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Effect of hot air, UV-C, white light and modified atmosphere treatments on expression of chlorophyll degrading genes in postharvest broccoli (Brassica oleracea L.) florets
Scientia Horticulturae 127 (2011) 214–219
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Effect of hot air, UV-C, white light and modified atmosphere treatments on expression of chlorophyll degrading genes in postharvest broccoli (Brassica oleracea L.) florets Agustín M. Büchert a , Pedro M. Civello a,b , Gustavo A. Martínez a,b,∗ a
Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús (IIB-INTECH) UNSAM-CONICET, Camino Circunvalación Laguna Km 6, Chascomús (B7130IWA), Buenos Aires, Argentina b Facultad de Ciencias Exactas. Universidad Nacional de La Plata (UNLP), 47 and 115, (1900) La Plata, Argentina
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Article history: Received 2 August 2010 Received in revised form 2 November 2010 Accepted 4 November 2010 Keywords: Chlorophyllase Pheophytinase Senescence Heat treatment UV-C Modified atmosphere
a b s t r a c t Several treatments were applied in order to delay postharvest degreening in broccoli florets and investigate their effects on genes associated with chlorophyll catabolism. Degradation of chlorophylls is the most evident visual manifestation of broccoli postharvest deterioration, occurring rapidly due to the immature stage in which the material is harvested. Treatments such as storage in modified atmosphere, exposure to hot air, UV-C and white lamps were employed in the current work to induce a delay in degreening and chlorophyll degradation. Expression of genes possibly related to chlorophyll catabolism was analyzed in these samples and discussed. Chlorophyllases, the enzymes traditionally believed to remove the phytol side chain from chlorophyll appear to have a gene expression that was not regulated by postharvest treatments. Pheophytinase, a recently discovered new enzyme in this metabolic pathway, correlated chlorophyll loss accurately in heat, UV-C and white-light treatments, but not in modified atmospheres. Results presented in this work indicate that postharvest treatments that delay chlorophyll degradation have a higher effect on the expression of pheophytinase rather than on chlorophyllase genes. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Broccoli (Brassica oleracea L. var. Italica) is a floral vegetable employed worldwide for human ingestion due to its high levels of antioxidants and anticarcinogenic compounds, ascorbic acid and dietary fiber (King and Morris, 1994). Epidemiological studies suggest that a diet rich in cruciferous vegetables, such as broccoli, has a higher effectiveness in decreasing the risk for a number of cancers, including prostate, colon and breast, in comparison to the ingestion of other types of vegetables (Verhoeven et al., 1996). Broccoli plants are cultivated for consumption of its inflorescences, which are harvested previous to sepal opening, while still immature. The harvest process implies loss of water and changes in hormone and nutrient contents, inducing severe stress conditions and an early onset of senescence. The main indicator of senescence
Abbreviations: Chl, chlorophyll; Chlide, chlorophyllide; CLH, chlorophyllase; EST, expressed sequence tags; LDPE, low density polyethylene; Pheide, pheophorbide; Phein, pheophytin; PPH, pheophytinase; RCC, red chlorophyll catabolyte; RT-qPCR, reverse transcription quantitative real-time PCR. ∗ Corresponding author at: Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús (IIB-INTECH) UNSAM-CONICET, Camino Circunvalación Laguna Km 6, Chascomús (B7130IWA), Buenos Aires, Argentina. E-mail address: [email protected] (G.A. Martínez). 0304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.11.001
in vegetables is loss of green color, as a consequence of chlorophyll (Chl) degradation, which is detrimental to the commercial approval of the product. Furthermore, an accelerated senescence will hasten processes such as loss of proteins and sugars and lipid peroxidation, developing in a loss of nutritional quality (King and Morris, 1994; Page et al., 2001). Several attempts have been done to extend broccoli postharvest life, including the use of refrigerated storage (Toivonen, 1997) controlled and modified atmospheres (Jacobsson et al., 2004), heat treatments (Funamoto et al., 2002; Costa et al., 2005b), UV-C applications (Costa et al., 2006a,b), 1-MCP (Ku and Wills, 1999; Able et al., 2002) and ethanol (Suzuki et al., 2004). In all cases, the main objective of the treatment was to regulate senescence and delay Chl degradation in order to maintain organoleptic and nutritional quality of broccoli. The Chl degradation process is essential in processes such as leaf senescence and fruit ripening, due to the much needed elimination of this molecule and its derivatives in order to avoid accumulation of phototoxic pigments (Matile et al., 1999; Hörtensteiner, 2006). A pathway has been established for Chl degradation, involving the concerted action of several enzymes on green pigments starting with the dephytilation of the Chl molecule, catalyzed by the enzyme chlorophyllase (CLH, EC 3.1.1.14), followed by removal of the central Mg2+ ion (Suzuki et al., 2005; Harpaz-Saad et al.,
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2007). CLH activity, producing chlorophyllide (Chlide) and phytol, has preferential action toward Chl a, but it can also accept its b form and pheophytins (Phein) as substrate (Hörtensteiner, 1999; Benedetti and Arruda, 2002). The direct intermediate resulting from Mg-dechelation, pheophorbide (Pheide), has its porphyrin ring oxygenolytically opened by Pheide a oxygenase, resulting in loss of the green color of the molecule (Hörtensteiner et al., 1998; Matile et al., 1999). Afterwards, red chlorophyll catabolyte (RCC) is site-specifically reduced by RCC reductase (Rodoni et al., 1997b; Wüthrich et al., 2000; Mach et al., 2001). Further downstream products are transported to the vacuole and degraded (Hinder et al., 1996; Rodoni et al., 1997a; Oberhuber et al., 2003). Removal of the phytol side chain from Chl has been subject to debate recently. The dephytilation step has been traditionally related to CLH, considered as the first enzymatic step in the catabolic process (Takamiya et al., 2000; Harpaz-Saad et al., 2007). Several studies have focused on CLH and genes have been isolated from diverse sources, such as Ginko biloba, wheat (Arkus et al., 2005), Citrus (Jacob-Wilk et al., 1999), Chenopodium album, arabidopsis (Tsuchiya et al., 1999) and Brassica oleracea (Tang et al., 2004). Traditionally, CLH genes have been located into two distinct phylogenetic clusters: expression of the first group (arabidopsis AtCLH1 and Citrus sinensis CsCLH) is regulated by ethylene and methyl jasmonate, and expression of the second group (Arabidopsis AtCLH2 and C. album CaCLH) occurs at low, constitutive levels (Tsuchiya et al., 1999; Jacob-Wilk et al., 1999). However, since not all CLH genes isolated have a chloroplast transit peptide, recent publications have questioned the participation of CLH in Chl breakdown, suggesting alternative pathways involving enzymes other than CLH (Takamiya et al., 2000; Hörtensteiner, 2006). Schenk et al. (2007) have shown that double knock-out arabidopsis mutants impaired in expression of both known CLH genes are still able to degrade Chl during senescence. Based on the assumption that CLH genes are not essential for Chl breakdown in arabidopsis, Schelbert et al. (2009) attempted to reveal a true CLH which would be responsible for Chl dephytilation. However, their results derived in the finding of a new enzyme, termed pheophytinase (PPH), which would act as a Phein hydrolase. In this given scenario, dephytilation would occur on Mg-free Chl and removal of the central Mg2+ ion would take place on intact Chl; therefore, the traditional Chl degradation pathway must be revised, particularly concerning the early steps (Schelbert et al., 2009). Knowledge of chlorophyll degradation pathway during postharvest senescence of broccoli would help to possible exploitation to breed genotypes/cultivars having a higher green color retention. In the present work, we applied several postharvest physical treatments that modified senescence rate of broccoli florets and evaluated their effects on the expression of a putative PPH and two CHL genes. 2. Materials and methods 2.1. Plant material and postharvest treatments Broccoli (Brassica oleracea L. var. Italica cv. Iron) heads were obtained from a local producer in La Plata, Buenos Aires, Argentina, and immediately transported and processed. For heat treatments, whole broccoli heads were placed in a laboratory stove and heated by air at 48 ◦ C for 3 h. For UV-C treatments, broccoli florets were placed vertically in plastic trays, in order to assure homogeneous irradiation and exposure to UV-C lamps (TUV G30T8, 30W, Philips). Irradiation was performed at a distance of 30 cm and a dose of 10 kJ m−2 . Immediately after heat or UV-C treatments, florets were stored in darkness at 22 ◦ C to induce senescence along with untreated samples as controls. For modified atmosphere treatments, whole florets were placed individually inside LDPE (low
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density polyethylene) bags hermetically sealed and stored in darkness at 22 ◦ C to induce senescence. Florets without LDPE bags were used as controls. For visible light treatments, broccoli heads were placed in a well-ventilated chamber isolated from external light sources at 22 ◦ C. One half of the chamber was kept in complete dark (<1 mol m−2 s−1 ) and the other half was exposed to a continuous dose of 12 mol m−2 s−1 , using 40 W white light fluorescent tubes. In all cases, at least 35 broccoli heads were used per treatment or control, samples were taken daily, heads were segmented and the inflorescences were immediately frozen using liquid nitrogen and stored at −20 ◦ C until use. 2.2. Superficial color measurement Superficial color was measured daily for all broccoli heads in all treatments, employing L*, a* and b* parameters on each sample at five different positions with a Minolta CR300 chromameter (Osaka, Japan). Hue angle (h◦ ) was calculated as h◦ = tan−1 (b* /a* ) when a* > 0 and b* > 0, or as h◦ = 180◦ − tan−1 (b*/a*) when a* < 0 and b* > 0. Modified atmosphere treatments only show data from day 3 onwards, since color was not measured before in order to preserve tightness of sealed bags. 2.3. Chlorophyll content Frozen broccoli florets were ground in liquid nitrogen and 0.5 g of the resulting powder was mixed with 5 ml 80% (v/v) acetone and centrifuged at 10,000 × g for 10 min at 4 ◦ C. The content of Chl was measured in the supernatant according to Inskeep and Bloom (1985) and results were expressed as g total chlorophyll per kg fresh weight tissue. All measurements were performed by triplicate. 2.4. RNA extraction and qPCR assays Total RNA was extracted from broccoli tissue using a modification of the phenol method (Kirby, 1968) and quantified via UV spectrophotometry. Approximately 6 g of frozen broccoli tissue was grounded and added to 10 ml TLE buffer (0.2 M Tris, 0.1 M LiCl, 5 mM EDTA, pH 8.2), 10 l -mercaptoethanol, 0.9 ml 2 M sodium acetate, pH 4, 10 ml saturated phenol, 2 ml chlorophorm. After centrifugation and washing with a phenol, chlorophorm, isoamyl alcohol mixture, RNA was precipitated using 8 M LiCl and resuspended in DEPC-treated double-distilled sterile water. The integrity of extracted RNA was observed by gel-electrophoresis. An amount corresponding to 4 g of total RNA was treated with RQ1 DNAse (Promega) according to the manufacturer’s protocol, purified with chlorophorm:1-octanol (24:1), precipitated with 3 M sodium acetate and reverse-transcribed using MML-V reverse transcriptase (Promega) and random primers (Hexamers), according to manufacturer’s suggestions. The absence of DNA contamination was assessed by PCR and gel-electrophoresis. Melting curve analysis was performed to discard possible contamination or unwanted amplification. Also, qPCR products were observed by gel electrophoresis and a single band of the expected size was observed for each reaction. Resulting cDNA was stored at −20 ◦ C and employed as template for two-step qPCR reactions using an Mx3005P qPCR system (Stratagene) and FastStart Universal SYBR Green Master (Roche), using recommended conditions. Sequences of primers employed are described below. Every RT-qPCR measurement was performed at least 4 times. 2.5. Primer design The following primers were designed in order to assess relative expression by RT-qPCR of two known broccoli CLH genes: BoCLH1 (AF337544): 5 -agacccatccatcaagttttcagc-3
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and 5 -agatttcgggatcggttcttatgc-3 , amplifying a fragment of 85 bp; BoCLH2 (AF337545): 5 -agatgcctgttctagttattgg-3 and 5 -cacgctggaccttgacattc-3 , amplifying a fragment of 125 bp. All qPCR assays employed -actin (AF044573) as normalizer, with the following primers: 5 -ccagaggtcttgttccagccatc-3 and 5 -gttccaccactgagcacaatgttac-3 , amplifying a fragment of 136 bp. The following specific primers were designed to amplify and quantify the expression of a 91-bp fragment of a broccoli EST similar to AtPPH (from B. oleracea var. alboglabra, OL386R): 5 -agaggttatcggtgagcca-3 and 5 -gacgagatgaggatggg-3 . 2.6. Statistical analysis The experiment was performed according to a factorial design. Data were analyzed using analysis of variance (ANOVA), and means were compared by the least significant difference (LSD) test at a significance level of P < 0.05. 3. Results and discussion 3.1. Effect of different postharvest treatments on broccoli senescence As was previously reported, broccoli senescence and degreening can be arrested by heat treatments, such as hot water dips of florets (Forney, 1995; Tian et al., 1997) or exposure to hot air (Funamoto et al., 2002; Costa et al., 2005b,a; Lemoine et al., 2007; Lemoine et
Fig. 1. Hue angle (A) and total Chl content (B) of broccoli florets subjected to different physical treatments during 5 days of induced senescence. Hue angle was calculated as described in Section 2.3 and Chl content is expressed as grams total Chl per kilogram of fresh weight tissue. Asterisks show statistical differences between all treated samples and controls (P < 0.005).
al., 2008). In our case, whole broccoli florets were treated by hot air at 48 ◦ C for 3 h, and then stored in darkness. As seen in Fig. 1, heat treatment delayed Chl degradation in florets, which was also evidenced by a higher retention of green color at the end of the experiment. Samples stored inside hermetically sealed LDPE bags also showed higher Chl retention in comparison to controls. Isolation from external environment inside LDPE bags, which have a partial permeability to gases, allowed for a modified atmosphere, originating a higher CO2 and lower O2 concentration inside the bags (Ballantyne et al., 2007). Treatments with UV-C light have been used to prevent or decrease the harshness of postharvest diseases (Stevens et al., 1996), to improve postharvest quality of crops (Vicente et al., 2005) and to delay fruit ripening (Barka et al., 2000), among other purposes. Particularly in broccoli, several reports have shown a delay in Chl degradation, lower respiration rates, reduced degradation of phenols, sugars and proteins (Costa et al., 2006b; Lemoine et al., 2007; Lemoine et al., 2008). As expected and seen in Fig. 1, samples exposed to UV-C light maintained higher Chl levels and Hue (◦ ) values related to controls throughout the storage time period. One of the most important factors involved in plant growth and development is exposure to light and its effect on pigment accumulation has been documented (Giovannoni, 2001; Chen et al., 2004; Franklin et al., 2005). However, there is scarce evidence regarding the effect of exposure to visible white light on postharvest life of horticultural crops during storage. As seen in Fig. 2, samples stored
Fig. 2. Hue angle (A) and total Chl content (B) of white light-treated broccoli florets during 5 days of induced senescence. Hue angle was calculated as described in Section 2 and Chl content is expressed as grams total chl per kilogram of fresh weight tissue. Asterisks show statistical differences between all treated samples and controls (P < 0.005).
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under a continuous dose of white light at 12 mol m−2 s−1 displayed higher greenness and Chl levels in comparison to controls from day 3 onwards. Hue (◦ ) and Chl content levels from treated samples at day 5 were comparable to controls at day 4, indicating a 1-day delay in postharvest degreening. 3.2. Effect of different postharvest treatments on chlorophyllase and pheophytinase gene expression Available data regarding the water solubility and cellular localization of Chl breakdown products indicate the need for a dephytilation step in the Chl degradation pathway. Traditionally, this step has been considered an early reaction, since mutants defective in expression of pheophorbide a oxygenase (PaO1) or red chlorophyll catabolyte reductase (acd2) accumulate dephytilated pigments (Tanaka et al., 2003; Pruˇzinská et al., 2003, 2005). Traditionally, it was considered that CLH was the first enzyme in chlorophyll catabolism and Mg-dechelatase the second one, producing consecutively Chlide and Pheide. However, the recent report by Schelbert et al. (2009) suggests PPH as the responsible enzyme for the dephytilation step, preceded by Mg-dechelation. The possibility of a Chl degradation pathway involving an initial Mg-dechelation step and formation of Phein had been addressed previously, even before PPH was brought into the light. In cabbage, Heaton et al. (1996) indicated the existence of two Chl degradation pathways in green plant tissues, one through Phein to Pheide and other though Chlide to Pheide. Also, pathway differences have been suggested in both Citrus and parsley senescence (Amir-Shapira et al., 1987) and in Brassica napus (Langmeier et al., 1993). Given the potential importance of PPH in Chl degradation during senescence, we considered analyzing the expression of CLH as well as that of PPH in broccoli tissue subjected to several postharvest treatments. Until this time, three broccoli CLH genes had been identified (BoCLH1, BoCLH2 and BoCLH3). Nevertheless, it has been reported that only mRNA transcripts of BoCLH1 are accumulated in broccoli tissue, both senescent and freshly harvested (Chen et al., 2008). However, in a previous work, we detected both BoCLH1 and BoCLH2 by RT-qPCR in senescent as well as presenescent broccoli florets (Büchert et al., in press). To some extent, CLH activity and its relation to Chl degradation during postharvest senescence are contradictory. Funamoto et al. (2002) reported that no changes were found in CLH activity during senescence, although both activity and Chl degradation were reduced by heat treatment. Also, Costa et al. (2005a) found an increment in activity during postharvest senescence, as well as regulation by hormone treatments. In our case, BoCLH1 expression was decreased during the course of postharvest senescence (Fig. 3A), showing values approximately 10 times lower after day 3 compared to the day of harvest. Expression of BoCLH2, on the other hand, was greatly enhanced with induced senescence (Fig. 3B). Expression values between 2.8- and 4.3-fold the initial value was found for all treatments after 3–4 days of senescence induction. Controls from light treated samples exhibited tendencies similar to controls of other treatments for both CLH genes analyzed, with slight differences probably due to different harvesting times (Fig. 4A, B). Concerning postharvest treatments assayed, in all cases expression of BoCLH1 was greatly reduced during incubation. Compared to controls at days 3–5, modified atmosphere samples showed higher expression values, contrary to what would be expected considering the delay in degreening found with this treatment. The other treatments had a behavior similar to controls and had no significant effect on the expression of this gene. On the other hand, BoCLH2 expression showed an important increase in heat-treated samples, being values 4-fold higher than controls. Samples exposed to UVC light expressed this gene twice as much as controls at the final
Fig. 3. Relative expression as measured by qPCR of (A) BoCLH1, (B) BoCLH2 and (C) BoPPH in broccoli florets subjected to different treatments, during 5 days of induced senescence.
day of the experiment and light-treated broccoli florets exhibit an inhibition in expression throughout the experiment. In the case of PPH, we recently cloned a fragment encoding a sequence with similarities to the published sequence for arabidopsis PPH (AtPPH, At5g13800), which was named BoPPH (Büchert et al., in press). Expression of BoPPH during induced senescence exhibits an increase in all samples (Figs. 3 and 4CFigs. 3C and 4C). In the case of light treatment controls, this increase is presented gradually while on the control from other treatments this increase occurs faster, which could be due to different samples employed, harvested at different times. Samples exposed to UV-C presented a lower expression, being values of treated samples half of those corresponding to controls. Light-treated material showed lower expression values than controls at day 5. Expression in heattreated samples appears to be lower than controls at day 4 and
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in postharvest yellowing and authors suggested that other genes might also be essential to this process. On the contrary, most treatments utilized in this work which delayed Chl degradation, such as heat-treatment, UV-C and visible light exposure, also seemed to regulate PPH expression. Thus, PPH expression in broccoli appears to be more closely related to Chl breakdown during senescence. Similar results were found by our group previously using hormonal treatments. Hormones which delay or accelerate senescence also inhibit or increase expression of BoPPH, but do not seem to have a clear effect on expression of CLH genes (Büchert et al., in press). In the case of modified atmospheres, the fact that Chl degradation was almost completely blocked by this treatment, a similar inhibition of BoPPH expression was not found, suggests that the expression of other Chl catabolic related genes could be inhibited by the treatment. In conclusion, postharvest treatments that delay senescence and chlorophyll degradation did not have a clear and relevant effect on expression of CHL related genes while treatments like heat, UV-C and light effectively had an inhibitory effect on BoPPH expression. This and previous results (Büchert et al., in press) indicates that PPH could serve as a reliable biochemical marker or target to identify genotypes or manipulate broccoli by molecular biology in order to obtain lines with reduced chlorophyll degradation. Acknowledgement This work was based on funding from Agencia Nacional de Promoción Científica y Tecnológica (Argentina) PICT 25283. References
Fig. 4. Relative expression as measured by qPCR of (A) BoCLH1, (B) BoCLH2 and (C) BoPPH in white light-treated broccoli florets, during 5 days of induced senescence.
florets stored in modified atmospheres display values similar to controls. Results mentioned above point toward a negative regulation of BoCLH1 and an enhancement in BoCLH2 expression during senescence. Nevertheless, while postharvest treatments delayed Chl degradation and degreening considerably, changes in CLH gene expression did not seem to correlate with Chl content values clearly. In addition, our results suggest that BoCLH2 and Chl degradation may not be directly related either, since even though BoCLH2 expression is enhanced during senescence; it is not modified by treatments assayed. Taken together, these results suggest that both BoCLH1 and BoCLH2 probably do not have a relevant role in Chl catabolism as it was suggested previously (Büchert et al., in press). In this sense, Chen et al. (2008) developed transgenic broccoli plants expressing antisense BoCLH1, which showed only 1–2 day delay
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Effect of hot air, UV-C, white light and modified atmosphere treatments on expression of chlorophyll degrading genes in postharvest broccoli (Brassica oleracea L.) florets Agustín M. Büchert a , Pedro M. Civello a,b , Gustavo A. Martínez a,b,∗ a
Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús (IIB-INTECH) UNSAM-CONICET, Camino Circunvalación Laguna Km 6, Chascomús (B7130IWA), Buenos Aires, Argentina b Facultad de Ciencias Exactas. Universidad Nacional de La Plata (UNLP), 47 and 115, (1900) La Plata, Argentina
a r t i c l e
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Article history: Received 2 August 2010 Received in revised form 2 November 2010 Accepted 4 November 2010 Keywords: Chlorophyllase Pheophytinase Senescence Heat treatment UV-C Modified atmosphere
a b s t r a c t Several treatments were applied in order to delay postharvest degreening in broccoli florets and investigate their effects on genes associated with chlorophyll catabolism. Degradation of chlorophylls is the most evident visual manifestation of broccoli postharvest deterioration, occurring rapidly due to the immature stage in which the material is harvested. Treatments such as storage in modified atmosphere, exposure to hot air, UV-C and white lamps were employed in the current work to induce a delay in degreening and chlorophyll degradation. Expression of genes possibly related to chlorophyll catabolism was analyzed in these samples and discussed. Chlorophyllases, the enzymes traditionally believed to remove the phytol side chain from chlorophyll appear to have a gene expression that was not regulated by postharvest treatments. Pheophytinase, a recently discovered new enzyme in this metabolic pathway, correlated chlorophyll loss accurately in heat, UV-C and white-light treatments, but not in modified atmospheres. Results presented in this work indicate that postharvest treatments that delay chlorophyll degradation have a higher effect on the expression of pheophytinase rather than on chlorophyllase genes. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Broccoli (Brassica oleracea L. var. Italica) is a floral vegetable employed worldwide for human ingestion due to its high levels of antioxidants and anticarcinogenic compounds, ascorbic acid and dietary fiber (King and Morris, 1994). Epidemiological studies suggest that a diet rich in cruciferous vegetables, such as broccoli, has a higher effectiveness in decreasing the risk for a number of cancers, including prostate, colon and breast, in comparison to the ingestion of other types of vegetables (Verhoeven et al., 1996). Broccoli plants are cultivated for consumption of its inflorescences, which are harvested previous to sepal opening, while still immature. The harvest process implies loss of water and changes in hormone and nutrient contents, inducing severe stress conditions and an early onset of senescence. The main indicator of senescence
Abbreviations: Chl, chlorophyll; Chlide, chlorophyllide; CLH, chlorophyllase; EST, expressed sequence tags; LDPE, low density polyethylene; Pheide, pheophorbide; Phein, pheophytin; PPH, pheophytinase; RCC, red chlorophyll catabolyte; RT-qPCR, reverse transcription quantitative real-time PCR. ∗ Corresponding author at: Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús (IIB-INTECH) UNSAM-CONICET, Camino Circunvalación Laguna Km 6, Chascomús (B7130IWA), Buenos Aires, Argentina. E-mail address: [email protected] (G.A. Martínez). 0304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.11.001
in vegetables is loss of green color, as a consequence of chlorophyll (Chl) degradation, which is detrimental to the commercial approval of the product. Furthermore, an accelerated senescence will hasten processes such as loss of proteins and sugars and lipid peroxidation, developing in a loss of nutritional quality (King and Morris, 1994; Page et al., 2001). Several attempts have been done to extend broccoli postharvest life, including the use of refrigerated storage (Toivonen, 1997) controlled and modified atmospheres (Jacobsson et al., 2004), heat treatments (Funamoto et al., 2002; Costa et al., 2005b), UV-C applications (Costa et al., 2006a,b), 1-MCP (Ku and Wills, 1999; Able et al., 2002) and ethanol (Suzuki et al., 2004). In all cases, the main objective of the treatment was to regulate senescence and delay Chl degradation in order to maintain organoleptic and nutritional quality of broccoli. The Chl degradation process is essential in processes such as leaf senescence and fruit ripening, due to the much needed elimination of this molecule and its derivatives in order to avoid accumulation of phototoxic pigments (Matile et al., 1999; Hörtensteiner, 2006). A pathway has been established for Chl degradation, involving the concerted action of several enzymes on green pigments starting with the dephytilation of the Chl molecule, catalyzed by the enzyme chlorophyllase (CLH, EC 3.1.1.14), followed by removal of the central Mg2+ ion (Suzuki et al., 2005; Harpaz-Saad et al.,
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2007). CLH activity, producing chlorophyllide (Chlide) and phytol, has preferential action toward Chl a, but it can also accept its b form and pheophytins (Phein) as substrate (Hörtensteiner, 1999; Benedetti and Arruda, 2002). The direct intermediate resulting from Mg-dechelation, pheophorbide (Pheide), has its porphyrin ring oxygenolytically opened by Pheide a oxygenase, resulting in loss of the green color of the molecule (Hörtensteiner et al., 1998; Matile et al., 1999). Afterwards, red chlorophyll catabolyte (RCC) is site-specifically reduced by RCC reductase (Rodoni et al., 1997b; Wüthrich et al., 2000; Mach et al., 2001). Further downstream products are transported to the vacuole and degraded (Hinder et al., 1996; Rodoni et al., 1997a; Oberhuber et al., 2003). Removal of the phytol side chain from Chl has been subject to debate recently. The dephytilation step has been traditionally related to CLH, considered as the first enzymatic step in the catabolic process (Takamiya et al., 2000; Harpaz-Saad et al., 2007). Several studies have focused on CLH and genes have been isolated from diverse sources, such as Ginko biloba, wheat (Arkus et al., 2005), Citrus (Jacob-Wilk et al., 1999), Chenopodium album, arabidopsis (Tsuchiya et al., 1999) and Brassica oleracea (Tang et al., 2004). Traditionally, CLH genes have been located into two distinct phylogenetic clusters: expression of the first group (arabidopsis AtCLH1 and Citrus sinensis CsCLH) is regulated by ethylene and methyl jasmonate, and expression of the second group (Arabidopsis AtCLH2 and C. album CaCLH) occurs at low, constitutive levels (Tsuchiya et al., 1999; Jacob-Wilk et al., 1999). However, since not all CLH genes isolated have a chloroplast transit peptide, recent publications have questioned the participation of CLH in Chl breakdown, suggesting alternative pathways involving enzymes other than CLH (Takamiya et al., 2000; Hörtensteiner, 2006). Schenk et al. (2007) have shown that double knock-out arabidopsis mutants impaired in expression of both known CLH genes are still able to degrade Chl during senescence. Based on the assumption that CLH genes are not essential for Chl breakdown in arabidopsis, Schelbert et al. (2009) attempted to reveal a true CLH which would be responsible for Chl dephytilation. However, their results derived in the finding of a new enzyme, termed pheophytinase (PPH), which would act as a Phein hydrolase. In this given scenario, dephytilation would occur on Mg-free Chl and removal of the central Mg2+ ion would take place on intact Chl; therefore, the traditional Chl degradation pathway must be revised, particularly concerning the early steps (Schelbert et al., 2009). Knowledge of chlorophyll degradation pathway during postharvest senescence of broccoli would help to possible exploitation to breed genotypes/cultivars having a higher green color retention. In the present work, we applied several postharvest physical treatments that modified senescence rate of broccoli florets and evaluated their effects on the expression of a putative PPH and two CHL genes. 2. Materials and methods 2.1. Plant material and postharvest treatments Broccoli (Brassica oleracea L. var. Italica cv. Iron) heads were obtained from a local producer in La Plata, Buenos Aires, Argentina, and immediately transported and processed. For heat treatments, whole broccoli heads were placed in a laboratory stove and heated by air at 48 ◦ C for 3 h. For UV-C treatments, broccoli florets were placed vertically in plastic trays, in order to assure homogeneous irradiation and exposure to UV-C lamps (TUV G30T8, 30W, Philips). Irradiation was performed at a distance of 30 cm and a dose of 10 kJ m−2 . Immediately after heat or UV-C treatments, florets were stored in darkness at 22 ◦ C to induce senescence along with untreated samples as controls. For modified atmosphere treatments, whole florets were placed individually inside LDPE (low
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density polyethylene) bags hermetically sealed and stored in darkness at 22 ◦ C to induce senescence. Florets without LDPE bags were used as controls. For visible light treatments, broccoli heads were placed in a well-ventilated chamber isolated from external light sources at 22 ◦ C. One half of the chamber was kept in complete dark (<1 mol m−2 s−1 ) and the other half was exposed to a continuous dose of 12 mol m−2 s−1 , using 40 W white light fluorescent tubes. In all cases, at least 35 broccoli heads were used per treatment or control, samples were taken daily, heads were segmented and the inflorescences were immediately frozen using liquid nitrogen and stored at −20 ◦ C until use. 2.2. Superficial color measurement Superficial color was measured daily for all broccoli heads in all treatments, employing L*, a* and b* parameters on each sample at five different positions with a Minolta CR300 chromameter (Osaka, Japan). Hue angle (h◦ ) was calculated as h◦ = tan−1 (b* /a* ) when a* > 0 and b* > 0, or as h◦ = 180◦ − tan−1 (b*/a*) when a* < 0 and b* > 0. Modified atmosphere treatments only show data from day 3 onwards, since color was not measured before in order to preserve tightness of sealed bags. 2.3. Chlorophyll content Frozen broccoli florets were ground in liquid nitrogen and 0.5 g of the resulting powder was mixed with 5 ml 80% (v/v) acetone and centrifuged at 10,000 × g for 10 min at 4 ◦ C. The content of Chl was measured in the supernatant according to Inskeep and Bloom (1985) and results were expressed as g total chlorophyll per kg fresh weight tissue. All measurements were performed by triplicate. 2.4. RNA extraction and qPCR assays Total RNA was extracted from broccoli tissue using a modification of the phenol method (Kirby, 1968) and quantified via UV spectrophotometry. Approximately 6 g of frozen broccoli tissue was grounded and added to 10 ml TLE buffer (0.2 M Tris, 0.1 M LiCl, 5 mM EDTA, pH 8.2), 10 l -mercaptoethanol, 0.9 ml 2 M sodium acetate, pH 4, 10 ml saturated phenol, 2 ml chlorophorm. After centrifugation and washing with a phenol, chlorophorm, isoamyl alcohol mixture, RNA was precipitated using 8 M LiCl and resuspended in DEPC-treated double-distilled sterile water. The integrity of extracted RNA was observed by gel-electrophoresis. An amount corresponding to 4 g of total RNA was treated with RQ1 DNAse (Promega) according to the manufacturer’s protocol, purified with chlorophorm:1-octanol (24:1), precipitated with 3 M sodium acetate and reverse-transcribed using MML-V reverse transcriptase (Promega) and random primers (Hexamers), according to manufacturer’s suggestions. The absence of DNA contamination was assessed by PCR and gel-electrophoresis. Melting curve analysis was performed to discard possible contamination or unwanted amplification. Also, qPCR products were observed by gel electrophoresis and a single band of the expected size was observed for each reaction. Resulting cDNA was stored at −20 ◦ C and employed as template for two-step qPCR reactions using an Mx3005P qPCR system (Stratagene) and FastStart Universal SYBR Green Master (Roche), using recommended conditions. Sequences of primers employed are described below. Every RT-qPCR measurement was performed at least 4 times. 2.5. Primer design The following primers were designed in order to assess relative expression by RT-qPCR of two known broccoli CLH genes: BoCLH1 (AF337544): 5 -agacccatccatcaagttttcagc-3
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and 5 -agatttcgggatcggttcttatgc-3 , amplifying a fragment of 85 bp; BoCLH2 (AF337545): 5 -agatgcctgttctagttattgg-3 and 5 -cacgctggaccttgacattc-3 , amplifying a fragment of 125 bp. All qPCR assays employed -actin (AF044573) as normalizer, with the following primers: 5 -ccagaggtcttgttccagccatc-3 and 5 -gttccaccactgagcacaatgttac-3 , amplifying a fragment of 136 bp. The following specific primers were designed to amplify and quantify the expression of a 91-bp fragment of a broccoli EST similar to AtPPH (from B. oleracea var. alboglabra, OL386R): 5 -agaggttatcggtgagcca-3 and 5 -gacgagatgaggatggg-3 . 2.6. Statistical analysis The experiment was performed according to a factorial design. Data were analyzed using analysis of variance (ANOVA), and means were compared by the least significant difference (LSD) test at a significance level of P < 0.05. 3. Results and discussion 3.1. Effect of different postharvest treatments on broccoli senescence As was previously reported, broccoli senescence and degreening can be arrested by heat treatments, such as hot water dips of florets (Forney, 1995; Tian et al., 1997) or exposure to hot air (Funamoto et al., 2002; Costa et al., 2005b,a; Lemoine et al., 2007; Lemoine et
Fig. 1. Hue angle (A) and total Chl content (B) of broccoli florets subjected to different physical treatments during 5 days of induced senescence. Hue angle was calculated as described in Section 2.3 and Chl content is expressed as grams total Chl per kilogram of fresh weight tissue. Asterisks show statistical differences between all treated samples and controls (P < 0.005).
al., 2008). In our case, whole broccoli florets were treated by hot air at 48 ◦ C for 3 h, and then stored in darkness. As seen in Fig. 1, heat treatment delayed Chl degradation in florets, which was also evidenced by a higher retention of green color at the end of the experiment. Samples stored inside hermetically sealed LDPE bags also showed higher Chl retention in comparison to controls. Isolation from external environment inside LDPE bags, which have a partial permeability to gases, allowed for a modified atmosphere, originating a higher CO2 and lower O2 concentration inside the bags (Ballantyne et al., 2007). Treatments with UV-C light have been used to prevent or decrease the harshness of postharvest diseases (Stevens et al., 1996), to improve postharvest quality of crops (Vicente et al., 2005) and to delay fruit ripening (Barka et al., 2000), among other purposes. Particularly in broccoli, several reports have shown a delay in Chl degradation, lower respiration rates, reduced degradation of phenols, sugars and proteins (Costa et al., 2006b; Lemoine et al., 2007; Lemoine et al., 2008). As expected and seen in Fig. 1, samples exposed to UV-C light maintained higher Chl levels and Hue (◦ ) values related to controls throughout the storage time period. One of the most important factors involved in plant growth and development is exposure to light and its effect on pigment accumulation has been documented (Giovannoni, 2001; Chen et al., 2004; Franklin et al., 2005). However, there is scarce evidence regarding the effect of exposure to visible white light on postharvest life of horticultural crops during storage. As seen in Fig. 2, samples stored
Fig. 2. Hue angle (A) and total Chl content (B) of white light-treated broccoli florets during 5 days of induced senescence. Hue angle was calculated as described in Section 2 and Chl content is expressed as grams total chl per kilogram of fresh weight tissue. Asterisks show statistical differences between all treated samples and controls (P < 0.005).
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under a continuous dose of white light at 12 mol m−2 s−1 displayed higher greenness and Chl levels in comparison to controls from day 3 onwards. Hue (◦ ) and Chl content levels from treated samples at day 5 were comparable to controls at day 4, indicating a 1-day delay in postharvest degreening. 3.2. Effect of different postharvest treatments on chlorophyllase and pheophytinase gene expression Available data regarding the water solubility and cellular localization of Chl breakdown products indicate the need for a dephytilation step in the Chl degradation pathway. Traditionally, this step has been considered an early reaction, since mutants defective in expression of pheophorbide a oxygenase (PaO1) or red chlorophyll catabolyte reductase (acd2) accumulate dephytilated pigments (Tanaka et al., 2003; Pruˇzinská et al., 2003, 2005). Traditionally, it was considered that CLH was the first enzyme in chlorophyll catabolism and Mg-dechelatase the second one, producing consecutively Chlide and Pheide. However, the recent report by Schelbert et al. (2009) suggests PPH as the responsible enzyme for the dephytilation step, preceded by Mg-dechelation. The possibility of a Chl degradation pathway involving an initial Mg-dechelation step and formation of Phein had been addressed previously, even before PPH was brought into the light. In cabbage, Heaton et al. (1996) indicated the existence of two Chl degradation pathways in green plant tissues, one through Phein to Pheide and other though Chlide to Pheide. Also, pathway differences have been suggested in both Citrus and parsley senescence (Amir-Shapira et al., 1987) and in Brassica napus (Langmeier et al., 1993). Given the potential importance of PPH in Chl degradation during senescence, we considered analyzing the expression of CLH as well as that of PPH in broccoli tissue subjected to several postharvest treatments. Until this time, three broccoli CLH genes had been identified (BoCLH1, BoCLH2 and BoCLH3). Nevertheless, it has been reported that only mRNA transcripts of BoCLH1 are accumulated in broccoli tissue, both senescent and freshly harvested (Chen et al., 2008). However, in a previous work, we detected both BoCLH1 and BoCLH2 by RT-qPCR in senescent as well as presenescent broccoli florets (Büchert et al., in press). To some extent, CLH activity and its relation to Chl degradation during postharvest senescence are contradictory. Funamoto et al. (2002) reported that no changes were found in CLH activity during senescence, although both activity and Chl degradation were reduced by heat treatment. Also, Costa et al. (2005a) found an increment in activity during postharvest senescence, as well as regulation by hormone treatments. In our case, BoCLH1 expression was decreased during the course of postharvest senescence (Fig. 3A), showing values approximately 10 times lower after day 3 compared to the day of harvest. Expression of BoCLH2, on the other hand, was greatly enhanced with induced senescence (Fig. 3B). Expression values between 2.8- and 4.3-fold the initial value was found for all treatments after 3–4 days of senescence induction. Controls from light treated samples exhibited tendencies similar to controls of other treatments for both CLH genes analyzed, with slight differences probably due to different harvesting times (Fig. 4A, B). Concerning postharvest treatments assayed, in all cases expression of BoCLH1 was greatly reduced during incubation. Compared to controls at days 3–5, modified atmosphere samples showed higher expression values, contrary to what would be expected considering the delay in degreening found with this treatment. The other treatments had a behavior similar to controls and had no significant effect on the expression of this gene. On the other hand, BoCLH2 expression showed an important increase in heat-treated samples, being values 4-fold higher than controls. Samples exposed to UVC light expressed this gene twice as much as controls at the final
Fig. 3. Relative expression as measured by qPCR of (A) BoCLH1, (B) BoCLH2 and (C) BoPPH in broccoli florets subjected to different treatments, during 5 days of induced senescence.
day of the experiment and light-treated broccoli florets exhibit an inhibition in expression throughout the experiment. In the case of PPH, we recently cloned a fragment encoding a sequence with similarities to the published sequence for arabidopsis PPH (AtPPH, At5g13800), which was named BoPPH (Büchert et al., in press). Expression of BoPPH during induced senescence exhibits an increase in all samples (Figs. 3 and 4CFigs. 3C and 4C). In the case of light treatment controls, this increase is presented gradually while on the control from other treatments this increase occurs faster, which could be due to different samples employed, harvested at different times. Samples exposed to UV-C presented a lower expression, being values of treated samples half of those corresponding to controls. Light-treated material showed lower expression values than controls at day 5. Expression in heattreated samples appears to be lower than controls at day 4 and
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in postharvest yellowing and authors suggested that other genes might also be essential to this process. On the contrary, most treatments utilized in this work which delayed Chl degradation, such as heat-treatment, UV-C and visible light exposure, also seemed to regulate PPH expression. Thus, PPH expression in broccoli appears to be more closely related to Chl breakdown during senescence. Similar results were found by our group previously using hormonal treatments. Hormones which delay or accelerate senescence also inhibit or increase expression of BoPPH, but do not seem to have a clear effect on expression of CLH genes (Büchert et al., in press). In the case of modified atmospheres, the fact that Chl degradation was almost completely blocked by this treatment, a similar inhibition of BoPPH expression was not found, suggests that the expression of other Chl catabolic related genes could be inhibited by the treatment. In conclusion, postharvest treatments that delay senescence and chlorophyll degradation did not have a clear and relevant effect on expression of CHL related genes while treatments like heat, UV-C and light effectively had an inhibitory effect on BoPPH expression. This and previous results (Büchert et al., in press) indicates that PPH could serve as a reliable biochemical marker or target to identify genotypes or manipulate broccoli by molecular biology in order to obtain lines with reduced chlorophyll degradation. Acknowledgement This work was based on funding from Agencia Nacional de Promoción Científica y Tecnológica (Argentina) PICT 25283. References
Fig. 4. Relative expression as measured by qPCR of (A) BoCLH1, (B) BoCLH2 and (C) BoPPH in white light-treated broccoli florets, during 5 days of induced senescence.
florets stored in modified atmospheres display values similar to controls. Results mentioned above point toward a negative regulation of BoCLH1 and an enhancement in BoCLH2 expression during senescence. Nevertheless, while postharvest treatments delayed Chl degradation and degreening considerably, changes in CLH gene expression did not seem to correlate with Chl content values clearly. In addition, our results suggest that BoCLH2 and Chl degradation may not be directly related either, since even though BoCLH2 expression is enhanced during senescence; it is not modified by treatments assayed. Taken together, these results suggest that both BoCLH1 and BoCLH2 probably do not have a relevant role in Chl catabolism as it was suggested previously (Büchert et al., in press). In this sense, Chen et al. (2008) developed transgenic broccoli plants expressing antisense BoCLH1, which showed only 1–2 day delay
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