- Email: [email protected]
Inhibition of PAL, CHS, and ERS1 in ‘Red d’Anjou’ Pear (Pyrus communis L.) by 1-MCP
Postharvest Biology and Technology 45 (2007) 46–55
Inhibition of PAL, CHS, and ERS1 in ‘Red d’Anjou’ Pear (Pyrus communis L.) by 1-MCP D.D. MacLean a,∗ , D.P. Murr a , J.R. DeEll b , A.B. Mackay a , E.M. Kupferman c a
Department of Plant Agriculture, E.C. Bovey Building, University of Guelph, Guelph, Ontario N1G 2W1, Canada b Ontario Ministry of Agriculture, Food and Rural Affairs, Simcoe, Ontario N3Y 4N5, Canada c WSU TFREC, Wenatchee, WA 98801, USA Received 17 August 2006; accepted 20 January 2007
Abstract The ethylene antagonist 1-MCP was investigated for its potential impact on the transcription of two key flavonoid biosynthetic (phenylalanine ammonia-lyase, PAL, E.C. 4.3.1.5; and chalcone synthase, CHS, E.C. 2.3.1.74), flavonoid transport (glutathione S-transferase, GST, E.C. 2.5.1.18) and ethylene perception (ethylene response sensor 1, ERS1) transcripts during the postharvest storage and ripening of pear (Pyrus communis L.). ‘Red d’Anjou’ pear fruit were harvested from Wenatchee, WA, USA, transported to Guelph, Ont., Canada, then treated with 1 L L−1 1-MCP, and subsequently placed in cold storage (0–1 ◦ C, 90–95% RH) for up to 126 days. After removal, fruit were warmed to room temperature (1 or 7 days) then tissue samples were collected for Northern blot analysis and determination of flavonoid and chlorogenic acid concentration by HPLC. In general, PAL content decreased during storage, with content increasing during the post-storage ripening period in parallel with the increase in respiration rate and ethylene content. In contrast, CHS content decreased dramatically during the 1-week ripening period, while ERS1 remained constant. The expression of PAL, CHS and ERS1 transcripts were all inhibited by 1-MCP. GST transcript abundance decreased during storage, and was largely unaffected by the 1-MCP treatment. The flavonoid concentration remained constant throughout storage and subsequent ripening. However, after the first removal and warming to room temperature, chlorogenic acid concentration increased in the untreated, but not in the 1-MCP-treated fruit. These results suggest that 1-MCP significantly inhibits the transcription of key flavonoid biosynthetic enzymes and ethylene perception proteins, but not the flavonoid transport enzyme. The increase in PAL with the concomitant post-storage decrease of CHS and postharvest decrease of GST suggests a diversion of carbon from flavonoid compounds into chlorogenic acid. © 2007 Elsevier B.V. All rights reserved. Keywords: Pear; 1-Methylcyclopropene; Flavonoid; Chlorogenic acid; Phenylalanine ammonia-lyase; Chalcone synthase
1. Introduction In recent years there has been a growing interest in all classes of flavonoids as integral antioxidants in the human diet, due in part to their demonstrated anticarcinogenic activity, inhibition of tumor cell proliferation, antioxidant and free radical scavenging capabilities, as well as their effectiveness as metal chelators (Harborne and Williams, 2000). The major flavonoids present in pear have been implicated in the prevention of lipid peroxidation of cell membranes in mammalian systems (Tsuda et al., 1994), while other studies have investigated the bioavailability of these flavonoid metabolites in human systems (Kay et al., 2004).
∗
Corresponding author. Tel.: +1 519 824 4120x58255; fax: +1 519 767 0755. E-mail address: [email protected] (D.D. MacLean).
0925-5214/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2007.01.007
The activity of PAL has been considered a critical step in the divergence of carbon into the phenylpropanoid biosynthetic pathway, and it has been demonstrated in many plant species, including carrot roots (Chalutz, 1973) and citrus (Riov et al., 1969), that ethylene is a key regulator of PAL activity. Numerous studies using apple have demonstrated the dependence of anthocyanin accumulation on the presence of ethylene and the concomitant increase in PAL activity (Faragher and Chalmers, 1977; Blankenship and Unrath, 1988). As well, factors other than the level of ethylene in the tissue contribute to the de novo synthesis of anthocyanins, including low temperature (Tan, 1980; Arawaka, 1991), fruit maturity (Murphey and Dilley, 1988), and storage duration (Jiang and Joyce, 2003), all of which are important considerations in the postharvest storage of pear fruit. Pears contain high levels of some hydroxycinnamic acid derivatives, with chlorogenic acid being the predominant structure (Oleszek et al., 1994). These classes of structures diverge
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
Fig. 1. An abbreviated phenylpropanoid pathway starting with the activity of phenylalanine ammonia-lyase (PAL), with products diverging either to the flavonoid biosynthetic pathway (left) through chalcone synthase (CHS), or to the purported chlorogenic acid biosynthetic pathway (right) through hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyltransferase (HCT, see Niggeweg et al., 2004). Both cyanidin 3-galactoside and chlorogenic acid are believed to be endogenous substrates for glutathione S-transferase, an enzyme involved in the transport of flavonoids through the cytoplasm to the vacuolar membrane (Walbot et al., 2000).
from the flavonoid biosynthetic pathway downstream of PAL, but upstream from CHS (Fig. 1). Pears also contain high amounts of flavonoids from the flavonol (e.g. quercetin 3-glycosides), flavan-3-ol (e.g. catechin), and anthocyanin (e.g. cyanidin 3glycosides) classes of structures (Mosel and Herrmann, 1974; Scheiber et al., 2001). These classes all differentiate downstream of CHS, a critical step in the divergence of carbon into the flavonoid biosynthetic pathway. One of the last defined enzymes of the flavonoid biosynthetic pathway is GST. The GST enzymes have a central role in the transport of flavonoids through the cytoplasm to the vacuolar membrane (Walbot et al., 2000). GSTs may also play a role in the regulation and signaling of the flavonoid biosynthetic pathway (Loyall et al., 2000). The imposition of a postharvest chilling period and the presence of ethylene is required for the successful ripening of pear. More specifically, ‘d’Anjou’ pear requires anywhere from 46 days (Blankenship and Richardson, 1985) to 60 days (Chen and Mellenthin, 1982) at −1 ◦ C to induce ethylene biosynthesis. When pear fruit are treated with 1-MCP prior to the burst of ethy-
47
lene production, the onset of fruit ripening is delayed (Ekman et al., 2004; Trinchero et al., 2004). For example, ‘d’Anjou’ fruit exposed to 1-MCP displayed a 1000- and 4-fold reduction in ethylene production and respiration rate, respectively, and also prevented the flesh from softening (Argenta et al., 2003). The ERS1 is an ethylene-binding protein, and a member of the 5-gene family of ethylene receptors that includes ETR1/2, EIN4, and ERS1/2 (Hall et al., 2000). Although the hierarchy or complementary function of the family of ethylene binding proteins is unclear, the ERS1 protein is interesting because it is constitutively present, but is also associated with system 2, or autocatalytic production of ethylene. As seen in ripening peach fruit, the mRNA transcript abundance of ERS1 increased in parallel with the climacteric burst in ethylene (Trainotti et al., 2006). A similar study investigating the transcript abundance of ERS1 in avocado also found a background level of ERS1 present in tissue prior to the onset of autocatalytic ethylene, followed by a very rapid and large increase in the abundance of transcript as the fruit neared the climacteric peak (Owino et al., 2002). Finally, using ‘Passe-Crasssane’ pear fruit it was found that PcERS1 increased in response to both ripening, and an exogenous treatment of ethylene (El-Sharkawy et al., 2003). It is evident that the ERS1 protein plays a role in the perception of ethylene during climacteric fruit ripening. Therefore, the down-regulation of this protein by a treatment with 1-MCP may result in the down-stream inhibition of numerous ripening-related transcription factors and enzymes, including PAL, which is regulated by ethylene. The dependency of PAL activity on the presence of ethylene raises concerns that treatment with 1-MCP may adversely affect total phenolic concentration of the ripening fruit. For example, in strawberry a decrease in PAL activity and the concomitant inhibition of the increase in flavonoid and phenolic concentration of the fruit resulted from a treatment with 1-MCP (Jiang et al., 2001). 1-MCP is now being used extensively at both a research and commercial level around the world as a postharvest tool to extend shelf life in numerous nutraceutical-producing crops, including pear. Therefore, the goal of the present study was to determine the effect of 1-MCP on the transcription of flavonoid biosynthetic (PAL and CHS), flavonoid transport (GST) and ethylene perception (ERS1) proteins in ‘Red d’Anjou’ pear, over the course of a 126-day cold storage period and a 1-week ripening period. 2. Materials and methods 2.1. Fruit harvest and 1-MCP treatment ‘Red d’Anjou’ pears were harvested at commercial maturity (flesh firmness of 58–67 N) from Guthrie Pear Orchard, Wenatchee, WA, USA, on 21 September 2004. Immediately after harvest, fruit were hand-packed in commercial shipping boxes and transported to Stemilt Growers Inc. and placed in cold storage at −0.5 ◦ C for 4 weeks. Fruit were subsequently transported to Canada in a refrigerated transport truck (3–4 ◦ C), and arrived at the University of Guelph after 35 days. Upon arrival, fruit were removed from packaging and placed into bushel boxes and were
48
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
treated twice, on consecutive days, with 1 L L−1 of 1-MCP (0.14% a.i.; SmartFreshTM , Agrofresh Inc. Spring House, PA) (0–1 ◦ C) with each treatment lasting 24 h. Similar non-treated control fruit were held in air for 24 h at 0–1 ◦ C. Following treatment, fruit were held at 0–1 ◦ C and 90–95% RH and removed at 21 day intervals for a total storage time of 126 days. After each removal time, fruit samples were allowed to equilibrate at room temperature for 1 or 7 days, then the epidermal and some underlying cortical tissue (∼1–2 mm) from 5 fruit (3 repetitions, 15 fruit total) were removed with a hand peeler, combined, flash frozen and ground under liquid nitrogen. The resulting frozen tissue samples were then held at −80 ◦ C until preparation for flavonoid determination and RNA extraction. 2.2. Fruit quality and maturity indices After completion of the two 1-MCP treatments, three replications of five fruit each were removed and evaluated for ethylene production and respiration rate every 3 days for 10 days, while flesh firmness and TSS were monitored after 1 and 7 days. These same tests were performed after every storage removal date. Ethylene was measured using a Varian CP-3380 GC (Varian Inc., Mississauga, ON) equipped with a 15 m × 0.32 mm Restek RT-S-Plot fused-silica column (Chromatographic Specialties, Inc., Brockville, Ont., Canada), and a flame ionization detector (FID). The injector, column and detector temperatures were 120, 35 and 225 ◦ C, respectively, while high-grade helium was used as the carrier gas at a flow rate of 22 mL min−1 . Respiration rate was determined using an ADC-EGA infrared CO2 gas analyzer (Nortech Control Equipment Inc. Etibicoke, Ont., Canada). Flesh firmness was determined using a hand-held Effegi penetrometer (Facchini 48011 Alfonsine, Italy) fitted with an 8-mm tip, and measurements were taken from pared opposite sides of the fruit. The exudate of five fruit from the firmness test was collected in a petri dish for determination of TSS using a handheld temperature-compensated refractometer (Fisher Scientific, Nepean, Ontario). 2.3. Template RNA isolation and cDNA synthesis Fruit that were stored in air for 63 days at 0–1 ◦ C and 1 day at 21 ◦ C were used for the extraction of total RNA and synthesis of cDNA. Approximately 2–3 g of the frozen pear skin tissue was ground to a fine powder with a mortar and pestle that was pre-chilled with liquid nitrogen. Total RNA was extracted using the Qiagen RNeasy Plant Mini Kit® (Mississauga, Ont., Canada), using the supplied RLT extraction buffer, according to the instructions of the manufacturer. RNA was eluted from the supplied column using 50 L sterile water, and the quantity and quality was estimated using a Biochrom Ultrospec® 2100 pro spectrophotometer set at 260 nm (Biochrom Ltd., Cambridge, UK). First-strand cDNA synthesis from the reverse-transcription of RNA was performed using the ‘two-step’ protocol of the RETROscript® Kit from Ambion (Austin, TX, USA), using oligo (dT) as the primer. For the RT reaction, 2 g RNA, 2 L oligo (dT) and sterile water to 12 L was mixed and heated to 80 ◦ C for 3 min to denature the RNA. After placing the reaction
on ice, the remaining RT components were added; 2 L 10× RT buffer (500 mM Tris–HCl, pH 8.3, 750 mM KCl, 30 mM MgCl2 , and 50 mM DTT), 4 L dNTP mix, 1 L RNase inhibitor, and 1 L MMLV-RT reverse transcriptase enzyme. The 20 L RT reaction was then incubated at 44 ◦ C for 2 h in a thermocycler, and then inactivated by a 10-min incubation at 92 ◦ C. The cDNA was either stored at −20 ◦ C or used immediately for reverse transcriptase polymerase chain reaction (RT-PCR). 2.4. Cloning of PAL, CHS, GST and ERS1 Selected PAL genes were identified from the NCBI database (accession numbers: Arabidopsis thaliana, P45724 and P35510; and Malus domestica, CAA48231 and P35512) and aligned using the ClustalW multiple sequence alignment software (Chenna et al., 2003). After conserved amino acid regions were identified, the PAL gene was amplified from pear cDNA by PCR using the following degenerate oligonucleotide primers: sense 5 -GG(A/C/G/T)GG(A/C/G/T)-AA(C/T)TT(C/T)-CA (A/G)GG-3 corresponding to the amino acid sequence (GGNFQG) and antisense 5 -AA(A/C/G/T)AC(C/T)TT(A/G)TC (A/G)CA(C/T)TC(C/T)TC-3 corresponding to the amino acid sequence (EECDKVF). The primers for Pc-CHS1 were designed based on the conserved regions from numerous species (accession numbers: M. domestica, BAB92996; A. thaliana, CAI30418; Lycopersicon esculentum, CAA38980; and Zea mays, CAA42763). The sense primer used was 5 -ATGATGTA(C/T)CA(A/G)CA(A/G)GG (A/C/G/T)-TG(C/T)TT-3 corresponding to the amino acid sequence (MMYQQGCF), while the antisense primer was 5 AC(A/C/G/T)GG(A/C/G/T)CC(A/G)AA(A/C/G/T)CC(A/G) AA-3 corresponding to the amino acid sequence (FGFGPG). The full-length cDNA clone was determined using Ambion’s FirstChoice ® RLM-RACE kit (Austin, TX, USA). The sense primer for Pc-GST was designed from the Malus EST (accession number: DY255365) and was 5 -ATGGTGGTGAAGGTGTACGG-3 corresponding to the amino acid sequence (MVVKVY). The antisense primer 5 TTCTTCCAAGC(C/T)-GG(A/C/G/T)CT-3 corresponding to the amino acid sequence (RPAWK), was selected based on conserved regions from numerous other GSTs (accession numbers: Glycine max, AAG34812; Citrus sinensis, ABA42224; Phaseolus acutifolius, AAM34480; Petunia hybrida, CAA68993; and Vitis vinifera, AAX81329). The primers for Pc-ERS1 were designed based on the previously published PcERS1 from Pyrus species (accession number: AF386517) (El-Sharkawy et al., 2003). The sense primer used was 5 -AATGGAGTCATGTGATTGC-3 corresponding to the amino acid sequence (MESCDC), while the antisense primer was 5 -GCAATCCATGCTCGCAAT-3 corresponding to the amino acid sequence (AIHARN). PCR reactions for all clones were performed using the Biorad iCycler thermocycler (firmware version 3.032), using 2.5 U of Promega Taq DNA polymerase (Madison, WI, USA) under the following conditions: hot start 3 min at 94 ◦ C, then 35 cycles at 94 ◦ C (1 min), anneal at 52 ◦ C (PAL), 50 ◦ C (CHS), or 55 ◦ C (GST and ERS1) (1 min), and extension at 72 ◦ C (1.5 min), with a final exten-
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
sion at 72 ◦ C for 5 min. An aliquot of the PCR reaction was loaded onto a 1% (w/v) agarose gel (80 V) in order to confirm a single product was amplified by the reaction. The remaining PCR product was cleaned using the GeneClean® Turbo PCR kit from QBiogene (Carlsbad, CA, USA) to remove any potential contaminants and subsequently cloned using the Qiagen® PCR Cloningplus Kit (Mississauga, Ont., Canada) into the supplied pDrive vector using a 10× molar excess of template. The ligation reaction was allowed to proceed for 1 h at 14 ◦ C, and subsequently transformed into Qiagen® EZ Competent Cells (Mississauga, Ont., Canada) following the recommended protocol. Colonies that successfully incorporated the PCR product were amplified using the M13 universal and gene-specific primers, and then sequenced using the T7 promoter region as the forward primer using a model 3730 Genie sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). The sequences that were obtained were edited to remove any vector sequence, and identified using Blastn program from NCBI (Altschul et al., 1997).
49
fixed by exposing the membrane to 700 kJ of UV light for 20 s in a UV cross-linker (Hoefer Pharmacia Biotech, Inc., San Francisco, CA, USA). The membrane was pre-hybridized for 30 min in freshly prepared and pre-heated (68 ◦ C) Roche DIG Easy Hyb granules (Roche Diagnostics, Laval, Que., Canada), then hybridized overnight (68 ◦ C) with 20 ng of digoxigeninlabelled gene specific RNA probe. The following morning, the membrane was washed twice in 2× SSC, 0.1% SDS for 5 min each at room temperature, followed by a higher stringency wash of 0.5× SSC, 0.1% SDS for 15 min each at the hybridization temperature (68 ◦ C). Blocking reagent, anti-Dig AP conjugate (1:20,000) and other washes were performed according to the recommended protocol supplied with the blocking and detection reagents (Roche Diagnostics, Laval, Que., Canada). Chemiluminescent detection was performed using CDP-Star (ready to use) system (Roche Diagnostics, Laval, Que., Canada) and the membrane was subsequently exposed to Kodak® BioMax Light film. 2.8. Tissue preparation for HPLC analysis
2.5. DIG-labeling of RNA probes The partial cDNA gene fragments of PcPAL, Pc-GST, and Pc-CHS1 were all cloned into the pDrive vector of Qiagen (Mississauga, Ont., Canada) and amplified and sequenced using a gene-specific primer in combination with the universal M13 primers. Probes were designed from these sequences, with the reverse complement primer having a T7 promoter region added to the 5 -end. The DIG-labeling transcription reaction made use of the MAXIscriptTM T7 in vitro Transcription Kit of Ambion (Austin, TX, USA). For the reaction, 1 g template, along with 2 L of each the DIG RNA labeling mix, T7 enzyme, and transcription buffer were used. Optional steps including the addition of DNase and EDTA were performed. 2.6. RNA extraction for Northern blot analysis Total RNA was extracted from pears that were treated with/without 1-MCP after each cold storage removal (0–126 days) and shelf-life period (+1 or +7 days) using the Qiagen RNeasy Plant Mini Kit® (Mississauga, Ont., Canada). Total RNA content was determined spectrophotometrically at 260 nm as described previously, and quality was estimated by monitoring the 260:280 nm ratio and by observing the 18s and 28s ribosomal RNA bands stained with ethidium bromide (EtBr) after performing formaldehyde denaturing agarose gel electrophoresis (see below). 2.7. Northern blot analysis For Northern blot analysis, 1 g total RNA was denatured at 80 ◦ C for 3 min in 3× RNA sample loading buffer (containing EtBr), and loaded onto a 1.5% (w/v) agarose gel containing 0.22 M formaldehyde in MOPS running buffer. RNA was separated at 60 V for 3–4 h prior to being blotted onto an Ambion BrightStar-PlusTM positively charged membrane (Austin, TX, USA). RNA was transferred overnight using 20× SSC, then
Approximately 1 g (±0.02 g) of finely ground frozen tissue from 5 fruit per replication was placed in a 12.5-mL disposable glass culture vial containing 5 mL MeOH (0.1% HCl). The sample was sonicated at 20 ◦ C for 30 min, then the headspace of the vial was flushed with N2(g) , capped with a rubber septa-seal, and subsequently placed in complete darkness at 4 ◦ C for overnight extraction. The following morning, the sample was centrifuged at 1000 × g for 5 min at 4 ◦ C. The supernatant was decanted and set aside, while the pelleted residue was further extracted with a 3-mL wash of MeOH (0.1% HCl). After a 15-min sonication, the sample was placed in complete darkness for 3 h then centrifuged at 1000 × g for 5 min at 4 ◦ C. The supernatant was added to the first supernatant for a final total extraction volume of ∼8 mL. The headspace of the vial was then flushed with N2(g) , capped with a rubber septa-seal and placed at −20 ◦ C for no more than 12 h. Flavonoids in the supernatant extract were concentrated to near dryness using a rotary evaporator held at 60 ◦ C, resuspended with 0.7-mL MeOH (0.1% HCl), vortexed for 10 s, and centrifuged at 16,000 × g for 20 min. After centrifugation, the sample was decanted directly into a 1.5-mL amber autosampler vial where the final volume was brought to 1-mL with MeOH (0.1% HCl), sealed with a PTFE autosampler vial cap, and stored at −80 ◦ C until analyzed by HPLC. 2.9. HPLC-DAD analysis Polyphenols were separated and identified using an Agilent 1100 series HPLC (Mississauga, Ont., Canada) system equipped with an inline continuous vacuum solvent degasser, binary pump, temperature controlled autosampler and column compartments, and a PDA, all controlled by a Chemstation (ver. 4.0) software package. Solvents used were A: 5% formic acid and B: ACN at a flow rate of 0.3 mL min−1 . The gradient (expressed as %B) was: 0–1 min, 5%; 1–8 min, 5–13%; 8–12 min, 13–15%; 12–26 min, 15–22%; 26–26.5 min, 22–40%; 26.5–30.5 min, 100%; 30.5–35 min, 5%. The autosampler compartment was
50
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
maintained at 4 ◦ C. The volume of injection for both samples and standards was 5 L. The polyphenolic compounds were retained using a Waters Symmetry Shield RP-18 column (100 mm × 2.1 mm × 3.5 m; Mississauga, Ont., Canada) protected by a guard column (10 mm × 2.1 mm × 3.5 m) all held at 30 ◦ C within the column compartment. Eluted compounds were detected using the PDA equipped with a semi-micro flow cell with a full spectral scan set from 210 to 700 nm (1 nm steps), and monitored at 530 nm for the detection of anthocyanins, 350 nm for flavonols, 280 nm for flavan-3-ols, and 330 nm for chlorogenic acid, all with a bandwidth of 4 nm. 2.10. Statistical analysis Each main effect of removal × shelf life × 1-MCP treatment contained three replications of five fruit per replication. The experimental setup was a completely randomized design with storage removal, shelf life, and 1-MCP treatment designated as fixed effects. Least squares means, analysis of variance, tests for normality and outliers were performed using the General Linear Model Procedure (Proc GLM) of the Statistical Analysis System (SAS) software (version 8.2, SAS Institute, Cary, NC, USA) at a significance level of P ≤ 0.05. 3. Results 3.1. Fruit harvest and 1-MCP treatment The pear fruit were harvested at commercial maturity on 21 September 2004. However, due to unforeseen circumstances involving the transportation of fruit across the continent and
the Canadian border, fruit were not received at the University of Guelph until 26 October 2004, 35 days after being harvested. The condition of the fruit upon arrival was excellent; however, based on the flesh firmness (53.1 N), TSS (13.7%), and headspace ethylene concentration (2.31 L kg−1 h−1 ), it is likely that the fruit had commenced ripening. As a consequence, there was a good possibility that fruit would not be as responsive to a 1-MCP treatment as they would have if they had been treated prior to the onset of fruit ripening. Therefore, as a preemptive measure, fruit were exposed to two successive 1-day treatments of 1 L L−1 1MCP (0–1 ◦ C) in order to inhibit the rise in ethylene production relative to the untreated control fruit. Based on ethylene production and respiration rates of fruit monitored over a 10-day period at room temperature immediately after exposure to 1-MCP, the treatment was effective at inhibiting the endogenous production of ethylene and general fruit metabolism despite the long delay between harvest and 1MCP treatment. No ethylene was detectable (<50 ppb) in fruit held at room temperature for up to 10 day post 1-MCP treatment (Fig. 2A). Similar inhibition of ethylene production was observed in fruit after 126 days of cold storage, where percent inhibition varied between 96 and 99% (Fig. 2C). With respect to respiration rate, as measured by CO2 production, in both the 1-MCP treated and non-treated control fruit, respiration rate was high after the initial removal from cold storage, and decreased by the second evaluation date 4 days after removal from storage (Fig. 2B and D). However, after the initial decrease in respiration rate, the control fruit displayed an increasing trend up to 10 days at room temperature, while the 1-MCP-treated fruit continued to decrease. By day 10, the inhibition was 65% for the initial fruit, and 70% for fruit after 126 days of storage. These results suggest
Fig. 2. The analysis of headspace ethylene (A and C: L kg−1 h−1 ) and CO2 (B and D: mL kg−1 h−1 ) production rates of ‘Red d’Anjou’ pear immediately following a treatment with 1-MCP (A and B) or after 126 days of storage (0–1 ◦ C) (B and D). Fruit were exposed to two successive treatments of 1 L L−1 1-MCP (24 h, 0–1 ◦ C) 35 days after harvest, storage and transportation of the fruit from Washington State. Legend is the same for all figures. Each point represents the mean of three values (P ≤ 0.05).
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
the 1-MCP treatment remained effective at inhibiting ethylene production and respiration rate, despite the higher metabolic rate expected at the warmer temperature. 3.2. Flavonoid and chlorogenic acid biosynthesis The average concentration of chlorogenic acid, across all storage and shelf life factors was 283 and 275 g g−1 f.w. for the control and 1-MCP-treated fruit, respectively. In contrast to the stable concentration of flavonoids during postharvest handling of the fruit, chlorogenic acid concentration tended to increase. This is especially evident after the initial fruit evaluation, where chlorogenic acid concentration in non-treated fruit increased 187% during the first week at room temperature (Fig. 3B). This increase was not present in fruit treated with 1-MCP. However, the effect of the 1-MCP treatment on chlorogenic acid biosynthesis appears to have been lost by the second removal, where chlorogenic acid concentration in 1-MCP-treated fruit was not significantly different from the untreated fruit. Based on regression equations for control (y = 23.942x + 175.04) and 1-MCP-treated fruit (y = 42.855x + 82.324), chlorogenic acid concentration increased during the storage and 1-week ripening period. The average concentration of cyanidin 3-galactoside across all storage and shelf life factors was 61 and 69 g g−1 f.w. for the control and 1-MCP treated fruit, respectively. Based on the regression equations for control (y = −0.3079x + 62.842)
Fig. 3. Changes in the concentration (g g−1 f.w.) of (A) chlorogenic acid and (B) cyanidin 3-galactoside in the skin of ‘Red d’Anjou’ pear, with shelf-life periods (+1 or +7 days), storage (0, 21, 63 and 126 days) and 1-MCP treatment effects. Legend is the same for both (A) and (B). Each point represents the mean of three values (P ≤ 0.05).
51
and 1-MCP-treated fruit (y = 1.2055x + 63.498), cyanidin 3galactoside concentration did not vary significantly during storage or after removal and warming to room temperature for one week (Fig. 3B). Also, the treatment with 1-MCP did not significantly affect the concentration of anthocyanin. Other flavonoids from the classes of anthocyanins, flavonols and flavan-3-ols, were also analyzed and they all exhibited a similar response to the storage, shelf life and 1-MCP treatment as cyanidin 3-galactoside (data not presented). 3.3. Gene cloning and sequence analysis Degenerate oligonucleotide primers were used successfully to isolate the PCR fragments of the expected size for Pc-PAL, PcCHS1, Pc-GST and Pc-ERS1. The full-length cDNA clone for chalcone synthase was determined by first using a set of degenerate oligonucleotide primers designed from conserved amino acid regions from the alignment of M. domestica (BAB92996), L. esculentum (CAA38980), A. thaliana (CAI30418) and Z. mays (CAA42763). This partial cDNA fragment of 660 bp was subsequently used to determine the full-length coding region, as well as the 3 and 5 UTRs. The full-length clone, designated PcCHS1, had a coding region of 1170 bp, with a predicted amino acid sequence of 389 residues, and predicted molecular mass of 42.5 kDa (Fig. 4). The deduced amino acid sequence displayed 98.4% identity with M. domestica (BAB92996), 98.2% identity with Sorbus aucuparia (ABB89213), 92.5% identity with Fragaria × ananassa (BAE17124), and 92.8% identity with Rosa hybrida (BAC66467). The three partial cDNA fragments for Pc-PAL, Pc-GST and Pc-ERS1 were also isolated in this study. For the Pc-PAL fragment, a single band of expected size (0.8 kbp) was amplified using degenerate primers from cDNA that was synthesized from total RNA using the oligo d(t) primer. Degenerate primers were designed based on sequence homology with M. domestica (P35512 and CAA48231), A. thaliana (P45724 and P35510), and Pyrus pyrifolia (AAQ15284). After cloning, the resulting PCR product encoded an 846 bp and 282 predicted amino acid sequence homologous to other PAL genes. The product displayed 92.7% identity with Prunus persica (AAF17247), 88.3% identity with Daucus carota (BAC56977), 86.5% identity with Rubus idaeus (AAF40224), and 57% identity over the entire length with M. domestica (CAA48231). The Pc-GST partial cDNA fragment was isolated using a sense primer designed using the Malus GST EST (accession number DY255365), while the reverse primer was based on a previously isolated polypeptide (DDISSRPAW). The PCR reaction resulted in a single band at the expected size of 0.6 kbp. After cloning, a 612 bp fragment was sequenced, and a 204 amino acid sequence was deduced. The highly divergent class of proteins had 73% amino acid identity over the entire length with G. max (AAG34814), 72% identity with A. thaliana (NP 180643), 60% identity with C. sinensis (ABA42224), and 54% identity with Petunia × hybrida (CAA68993). Analysis of domain architecture indicated that the isolated Pc-GST is a member of the phi class of GST proteins, and is involved in the ligandin-binding and transport of flavonoids.
52
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
Fig. 4. Alignment of the deduced amino acid sequence of chalcone synthase (CHS) genes from Pyrus communis (DQ901397), Malus domestica (BAB92996), Lycopersicon esculentum (CAA38980), Arabidopsis thaliana (CAI30418), and Zea mays (CAA42763). Conserved amino acids residues are denoted by (*), conserved substitutes by (:), and semi-conserved substitutes by (.). The low-scoring segments are in dark shading, and exceptional residues are in gray shading.
The Pc-ERS1 partial cDNA fragment was isolated using primers designed from the previously published ERS1 sequence from P. communis (El-Sharkawy et al., 2003). The PCR reaction resulted in a single band at the expected size (1.0 kbp). After cloning, a 741 bp fragment was sequenced from the PCR product, with a 247-deduced amino acid sequence, corresponding to 38.7% of the coding region of the gene, from the start codon forward. The Pc-ERS1 fragment showed 99.6% identity to the previously published Pc-ERS1a sequence (AF386517). The clones for Pc-PAL, Pc-CHS1, Pc-GST, and Pc-ERS1 have been deposited into GenBank under the accession numbers DQ901399, DQ901397, DQ901400, and DQ901398, respectively.
3.4. Northern blot analysis of PAL, CHS, GST and ERS1 The Pc-PAL and Pc-ERS1 transcripts accumulated in a similar manner in response to cold storage, during ripening at 21 ◦ C for 1 week, and 1-MCP treatment (Fig. 5). In the non-treated fruit, transcript abundance for Pc-PAL appears to increase (initial) or remain constant (after 63 and 126 days), during the 1-week ripening period. In contrast, the transcript abundance for Pc-PAL in the 1-MCP-treated fruit was much lower, but constant throughout cold storage and ripening period. Similarily, the Pc-ERS1 transcript abundance was constant and higher in the non-treated control fruit than in the 1-MCP fruit throughout storage and ripening. In contrast, the Pc-CHS1 transcript abun-
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
Fig. 5. Northern blot analysis of two flavonoid biosynthetic genes (Pc-PAL and Pc-CHS1), flavonoid transport (Pc-GST), and one ethylene binding protein gene associated with fruit ripening (Pc-ERS1). ’Red d’Anjou’ pear fruit were harvested and stored for 35 days prior to two successive treatments with 1 L L−1 of 1-MCP (0–1 ◦ C). Dates (i.e. 0, 63 or 126, and +1 or +7 days) indicate the time after 1-MCP treatment. A separate gel and blot was performed for each probe, with each lane containing 1 g of total RNA. The 18s ribosomal RNA band stained with ethidium bromide is included to confirm equivalent loading of the lanes.
dance was very high in fruit tissue 1-day after removal from cold storage. However, after 1-week at room temperature, the transcript abundance for chalcone synthase was not detectable. The treatment of 1-MCP further exacerbated this decreasing trend in Pc-CHS1 abundance. The expression pattern of the Pc-GST indicates a gradual decline in transcript abundance up to the 126 days of storage. Also, there was no change in expression pattern after fruit were removed from storage and allowed to ripen for a week. Interestingly, aside from a moderate retention of transcript after removal from 126 days of storage (which disappeared over the 7-day period at 21 ◦ C), the 1-MCP treatment had no effect on Pc-GST mRNA abundance. Therefore, due to the similar abundance of Pc-GST transcript between the control and 1-MCP-treated fruit, and the relatively high abundance early in the experiment with similar rates of transcript disappearance between the control and 1-MCP-treated fruit, it is likely that there is no de novo biosynthesis of GST during storage and ripening. Therefore, these results suggest that even though PAL is active in control fruit, the carbon is not being directed into the production of flavonoid compounds, as suggested by the rapid decrease in PcCHS1 transcript abundance during the 1-week ripening period. Furthermore, a treatment of 1-MCP appears to inhibit the transcription of key phenylpropanoid biosynthetic genes, and the ethylene binding protein ERS1. 4. Discussion In apple, the increase in PAL activity and the resulting accumulation of anthocyanins is dependent on the presence of ethylene (Faragher and Chalmers, 1977; Blankenship and
53
Unrath, 1988; Gomez-Cordoves et al., 1996). In the present study, the parallel pattern of transcript abundance between PcPAL and Pc-ERS1 supports the notion that PAL is regulated (whole or in part) by ethylene. When fruit were treated with 1MCP, the expression of both Pc-ERS1 and Pc-PAL transcripts, as well as the ethylene production rate all decreased over the 1-week at room temperature. However, in untreated fruit both the ethylene production and the Pc-PAL transcript abundance increased, while the expression of Pc-ERS1 remained constant, thereby suggesting that the accumulation of Pc-PAL transcript is dependent on the presence of ethylene and an active ethylene receptor. A similar effect was observed in strawberry fruit where a treatment of 1-MCP resulted in a decrease in PAL activity (Jiang et al., 2001). The expression of the Pc-ERS1 transcript was markedly reduced by the 1-MCP treatment. Aside from the inhibition of ethylene perception and production, a treatment of 1-MCP often results in a reduced rate of respiration, as well as the inhibition of many other processes associated with fruit ripening, such as flesh softening, starch catabolism, and volatile production (Argenta et al., 2003; Blankenship and Dole, 2003). Thus, the reduced expression of PC-ERS1 may be due to the reduction in overall metabolism of the fruit after the 1-MCP treatment. However, the up-regulation of ERS1 mRNA transcripts has been correlated with the presence of ethylene (Hua et al., 1998), and has been found to increase during fruit ripening, and in response to exogenous ethylene (El-Sharkawy et al., 2003), indicating that ethylene acts as a regulator of ERS1 gene expression. Thus, it is suggested that the binding of 1-MCP by the ERS1 receptor directly alters the regulation of the PcERS1 transcript by inhibiting the regulatory ability of ethylene, as opposed to the reduced metabolic rate that may account for the reduced expression of other genes not directly regulated by ethylene. Based on the northern blot analysis of Pc-PAL and PcCHS1 transcripts, and the propensity of ‘Red d’Anjou’ fruit to preferentially accumulate chlorogenic acid over flavonoid compounds, it is suggested that pear fruit lose the capacity to synthesize flavonoids postharvest. It was found that high-pressure sodium lights stimulated anthocyanin synthesis in ‘Cripps’ Pink apple, but not in ‘Forelle’, ‘Bon Rouge’ or ‘Red d’Anjou’ pear fruit (Marais et al., 2001). As determined in the present study, the reason pear fruit were not responsive to light stimulation might be attributable to the postharvest down-regulation of PcCHS1. Similarly, this down-regulation of Pc-CHS1 may also be attributable to fruit maturity. A study using red ‘Bon Rouge’ and blush ‘Rosemarie’ pear fruit, found that both attained their maximum anthocyanin concentration when the fruit were immature, with pigmentation fading as the fruit matured (Steyn et al., 2004). Thus, it is unlikely that pear fruit have the capacity to biosynthesize flavonoids postharvest. This statement is consistent with the observed stability of cyanidin 3-galactoside, as no significant changes in concentration were detected throughout the storage and 1-week ripening periods, or due to treatment with 1-MCP. In contrast to the flavonoids, the one hydroxycinnamic acid quantified in this study, chlorogenic acid, tended to increase
54
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
during postharvest storage and ripening. This increase has also been observed in ‘Rocha’ pear (Galvis-Sanchez et al., 2003). The general increase in chlorogenic acid content was temporarily inhibited in fruit immediately after the 1-MCP treatment, but this effect was quickly lost by the first removal 21 days into the experiment. After the inhibitory effect was lost, both the control and 1-MCP-treated fruit continued to accumulate chlorogenic acid in parallel for the duration of the study. Thus, the downregulation of Pc-CHS1 transcript and the lack of appreciable changes in flavonoid content postharvest, suggests that carbon is not flowing through CHS into the production of flavonoids. However, the post-storage up-regulation of Pc-PAL transcript and the increase in chlorogenic acid content suggests that carbon is being shunted into the phenylpropanoid biosynthetic pathway, and is being diverted into the production of simple phenols prior to CHS. Glutathione S-transferases are the last genetically defined step in the flavonoid biosynthetic pathway, where they act as flavonoid transport enzymes (Walbot et al., 2000). Unlike PcPAL and Pc-CHS1, the lack of inhibition of Pc-GST transcript by 1-MCP, in conjunction with approximately uniform band intensities within a storage removal (0, 63 or 126 days), suggests that Pc-GST is not actively metabolized postharvest. This is in agreement with the study of Steyn et al. (2004), who demonstrated that anthocyanin concentration is greatest in immature pear fruit. Thus, GST mRNA transcript is most abundant in immature fruit, when fruit are actively synthesizing flavonoids. Once the fruit are harvested, only inhibition of catabolic activities through cold storage and a 1-MCP treatment will assist in the retention of the GST proteins present at the time of harvest. This is evident in the 126-day removal, where the only Pc-GST transcript detected was in 1-MCP-treated fruit 1-day after being removed from cold storage. 5. Conclusions A treatment with 1-MCP inhibited the postharvest abundance of PAL, CHS and ERS1 mRNA transcripts. In non-1-MCPtreated fruit, the up-regulation of PAL mRNA transcript during the 1-week post-storage ripening period in conjunction with the simultaneous down-regulation of CHS over the same period suggests a diversion of carbon from the flavonoid biosynthesis into the production of hydroxycinnamic acids, such as chlorogenic acid. Therefore, it is concluded that a postharvest treatment with 1-MCP will not have an effect on the flavonoid concentration of pear fruit, but may adversely effect the concentration of chlorogenic acid. Acknowledgements This research was supported by AgroFresh Inc., the Natural Sciences and Engineering Research Council, Canada (NSERC), and the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA). We wish to thank AgroFresh Inc. (a Rohm & Haas Co.) for kindly supplying the 1-MCP, Chris Sater for harvesting and packaging of the fruit, and Stemilt Growers Inc., Wenatchee, WA for the transportation of the fruit to Canada.
References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389– 3402. Arawaka, O., 1991. Effect of temperature on anthocyanin accumulation in apple fruit as affected by cultivar, stage of fruit ripening and bagging. J. Hortic. Sci. 66, 763–768. Argenta, L., Fan, X., Mattheis, J., 2003. Influence of 1-methylcyclopropene on ripening, storage life, and volatile production by d’Anjou cv. pear fruit. J. Agric. Food Chem. 51, 3858–3864. Blankenship, S.M., Dole, J.M., 2003. 1-Methylcyclopropene: a review. Postharvest Biol. Technol. 28, 1–25. Blankenship, S.M., Richardson, D.G., 1985. Development of ethylene biosynthesis and ethylene-induced ripening in ‘d’Anjou’ pears during the cold storage requirement for ripening. J. Am. Soc. Hortic. Sci. 110, 520–523. Blankenship, S.M., Unrath, C.R., 1988. PAL and ethylene content during maturation of red and Golden Delicious apples. Phytochemsitry 27, 1001–1003. Chalutz, E., 1973. Ethylene-induced phenylalanine ammonia-lyase activity in carrot roots. Plant Physiol. 51, 1033–1036. Chen, P.M., Mellenthin, W.M., 1982. Maturity, chilling requirement, and dessert quality of ’d’Anjou’ and ’Bosc’ pears. Acta Hortic. 124, 203–210. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T.J., Higgins, D.G., Thompson, J.D., 2003. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 31, 3497–3500. Ekman, J.H., Clayton, M., Biasi, W.V., Mitcham, E.J., 2004. Interactions between 1-MCP concentration, treatment interval and storage time for ‘Bartlett’ pears. Postharvest Biol. Technol. 31, 127–136. El-Sharkawy, I., Jones, B., Li, Z.G., Lelievre, J.M., Pech, J.-C., Latche, A., 2003. Isolation and characterization of four ethylene perception elements and their expression during ripening in pears (Pyrus communis L.) with/without cold requirement. J. Exp. Bot. 54, 1615–1625. Faragher, J.D., Chalmers, D.J., 1977. Regulation of anthocyanin synthesis in apple skin. III. Involvement of phenylalanine ammonia-lyase. Aust. J. Plant Physiol. 4, 133–141. Galvis-Sanchez, A.C., Morais, A.M.M.B., Gil, M.I., 2003. Quantification of chlorogenic acid and polyphenol oxidase activity in ’Rocha’ pear after CAlong storage. Acta Hortic. 600, 405–408. Gomez-Cordoves, C., Varela, F., Larrigaudiere, C., Vendrell, M., 1996. Effect of ethephon and seniphos treatments on the anthocyanin composition of Starking apples. J. Agric. Food Chem. 44, 3449–3452. Hall, A.E., Findell, J.L., Schaller, G.E., Sisler, E.C., Bleecker, A.B., 2000. Ethylene perception by the ERS1 protein in Arabidopsis. Plant Physiol. 123, 1449–1457. Harborne, J.B., Williams, C.A., 2000. Advances in flavonoid research since 1992. Phytochemistry 55, 481–504. Hua, J., Sakai, H., Nourizadeh, S., Chen, Q.G., Bleecker, A.B., Ecker, J.R., Meyerowitz, E.M., 1998. EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis. Plant Cell 10, 1321–1332. Jiang, Y., Joyce, D.C., 2003. ABA effects on ethylene production, PAL activity, anthocyanin and phenolic contents of strawberry fruit. Plant Growth Regul. 39, 171–174. Jiang, Y., Joyce, D.C., Terry, L.A., 2001. 1-Methylcyclopropene treatment affects strawberry fruit decay. Postharvest Biol. Technol. 23, 227–232. Kay, C.D., Mazza, G., Holub, B.J., Wang, J., 2004. Anthocyanin metabolites in human urine and serum. Br. J. Nutr. 91, 933–942. Loyall, L., Uchida, K., Braun, S., Furuya, M., Frohnmeyer, H., 2000. Glutathione and a UV light-induced glutathione S-transferase are involved in signalling to chalcone synthase in cell cultures. Plant Cell 12, 1939–1950. Marais, E., Jacobs, G., Holcroft, D.M., 2001. Postharvest irradiation enhances anthocyanin synthesis in apples but not in pears. HortScience 36, 738–740. Mosel, H.-D., Herrmann, K., 1974. Changes in catechins and hydroxycinnamic acid derivatives during development of apples and pears. J. Sci. Food Agric. 25, 156–251. Murphey, A.S., Dilley, D.R., 1988. Anthocyanin biosynthesis and maturity of ’McIntosh’ apples as influenced by ethylene-releasing compounds. J. Am. Soc. Hortic. Sci. 113, 718–723.
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55 Niggeweg, R., Micheal, A.J., Martin, C., 2004. Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat. Biotechnol. 22, 746–754. Oleszek, W., Amiot, M.J., Aubert, S., 1994. Identification of some phenolics in pear fruit. J. Agric. Food Chem. 42, 1261–1265. Owino, W.Z.O., Nakano, R., Kubo, Y., Inaba, A., 2002. Differential regulation of genes encoding ethylene biosynthesis enzymes and ethylene response sensor ortholog during ripening and in response to wounding in avocados. J. Am. Soc. Hortic. Sci. 127, 520–527. Riov, J., Monselise, S.P., Kahan, R.S., 1969. Ethylene-controlled induction of phenylalanine ammonia-lyase in citrus fruit peel. Plant Physiol. 44, 631– 635. Scheiber, A., Keller, P., Carle, R., 2001. Determination of phenolic acids and flavonoids of apple and pear by HPLC. J. Chromatogr. A 910, 265–273. Steyn, W.J., Holcroft, D.M., Wand, S.J.E., Jacobs, G., 2004. Regulation of pear color development in relation to activity of flavonoid enzymes. J. Am. Soc. Hortic. Sci. 129, 6–12.
55
Tan, S.C., 1980. Phenylalanine ammonia-lyase and the phenylalanine ammonialyase inactivating system: effects of light, temperature and mineral deficiencies. Aust. J. Plant Physiol. 7, 159–167. Trainotti, L., Bonghi, C., Ziliotto, F., Zanin, D., Rasori, A., Casadoro, G., Ramina, A., Tonutti, P., 2006. The use of microarray microPEACH 1.0 to investigate transcriptome changes during transition from pre-climacteric to climacteric phase in peach fruit. Plant Sci. 170, 606–613. Trinchero, G.D., Sozzi, G.O., Covatta, F., Fraschina, A.A., 2004. Inhibition of ethylene action by 1-methylcyclopropene extends postharvest life of “Bartlett” pears. Postharvest Biol. Technol. 32, 193–204. Tsuda, T., Watanabe, M., Ohshima, K., Norinobu, S., Choi, S.W., Kawakishi, S., Osawa, T., 1994. Antioxidative activity of the anthocyanin pigments cyanidin 3-glucoside and cyanidin. J. Agric. Food Chem. 42, 2407–2410. Walbot, V., Mueller, L.A., Silady, R.A., Goodman, C.D., 2000. Do glutathione s-transferases act as enzymes or carrier proteins for their substrates. Sulfur Nutr. Sulfur Assimilation in Higher Plants, 155–165.
Inhibition of PAL, CHS, and ERS1 in ‘Red d’Anjou’ Pear (Pyrus communis L.) by 1-MCP D.D. MacLean a,∗ , D.P. Murr a , J.R. DeEll b , A.B. Mackay a , E.M. Kupferman c a
Department of Plant Agriculture, E.C. Bovey Building, University of Guelph, Guelph, Ontario N1G 2W1, Canada b Ontario Ministry of Agriculture, Food and Rural Affairs, Simcoe, Ontario N3Y 4N5, Canada c WSU TFREC, Wenatchee, WA 98801, USA Received 17 August 2006; accepted 20 January 2007
Abstract The ethylene antagonist 1-MCP was investigated for its potential impact on the transcription of two key flavonoid biosynthetic (phenylalanine ammonia-lyase, PAL, E.C. 4.3.1.5; and chalcone synthase, CHS, E.C. 2.3.1.74), flavonoid transport (glutathione S-transferase, GST, E.C. 2.5.1.18) and ethylene perception (ethylene response sensor 1, ERS1) transcripts during the postharvest storage and ripening of pear (Pyrus communis L.). ‘Red d’Anjou’ pear fruit were harvested from Wenatchee, WA, USA, transported to Guelph, Ont., Canada, then treated with 1 L L−1 1-MCP, and subsequently placed in cold storage (0–1 ◦ C, 90–95% RH) for up to 126 days. After removal, fruit were warmed to room temperature (1 or 7 days) then tissue samples were collected for Northern blot analysis and determination of flavonoid and chlorogenic acid concentration by HPLC. In general, PAL content decreased during storage, with content increasing during the post-storage ripening period in parallel with the increase in respiration rate and ethylene content. In contrast, CHS content decreased dramatically during the 1-week ripening period, while ERS1 remained constant. The expression of PAL, CHS and ERS1 transcripts were all inhibited by 1-MCP. GST transcript abundance decreased during storage, and was largely unaffected by the 1-MCP treatment. The flavonoid concentration remained constant throughout storage and subsequent ripening. However, after the first removal and warming to room temperature, chlorogenic acid concentration increased in the untreated, but not in the 1-MCP-treated fruit. These results suggest that 1-MCP significantly inhibits the transcription of key flavonoid biosynthetic enzymes and ethylene perception proteins, but not the flavonoid transport enzyme. The increase in PAL with the concomitant post-storage decrease of CHS and postharvest decrease of GST suggests a diversion of carbon from flavonoid compounds into chlorogenic acid. © 2007 Elsevier B.V. All rights reserved. Keywords: Pear; 1-Methylcyclopropene; Flavonoid; Chlorogenic acid; Phenylalanine ammonia-lyase; Chalcone synthase
1. Introduction In recent years there has been a growing interest in all classes of flavonoids as integral antioxidants in the human diet, due in part to their demonstrated anticarcinogenic activity, inhibition of tumor cell proliferation, antioxidant and free radical scavenging capabilities, as well as their effectiveness as metal chelators (Harborne and Williams, 2000). The major flavonoids present in pear have been implicated in the prevention of lipid peroxidation of cell membranes in mammalian systems (Tsuda et al., 1994), while other studies have investigated the bioavailability of these flavonoid metabolites in human systems (Kay et al., 2004).
∗
Corresponding author. Tel.: +1 519 824 4120x58255; fax: +1 519 767 0755. E-mail address: [email protected] (D.D. MacLean).
0925-5214/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2007.01.007
The activity of PAL has been considered a critical step in the divergence of carbon into the phenylpropanoid biosynthetic pathway, and it has been demonstrated in many plant species, including carrot roots (Chalutz, 1973) and citrus (Riov et al., 1969), that ethylene is a key regulator of PAL activity. Numerous studies using apple have demonstrated the dependence of anthocyanin accumulation on the presence of ethylene and the concomitant increase in PAL activity (Faragher and Chalmers, 1977; Blankenship and Unrath, 1988). As well, factors other than the level of ethylene in the tissue contribute to the de novo synthesis of anthocyanins, including low temperature (Tan, 1980; Arawaka, 1991), fruit maturity (Murphey and Dilley, 1988), and storage duration (Jiang and Joyce, 2003), all of which are important considerations in the postharvest storage of pear fruit. Pears contain high levels of some hydroxycinnamic acid derivatives, with chlorogenic acid being the predominant structure (Oleszek et al., 1994). These classes of structures diverge
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
Fig. 1. An abbreviated phenylpropanoid pathway starting with the activity of phenylalanine ammonia-lyase (PAL), with products diverging either to the flavonoid biosynthetic pathway (left) through chalcone synthase (CHS), or to the purported chlorogenic acid biosynthetic pathway (right) through hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyltransferase (HCT, see Niggeweg et al., 2004). Both cyanidin 3-galactoside and chlorogenic acid are believed to be endogenous substrates for glutathione S-transferase, an enzyme involved in the transport of flavonoids through the cytoplasm to the vacuolar membrane (Walbot et al., 2000).
from the flavonoid biosynthetic pathway downstream of PAL, but upstream from CHS (Fig. 1). Pears also contain high amounts of flavonoids from the flavonol (e.g. quercetin 3-glycosides), flavan-3-ol (e.g. catechin), and anthocyanin (e.g. cyanidin 3glycosides) classes of structures (Mosel and Herrmann, 1974; Scheiber et al., 2001). These classes all differentiate downstream of CHS, a critical step in the divergence of carbon into the flavonoid biosynthetic pathway. One of the last defined enzymes of the flavonoid biosynthetic pathway is GST. The GST enzymes have a central role in the transport of flavonoids through the cytoplasm to the vacuolar membrane (Walbot et al., 2000). GSTs may also play a role in the regulation and signaling of the flavonoid biosynthetic pathway (Loyall et al., 2000). The imposition of a postharvest chilling period and the presence of ethylene is required for the successful ripening of pear. More specifically, ‘d’Anjou’ pear requires anywhere from 46 days (Blankenship and Richardson, 1985) to 60 days (Chen and Mellenthin, 1982) at −1 ◦ C to induce ethylene biosynthesis. When pear fruit are treated with 1-MCP prior to the burst of ethy-
47
lene production, the onset of fruit ripening is delayed (Ekman et al., 2004; Trinchero et al., 2004). For example, ‘d’Anjou’ fruit exposed to 1-MCP displayed a 1000- and 4-fold reduction in ethylene production and respiration rate, respectively, and also prevented the flesh from softening (Argenta et al., 2003). The ERS1 is an ethylene-binding protein, and a member of the 5-gene family of ethylene receptors that includes ETR1/2, EIN4, and ERS1/2 (Hall et al., 2000). Although the hierarchy or complementary function of the family of ethylene binding proteins is unclear, the ERS1 protein is interesting because it is constitutively present, but is also associated with system 2, or autocatalytic production of ethylene. As seen in ripening peach fruit, the mRNA transcript abundance of ERS1 increased in parallel with the climacteric burst in ethylene (Trainotti et al., 2006). A similar study investigating the transcript abundance of ERS1 in avocado also found a background level of ERS1 present in tissue prior to the onset of autocatalytic ethylene, followed by a very rapid and large increase in the abundance of transcript as the fruit neared the climacteric peak (Owino et al., 2002). Finally, using ‘Passe-Crasssane’ pear fruit it was found that PcERS1 increased in response to both ripening, and an exogenous treatment of ethylene (El-Sharkawy et al., 2003). It is evident that the ERS1 protein plays a role in the perception of ethylene during climacteric fruit ripening. Therefore, the down-regulation of this protein by a treatment with 1-MCP may result in the down-stream inhibition of numerous ripening-related transcription factors and enzymes, including PAL, which is regulated by ethylene. The dependency of PAL activity on the presence of ethylene raises concerns that treatment with 1-MCP may adversely affect total phenolic concentration of the ripening fruit. For example, in strawberry a decrease in PAL activity and the concomitant inhibition of the increase in flavonoid and phenolic concentration of the fruit resulted from a treatment with 1-MCP (Jiang et al., 2001). 1-MCP is now being used extensively at both a research and commercial level around the world as a postharvest tool to extend shelf life in numerous nutraceutical-producing crops, including pear. Therefore, the goal of the present study was to determine the effect of 1-MCP on the transcription of flavonoid biosynthetic (PAL and CHS), flavonoid transport (GST) and ethylene perception (ERS1) proteins in ‘Red d’Anjou’ pear, over the course of a 126-day cold storage period and a 1-week ripening period. 2. Materials and methods 2.1. Fruit harvest and 1-MCP treatment ‘Red d’Anjou’ pears were harvested at commercial maturity (flesh firmness of 58–67 N) from Guthrie Pear Orchard, Wenatchee, WA, USA, on 21 September 2004. Immediately after harvest, fruit were hand-packed in commercial shipping boxes and transported to Stemilt Growers Inc. and placed in cold storage at −0.5 ◦ C for 4 weeks. Fruit were subsequently transported to Canada in a refrigerated transport truck (3–4 ◦ C), and arrived at the University of Guelph after 35 days. Upon arrival, fruit were removed from packaging and placed into bushel boxes and were
48
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
treated twice, on consecutive days, with 1 L L−1 of 1-MCP (0.14% a.i.; SmartFreshTM , Agrofresh Inc. Spring House, PA) (0–1 ◦ C) with each treatment lasting 24 h. Similar non-treated control fruit were held in air for 24 h at 0–1 ◦ C. Following treatment, fruit were held at 0–1 ◦ C and 90–95% RH and removed at 21 day intervals for a total storage time of 126 days. After each removal time, fruit samples were allowed to equilibrate at room temperature for 1 or 7 days, then the epidermal and some underlying cortical tissue (∼1–2 mm) from 5 fruit (3 repetitions, 15 fruit total) were removed with a hand peeler, combined, flash frozen and ground under liquid nitrogen. The resulting frozen tissue samples were then held at −80 ◦ C until preparation for flavonoid determination and RNA extraction. 2.2. Fruit quality and maturity indices After completion of the two 1-MCP treatments, three replications of five fruit each were removed and evaluated for ethylene production and respiration rate every 3 days for 10 days, while flesh firmness and TSS were monitored after 1 and 7 days. These same tests were performed after every storage removal date. Ethylene was measured using a Varian CP-3380 GC (Varian Inc., Mississauga, ON) equipped with a 15 m × 0.32 mm Restek RT-S-Plot fused-silica column (Chromatographic Specialties, Inc., Brockville, Ont., Canada), and a flame ionization detector (FID). The injector, column and detector temperatures were 120, 35 and 225 ◦ C, respectively, while high-grade helium was used as the carrier gas at a flow rate of 22 mL min−1 . Respiration rate was determined using an ADC-EGA infrared CO2 gas analyzer (Nortech Control Equipment Inc. Etibicoke, Ont., Canada). Flesh firmness was determined using a hand-held Effegi penetrometer (Facchini 48011 Alfonsine, Italy) fitted with an 8-mm tip, and measurements were taken from pared opposite sides of the fruit. The exudate of five fruit from the firmness test was collected in a petri dish for determination of TSS using a handheld temperature-compensated refractometer (Fisher Scientific, Nepean, Ontario). 2.3. Template RNA isolation and cDNA synthesis Fruit that were stored in air for 63 days at 0–1 ◦ C and 1 day at 21 ◦ C were used for the extraction of total RNA and synthesis of cDNA. Approximately 2–3 g of the frozen pear skin tissue was ground to a fine powder with a mortar and pestle that was pre-chilled with liquid nitrogen. Total RNA was extracted using the Qiagen RNeasy Plant Mini Kit® (Mississauga, Ont., Canada), using the supplied RLT extraction buffer, according to the instructions of the manufacturer. RNA was eluted from the supplied column using 50 L sterile water, and the quantity and quality was estimated using a Biochrom Ultrospec® 2100 pro spectrophotometer set at 260 nm (Biochrom Ltd., Cambridge, UK). First-strand cDNA synthesis from the reverse-transcription of RNA was performed using the ‘two-step’ protocol of the RETROscript® Kit from Ambion (Austin, TX, USA), using oligo (dT) as the primer. For the RT reaction, 2 g RNA, 2 L oligo (dT) and sterile water to 12 L was mixed and heated to 80 ◦ C for 3 min to denature the RNA. After placing the reaction
on ice, the remaining RT components were added; 2 L 10× RT buffer (500 mM Tris–HCl, pH 8.3, 750 mM KCl, 30 mM MgCl2 , and 50 mM DTT), 4 L dNTP mix, 1 L RNase inhibitor, and 1 L MMLV-RT reverse transcriptase enzyme. The 20 L RT reaction was then incubated at 44 ◦ C for 2 h in a thermocycler, and then inactivated by a 10-min incubation at 92 ◦ C. The cDNA was either stored at −20 ◦ C or used immediately for reverse transcriptase polymerase chain reaction (RT-PCR). 2.4. Cloning of PAL, CHS, GST and ERS1 Selected PAL genes were identified from the NCBI database (accession numbers: Arabidopsis thaliana, P45724 and P35510; and Malus domestica, CAA48231 and P35512) and aligned using the ClustalW multiple sequence alignment software (Chenna et al., 2003). After conserved amino acid regions were identified, the PAL gene was amplified from pear cDNA by PCR using the following degenerate oligonucleotide primers: sense 5 -GG(A/C/G/T)GG(A/C/G/T)-AA(C/T)TT(C/T)-CA (A/G)GG-3 corresponding to the amino acid sequence (GGNFQG) and antisense 5 -AA(A/C/G/T)AC(C/T)TT(A/G)TC (A/G)CA(C/T)TC(C/T)TC-3 corresponding to the amino acid sequence (EECDKVF). The primers for Pc-CHS1 were designed based on the conserved regions from numerous species (accession numbers: M. domestica, BAB92996; A. thaliana, CAI30418; Lycopersicon esculentum, CAA38980; and Zea mays, CAA42763). The sense primer used was 5 -ATGATGTA(C/T)CA(A/G)CA(A/G)GG (A/C/G/T)-TG(C/T)TT-3 corresponding to the amino acid sequence (MMYQQGCF), while the antisense primer was 5 AC(A/C/G/T)GG(A/C/G/T)CC(A/G)AA(A/C/G/T)CC(A/G) AA-3 corresponding to the amino acid sequence (FGFGPG). The full-length cDNA clone was determined using Ambion’s FirstChoice ® RLM-RACE kit (Austin, TX, USA). The sense primer for Pc-GST was designed from the Malus EST (accession number: DY255365) and was 5 -ATGGTGGTGAAGGTGTACGG-3 corresponding to the amino acid sequence (MVVKVY). The antisense primer 5 TTCTTCCAAGC(C/T)-GG(A/C/G/T)CT-3 corresponding to the amino acid sequence (RPAWK), was selected based on conserved regions from numerous other GSTs (accession numbers: Glycine max, AAG34812; Citrus sinensis, ABA42224; Phaseolus acutifolius, AAM34480; Petunia hybrida, CAA68993; and Vitis vinifera, AAX81329). The primers for Pc-ERS1 were designed based on the previously published PcERS1 from Pyrus species (accession number: AF386517) (El-Sharkawy et al., 2003). The sense primer used was 5 -AATGGAGTCATGTGATTGC-3 corresponding to the amino acid sequence (MESCDC), while the antisense primer was 5 -GCAATCCATGCTCGCAAT-3 corresponding to the amino acid sequence (AIHARN). PCR reactions for all clones were performed using the Biorad iCycler thermocycler (firmware version 3.032), using 2.5 U of Promega Taq DNA polymerase (Madison, WI, USA) under the following conditions: hot start 3 min at 94 ◦ C, then 35 cycles at 94 ◦ C (1 min), anneal at 52 ◦ C (PAL), 50 ◦ C (CHS), or 55 ◦ C (GST and ERS1) (1 min), and extension at 72 ◦ C (1.5 min), with a final exten-
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
sion at 72 ◦ C for 5 min. An aliquot of the PCR reaction was loaded onto a 1% (w/v) agarose gel (80 V) in order to confirm a single product was amplified by the reaction. The remaining PCR product was cleaned using the GeneClean® Turbo PCR kit from QBiogene (Carlsbad, CA, USA) to remove any potential contaminants and subsequently cloned using the Qiagen® PCR Cloningplus Kit (Mississauga, Ont., Canada) into the supplied pDrive vector using a 10× molar excess of template. The ligation reaction was allowed to proceed for 1 h at 14 ◦ C, and subsequently transformed into Qiagen® EZ Competent Cells (Mississauga, Ont., Canada) following the recommended protocol. Colonies that successfully incorporated the PCR product were amplified using the M13 universal and gene-specific primers, and then sequenced using the T7 promoter region as the forward primer using a model 3730 Genie sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). The sequences that were obtained were edited to remove any vector sequence, and identified using Blastn program from NCBI (Altschul et al., 1997).
49
fixed by exposing the membrane to 700 kJ of UV light for 20 s in a UV cross-linker (Hoefer Pharmacia Biotech, Inc., San Francisco, CA, USA). The membrane was pre-hybridized for 30 min in freshly prepared and pre-heated (68 ◦ C) Roche DIG Easy Hyb granules (Roche Diagnostics, Laval, Que., Canada), then hybridized overnight (68 ◦ C) with 20 ng of digoxigeninlabelled gene specific RNA probe. The following morning, the membrane was washed twice in 2× SSC, 0.1% SDS for 5 min each at room temperature, followed by a higher stringency wash of 0.5× SSC, 0.1% SDS for 15 min each at the hybridization temperature (68 ◦ C). Blocking reagent, anti-Dig AP conjugate (1:20,000) and other washes were performed according to the recommended protocol supplied with the blocking and detection reagents (Roche Diagnostics, Laval, Que., Canada). Chemiluminescent detection was performed using CDP-Star (ready to use) system (Roche Diagnostics, Laval, Que., Canada) and the membrane was subsequently exposed to Kodak® BioMax Light film. 2.8. Tissue preparation for HPLC analysis
2.5. DIG-labeling of RNA probes The partial cDNA gene fragments of PcPAL, Pc-GST, and Pc-CHS1 were all cloned into the pDrive vector of Qiagen (Mississauga, Ont., Canada) and amplified and sequenced using a gene-specific primer in combination with the universal M13 primers. Probes were designed from these sequences, with the reverse complement primer having a T7 promoter region added to the 5 -end. The DIG-labeling transcription reaction made use of the MAXIscriptTM T7 in vitro Transcription Kit of Ambion (Austin, TX, USA). For the reaction, 1 g template, along with 2 L of each the DIG RNA labeling mix, T7 enzyme, and transcription buffer were used. Optional steps including the addition of DNase and EDTA were performed. 2.6. RNA extraction for Northern blot analysis Total RNA was extracted from pears that were treated with/without 1-MCP after each cold storage removal (0–126 days) and shelf-life period (+1 or +7 days) using the Qiagen RNeasy Plant Mini Kit® (Mississauga, Ont., Canada). Total RNA content was determined spectrophotometrically at 260 nm as described previously, and quality was estimated by monitoring the 260:280 nm ratio and by observing the 18s and 28s ribosomal RNA bands stained with ethidium bromide (EtBr) after performing formaldehyde denaturing agarose gel electrophoresis (see below). 2.7. Northern blot analysis For Northern blot analysis, 1 g total RNA was denatured at 80 ◦ C for 3 min in 3× RNA sample loading buffer (containing EtBr), and loaded onto a 1.5% (w/v) agarose gel containing 0.22 M formaldehyde in MOPS running buffer. RNA was separated at 60 V for 3–4 h prior to being blotted onto an Ambion BrightStar-PlusTM positively charged membrane (Austin, TX, USA). RNA was transferred overnight using 20× SSC, then
Approximately 1 g (±0.02 g) of finely ground frozen tissue from 5 fruit per replication was placed in a 12.5-mL disposable glass culture vial containing 5 mL MeOH (0.1% HCl). The sample was sonicated at 20 ◦ C for 30 min, then the headspace of the vial was flushed with N2(g) , capped with a rubber septa-seal, and subsequently placed in complete darkness at 4 ◦ C for overnight extraction. The following morning, the sample was centrifuged at 1000 × g for 5 min at 4 ◦ C. The supernatant was decanted and set aside, while the pelleted residue was further extracted with a 3-mL wash of MeOH (0.1% HCl). After a 15-min sonication, the sample was placed in complete darkness for 3 h then centrifuged at 1000 × g for 5 min at 4 ◦ C. The supernatant was added to the first supernatant for a final total extraction volume of ∼8 mL. The headspace of the vial was then flushed with N2(g) , capped with a rubber septa-seal and placed at −20 ◦ C for no more than 12 h. Flavonoids in the supernatant extract were concentrated to near dryness using a rotary evaporator held at 60 ◦ C, resuspended with 0.7-mL MeOH (0.1% HCl), vortexed for 10 s, and centrifuged at 16,000 × g for 20 min. After centrifugation, the sample was decanted directly into a 1.5-mL amber autosampler vial where the final volume was brought to 1-mL with MeOH (0.1% HCl), sealed with a PTFE autosampler vial cap, and stored at −80 ◦ C until analyzed by HPLC. 2.9. HPLC-DAD analysis Polyphenols were separated and identified using an Agilent 1100 series HPLC (Mississauga, Ont., Canada) system equipped with an inline continuous vacuum solvent degasser, binary pump, temperature controlled autosampler and column compartments, and a PDA, all controlled by a Chemstation (ver. 4.0) software package. Solvents used were A: 5% formic acid and B: ACN at a flow rate of 0.3 mL min−1 . The gradient (expressed as %B) was: 0–1 min, 5%; 1–8 min, 5–13%; 8–12 min, 13–15%; 12–26 min, 15–22%; 26–26.5 min, 22–40%; 26.5–30.5 min, 100%; 30.5–35 min, 5%. The autosampler compartment was
50
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
maintained at 4 ◦ C. The volume of injection for both samples and standards was 5 L. The polyphenolic compounds were retained using a Waters Symmetry Shield RP-18 column (100 mm × 2.1 mm × 3.5 m; Mississauga, Ont., Canada) protected by a guard column (10 mm × 2.1 mm × 3.5 m) all held at 30 ◦ C within the column compartment. Eluted compounds were detected using the PDA equipped with a semi-micro flow cell with a full spectral scan set from 210 to 700 nm (1 nm steps), and monitored at 530 nm for the detection of anthocyanins, 350 nm for flavonols, 280 nm for flavan-3-ols, and 330 nm for chlorogenic acid, all with a bandwidth of 4 nm. 2.10. Statistical analysis Each main effect of removal × shelf life × 1-MCP treatment contained three replications of five fruit per replication. The experimental setup was a completely randomized design with storage removal, shelf life, and 1-MCP treatment designated as fixed effects. Least squares means, analysis of variance, tests for normality and outliers were performed using the General Linear Model Procedure (Proc GLM) of the Statistical Analysis System (SAS) software (version 8.2, SAS Institute, Cary, NC, USA) at a significance level of P ≤ 0.05. 3. Results 3.1. Fruit harvest and 1-MCP treatment The pear fruit were harvested at commercial maturity on 21 September 2004. However, due to unforeseen circumstances involving the transportation of fruit across the continent and
the Canadian border, fruit were not received at the University of Guelph until 26 October 2004, 35 days after being harvested. The condition of the fruit upon arrival was excellent; however, based on the flesh firmness (53.1 N), TSS (13.7%), and headspace ethylene concentration (2.31 L kg−1 h−1 ), it is likely that the fruit had commenced ripening. As a consequence, there was a good possibility that fruit would not be as responsive to a 1-MCP treatment as they would have if they had been treated prior to the onset of fruit ripening. Therefore, as a preemptive measure, fruit were exposed to two successive 1-day treatments of 1 L L−1 1MCP (0–1 ◦ C) in order to inhibit the rise in ethylene production relative to the untreated control fruit. Based on ethylene production and respiration rates of fruit monitored over a 10-day period at room temperature immediately after exposure to 1-MCP, the treatment was effective at inhibiting the endogenous production of ethylene and general fruit metabolism despite the long delay between harvest and 1MCP treatment. No ethylene was detectable (<50 ppb) in fruit held at room temperature for up to 10 day post 1-MCP treatment (Fig. 2A). Similar inhibition of ethylene production was observed in fruit after 126 days of cold storage, where percent inhibition varied between 96 and 99% (Fig. 2C). With respect to respiration rate, as measured by CO2 production, in both the 1-MCP treated and non-treated control fruit, respiration rate was high after the initial removal from cold storage, and decreased by the second evaluation date 4 days after removal from storage (Fig. 2B and D). However, after the initial decrease in respiration rate, the control fruit displayed an increasing trend up to 10 days at room temperature, while the 1-MCP-treated fruit continued to decrease. By day 10, the inhibition was 65% for the initial fruit, and 70% for fruit after 126 days of storage. These results suggest
Fig. 2. The analysis of headspace ethylene (A and C: L kg−1 h−1 ) and CO2 (B and D: mL kg−1 h−1 ) production rates of ‘Red d’Anjou’ pear immediately following a treatment with 1-MCP (A and B) or after 126 days of storage (0–1 ◦ C) (B and D). Fruit were exposed to two successive treatments of 1 L L−1 1-MCP (24 h, 0–1 ◦ C) 35 days after harvest, storage and transportation of the fruit from Washington State. Legend is the same for all figures. Each point represents the mean of three values (P ≤ 0.05).
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
the 1-MCP treatment remained effective at inhibiting ethylene production and respiration rate, despite the higher metabolic rate expected at the warmer temperature. 3.2. Flavonoid and chlorogenic acid biosynthesis The average concentration of chlorogenic acid, across all storage and shelf life factors was 283 and 275 g g−1 f.w. for the control and 1-MCP-treated fruit, respectively. In contrast to the stable concentration of flavonoids during postharvest handling of the fruit, chlorogenic acid concentration tended to increase. This is especially evident after the initial fruit evaluation, where chlorogenic acid concentration in non-treated fruit increased 187% during the first week at room temperature (Fig. 3B). This increase was not present in fruit treated with 1-MCP. However, the effect of the 1-MCP treatment on chlorogenic acid biosynthesis appears to have been lost by the second removal, where chlorogenic acid concentration in 1-MCP-treated fruit was not significantly different from the untreated fruit. Based on regression equations for control (y = 23.942x + 175.04) and 1-MCP-treated fruit (y = 42.855x + 82.324), chlorogenic acid concentration increased during the storage and 1-week ripening period. The average concentration of cyanidin 3-galactoside across all storage and shelf life factors was 61 and 69 g g−1 f.w. for the control and 1-MCP treated fruit, respectively. Based on the regression equations for control (y = −0.3079x + 62.842)
Fig. 3. Changes in the concentration (g g−1 f.w.) of (A) chlorogenic acid and (B) cyanidin 3-galactoside in the skin of ‘Red d’Anjou’ pear, with shelf-life periods (+1 or +7 days), storage (0, 21, 63 and 126 days) and 1-MCP treatment effects. Legend is the same for both (A) and (B). Each point represents the mean of three values (P ≤ 0.05).
51
and 1-MCP-treated fruit (y = 1.2055x + 63.498), cyanidin 3galactoside concentration did not vary significantly during storage or after removal and warming to room temperature for one week (Fig. 3B). Also, the treatment with 1-MCP did not significantly affect the concentration of anthocyanin. Other flavonoids from the classes of anthocyanins, flavonols and flavan-3-ols, were also analyzed and they all exhibited a similar response to the storage, shelf life and 1-MCP treatment as cyanidin 3-galactoside (data not presented). 3.3. Gene cloning and sequence analysis Degenerate oligonucleotide primers were used successfully to isolate the PCR fragments of the expected size for Pc-PAL, PcCHS1, Pc-GST and Pc-ERS1. The full-length cDNA clone for chalcone synthase was determined by first using a set of degenerate oligonucleotide primers designed from conserved amino acid regions from the alignment of M. domestica (BAB92996), L. esculentum (CAA38980), A. thaliana (CAI30418) and Z. mays (CAA42763). This partial cDNA fragment of 660 bp was subsequently used to determine the full-length coding region, as well as the 3 and 5 UTRs. The full-length clone, designated PcCHS1, had a coding region of 1170 bp, with a predicted amino acid sequence of 389 residues, and predicted molecular mass of 42.5 kDa (Fig. 4). The deduced amino acid sequence displayed 98.4% identity with M. domestica (BAB92996), 98.2% identity with Sorbus aucuparia (ABB89213), 92.5% identity with Fragaria × ananassa (BAE17124), and 92.8% identity with Rosa hybrida (BAC66467). The three partial cDNA fragments for Pc-PAL, Pc-GST and Pc-ERS1 were also isolated in this study. For the Pc-PAL fragment, a single band of expected size (0.8 kbp) was amplified using degenerate primers from cDNA that was synthesized from total RNA using the oligo d(t) primer. Degenerate primers were designed based on sequence homology with M. domestica (P35512 and CAA48231), A. thaliana (P45724 and P35510), and Pyrus pyrifolia (AAQ15284). After cloning, the resulting PCR product encoded an 846 bp and 282 predicted amino acid sequence homologous to other PAL genes. The product displayed 92.7% identity with Prunus persica (AAF17247), 88.3% identity with Daucus carota (BAC56977), 86.5% identity with Rubus idaeus (AAF40224), and 57% identity over the entire length with M. domestica (CAA48231). The Pc-GST partial cDNA fragment was isolated using a sense primer designed using the Malus GST EST (accession number DY255365), while the reverse primer was based on a previously isolated polypeptide (DDISSRPAW). The PCR reaction resulted in a single band at the expected size of 0.6 kbp. After cloning, a 612 bp fragment was sequenced, and a 204 amino acid sequence was deduced. The highly divergent class of proteins had 73% amino acid identity over the entire length with G. max (AAG34814), 72% identity with A. thaliana (NP 180643), 60% identity with C. sinensis (ABA42224), and 54% identity with Petunia × hybrida (CAA68993). Analysis of domain architecture indicated that the isolated Pc-GST is a member of the phi class of GST proteins, and is involved in the ligandin-binding and transport of flavonoids.
52
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
Fig. 4. Alignment of the deduced amino acid sequence of chalcone synthase (CHS) genes from Pyrus communis (DQ901397), Malus domestica (BAB92996), Lycopersicon esculentum (CAA38980), Arabidopsis thaliana (CAI30418), and Zea mays (CAA42763). Conserved amino acids residues are denoted by (*), conserved substitutes by (:), and semi-conserved substitutes by (.). The low-scoring segments are in dark shading, and exceptional residues are in gray shading.
The Pc-ERS1 partial cDNA fragment was isolated using primers designed from the previously published ERS1 sequence from P. communis (El-Sharkawy et al., 2003). The PCR reaction resulted in a single band at the expected size (1.0 kbp). After cloning, a 741 bp fragment was sequenced from the PCR product, with a 247-deduced amino acid sequence, corresponding to 38.7% of the coding region of the gene, from the start codon forward. The Pc-ERS1 fragment showed 99.6% identity to the previously published Pc-ERS1a sequence (AF386517). The clones for Pc-PAL, Pc-CHS1, Pc-GST, and Pc-ERS1 have been deposited into GenBank under the accession numbers DQ901399, DQ901397, DQ901400, and DQ901398, respectively.
3.4. Northern blot analysis of PAL, CHS, GST and ERS1 The Pc-PAL and Pc-ERS1 transcripts accumulated in a similar manner in response to cold storage, during ripening at 21 ◦ C for 1 week, and 1-MCP treatment (Fig. 5). In the non-treated fruit, transcript abundance for Pc-PAL appears to increase (initial) or remain constant (after 63 and 126 days), during the 1-week ripening period. In contrast, the transcript abundance for Pc-PAL in the 1-MCP-treated fruit was much lower, but constant throughout cold storage and ripening period. Similarily, the Pc-ERS1 transcript abundance was constant and higher in the non-treated control fruit than in the 1-MCP fruit throughout storage and ripening. In contrast, the Pc-CHS1 transcript abun-
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
Fig. 5. Northern blot analysis of two flavonoid biosynthetic genes (Pc-PAL and Pc-CHS1), flavonoid transport (Pc-GST), and one ethylene binding protein gene associated with fruit ripening (Pc-ERS1). ’Red d’Anjou’ pear fruit were harvested and stored for 35 days prior to two successive treatments with 1 L L−1 of 1-MCP (0–1 ◦ C). Dates (i.e. 0, 63 or 126, and +1 or +7 days) indicate the time after 1-MCP treatment. A separate gel and blot was performed for each probe, with each lane containing 1 g of total RNA. The 18s ribosomal RNA band stained with ethidium bromide is included to confirm equivalent loading of the lanes.
dance was very high in fruit tissue 1-day after removal from cold storage. However, after 1-week at room temperature, the transcript abundance for chalcone synthase was not detectable. The treatment of 1-MCP further exacerbated this decreasing trend in Pc-CHS1 abundance. The expression pattern of the Pc-GST indicates a gradual decline in transcript abundance up to the 126 days of storage. Also, there was no change in expression pattern after fruit were removed from storage and allowed to ripen for a week. Interestingly, aside from a moderate retention of transcript after removal from 126 days of storage (which disappeared over the 7-day period at 21 ◦ C), the 1-MCP treatment had no effect on Pc-GST mRNA abundance. Therefore, due to the similar abundance of Pc-GST transcript between the control and 1-MCP-treated fruit, and the relatively high abundance early in the experiment with similar rates of transcript disappearance between the control and 1-MCP-treated fruit, it is likely that there is no de novo biosynthesis of GST during storage and ripening. Therefore, these results suggest that even though PAL is active in control fruit, the carbon is not being directed into the production of flavonoid compounds, as suggested by the rapid decrease in PcCHS1 transcript abundance during the 1-week ripening period. Furthermore, a treatment of 1-MCP appears to inhibit the transcription of key phenylpropanoid biosynthetic genes, and the ethylene binding protein ERS1. 4. Discussion In apple, the increase in PAL activity and the resulting accumulation of anthocyanins is dependent on the presence of ethylene (Faragher and Chalmers, 1977; Blankenship and
53
Unrath, 1988; Gomez-Cordoves et al., 1996). In the present study, the parallel pattern of transcript abundance between PcPAL and Pc-ERS1 supports the notion that PAL is regulated (whole or in part) by ethylene. When fruit were treated with 1MCP, the expression of both Pc-ERS1 and Pc-PAL transcripts, as well as the ethylene production rate all decreased over the 1-week at room temperature. However, in untreated fruit both the ethylene production and the Pc-PAL transcript abundance increased, while the expression of Pc-ERS1 remained constant, thereby suggesting that the accumulation of Pc-PAL transcript is dependent on the presence of ethylene and an active ethylene receptor. A similar effect was observed in strawberry fruit where a treatment of 1-MCP resulted in a decrease in PAL activity (Jiang et al., 2001). The expression of the Pc-ERS1 transcript was markedly reduced by the 1-MCP treatment. Aside from the inhibition of ethylene perception and production, a treatment of 1-MCP often results in a reduced rate of respiration, as well as the inhibition of many other processes associated with fruit ripening, such as flesh softening, starch catabolism, and volatile production (Argenta et al., 2003; Blankenship and Dole, 2003). Thus, the reduced expression of PC-ERS1 may be due to the reduction in overall metabolism of the fruit after the 1-MCP treatment. However, the up-regulation of ERS1 mRNA transcripts has been correlated with the presence of ethylene (Hua et al., 1998), and has been found to increase during fruit ripening, and in response to exogenous ethylene (El-Sharkawy et al., 2003), indicating that ethylene acts as a regulator of ERS1 gene expression. Thus, it is suggested that the binding of 1-MCP by the ERS1 receptor directly alters the regulation of the PcERS1 transcript by inhibiting the regulatory ability of ethylene, as opposed to the reduced metabolic rate that may account for the reduced expression of other genes not directly regulated by ethylene. Based on the northern blot analysis of Pc-PAL and PcCHS1 transcripts, and the propensity of ‘Red d’Anjou’ fruit to preferentially accumulate chlorogenic acid over flavonoid compounds, it is suggested that pear fruit lose the capacity to synthesize flavonoids postharvest. It was found that high-pressure sodium lights stimulated anthocyanin synthesis in ‘Cripps’ Pink apple, but not in ‘Forelle’, ‘Bon Rouge’ or ‘Red d’Anjou’ pear fruit (Marais et al., 2001). As determined in the present study, the reason pear fruit were not responsive to light stimulation might be attributable to the postharvest down-regulation of PcCHS1. Similarly, this down-regulation of Pc-CHS1 may also be attributable to fruit maturity. A study using red ‘Bon Rouge’ and blush ‘Rosemarie’ pear fruit, found that both attained their maximum anthocyanin concentration when the fruit were immature, with pigmentation fading as the fruit matured (Steyn et al., 2004). Thus, it is unlikely that pear fruit have the capacity to biosynthesize flavonoids postharvest. This statement is consistent with the observed stability of cyanidin 3-galactoside, as no significant changes in concentration were detected throughout the storage and 1-week ripening periods, or due to treatment with 1-MCP. In contrast to the flavonoids, the one hydroxycinnamic acid quantified in this study, chlorogenic acid, tended to increase
54
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55
during postharvest storage and ripening. This increase has also been observed in ‘Rocha’ pear (Galvis-Sanchez et al., 2003). The general increase in chlorogenic acid content was temporarily inhibited in fruit immediately after the 1-MCP treatment, but this effect was quickly lost by the first removal 21 days into the experiment. After the inhibitory effect was lost, both the control and 1-MCP-treated fruit continued to accumulate chlorogenic acid in parallel for the duration of the study. Thus, the downregulation of Pc-CHS1 transcript and the lack of appreciable changes in flavonoid content postharvest, suggests that carbon is not flowing through CHS into the production of flavonoids. However, the post-storage up-regulation of Pc-PAL transcript and the increase in chlorogenic acid content suggests that carbon is being shunted into the phenylpropanoid biosynthetic pathway, and is being diverted into the production of simple phenols prior to CHS. Glutathione S-transferases are the last genetically defined step in the flavonoid biosynthetic pathway, where they act as flavonoid transport enzymes (Walbot et al., 2000). Unlike PcPAL and Pc-CHS1, the lack of inhibition of Pc-GST transcript by 1-MCP, in conjunction with approximately uniform band intensities within a storage removal (0, 63 or 126 days), suggests that Pc-GST is not actively metabolized postharvest. This is in agreement with the study of Steyn et al. (2004), who demonstrated that anthocyanin concentration is greatest in immature pear fruit. Thus, GST mRNA transcript is most abundant in immature fruit, when fruit are actively synthesizing flavonoids. Once the fruit are harvested, only inhibition of catabolic activities through cold storage and a 1-MCP treatment will assist in the retention of the GST proteins present at the time of harvest. This is evident in the 126-day removal, where the only Pc-GST transcript detected was in 1-MCP-treated fruit 1-day after being removed from cold storage. 5. Conclusions A treatment with 1-MCP inhibited the postharvest abundance of PAL, CHS and ERS1 mRNA transcripts. In non-1-MCPtreated fruit, the up-regulation of PAL mRNA transcript during the 1-week post-storage ripening period in conjunction with the simultaneous down-regulation of CHS over the same period suggests a diversion of carbon from the flavonoid biosynthesis into the production of hydroxycinnamic acids, such as chlorogenic acid. Therefore, it is concluded that a postharvest treatment with 1-MCP will not have an effect on the flavonoid concentration of pear fruit, but may adversely effect the concentration of chlorogenic acid. Acknowledgements This research was supported by AgroFresh Inc., the Natural Sciences and Engineering Research Council, Canada (NSERC), and the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA). We wish to thank AgroFresh Inc. (a Rohm & Haas Co.) for kindly supplying the 1-MCP, Chris Sater for harvesting and packaging of the fruit, and Stemilt Growers Inc., Wenatchee, WA for the transportation of the fruit to Canada.
References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389– 3402. Arawaka, O., 1991. Effect of temperature on anthocyanin accumulation in apple fruit as affected by cultivar, stage of fruit ripening and bagging. J. Hortic. Sci. 66, 763–768. Argenta, L., Fan, X., Mattheis, J., 2003. Influence of 1-methylcyclopropene on ripening, storage life, and volatile production by d’Anjou cv. pear fruit. J. Agric. Food Chem. 51, 3858–3864. Blankenship, S.M., Dole, J.M., 2003. 1-Methylcyclopropene: a review. Postharvest Biol. Technol. 28, 1–25. Blankenship, S.M., Richardson, D.G., 1985. Development of ethylene biosynthesis and ethylene-induced ripening in ‘d’Anjou’ pears during the cold storage requirement for ripening. J. Am. Soc. Hortic. Sci. 110, 520–523. Blankenship, S.M., Unrath, C.R., 1988. PAL and ethylene content during maturation of red and Golden Delicious apples. Phytochemsitry 27, 1001–1003. Chalutz, E., 1973. Ethylene-induced phenylalanine ammonia-lyase activity in carrot roots. Plant Physiol. 51, 1033–1036. Chen, P.M., Mellenthin, W.M., 1982. Maturity, chilling requirement, and dessert quality of ’d’Anjou’ and ’Bosc’ pears. Acta Hortic. 124, 203–210. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T.J., Higgins, D.G., Thompson, J.D., 2003. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 31, 3497–3500. Ekman, J.H., Clayton, M., Biasi, W.V., Mitcham, E.J., 2004. Interactions between 1-MCP concentration, treatment interval and storage time for ‘Bartlett’ pears. Postharvest Biol. Technol. 31, 127–136. El-Sharkawy, I., Jones, B., Li, Z.G., Lelievre, J.M., Pech, J.-C., Latche, A., 2003. Isolation and characterization of four ethylene perception elements and their expression during ripening in pears (Pyrus communis L.) with/without cold requirement. J. Exp. Bot. 54, 1615–1625. Faragher, J.D., Chalmers, D.J., 1977. Regulation of anthocyanin synthesis in apple skin. III. Involvement of phenylalanine ammonia-lyase. Aust. J. Plant Physiol. 4, 133–141. Galvis-Sanchez, A.C., Morais, A.M.M.B., Gil, M.I., 2003. Quantification of chlorogenic acid and polyphenol oxidase activity in ’Rocha’ pear after CAlong storage. Acta Hortic. 600, 405–408. Gomez-Cordoves, C., Varela, F., Larrigaudiere, C., Vendrell, M., 1996. Effect of ethephon and seniphos treatments on the anthocyanin composition of Starking apples. J. Agric. Food Chem. 44, 3449–3452. Hall, A.E., Findell, J.L., Schaller, G.E., Sisler, E.C., Bleecker, A.B., 2000. Ethylene perception by the ERS1 protein in Arabidopsis. Plant Physiol. 123, 1449–1457. Harborne, J.B., Williams, C.A., 2000. Advances in flavonoid research since 1992. Phytochemistry 55, 481–504. Hua, J., Sakai, H., Nourizadeh, S., Chen, Q.G., Bleecker, A.B., Ecker, J.R., Meyerowitz, E.M., 1998. EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis. Plant Cell 10, 1321–1332. Jiang, Y., Joyce, D.C., 2003. ABA effects on ethylene production, PAL activity, anthocyanin and phenolic contents of strawberry fruit. Plant Growth Regul. 39, 171–174. Jiang, Y., Joyce, D.C., Terry, L.A., 2001. 1-Methylcyclopropene treatment affects strawberry fruit decay. Postharvest Biol. Technol. 23, 227–232. Kay, C.D., Mazza, G., Holub, B.J., Wang, J., 2004. Anthocyanin metabolites in human urine and serum. Br. J. Nutr. 91, 933–942. Loyall, L., Uchida, K., Braun, S., Furuya, M., Frohnmeyer, H., 2000. Glutathione and a UV light-induced glutathione S-transferase are involved in signalling to chalcone synthase in cell cultures. Plant Cell 12, 1939–1950. Marais, E., Jacobs, G., Holcroft, D.M., 2001. Postharvest irradiation enhances anthocyanin synthesis in apples but not in pears. HortScience 36, 738–740. Mosel, H.-D., Herrmann, K., 1974. Changes in catechins and hydroxycinnamic acid derivatives during development of apples and pears. J. Sci. Food Agric. 25, 156–251. Murphey, A.S., Dilley, D.R., 1988. Anthocyanin biosynthesis and maturity of ’McIntosh’ apples as influenced by ethylene-releasing compounds. J. Am. Soc. Hortic. Sci. 113, 718–723.
D.D. MacLean et al. / Postharvest Biology and Technology 45 (2007) 46–55 Niggeweg, R., Micheal, A.J., Martin, C., 2004. Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat. Biotechnol. 22, 746–754. Oleszek, W., Amiot, M.J., Aubert, S., 1994. Identification of some phenolics in pear fruit. J. Agric. Food Chem. 42, 1261–1265. Owino, W.Z.O., Nakano, R., Kubo, Y., Inaba, A., 2002. Differential regulation of genes encoding ethylene biosynthesis enzymes and ethylene response sensor ortholog during ripening and in response to wounding in avocados. J. Am. Soc. Hortic. Sci. 127, 520–527. Riov, J., Monselise, S.P., Kahan, R.S., 1969. Ethylene-controlled induction of phenylalanine ammonia-lyase in citrus fruit peel. Plant Physiol. 44, 631– 635. Scheiber, A., Keller, P., Carle, R., 2001. Determination of phenolic acids and flavonoids of apple and pear by HPLC. J. Chromatogr. A 910, 265–273. Steyn, W.J., Holcroft, D.M., Wand, S.J.E., Jacobs, G., 2004. Regulation of pear color development in relation to activity of flavonoid enzymes. J. Am. Soc. Hortic. Sci. 129, 6–12.
55
Tan, S.C., 1980. Phenylalanine ammonia-lyase and the phenylalanine ammonialyase inactivating system: effects of light, temperature and mineral deficiencies. Aust. J. Plant Physiol. 7, 159–167. Trainotti, L., Bonghi, C., Ziliotto, F., Zanin, D., Rasori, A., Casadoro, G., Ramina, A., Tonutti, P., 2006. The use of microarray microPEACH 1.0 to investigate transcriptome changes during transition from pre-climacteric to climacteric phase in peach fruit. Plant Sci. 170, 606–613. Trinchero, G.D., Sozzi, G.O., Covatta, F., Fraschina, A.A., 2004. Inhibition of ethylene action by 1-methylcyclopropene extends postharvest life of “Bartlett” pears. Postharvest Biol. Technol. 32, 193–204. Tsuda, T., Watanabe, M., Ohshima, K., Norinobu, S., Choi, S.W., Kawakishi, S., Osawa, T., 1994. Antioxidative activity of the anthocyanin pigments cyanidin 3-glucoside and cyanidin. J. Agric. Food Chem. 42, 2407–2410. Walbot, V., Mueller, L.A., Silady, R.A., Goodman, C.D., 2000. Do glutathione s-transferases act as enzymes or carrier proteins for their substrates. Sulfur Nutr. Sulfur Assimilation in Higher Plants, 155–165.