facilities.
946
REFERENCES
[1] Bach T.J., Hydroxymethylglutaryl-CoA reductase, a key enzyme in phytosterol synthesis, Lipids 21 (1986) 8288. [2] Brooker J.D., Russell D.W., Properties of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from Pisum sativum seedlings, Arch. Biochem. Biophys. 167 (1975) 723729. [3] Burnett R.J., Maldonado-Mendoza I.E., Mc Knight T.D., Nessler C.L., Expression of 3-hydroxy3-methylglutaryl coenzyme A reductase gene from Camptotheca acuminata is differentially regulated by wounding and methyl jasmonate, Plant Physiol. 103 (1993) 4148. [4] Caelles C., Ferrer A., Balcells L., Hegardt F.G., Boronat A., Isolation and structural characterization of a cDNA encoding Arabidopsis thaliana 3-hydroxy-3methylglutaryl coenzyme A reductase, Plant Mol. Biol. 13 (1989) 627638. [5] Campos N., Boronat A., Targeting and topology in the membrane of plant 3-hydroxy-3-methylglutaryl coenzyme A reductase, Plant Cell 7 (2000) 21632174. [6] Chappell J., The biochemistry and molecular biology of isoprenoid biosynthetic pathway in plants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46 (1995) 521547. [7] Chappell J., Wolf F., Proulx J., Cuellar R., Saunders C., Is the reaction catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase a rate-limiting step for isoprenoid biosynthesis in plants?, Plant Physiol. 109 (1995) 13371343. [8] Chen P.M., Varga D.M., Mielke E.A., Facteau T.J., Drake S.R., Control of supercial scald on dAnjou pears by ethoxyquin: oxidation of -farnesene and its inhibition, J. Food Sci. 55 (1990) 171180. [9] Choi D., Wards B.L., Bostock R.M., Differential induction and suppression of potato 3-hydroxy-3methylglutaryl coenzymeA reductase genes in response to Phytophthora infestans and to its elicitor arachidonic acid, Plant Cell 4 (1992) 13331344. [10] Chye M.L., Kush A., Tan C.T., Chua N.H., Characterization of cDNA and genomic clones encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Hevea brasiliensis, Plant Mol. Biol. 16 (1991) 567577. [11] Chye M.L., Tan C.T., Chua N.H., Three genes encode 3-hydroxy-3-methylglutaryl coenzyme A reductase in Hevea brasiliensis: hmg1 and hmg3 are differentially expressed, Plant Mol. Biol. 19 (1992) 473484. [12] Dale S., Arro M., Becerra B., Morrice N.G., Boronat A., Hardie D.G., Ferrer A., Bacterial expression of the catalytic domain of 3-hydroxy-3-methylglutarylCoA reductase (isoform HMGR1) from Arabidopsis thaliana, and its inactivation by phosphorylation at Ser577 by Brassica oleracea 3-hydroxy-3-methylglutaryl-CoA reductase kinase, Eur. J. Biochem. 233 (1995) 506513.
[13] Eisenreich W., Rohdich F., Bacher A., Deoxyxylulose phosphate pathway to terpenoids, Trends Plant Sci. 6 (2001) 7884. [14] Enjuto M., Balcells L., Campos M., Caelles C., Arro M., Boronat A., Arabidopsis thaliana contains two differentially expressed 3-hydroxy-3-methylglutaryl-CoA reductase genes, which encode microsomal forms of the enzyme, Proc. Natl. Acad. Sci. USA 91 (1994) 927931. [15] Gray J.C., Control of isoprenoid biosynthesis in higher plants, Adv. Bot. Res. 14 (1987) 2591. [16] Huelin F.E., Coggiola I.M., Supercial scald, a functional disorder of apples. V. Oxidation of -farnesene and its inhibition by diphenylamine, J. Sci. Food Agric. 21 (1970) 4448. [17] Jelesko J.G., Jenkins S.M., Rodriguez-Concepcion M., Gruissem W., Regulation of tomato HMG1 during cell proliferation and growth, Planta 208 (1999) 310318. [18] Ju Z., Curry E.A., Lovastatin inhibits -farnesene biosynthesis and scald development in Delicious and Granny Smith apples and dAnjou pears, J. Am. Soc. Hort. Sci. 125 (2000) 626629. [19] Kreuzwieser J., Schnitzler J.P., Steinbrecher R., Biosynthesis of organic compounds emitted by plants, Plant Biol. 1 (1999) 149159. [20] Kyte J., Doolittle R.F., A simple method of displaying the hydropathic character of a protein, J. Mol. Biol. 157 (1982) 105132. [21] Lange M.B., Rujan T., Martin W., Croteau R., Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes, Proc. Natl. Acad. Sci. USA 97 (2000) 1317213177. [22] Learned R.M., Fink G.R., 3-Hydroxy-3-methylglutarylcoenzyme A reductase from Arabidopsis thaliana is structurally distinct from the yeast and animal enzymes, Proc. Natl. Acad. Sci. USA 86 (1989) 27792783. [23] Lichtenthaler H.K., The plant prenyllipids including carotenoids, chlorophylls and prenylquinones, in: Moore T.S. (Ed.), Lipid Metabolism in Plants, CRC Press, Boca Raton FL, 1993, pp. 427470. [24] Lichtenthaler H.K., Rohmer M., Schwender J., Two independent biochemical pathways for isopentenyl diphosphate and isoprenoid biosynthesis in higher plants, Physiol. Plant. 101 (1997) 643652. [25] Loguercio L.L., Wilkins T.A., The genomic clones encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase from cotton (Gossypium hirsutum L.), Plant Physiol. 116 (1998) 869. [26] Loguercio L.L., Scott H.C., Trolinder N.L., Wilkins T.A., HMG-CoA reductase gene family in Cotton (Gossypium hirsutum L.): Unique structural features and differential expression of hmg2 potentially associated with synthesis of specic isoprenoids in developing embryos, Plant Cell Physiol. 40 (1999) 750761. [27] Maldonado-Mendoza I.E., Vincent R.M., Nessler C.L., Molecular characterization of three differentially expressed members of the Camptotheca acuminata 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) gene family, Plant Mol. Biol. 34 (1997) 781790.
947
[28] Meir S., Bramlage W.J., Antioxidant activity in Cortland apple peel and susceptibility to supercial scald after storage, J. Amer. Soc. Hort. Sci. 113 (1988) 412418. [29] Moore K.B., Oishi K.K., Characterization of 3-hydroxy3-methylglutaryl Coenzyme A reductase activity during maize seed development, germination and seedling emergence, Plant Physiol. 101 (1993) 485491. [30] Narita J.O., Gruissem W., Tomato hydroxymethylglutaryl-CoA reductase is required early in fruit development but not during ripening, Plant Cell 1 (1989) 181190. [31] Nelson A.J., Doerner P.W., Zhu Q., Lamb C.J., Isolation of a monocot 3-hydroxy-3-methylglutaryl coenzyme A reductase gene that is elicitor-inducible, Plant Mol. Biol. 25 (1994) 401412. [32] Rodriguez-Concepcion M., Gruissem W., Arachidonic acid alters tomato HMG expression and fruit growth and induces 1-hydroxy-3-methylglutaryl coenzyme A reductase-independent lycopene accumulation, Plant Physiol. 119 (1999) 441448. [33] Rose T.M., Schultz E.R., Henikoff J.G., Pietrokovski S., McCallum C.M., Henikoff S., Consensusdegenerate hybrid oligonucleotide primers for amplication of distantly related sequences, Nucleic Acids Res. 26 (1998) 16281635. [34] Rupasinghe H.P.V., Murr D.P., Paliyath G., DeEll J.R.,
[35]
[36]
[37]
Suppression of -farnesene synthesis in Delicious apples by aminoethoxyvinylglycine (AVG) and 1-methylcyclopropene (1-MCP), Physiol. Mol. Biol. Plant. 6 (2000) 195198. Rupasinghe H.P.V., Murr D.P., Paliyath G., Skog L., Inhibitory effect of 1-MCP on ripening and supercial scald development in Delicious and McIntosh apples, J. Hort. Sci. Biotech. 74 (2000) 271276. Rupasinghe H.P.V., Paliyath G., Murr D.P., Biosynthesis of -farnesene and its relation to supercial scald in Delicious apples, J. Am. Soc. Hort. Sci. 123 (1998) 882886. Rupasinghe H.P.V., Paliyath G., Murr D.P., Sesquiterpene -farnesene synthase: partial purication, characterization, and activity in relation to supercial scald development in Delicious apples, J. Am. Soc. Hort. Sci. 125 (2000) 111119. Sanger F., Nicklen S., Coulson A.R., DNA sequencing with chain-terminating inhibitors, Proc. Natl. Acad. Sci. USA 74 (1977) 54635467. Stermer B.A., Bianchini G.M., Korth K.L., Regulation of HMG-CoA reductase activity in plants, J. Lipid Res. 35 (1994) 11331140. Yang Z., Park H., Lacy G.H., Cramer C.L., Differential activation of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes by wounding and pathogen challenge, Plant Cell 3 (1991) 397405.