RESEARCH ARTICLE
Effects of low light on photosynthetic properties, antioxidant enzyme activity, and anthocyanin accumulation in purple pak-choi (Brassica campestris ssp. Chinensis Makino) Hongfang Zhu1,2, Xiaofeng Li2, Wen Zhai2, Yang Liu1, Qianqian Gao2, Jinping Liu3, Li Ren1, Huoying Chen1*, Yuying Zhu2*
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1 School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai, China, 2 Shanghai Key Lab of Protected Horticultural Technology, Horticultural Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, China, 3 College of Horticulture, Nanjing Agricultural University, Nanjing, China *
[email protected] (HC);
[email protected] (YZ)
Abstract OPEN ACCESS Citation: Zhu H, Li X, Zhai W, Liu Y, Gao Q, Liu J, et al. (2017) Effects of low light on photosynthetic properties, antioxidant enzyme activity, and anthocyanin accumulation in purple pak-choi (Brassica campestris ssp. Chinensis Makino). PLoS ONE 12(6): e0179305. https://doi.org/ 10.1371/journal.pone.0179305 Editor: Zhihui Cheng, Northwest Agriculture and Forestry University, CHINA Received: October 27, 2016 Accepted: May 26, 2017 Published: June 13, 2017 Copyright: © 2017 Zhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Anthocyanins are secondary metabolites that contribute to red, blue, and purple colors in plants and are affected by light, but the effects of low light on the physiological responses of purple pak-choi plant leaves are still unclear. In this study, purple pak-choi seedlings were exposed to low light by shading with white gauze and black shading in a phytotron. The responses in terms of photosynthetic properties, carbohydrate metabolism, antioxidant enzyme activity, anthocyanin biosynthetic enzyme activity, and the relative chlorophyll and anthocyanin content of leaves were measured. The results showed that chlorophyll b, intracellular CO2 content, stomatal conductance and antioxidant activities of guaiacol peroxidase, catalase and superoxide dismutase transiently increased in the shade treatments at 5 d. The malondialdehyde content also increased under low light stress, which damages plant cells. With the extension of shading time (at 15 d), the relative chlorophyll a, anthocyanin and soluble protein contents, net photosynthetic rate, transpiration rate, stomata conductance, antioxidant enzyme activities, and activities of four anthocyanin biosynthetic enzymes decreased significantly. Thus, at the early stage of low light treatment, the chlorophyll b content increased to improve photosynthesis. When the low light treatment was extended, antioxidant enzyme activity and the activity of anthocyanin biosynthesis enzymes were inhibited, causing the purple pak-choi seedlings to fade from purple to green. This study provides valuable information for further deciphering genetic mechanisms and improving agronomic traits in purple pak-choi under optimal light requirements.
Data Availability Statement: All relevant data are within the paper and its Supporting Information file. Funding: This work was supported by the Shanghai green vegetable industry system, and SAAS Program for Excellent Research Team (SPERT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Introduction Light is one of the most important environmental factors and plays a critical function in plant development and metabolism [1,2]. Additionally, light is indispensable for photosynthesis and photomorphogenesis. Low light is a pervasive abiotic stress in plant breeding and cultivation
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Competing interests: The authors have declared that no competing interests exist.
due to light block from horticulture facilities, clouds and snow. Low light was shown to substantially affect the agronomic traits of plants and inhibit physiological metabolic processes, including photosynthesis and antioxidant characteristics, as well as carbon and nitrogen fixation [3–6]. It causes slow growth, decrease of leaf weight and flower bud number. Furthermore, this stressor reduces sugar and starch contents in eggplant, grape and rice [7–9], and changes the coloration and extends the maturity time in cherry [10]. Chlorophyll is an important pigment involved in absorbing, transmitting and converting solar energy into electrochemical energy [11]. It was reported [12] that low light-tolerant hybrid rice -exhibited a higher content of chlorophyll b following exposure to low light. Low light negatively affects stomata conductance and results in enhanced concentration of intercellular CO2 in rice leaves [13,14]. Moreover, stomata conductance and photosynthetic efficiency under low light decreases by the number of 24.31% and 79.84%, respectively compared to that of natural light [15]. Antioxidant metabolism plays an important role in protecting plants from a wide variety of environmental stresses, such as drought, extreme temperatures, pollutants, ultraviolet radiation and high levels of light [16,17]. Enhancement of antioxidant defense in plants can thus increase tolerance to different stresses. Antioxidants include the enzymes peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) [18]. Analyses of membrane lipid peroxidation in peach fruit showed that decreasing the light intensity decreased CAT, G-POD and APX activity but increased malondialdehyde (MDA) content with more cell membrane damage [6,19]. Pak-choi (Brassica campestris ssp. Chinensis Makino L.) originated from China is one of the most important vegetables worldwide in terms of its planting areas and annual yields. Purple pak-choi contains high levels of light-dependent anthocyanin in its leaves. This plant is very popular in China, Japan and surrounding countries. Anthocyanins, a class of secondary metabolites, contribute to the red, blue, and purple colors in flowers, fruits, and leaves [20]. They also act as antioxidants and protect DNA and the photosynthetic apparatus from damage due to high radiation fluxes [21]. Additionally anthocyanin protects plants against cold and drought stress [22]. Anthocyanin is accumulated in response to light in the seedlings of mustard and tomato. Phytochrome is an important photoreceptor controlling the accumulation of anthocyanins [23–26]. However, how phytochrome regulates anthocyanin and other key enzymes under low light remains unclear. Anthocyanin is synthesized via a branch of the phenylpropanoid pathway, i.e., the flavonoid pathway (Fig 1) [27]. Anthocyanin biosynthesis consists of sequential reactions leading to the production of different anthocyanins. The gene structure of 73 anthocyanin biosynthetic genes was identified in B. rapa [28]. These gene expression analyses showed that almost all late biosynthetic genes of anthocyanin were highly up-regulated in all purple leaves of Brassica [29]. The key enzymes in the anthocyanin biosynthetic pathway include chalcone synthase (CHS), chalconeisomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), leucoanthocyanidin oxygenase (LDOX), anthocyanidin synthase (ANS), and anthocyanidin reductase (ANR). CHI is the first identified key enzyme in the flavonoid metabolic pathway [30], while CHS is the first enzyme in the pathway [31]. Cominelli et al. [32] investigated different light treatments and found that the activities of ANS and ANR in Arabidopsis were related to their gene expression level. The regulation of anthocyanin accumulation under different light levels was shown to be due to transcriptional control or transcription factors [33–37]. The lowest light levels caused death and decreased anthocyanin content in Anacampseros rufescens, maize and perilla [38–40]. The shading of stems and leaves of Eustoma grandiflorum resulted in a significant anthocyanin reduction in petal color [41], while incubation in
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Fig 1. Diagram of the flavonoid pathway. The enzymes for each step are italicized, with the following enzymes required for anthocyanin biosynthesis: chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and UDP-glucose: flavonoid-3-O-glycosyltranferase (UFGT). https://doi.org/10.1371/journal.pone.0179305.g001
complete darkness was beneficial to the nutritional quality of the brassica sprouts [42]. When B. rapa was exposed to low light, the levels of phenolics and shoot biomass were reduced [43]. Still, a comprehensive study of physiological change after low light treatment is lacking. Thus, we investigated the responses of various plant parameters, such as photosynthesis, chlorophyll, and the activities of anthocyanin biosynthetic and antioxidant enzymes, in purple pak-choi under low light stress by shading the plants in a phytotron. Moreover, the examination of anthocyanin accumulation in this purple plant under different low light intensities provides invaluable guidance for artificially supplementing light intensity in agricultural facilities.
Materials and methods Ethics statement This study was carried out in a phytotron. No specific permissions were required. The study did not involve any endangered or protected species.
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Plant material and treatments The variety of purple pak-choi (Brassica campestris ssp. Chinensis Makino L. "ziyi") was selected by the Horticultural Research Institute of Shanghai Academy of Agricultural Sciences, China. Initially 192 purple pak-choi seeds were sown in 12 plastic plates growing at a temperature of 28/15˚C day/night in a greenhouse on October 20, 2015. Plants were watered and fertilized daily with a half-strength Hoagland nutrient solution. The low light treatments were started when the plant had three expanded leaves (four weeks after sowing on November 17). They were transferred and maintained in a phytotron with the temperature of 28/15˚C day/night and 60% humidity. They were divided into four groups and were exposed to low light treatment as follows (Table 1): (1) normal light (NL, 1000 μmol m–2 s–1), (2) low light 1 (TL1, 750 μmol m–2 s–1), (3) low light 2 (TL2, 500 μmol m–2 s–1), and (4) low light 3 (TL3, 250 μmol m–2 s–1). The light intensity was measured by a Specbos 4001 (JETI Company, Germany). The experiment was carried out in triplicate, and approximately 100 plants were used in each replicate.
Relative pigment levels Twenty-milligram samples of purple pak-choi leaves were incubated with 10 ml of 95% ethanol in the dark for 24 h and mixed by vortexing for 30 s after 12 h. The relative chlorophyll and carotenoid levels were measured with a spectrophotometer (DU 730, Beckman Coulter, Inc., Brea, CA, USA) at 649, 665, and 470 nm, and then the amount of chlorophyll a, chlorophyll b, and carotenoid was calculated using formulas 2.1, 2.2 and 2.3 [44]: Chlorophyll a ¼ 13:95 A665
6:88A649
ð2:1Þ
Chlorophyll b ¼ 24:96A649
7:32A665
ð2:2Þ
Carotenoid ¼ ð1000A470
2:05 chlorophyll a
114:8 chlorophyll bÞ=245
ð2:3Þ
The total anthocyanin content (TAC) of purple pak-choi was quantified with a modified pH differential method (AOAC official method 2005.2) [45,46]. The TAC was derived using cyanidin-3-glucoside, which has a molar extinction coefficient of 26,900 L cm-1 mol-1 and a molecular weight of 449.2 g mol-1. The results are expressed as milligrams of cyanidin-3-glucoside equivalent per gram of fresh weight sample. Twenty-milligram leaf samples were incubated with 10 ml buffer (95% ethanol and 1.5 mol l-1 HCl (v/v) 85:15) at room temperature in the dark for 24 h. Then, 1 ml of leaf supernatant was mixed separately with 2 ml of 0.025 M KCl buffer at pH 1.0 and 0.4 M sodium acetate (NaAc) buffer at pH 4.5. Absorbance was read by a nucleic acid/protein analyzer (Beckman Coulter, Inc., USA) at 536 nm and at 700 nm in the pH 1.0 and pH 4.5 buffers, respectively. TAC was calculated with the following equation (2.4): A ¼ ðA536
A700 Þ pH1:0
ðA536
A700 Þ pH4:5
ð2:4Þ
Table 1. Low light treatment of purple pak-choi seedlings in a phytotron. Category
Rate of light transmittance (%)
Treatment
Illumination intensity (μmol m–2 s–1)
NL
100
Normal illumination
1000
TL1
75
A layer of white gauze
750
TL2
50
A layer of black shading with 50% transmittance
500
TL3
25
A layer of black shading with 25% transmittance
250
https://doi.org/10.1371/journal.pone.0179305.t001
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Leaf gas exchange Leaf gas exchange was measured on a fully developed leaf from the middle of each seedling at 9:30 AM after 5 d, 10 d and 15 d of low light treatment by a Li-6400 Portable Photosynthesis System (Li-Cor Inc., Lincoln, NE, USA). The CO2 assimilation rate or net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular carbon dioxide (Ci) and transpiration rate (Tr) of purple pak-choi leaves were analyzed. After measurement, the largest leaves from each group in the same position were harvested. Three biological replicates were frozen immediately in liquid nitrogen and stored at -80˚C for further analysis.
Quantification of MDA and soluble protein The frozen leaf samples were ground to determine the MDA and soluble protein content. As described by Jiang and Zhang [47], the amount of MDA, which represents lipid peroxidation was calculated by its molar extinction coefficient (155 mM−1 cm−1) in the thiobarbituric acid reaction. Total soluble protein content was measured using the Bradford reagent [48].
Antioxidant enzyme activity assay For the enzyme assays, 0.2 g of leaf samples were ground in 3 ml of ice-cold 25 mM HEPES buffer (0.2 mM EDTA, 2 mM ASA, and 2% PVP, pH 7.8). The homogenates were centrifuged at 4˚C for 20 min at 12,000 g, and the supernatants were used to determine the enzymatic activities. The G-POD activity was measured by the modified method of Cakmak [49]. The reaction mixture had 25 mM phosphate buffer (pH 7.0), 0.05% guaiacol, 1.0 mM H2O2 and 100 μl of enzyme extract. The increase in absorbance at 470 nm caused by guaiacol oxidation (E = 26.6 mM cm-1) was used to determine the G-POD activity. CAT was assayed as described by Durner and Klessing [50], and the activity was determined as a decrease in the absorbance at 240 nm for 1 min following the decomposition of H2O2. APX was measured by monitoring the rate of ascorbate oxidation at 290 nm as described by Nakano and Asada [51]. SOD activity was measured in a mixture of 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM methionine, 75 μM nitroblue tetrazolium (NBT), 2 μM riboflavin, and 50 μl of enzyme [52]. One unit of SOD activity was defined as the amount of enzyme required to inhibit 50% of the p-nitro blue tetrazolium chloride reduction at 560 nm.
Anthocyanin biosynthetic enzyme activity assay For these assays, 0.2 g of leaf samples were ground in 2 ml of ice-cold 25 mM HEPES buffer (pH 7.4) containing 0.2 mM EDTA, 2 mM AsA, and 2% PVP. The homogenates were centrifuged at 4˚C for 20 min at 12,000 g, and the supernatants were used to determine the enzymatic activities. The activity of 6 anthocyanin biosynthetic enzymes, CHS, CHI, F3H, DFR, ANS and ANR, were assayed using an ELISA Kit (U.S.A TSZ Biological Trade Co., Ltd.) according to the manufacturer’s instructions. This experimental method was based on a laboratory protocol deposited in protocols io, which was obtained from doi:dx.doi.org/10.17504/ protocols.io.h2mb8c6.
Statistical analysis Statistical Product and Service Solutions (SPSS, version 20, IBM Corporation, U.S.A) was used to performed analysis of variance (ANOVA). The physiological variables are presented as the mean ± standard deviation (SD), with a minimum of three replicates. Differences between the control and treatments were considered significant at p = 0.05. Significance between
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Fig 2. Effects of low light treatment on chlorophyll a (A), chlorophyll b (B), carotenoid (C) and anthocyanin (D) content in leaves of purple pakchoi seedlings. The data are the mean of three replicates, and SDs are shown as vertical bars. The means marked with different letters indicate significant differences between treatments at p
