Article pubs.acs.org/JAFC
Manipulating Sensory and Phytochemical Profiles of Greenhouse Tomatoes Using Environmentally Relevant Doses of Ultraviolet Radiation Michael P. Dzakovich,*,† Mario G. Ferruzzi,‡ and Cary A. Mitchell† †
Department of Horticulture and Landscape Architecture, Purdue University, 625 Agriculture Mall Drive, West Lafayette, Indiana 47907-2010, United States ‡ Department of Food Science, Purdue University, 745 Agriculture Mall Drive, West Lafayette, Indiana 47907-2010, United States S Supporting Information *
ABSTRACT: Fruits harvested from off-season, greenhouse-grown tomato plants have a poor reputation compared to their inseason, garden-grown counterparts. Presently, there is a gap in knowledge with regard to the role of UV-B radiation (280−315 nm) in determining greenhouse tomato quality. Knowing that UV-B is a powerful elicitor of secondary metabolism and not transmitted through greenhouse glass and some greenhouse plastics, we tested the hypothesis that supplemental UV-B radiation in the greenhouse will impart quality attributes typically associated with garden-grown tomatoes. Environmentally relevant doses of supplemental UV-B radiation did not strongly affect antioxidant compounds of fruits, although the flavonol quercetin-3-Orutinoside (rutin) significantly increased in response to UV-B. Physicochemical metrics of fruit quality attributes and consumer sensory panels were used to determine if any such differences altered consumer perception of tomato quality. Supplemental UVA radiation (315−400 nm) pre-harvest treatments enhanced sensory perception of aroma, acidity, and overall approval, suggesting a compelling opportunity to environmentally enhance the flavor of greenhouse-grown tomatoes. The expression of the genes COP1 and HY5 were indicative of adaptation to UV radiation, which explains the lack of marked effects reported in these studies. To our knowledge, these studies represent the first reported use of environmentally relevant doses of UV radiation throughout the reproductive portion of the tomato plant life cycle to positively enhance the sensory and chemical properties of fruits. KEYWORDS: carotenoids, flavor, polyphenols, photobiology, Solanum lycopersicum, UV-B
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Similar trends have been reported for flavonoids, which are posited to reduce coronary artery disease,9 reduce intestinal inflammation,10 and improve the survival of nerve tissue in the brain.11 Additionally, concentrations of the predominant carotenoids found in tomatoes, lycopene and β-carotene, can be modified by UV-B exposure either in a postharvest setting12,13 or in greenhouses with special cladding materials that allow for UV-B transmission.8,14,15 Importantly, both flavonoids and carotenoids are derived from pathways that also produce volatile organic compounds (VOCs) important for tomato flavor.16,17 Altered flux through these pathways could in turn alter the VOCs produced by tomato fruits and affect sensory quality. Tomato flavor is determined by a complex interaction between sugars, acids, VOCs, appearance, texture, temperature, mouthfeel, and previous experiences.18,19 UV-B radiation has been reported to alter the expression of genes in the citric acid cycle,20,21 which provides substrate to the phenylpropanoid pathway as plants produce and accumulate photoprotectant flavonoids. The citric acid cycle also produces citric and malic acids, which contribute to the acidic component of tomato
INTRODUCTION Within the United States, about 40% of tomatoes (Solanum lycopersicum) are grown in greenhouses.1 However, off-season greenhouse tomatoes have a poor reputation relative to their inseason, field-grown counterparts, partially due to environmental and cultural differences.2 One noteworthy difference is the lack of ultraviolet-B (UV-B; 280−315 nm) and some ultraviolet-A (UV-A; 315−400 nm) in glass-glazed greenhouses as well as in polyhouses with UV-B-absorbing films.3 Although UV-B comprises only a small fraction of solar radiation that reaches the Earth’s surface (90% of fruit within a cluster were red; USDA Tomato Ripeness Classification) were harvested, and color measurements were done according to the method of Dzakovich et al.31 Fruits were blended under nitrogen gas and homogenates immediately stored at −80 °C. Samples from greenhouse and field experiments were an aggregate of 3 or 10 fruits, respectively, and all harvests took place within 3 weeks in mid to late August 2014 (field trial), mid to late October 2014 (expt 1, greenhouse), and early to mid April 2015 (expt 2, greenhouse). Brix, titratable acidity, and pH were determined according to the method of Dzakovich et al.31 Ascorbic acid concentration was determined according to the method of Nielsen.32 Fruit fresh weight, water content, and colorimetric data can be found in the Supporting Information. Carotenoid Extraction and Quantification. Samples were twice-extracted in near darkness (≤1 μmol m−2 s−1) using 10 mL of 2:3 (v/v) acetone/hexanes and analyzed with a spectrophotometer (Shimadzu UV160U; Shimadzu Corp., Kyoto, Japan).33 Values were converted into mg/g DW using a FW-to-DW conversion factor calculated from fresh and lyophilized tissue. Total Phenolics. Total phenolics were extracted according to the method of Luthria et al.24 Supernatants were pooled and analyzed using the Folin-Ciocalteu method according to Külen and others.34 The absorbance of samples at 765 nm was measured using a 96-well plate reader (SpectraMax 190 microplate reader; Molecular Devices, LLC, Sunnyvale, CA, USA), and gallic acid equivalents (GAE) were calculated on the basis of a five-point standard curve included on each plate. All samples were measured in triplicate.
MATERIALS AND METHODS
Plant Materials and Growing Conditions. In June 2014, 6week-old ‘Moneymaker’ (Baker Creek Heirloom Seed Co., Mansfield, MO, USA) tomato seedlings were transferred to 22.7 L pots containing Faffard 52 grow mix (Sun Gro Horticulture, Agawam, MA, USA) and were fertigated with an acidified fertilizer solution (pH 6.0; EC = 1.4 mS/cm) that provided (in mg L−1) 200 N-NO3, 26 P, 163 K, 50 Ca, 20 Mg, and micronutrients (The Scotts Co., Marysville, OH, USA). Plants were moved into a glass-glazed greenhouse in West Lafayette, IN, USA (latitude 40° N, longitude 86° W; USDA hardiness zone 5b) and trained to two heads per plant (planting density = 1.7 plants/m2). Fertigation was provided by pressure-compensating drippers connected to a computer-controlled irrigation system ensuring consistent plant water status among all greenhouse treatments (Priva, De Lier, The Netherlands). Greenhouse day/ night temperature set points were 25/15 °C. Plants were arranged in a randomized complete block design with three rows separated by 1.5m-tall polyethylene curtains running east−west (Supporting Information). Supplemental daily light integral (DLI) was provided by overhead high-pressure sodium (HPS) lamps and adjusted monthly on the basis of outdoor data collected by Korczynski and colleagues28 for different latitudes and adjusted for a 50% loss of incident solar DLI due to greenhouse glass and infrastructure with a target DLI of 25 mol m−2 day−1. Similar crop-culture conditions were used for expt 2, which began in mid-December 2014, with day/night temperatures of 25/23 °C. Plants in expt 2 were arranged in a completely randomized design, and the total DLI was approximately 12 mol m−2 day−1. Plants for outdoor field trials were cultured similarly, but grown to maturity at a nearby field site in a cambic-loam soil amended with organic compost during summer 2014. Heavy-duty weed cloth (FarmTek; Dyersville, B
DOI: 10.1021/acs.jafc.6b02983 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 1. Basic Physicochemical Attributes As Affected by Environment or Light Treatmenta treatment
sample size
Brixb
total sugarc (g/fruit)
pH
titratable acidity (g/L)
total acidsd (g/fruit)
sugar/acid
control UV-A UV-A+B
n=9 n=9 n=9
5.0 ± 0.05a*e 4.8 ± 0.05a* 5.1 ± 0.08a*
5.1 ± 0.15a* 4.45 ± 0.14a* 5.25 ± 0.13a
4.21 ± 0.02a 4.23 ± 0.02a* 4.18 ± 0.02a
5.28 ± 0.14a* 5.35 ± 0.11a* 5.97 ± 0.12a*
0.54 ± 0.02a 0.49 ± 0.02a* 0.62 ± 0.02a
9.54 ± 0.2a 9.04 ± 0.13a* 8.5 ± 0.13a*
control UV-A+B outdoor
n=3 n=3 n=3
4.1 ± 0.2a 4.6 ± 0.2a 4.2 ± 0.06
3.22 ± 0.23a* 3.58 ± 0.14a* 5.88 ± 0.08
4.17 ± 0.03a 4.17 ± 0.03a 4.2 ± 0
4.86 ± 0.13a 5.46 ± 0.21a* 4.44 ± 0.11
0.38 ± 0.01a* 0.42 ± 0.03a* 0.61 ± 0.01
8.36 ± 0.48a 8.43 ± 0.36a 9.47 ± 0.18
expt 1
expt 2
Values represent means ± standard error. bGrams sucrose/100 g sample. cTotal sugar represents Brix corrected for fruit fresh weight. dTotal acids represents titratable acidity corrected for fruit fresh weight. e*, significantly different at P ≤ 0.05 compared to outdoor controls within individual experiments. Values with the same letters within an experiment are statistically similar as determined by Tukey’s honestly significant difference (HSD) test (α = 0.05). a
Quantification of Fruit Tissue Flavonols by LC-MS. Lyophilized tomato fruit tissue was extracted according to the method of Muir and colleagues,35 with some modifications. Two hundred milligrams (±5 mg) of lyophilized tomato fruit was twice-extracted with 4 mL of 70% (v/v) methanol using a workflow similar to that for total phenolics. Pooled supernatants were dried using a nitrogen evaporator (N-EVAP 11250; Organomation Associates Inc., Berlin, MA, USA) with a water bath maintained at 37 °C. The residue was resuspended in 500 μL of 1:1 methanol/acidified water (2% glacial acetic acid), and the headspace was replaced with nitrogen gas and stored at −80 °C. Before analysis, samples were filtered through a 0.45-μm filter (Chromafil O-45/15 MS; Macherey-Nagel, Dü ren, Germany). Compounds were separated and analyzed according to the method of Neilson et al. with some modifications.36 Compound separation was achieved using a Waters 2695 separations module (Waters, Milford, MA, USA) with a Waters XBridge BEH Shield RP C18 column (2.1 × 100 mm, 2.5 μm particle size) at 40 °C. A flow rate of 0.25 mL/min with mobile phases of 0.4% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) was used. Separation lasted 24 min with the following gradient: 95 (A):5 (B) 0 min, 65 (A):35 (B) 15 min, 30 (A):70 (B) 17 min, 95 (A):5 (B) 19 min. The column was reequilibrated at 95 (A):5 (B) for an additional 5 min. The column effluent was split 1:1 into a Waters ZQ 2000 single-quadrupole mass spectrometer with an electrospray ionization (ESI) source operated in negative mode. Capillary and cone voltages were 3 kV and 40 V, respectively. Desolvation and cone-gas (N2 gas) flow rates were 400 and 60 L/h, respectively. Mass-to-charge ratios for analyte ions were 609 and 593 for quercetin-3-O-rutinoside and kaempferol-3-Orutinoside, respectively. Dwell time for each selected ion recording was 0.2 s with an interscan delay of 0.01 s. Consumer Sensory Panels. Consumer sensory panels were conducted for fruits harvested from the field and expt 1 plants according to the procedure of Dzakovich et al.31 with some modifications. Surveys included both 9-point objective (magnitude) and 9-point hedonic (preference) scales for the following attributes: color, aroma, texture/mouthfeel, acidity, sweetness, and aftertaste. Overall approval was scored using only the 9-point hedonic scale. Consumer panelists participated voluntarily and were presented samples of freshly diced tomatoes in random order. Water and plain crackers were provided as a palate cleanser between samples. All personnel involved in hosting the sensory panel were required to pass research ethics training for human subject research through the Collaborative Institution Training Initiative (CITI) Program, with Institutional Review Board approval. RNA Extraction, Purification, and cDNA Synthesis. Tomato fruit epidermis from fruit hemispheres facing UV-emitting lamps was peeled with a razor blade and stored in a −80 °C freezer. Fruit epidermis was submerged in liquid nitrogen and ground to a fine powder using a chilled mortar and pestle. RNA was extracted according to the method of Eggermont et al.37 RNA extracts were treated with DNase (DNase I; Zymo Research, Irvine, CA, USA) to remove genomic DNA. RNA was concentrated and purified (RNA
Clean & Concentrator; Zymo Research) according to the manufacturer’s directions. cDNA was generated using the Bio-Rad iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s directions and stored at −20 °C. Real-Time PCR. cDNA was diluted 20-fold, and quantitative realtime PCR was carried out according to the method of Nambeesan et al.38 using an Applied Biosystems StepOnePlus PCR system (Applied Biosystems, Foster City, CA, USA). Primer concentrations varied between 0.33 and 0.7 μM. Primers for COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC 1) were developed using Primer Express (v.3.0.1.; Applied Biosystems). Additionally, HY5 primers from Calvenzani et al.26 were optimized for our PCR system. The sequences were as follows: F 5′-ACGGGCTTGGAGTGTTGATT-3′ and R 5′CCTGCTTCGTGCACCAAACT-3′ for COP1 and F 5′-AAGCAAGGGTGAAGGAATTG-3′ and R 5′-ACAATCCACCCGAAACTAGC-3′ for HY5. As a reference gene, EF1 (tomato elongation factor 1α; F 5′-TGGCCCTACTGGTTTGACAACTG-3′ and R 5′-CACAGTTCACTTCCCCTTCTTCTG-3′) was used because of consistent expression in mature tomato fruits.39 For HY5, a melting temperature of 53 °C instead of 60 °C was used by enabling the VeriFlex Plus Block. Relative expression was calculated using the ΔΔCt method though StepOne software (v2.3; Applied Biosystems). Control fruits served as a calibrator for different light treatments. All measurements were done in triplicate. Statistical Analyses. For expt 1, data were analyzed as a randomized complete block design and subject to analysis of variance (ANOVA) and the general linear model procedure of SAS for pooling decisions (version 9.4; SAS Institute, Cary, NC, USA). Additionally, planned comparisons of means at α = 0.05 using Tukey’s honestly significant difference (HSD) test were used. Expt 2 was conducted as a completely randomized design and subjected to ANOVA and Tukey’s HSD test. Sensory panel data were arcsin transformed for analyses, and back-transformed data are presented. Greenhouse treatments were compared to outdoor controls using Fisher’s protected least significant difference (LSD; α = 0.05).
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RESULTS AND DISCUSSION Simple Physicochemical Characteristics. Brix, total sugar, pH, titratable acidity, and total acids were measured in all experiments (Table 1) to make inferences about how UV radiation might affect the perception of tomato flavor. Broadly speaking, none of these quality attributes was significantly changed by UV supplementation. Brix values in all three treatments of expt 1 were higher than those of outdoor-grown fruits. This may have been due to greenhouse irrigation intervals that elevated substrate EC, which has been shown to influence tomato fruit sugar, titratable acidity, and carotenoid contents.40-43 The fertigation solution was diluted by half in expt 2 to avoid this potentially confounding factor, and Brix values were comparatively lower as a result. Differences in soil C
DOI: 10.1021/acs.jafc.6b02983 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 2. Absolute and Hedonic Ratings of Select Consumer Sensory-Panel Tomato Fruit Attributes in Expt 1a treatment absolute control UV-A UV-A+B outdoor hedonic control UV-A UV-A+B outdoor
sample size
color
aroma
sweetness
acidity
aftertaste
texture
n n n n
= = = =
39 39 39 54
7.21 6.44 7.13 6.93
± ± ± ±
0.18a 0.24b 0.2ab 0.17
5.41 6.59 5.92 6.11
± ± ± ±
0.23b 0.21a 0.22b 0.22
4.51 4.85 4.87 4.48
± ± ± ±
0.24a 0.24a 0.23a 0.23
6.05 5.77 5.9 4.26
± ± ± ±
0.24a*c 0.24a* 0.2a* 0.2
5.31 5.59 5.51 5.19
± ± ± ±
0.21a 0.25a 0.24a 0.22
5.51 5.77 6.1 5.24
± ± ± ±
0.28a 0.24a 0.27a* 0.23
n n n n
= = = =
39 39 39 54
7.05 7.0 7.08 7.43
± ± ± ±
0.21a 0.24a 0.21a 0.17
5.82 7.05 6.08 6.87
± ± ± ±
0.21b* 0.23a 0.22b* 0.19
5.18 5.67 5.41 5.83
± ± ± ±
0.25a 0.23a 0.25a 0.26
5.59 6.31 5.82 6.2
± ± ± ±
0.25b 0.24a 0.25ab 0.25
5.56 6.02 5.56 6.74
± ± ± ±
0.23a* 0.24a 0.23a* 0.2
5.56 6.28 5.92 6.26
± ± ± ±
0.26a 0.26a 0.31a 0.3
overall approval N/Ab N/A N/A N/A 5.67 6.49 5.87 6.61
± ± ± ±
0.26b* 0.2a 0.24ab 0.22
a Values represent back-transformed means ± standard error of absolute (magnitude) and hedonic (preference) measures of select tomato fruit sensory attributes using 9-point scales. Increasing values indicate an increase in the magnitude or liking of an attribute. bNot applicable. c*, significantly different at P ≤ 0.05 compared to outdoor controls. Values with different letters are statistically different as determined by Tukey’s honestly significant difference (HSD) test (α = 0.05).
was unchanged by UV radiation in expt 1, but was increased in expt 2. This is likely due to the plants being grown at lower DLI while being completely surrounded by UV-B-emitting lamps. Similar trends were seen for fruit carotenoids, another major class of antioxidant phytochemicals in tomato fruits. Carotenoids. Although the role of UV-B in carotenogenesis is highly variable among species of plants,4 there is evidence that UV-B can modify carotenoid profiles in tomato fruits. Liu and colleagues12 reported an increase in the concentration of carotenoids in ‘Moneymaker’ tomato fruits in a postharvest setting with different doses of UV-B radiation. Similar findings were observed by Castagna et al.47 This effect likely was due to a down-regulation of lycopene β and ε-cyclases.48 The bluelight/UV-A receptor cryptochrome is also linked to carotenogenesis.49 Blue-light has increased carotenoids in lettuce (Lactuca sativa),46 kale (Brassica oleracea),50 and broccoli microgreens51 in growth-chamber studies. In our greenhouse UV-reconstitution studies, carotenoid content remained largely unchanged by either UV-A+B or UV-A alone (Table 4). However, a nonsignificant trend for increased carotenoid content with UV treatment was seen in expt 2. The promising results seen in postharvest studies may be somewhat artificial, because those postharvest fruits received UV-B radiation but not photosynthetically active radiation (PAR). In species such as soybean (Glycine max), UV-B responses are dependent on background levels of PAR,52 likely through competition between or among light-sensing proteins.6,53 The effects of our treatments also may have been diminished by supplemental HPS as well as solar PAR. To better understand how our tomato fruits perceived the radiation treatments at the molecular level, we used qPCR to quantify the expression of genes in the UV-B light-signal transduction cascade. UV-B Perception in Higher Plants. Although many proteins and transcription factors are involved in UV-B perception, our study focused on the expression of COP1 and HY5 and found similar trends in expression for both treatments in expt 1 compared to control fruits (Figure 1). Both treatments tended to lower the relative expression of HY5 and COP1 compared to controls, but only COP1 was significantly lower. We selected COP1 and HY5 because they are crucial in UV-B perception via the dimeric protein UVR8.6 After UV-B-induced monomerization, UVR8 monomers are transported into the nucleus, where the UVR8−COP1 complex interacts with the bZIP transcription factor HY5.5 However, limited data exist regarding the expression of these genes in
composition and EC were likely drivers of differences between greenhouse and field-grown fruits in our studies. Past studies have determined that UV-B radiation can affect the expression of genes in the Krebs cycle and divert organic acid precursors into the phenylpropanoid pathway.20,21 The doses of UV radiation used in our studies had little effect on tomato fruit titratable acidity in all experiments. Although these metrics were only subtly modified, consumer sensory panelists detected differences in aroma, acidity, and overall quality. Tomato Flavor. Human perception of tomato quality is due to not only the interaction of sugars, acids, and VOCs but also a complex phenomenon that also involves appearance, texture, temperature, mouthfeel, and past experiences.18,19 UV-Asupplemented fruits had the highest hedonic (preference) ratings for aroma, acidity, and overall approval compared to control or UV-A+B-supplemented fruits (Table 2). UV-A+Bsupplemented fruits were similar in overall approval rating compared to UV-A-supplemented fruits, but not different from controls. Because UV-A and UV-B are perceived by distinct proteins (cryptochrome and UVR8, respectively), perhaps the UV-B portion of the UV-A+B treatment inhibited the production of certain VOCs that generated high aroma and overall liking scores in the UV-A treatment. VOCs are crucial for shaping the flavor and aroma profiles of tomato fruits,44 and subtle changes in their concentration can greatly affect human perception of these compounds.27 Additionally, VOC concentrations are highly dependent on environmental conditions,45 which likely contributed to many of the sensory differences detected between greenhouse-grown and field-grown fruits. Water-Soluble Antioxidants. UV-A and UV-B both have been shown to affect the concentration of many water-soluble antioxidant compounds,26,46 and this effect is often governed by complex genetic × environment interactions.8,13 Postharvest treatment stimulated the production of ascorbic acid in ‘Moneymaker’ tomato13 and increased total phenolics and flavonoids in other varieties.12,47 Flavonoids in both flesh and peel tissues of ‘Esperanza’ and ‘DRW 5981’ fruits were highly modified by UV-B radiation, and expression of genes encoding flavonoid biosynthetic enzymes measured varied between cultivars.25 In our studies, UV radiation did not greatly modify most of the water-soluble antioxidant compounds that were measured (Table 3). However, rutin concentration was statistically higher in the UV-A+B treatments in both expts 1 and 2. In expt 2, this trend was conserved on a fruit-weight basis, confirming an increase in rutin biosynthesis. Ascorbic acid D
DOI: 10.1021/acs.jafc.6b02983 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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26.72 ± 1.8a* 29.97 ± 3.96a* 43.11 ± 0.35
59.37 ± 4.08b* 119.2 ± 6.21a 71.79 ± 3.94
84.4 ± 3.99b 95.17 ± 2.65ab* 113.93 ± 1.7a*
5.305 ± 0.74a 7.39 ± 0.24a* 5.05 ± 0.54
36.63 ± 0.4a 35.02 ± 0.3a 34.31 ± 0.23a
12.03 ± 1.13a* 15.7 ± 1.3a* 19.16 ± 0.66
4.63 ± 0.42a 5.63 ± 0.4a 6.07 ± 0.42a 18.15 ± 1.34a 16.92 ± 0.57a 16.64 ± 1.23a expt 2
control UV-A+B outdoor
n=3 n=3 n=3
2.34 ± 0.09b 3.95 ± 0.48a* 2.23 ± 0.01
2.67 ± 0.14a*e 2.92 ± 0.04a* 2.56 ± 0.17a* n=9 n=9 n=9 control UV-A UV-A+B expt 1
tomato fruits. Calvenzani and colleagues26 quantified genes including COP1 and HY5 in ‘Moneymaker’ tomato fruits grown in high tunnels that either blocked or transmitted UV-B radiation. COP1 expression increased in epidermal peels of redripe ‘Moneymaker’ fruits exposed to UV-B, whereas HY5 remained similar to control (the expression in mature-green fruit flesh). Studies using Arabidopsis in growth chambers have shown increases in both COP1 and HY5 in response to UV-B radiation.54-56 However, Morales et al.53 found that, although not significant, HY5 expression was lowest in outdoor-grown wild-type Arabidopsis exposed to UV-A+B compared to Arabidopsis exposed to UV-A or no UV for 3 weeks. Whereas HY5 expression was significant between UV treatments after 12 h, the difference was no longer significant after 36 h, indicating adaptation to UV-B. Similar trends in expression changing over time as a response to UV-B have been shown for COP155and HY5.57 Given these trends in the literature, it was surprising that COP1 expression decreased in both UV treatments in expt 1. Clearly, the expression of HY5, and likely other genes in light-signaling pathways, is highly sensitive to the environment and adapt its expression to meet the needs of the plant. Differences between controls and treatments indicated that plants adapted to supplemental UV treatments (Figure 1). Other factors, such as abundant solar and supplemental light, also contributed to UV treatment responses. COP1 is a critical mediator for both blue- and red-light signaling.58,59 It has been proposed that prolonged exposure to UV-B can limit the availability of COP1 to phytochrome and cryptochrome and that cryptochromes may outcompete UVR8 for COP1 under high-intensity solar light.6,53 Our studies were conducted in greenhouses with modest amounts of supplemental PAR in addition to background solar PAR, which may have minimized any major effects from our UV treatments due to competing light-signaling pathways. Similar findings have been noted for studies measuring the growth and development of soybean and bean plants (Phaseolus vulgaris) during highand low-light times of year.52,60 A more comprehensive analysis of genes in the light signal transduction pathways, and possibly of genes affected downstream, is needed to better understand how greenhouse-grown plants respond to supplemented UV radiation over their lifespan. Outside on a sunny summer day, it is common to measure PAR photon fluxes at or above 1600 μmol m−2 s−1 in addition to 1.5 μmol m−2 s−1 of UV-B radiation. Many fundamental studies that have shaped our understanding of UV-B signal transduction have used grossly different environmental settings than what a plant would be exposed to in nature. For example, part of a study by Brown and Jenkins5 included growth of Arabidopsis plants with 20 μmol m−2 s−1 of PAR from fluorescent lamps and 3 μmol m−2 s−1 of UV-B. Spectral quality aside, the instantaneous UV-B/PAR ratio was 160 times higher than what might be seen outdoors during the summer, which likely exaggerated plant responses to UV-B. Other studies that used unnatural PAR/UV-B ratios are generally those investigating photomorphogenic and molecular control mechanisms in seedlings where UV-B/PAR ratios have been up to 445 times higher than in outdoor conditions.6,7,54,55,57 Although these studies are valid within their context, they may oversimplify plant responses to UV-B and poorly represent how seedlings or mature plants would respond to UV-B in a greenhouse environment under high-intensity, broad-spectrum solar and/or supplemental light where all plant photoreceptors are operating concurrently. This may explain why environ-
a Values represent means ± standard error. bDW, dry weight. c“Per fruit” represents specific content corrected for fruit fresh weight. dGAE, gallic acid equivalents. e*, significantly different at P ≤ 0.05 compared to outdoor controls within individual experiments. Values with different letters within an experiment are statistically different as determined by Tukey’s honestly significant difference (HSD) test (α = 0.05).
78.62 ± 11.87a* 71.63 ± 10.88a* 12.83 ± 0.83 15.282 ± 1.98a* 17.74 ± 2.26a* 1.49 ± 0.06
0.58 ± 0.04b 0.55 ± 0.03b 0.79 ± 0.02a*
rutin per fruit (mg/fruit) rutin concentration (μg/g DW) phenolics per fruitc (mg GAE/fruit) phenolics (mg GAEd/g DW) ascorbic acid per fruitc (mg/fruit) ascorbic acid (mg/g DWb) sample size treatment
Table 3. Select Tomato-Fruit Phytochemicals As Affected by Environment or Light Treatmenta
0.3 ± 0.03b* 0.48 ± 0.07a 0.62 ± 0.03
8.77 ± 0.79a* 9.07 ± 1.09a* 8.57 ± 0.53a*
kaempferol-3-O-rutinoside concentration (μg/g DW)
59.71 ± 5.88a* 51.93 ± 5.99a* 59.04 ± 3.86a*
kaempferol-3-O-rutinoside per fruit (μg/fruit)
Journal of Agricultural and Food Chemistry
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DOI: 10.1021/acs.jafc.6b02983 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 4. Primary Tomato Fruit Carotenoids As Affected by Environment or Light Treatmenta treatment
sample size
lycopene concentration (mg/g DWb)
lycopene per fruitc (mg/ fruit)
β-carotene concentration (mg/g DW)
β-carotene per fruit (mg/fruit)
control UV-A UV-A+B
n=9 n=9 n=9
0.68 ± 0.03a*d 0.73 ± 0.05a 0.75 ± 0.03a
4.56 ± 0.26a* 4.29 ± 0.36ad 5.17 ± 0.27a*
0.22 ± 0.01a 0.24 ± 0.02a 0.25 ± 0.02a
1.51 ± 0.09a* 1.42 ± 0.11ad 1.7 ± 0.15a
control UV-A+B outdoor
n=3 n=3 n=3
0.56 ± 0.07a* 0.91 ± 0.15a 0.82 ± 0.04
2.84 ± 0.19a* 3.56 ± 0.27a* 7.0 ± 0.24
0.18 ± 0.02a 0.25 ± 0.06a 0.24 ± 0.06
0.92 ± 0.02a* 0.98 ± 0.15a* 2.05 ± 0.04
expt 1
expt 2
Values represent means ± standard error. bDW, dry weight. c“Per fruit” represents concentration corrected for fruit fresh weight. d*, significantly different at P ≤ 0.05 compared to outdoor controls within individual experiments. Values with the same letters within an experiment are statistically similar as determined by Tukey’s honestly significant difference (HSD) test (α = 0.05). a
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b02983. Photograph of one row from greenhouse expt 1 (S1), spectral scan of UV region of field and greenhouse environments as well as the two supplemental light treatments (S2), and fruit fresh mass, water content, and epidermal colorimetric attributes of tomato fruits from different treatments (S3) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*(M.P.D.) E-mail:
[email protected]. Funding
Funding was from the USDA NIFA-SCRI program (201051181-21369). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Judy Santini for statistical consulting, as well as Sydney Moser and Ben Redan for guidance with LC-MS. We also thank Rob Eddy, Dan Hahn, Dan Martin, and Eric Whitehead for help with greenhouse studies and Steve Hallett, Dan Martin, and Rachel Beyer for field studies. Finally, we thank Tanya Datsenko, Raheel Anwar, Norman Best, Matt Rudisill, and Roger Rozzi for assistance with qPCR.
Figure 1. Means and standard errors of relative expression of HY5 (a) and COP1 (b) in tomato fruit peel from expt 1 of three independent samples (n = 3) per treatment. Red-ripe control fruit peels were used as a calibrator and scaled to 1. EF1 was used as an endogenous control. Similar letters indicate non-significance by Tukey’s HSD test (α = 0.05). All samples were measured in triplicate, and repetitions of qPCR experiments yielded similar results.
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ABBREVIATIONS USED COP1, CONSTITUTIVE PHOTOMORPHOGENIC 1; DLI, daily light integral; DW, dry weight; EC, electrical conductivity; ESI, electrospray ionization; FW, fresh weight; GAE, gallic acid equivalents; expt, experiment; HPS, high-pressure sodium; HY5, ELONGATED HYPOCOTYL 5; LC-ESI(−)-MS, liquid chromatography electrospray ionization mass spectrometry in negative mode; PAR, photosynthetically active radiation; qPCR, real-time polymerase chain reaction; UV, ultraviolet radiation; UV-A, ultraviolet A; UV-B, ultraviolet B; UVR8, ULTRAVIOLET RESISTANCE LOCUS 8; VOC, volatile organic compound
mentally relevant doses of supplemental UV-B were unable to acutely modify secondary metabolic processes in our studies. Our results show that neither supplemental UV-A nor supplemental UV-A+B strongly affected the biosynthesis of potentially beneficial phytochemicals including carotenoids and phenolic compounds. However, our data do highlight the possibilities of leveraging UV responses as a novel method to alter the flavor of greenhouse-grown tomatoes. Still, many questions remain as to the exact mechanism of these effects. To our awareness, these studies are the only present work detailing the effects of using environmentally relevant doses of UV radiation throughout the reproductive stage of the tomato plant lifecycle to enhance fruit quality. More research is necessary to better understand plant−UV interactions in general, but especially in the context of production settings such as greenhouse environments.
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REFERENCES
(1) USDA-FAS. Fresh & processing tomatoes: world markets and trade and global agriculture trade system (GATS); http://www.fas. usda.gov/data.asp (accessed Nov 1, 2014).
F
DOI: 10.1021/acs.jafc.6b02983 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry (2) Muñoz, P.; Antón, A.; Nuñez, M.; Paranjpe, A.; Ariño, J.; Castells, X.; Montero, J. I. Comparing the environmental impacts of greenhouse versus open-field tomato production in the Mediterranean region. Acta Hortic. 2008, 801, 1591−1596. (3) Aphalo, P. J.; Albert, A.; Mcleod, A. R.; Robson, T. M.; Rosenqvist, E. Beyond the visible; COST Action FA0906 UV4growth, 2012. (4) Bassman, J. H. Ecosystem consequences of enhanced solar ultraviolet radiation: secondary plant metabolites as mediators of multiple trophic interactions in terrestrial plant communities. Photochem. Photobiol. 2004, 79 (5), 382−398. (5) Brown, B. A.; Jenkins, G. I. UV-B signaling pathways with different fluence-rate response profiles are distinguished in mature Arabidopsis leaf tissue by requirement for UVR8, HY5, and HYH. Plant Physiol. 2008, 146 (2), 576−588. (6) Favory, J.-J.; Stec, A.; Gruber, H.; Rizzini, L.; Oravecz, A.; Funk, M.; Albert, A.; Cloix, C.; Jenkins, G. I.; Oakeley, E. J.; et al. Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis. EMBO J. 2009, 28 (5), 591−601. (7) Rizzini, L.; Favory, J.-J.; Cloix, C.; Faggionato, D.; O’Hara, A.; Kaiserli, E.; Baumeister, R.; Schäfer, E.; Nagy, F.; Jenkins, G. I.; et al. Perception of UV-B by the Arabidopsis UVR8 protein. Science 2011, 332 (6025), 103−106. (8) Giuntini, D.; Graziani, G.; Lercari, B.; Fogliano, V.; Soldatini, G. F.; Ranieri, A. Changes in carotenoid and ascorbic acid contents in fruits of different tomato genotypes related to the depletion of UV-B radiation. J. Agric. Food Chem. 2005, 53 (8), 3174−3181. (9) Naderi, G. A.; Asgary, S.; Sarraf-Zadegan, N.; Shirvany, H. Antioxidant effect of flavonoids on the susceptibility of LDL oxidation. Mol. Cell. Biochem. 2003, 246 (1-2), 193−196. (10) González, R.; Ballester, I.; López-Posadas, R.; Suárez, M. D.; Zarzuelo, A.; Martínez-Augustin, O.; Medina, F. S. De. Effects of flavonoids and other polyphenols on inflammation. Crit. Rev. Food Sci. Nutr. 2011, 51 (4), 331−362. (11) Spencer, J. P. E. Flavonoids and brain health: multiple effects underpinned by common mechanisms. Genes Nutr. 2009, 4 (4), 243− 250. (12) Liu, C.; Han, X.; Cai, L.; Lu, X.; Ying, T.; Jiang, Z. Postharvest UV-B irradiation maintains sensory qualities and enhances antioxidant capacity in tomato fruit during storage. Postharvest Biol. Technol. 2011, 59 (3), 232−237. (13) Castagna, A.; Chiavaro, E.; Dall’Asta, C.; Rinaldi, M.; Galaverna, G.; Ranieri, A. Effect of postharvest UV-B irradiation on nutraceutical quality and physical properties of tomato fruits. Food Chem. 2013, 137 (1-4), 151−158. (14) Becatti, E.; Petroni, K.; Giuntini, D.; Castagna, A.; Calvenzani, V.; Serra, G.; Mensuali-Sodi, A.; Tonelli, C.; Ranieri, A. Solar UV-B radiation influences carotenoid accumulation of tomato fruit through both ethylene-dependent and -independent mechanisms. J. Agric. Food Chem. 2009, 57 (22), 10979−10989. (15) Lazzeri, V.; Calvenzani, V.; Petroni, K.; Tonelli, C.; Castagna, A.; Ranieri, A. Carotenoid profiling and biosynthetic gene expression in flesh and peel of wild-type and hp-1 tomato fruit under UV-B depletion. J. Agric. Food Chem. 2012, 60 (19), 4960−4969. (16) Simkin, A. J.; Schwartz, S. H.; Auldridge, M.; Taylor, M. G.; Klee, H. J. The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the flavor volatiles β-ionone, pseudoionone, and geranylacetone. Plant J. 2004, 40 (6), 882−892. (17) Tieman, D.; Taylor, M.; Schauer, N.; Fernie, A. R.; Hanson, A. D.; Klee, H. J. Tomato aromatic amino acid decarboxylases participate in synthesis of the flavor volatiles 2-phenylethanol and 2-phenylacetaldehyde. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (21), 8287− 8292. (18) Baldwin, E. A.; Goodner, K.; Plotto, A. Interaction of volatiles, sugars, and acids on perception of tomato aroma and flavor descriptors. J. Food Sci. 2008, 73 (6), S294−S307. (19) Goff, S. A.; Klee, H. J. Plant volatile compounds: sensory cues for health and nutritional value? Science 2006, 311 (5762), 815−819.
(20) Kusano, M.; Tohge, T.; Fukushima, A.; Kobayashi, M.; Hayashi, N.; Otsuki, H.; Kondou, Y.; Goto, H.; Kawashima, M.; Matsuda, F.; et al. Metabolomics reveals comprehensive reprogramming involving two independent metabolic responses of Arabidopsis to UV-B light. Plant J. 2011, 67 (2), 354−369. (21) Cavalcanti, J. H. F.; Esteves-Ferreira, A. A.; Quinhones, C. G. S.; Pereira-Lima, I. A.; Nunes-Nesi, A.; Fernie, A. R.; Araújo, W. L. Evolution and functional implications of the tricarboxylic acid cycle as revealed by phylogenetic analysis. Genome Biol. Evol. 2014, 6 (10), 2830−2848. (22) Lewinsohn, E.; Sitrit, Y.; Bar, E.; Azulay, Y.; Meir, A.; Zamir, D.; Tadmor, Y. Carotenoid pigmentation affects the volatile composition of tomato and watermelon fruits, as revealed by comparative genetic analyses. J. Agric. Food Chem. 2005, 53 (8), 3142−3148. (23) Baldwin, E.; Nisperos-Carriedo, M. O.; Baker, R.; Scott, J. W. Qualitative analysis of flavor parameters in six Florida tomato cultivars (Lycopersicon esculentum Mill). J. Agric. Food Chem. 1991, 39, 1135− 1140. (24) Luthria, D. L.; Mukhopadhyay, S.; Krizek, D. T. Content of total phenolics and phenolic acids in tomato (Lycopersicon esculentum Mill.) fruits as influenced by cultivar and solar UV radiation. J. Food Compos. Anal. 2006, 19 (8), 771−777. (25) Giuntini, D.; Lazzeri, V.; Calvenzani, V.; Dall’Asta, C.; Galaverna, G.; Tonelli, C.; Petroni, K.; Ranieri, A. Flavonoid profiling and biosynthetic gene expression in flesh and peel of two tomato genotypes grown under UV-B-depleted conditions during ripening. J. Agric. Food Chem. 2008, 56 (14), 5905−5915. (26) Calvenzani, V.; Martinelli, M.; Lazzeri, V.; Giuntini, D.; Dall’Asta, C.; Galaverna, G.; Tonelli, C.; Ranieri, A.; Petroni, K. Response of wild-type and high pigment-1 tomato fruit to UV-B depletion: flavonoid profiling and gene expression. Planta 2010, 231 (3), 755−765. (27) Tieman, D.; Bliss, P.; McIntyre, L. M. M.; Blandon-Ubeda, A.; Bies, D.; Odabasi, A. Z. Z.; Rodríguez, G. R. R.; Van Der Knaap, E.; Taylor, M. G. G.; Goulet, C.; et al. The chemical interactions underlying tomato flavor preferences. Curr. Biol. 2012, 22 (11), 1035− 1039. (28) Korczynski, P. C.; Logan, J.; Faust, J. E. Mapping monthly distribution of daily light integrals across the contiguous United States. Hortechnology 2002, 12 (1), 12−16. (29) Adamse, P.; Britz, S. J. Spectral quality of two fluorescent UV sources during long-term use. Photochem. Photobiol. 1992, 56 (5), 641−644. (30) Flint, S. D.; Caldwell, M. M. A biological spectral weighting function for ozone depletion research with higher plants. Physiol. Plant. 2003, 117 (1), 137−144. (31) Dzakovich, M. P.; Gómez, C.; Mitchell, C. A.; Gomez, C.; Mitchell, C. A. Tomatoes grown with light-emitting diodes or highpressure sodium supplemental lights have similar fruit-quality attributes. HortScience 2015, 50 (10), 1498−1502. (32) Nielsen, S. Food Analysis Laboratory Manual, Second; Springer: New York, 2010. (33) Nagata, M.; Yamashita, I. Simple method for simultaneous determination of chlorophyll and carotenoids in tomato fruit. Nippon Shokuhin Kogyo Gakkaishi 1992, 39 (102), 925−928. (34) Külen, O.; Stushnoff, C.; Holm, D. G.; Külen, O.; Stushnoff, C.; Holm, D. G. Effect of cold storage on total phenolics content, antioxidant activity and vitamin C level of selected potato clones. J. Sci. Food Agric. 2013, 93 (10), 2437−2444. (35) Muir, S. R.; Collins, G. J.; Robinson, S.; Hughes, S.; Bovy, A.; Ric De Vos, C. H.; van Tunen, A. J.; Verhoeyen, M. E. Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols. Nat. Biotechnol. 2001, 19 (5), 470−474. (36) Neilson, A. P.; Song, B. J.; Sapper, T. N.; Bomser, J. A.; Ferruzzi, M. G. Tea catechin auto-oxidation dimers are accumulated and retained by Caco-2 human intestinal cells. Nutr. Res. (N. Y., NY, U. S.) 2010, 30 (5), 327−340. (37) Eggermont, K.; Goderis, I. J.; Broekaert, W. F. High-throughput RNA extraction from plant samples based on homogenisation by G
DOI: 10.1021/acs.jafc.6b02983 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry reciprocal shaking in the presence of a mixture of sand and glass beads. Plant Mol. Biol. Rep. 1996, 14 (3), 273−279. (38) Nambeesan, S.; Datsenka, T.; Ferruzzi, M. G.; Malladi, A.; Mattoo, A. K.; Handa, A. K. Overexpression of yeast spermidine synthase impacts ripening, senescence and decay symptoms in tomato. Plant J. 2010, 63 (5), 836−847. (39) Bartley, G. E.; Ishida, B. K.; Giovannoni, J.; Grierson, D.; Fray, R.; Grierson, D.; Barry, C.; Llop-Tous, M.; Grierson, D.; Theologis, A.; et al. Developmental gene regulation during tomato fruit ripening and in-vitro sepal morphogenesis. BMC Plant Biol. 2003, 3 (1), 4. (40) Mitchell, J.; Shennan, C.; Grattan, S.; May, D.; Jones, R. A.; Lorenz, O. Tomato fruit yields and quality under water deficit and salinity. J. Am. Soc. Hortic. Sci. 1991, 116 (2), 215−221. (41) Wu, M.; Kubota, C. Effects of high electrical conductivity of nutrient solution and its application timing on lycopene, chlorophyll and sugar concentrations of hydroponic tomatoes during ripening. Sci. Hortic. (Amsterdam, Neth.) 2008, 116 (2), 122−129. (42) Yin, Y.-G.; Kobayashi, Y.; Sanuki, A.; Kondo, S.; Fukuda, N.; Ezura, H.; Sugaya, S.; Matsukura, C. Salinity induces carbohydrate accumulation and sugar-regulated starch biosynthetic genes in tomato (Solanum lycopersicum L. cv. ‘Micro-Tom’) fruits in an ABA- and osmotic stress-independent manner. J. Exp. Bot. 2010, 61 (2), 563− 574. (43) Kubota, C.; Kroggel, M.; Torabi, M.; Dietrich, K. A.; Kim, H.-J.; Fonseca, J.; Thomson, C. A. Changes in selected quality attributes of greenhouse tomato fruit as affected by pre- and postharvest environmental conditions in year-round production. HortScience 2012, 47 (12), 1698−1704. (44) Tieman, D. M.; Zeigler, M.; Schmelz, E. A.; Taylor, M. G.; Bliss, P.; Kirst, M.; Klee, H. J. Identification of loci affecting flavour volatile emissions in tomato fruits. J. Exp. Bot. 2006, 57 (4), 887−896. (45) Dalal, K. B.; Salunkhe, D. K.; Olson, L. E.; Do, J. Y.; Yu, M. H. Volatile components of developing tomato fruit grown under field and greenhouse conditions. Plant Cell Physiol. 1968, 9, 389−400. (46) Li, Q.; Kubota, C. Effects of supplemental light quality on growth and phytochemicals of baby leaf lettuce. Environ. Exp. Bot. 2009, 67 (1), 59−64. (47) Castagna, A.; Dall’Asta, C.; Chiavaro, E.; Galaverna, G.; Ranieri, A. Effect of post-harvest UV-B irradiation on polyphenol profile and antioxidant activity in flesh and peel of tomato fruits. Food Bioprocess Technol. 2014, 7 (8), 2241−2250. (48) Lazzeri, V.; Calvenzani, V.; Petroni, K.; Tonelli, C.; Castagna, A.; Ranieri, A. Carotenoid profiling and biosynthetic gene expression in flesh and peel of wild-type and hp-1 tomato fruit under UV-B depletion. J. Agric. Food Chem. 2012, 60 (19), 4960−4969. (49) Giliberto, L.; Perrotta, G.; Pallara, P.; Weller, J. L.; Fraser, P. D.; Bramley, P. M.; Fiore, A.; Tavazza, M.; Giuliano, G. Manipulation of the blue light photoreceptor cryptochrome 2 in tomato affects vegetative development, flowering time, and fruit antioxidant content. Plant Physiol. 2005, 137 (1), 199−208. (50) Lefsrud, M. G.; Kopsell, D. A.; Sams, C. E. Irradiance from distinct wavelength light-emitting diodes affect secondary metabolites in kale. HortScience 2008, 43 (7), 2243−2244. (51) Kopsell, D. A.; Sams, C. E. Increases in shoot tissue pigments, glucosinolates, and mineral elements in sprouting broccoli after exposure to short-duration blue light from light emitting diodes. J. Am. Soc. Hortic. Sci. 2013, 138 (1), 31−37. (52) Mirecki, R. M.; Teramura, A. H. Effects of ultraviolet-B irradiance on soybean: V. The dependence of plant sensitivity on the photosynthetic photon flux density during and after leaf expansion. Plant Physiol. 1984, 74 (3), 475−480. (53) Morales, L. O.; Brosché, M.; Vainonen, J.; Jenkins, G. I.; Wargent, J. J.; Sipari, N.; Strid, Å.; Lindfors, A. V.; Tegelberg, R.; Aphalo, P. J. Multiple roles for UV RESISTANCE LOCUS8 in regulating gene expression and metabolite accumulation in Arabidopsis under solar ultraviolet radiation. Plant Physiol. 2013, 161 (2), 744− 759. (54) Ulm, R.; Baumann, A.; Oravecz, A.; Máté, Z.; Adám, E.; Oakeley, E. J.; Schäfer, E.; Nagy, F. Genome-wide analysis of gene
expression reveals function of the bZIP transcription factor HY5 in the UV-B response of Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (5), 1397−1402. (55) Huang, X.; Ouyang, X.; Yang, P.; Lau, O. S.; Li, G.; Li, J.; Chen, H.; Deng, X. W. Arabidopsis FHY3 and HY5 positively mediate induction of COP1 transcription in response to photomorphogenic UV-B light. Plant Cell 2012, 24 (11), 4590−4606. (56) Huang, X.; Yang, P.; Ouyang, X.; Chen, L.; Deng, X. X. W. Photoactivated UVR8-COP1 module determines photomorphogenic UV-B signaling output in Arabidopsis. PLoS Genet. 2014, 10 (3), e1004218. (57) Oravecz, A.; Baumann, A.; Máté, Z.; Brzezinska, A.; Molinier, J.; Oakeley, E. J.; Adám , E.; Schäf er, E.; Nagy, F.; Ulm, R. CONSTITUTIVELY PHOTOMORPHOGENIC1 is required for the UV-B response in Arabidopsis. Plant Cell 2006, 18 (8), 1975−1990. (58) Holm, M.; Ma, L.-G.; Qu, L.-J.; Deng, X.-W. Two interacting bZIP proteins are direct targets of COP1-mediated control of lightdependent gene expression in Arabidopsis. Genes Dev. 2002, 16 (10), 1247−1259. (59) Sellaro, R.; Hoecker, U.; Yanovsky, M.; Chory, J.; Casal, J. J. Synergism of red and blue light in the control of Arabidopsis gene expression and development. Curr. Biol. 2009, 19 (14), 1216−1220. (60) Cen, Y.-P.; Bornman, J. F. The response of bean plants to UV-B radiation under different irradiances of background visible light. J. Exp. Bot. 1990, 41 (11), 1489−1495.
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DOI: 10.1021/acs.jafc.6b02983 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
