ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
THE STRESS FACTOR, EXOGENOUS ASCORBIC ACID, AFFECTS PLANT GROWTH AND THE ANTIOXIDANT SYSTEM in Arabidopsis thaliana1 © 2014 H. F. Qian, X. F. Peng, X. Han, J. Ren, K. Y. Zhan, M. Zhu
College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, P.R. China
Received March 19, 2013
Ascorbic acid (AsA) is one of the most important soluble antioxidant molecules in plants, but excess AsA is a type of stress factor that inhibits plant growth. The exposure of Arabidopsis thaliana seedlings to 2 and 8 mM AsA decreased fresh weight to 78.6 and 64.3% of the control, respectively, after 5 days of treatment. A more than fivefold increase in the MDA content following the exposure to AsA suggests that the plant cellular structure is severely damaged by an increase in the ROS content. We also found that the transcripts of several antioxidant genes were down-regulated, which resulted in decreased activities of several antioxidant enzymes. These events caused an imbalance between oxidants and the antioxidant system. Real-time PCR demonstrated that the exogenous AsA reduced the transcript abundance of several aquaporins, whereas 2 mM exogenous AsA increased the transcripts of four aquaporins after 5 days of exposure. A high concentration of AsA significantly down-regulated aquaporins compared to a low concentration of AsA, especially PIP 2;1 and PIP 2;2, which were only 6 and 10% of the control, respectively. These results demonstrated that exogenous AsA was a stress factor that caused ROS overproduction, inhibited antioxidant ability, regulated aquaporin gene expression, and inhibited plant growth.
Keywords: Arabidopsis thaliana - ascorbic acid - antioxidant system - ROS - aquaporins
DOI: 10.7868/S0015330314040149
INTRODUCTION
Ascorbic acid (AsA) is one of the most important and abundantly occurring water-soluble antioxidant molecules in plants: its concentration in plant species is in mil-limolar range, from 10 to 300 mM, and is 5-10 times higher than that of glutathione (GSH), another antioxidant molecule [1]. AsA is a substrate for ascorbate peroxidase in the detoxification of hydrogen peroxide
(H2O2), superoxide (O2-), hydroxyl radical (OH), and lipid hydroperoxides. Previous results showed that the AsA content decreased in various species when the plants were subjected to water, salt, and other stresses [2, 3]. Thus, a high endogenous level of AsA in plants is necessary to counteract oxidative stress. Furthermore, AsA is a multifunctional molecule, which plays an important role in photosynthesis, redox signaling,
1 This text was submitted by the authors in English.
Abbreviations'. APX - ascorbate peroxidase; AsA - ascorbic acid; CAT - catalase; Chl - chlorophyll; CSD1-3 - Cu/Zn-SOD gene; DAB - 3,3'-diaminobenzidine; FSD1-3 - Fe-SOD gene; GPX -glutathione peroxidase; MSD1 - Mn-SOD gene; NBT - ni-troblue tetrazolium; PIP - plasma membrane intrinsic protein; SIP - small intrinsic protein; SOD - superoxide dismutase; TIP -tonoplast intrinsic protein.
Corresponding author. H. F. Qian. College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, P.R. China. E-mail. hfqian@zjut.edu.cn
pathogen defense, and metal and xenobiotic detoxification. As an important cofactor of violaxanthin de-epoxidase, AsA is involved in the synthesis of hydrox-yproline-rich glycoproteins in the cell wall during the cell cycle [4]. AsA can promote cell proliferation and enable cells that are already competent to progress through the phases of the cell cycle [5]. By employing ascorbate-deficient mutants, Muller-Moule [6] demonstrated that AsA was involved in the electron transport, oxygen evolution rates, and the defense response for photooxidative stress in Arabidopsis thaliana.
Endogenous AsA content can be increased by the exogenous application of AsA through the root medium, foliar spray, or seed priming [7]. In recent years, many reports showed that exogenous AsA has a role in protecting against abiotic stresses, including low temperature, salt, ozone, drought stresses, and metal compounds. Exogenous AsA counteracted the growth inhibition of rice roots that was induced by AlCl3 and enhanced Cd tolerance [8]. Athar et al. [3] found that exogenous AsA induced the improvement of wheat growth under saline conditions, an effect that appeared to be associated with a higher endogenous AsA that triggered the antioxidant system and enhanced the photosynthetic capacity. Exogenous AsA improves the plant response to stress by increasing the level of endogenous AsA, which is generally considered to be
essential for maintaining the antioxidant defense system of various plants against the oxidative damage caused by abiotic stresses.
However, it is unclear how exogenous AsA treatment under normal growth conditions, without any environmental stress, would affect plant morphology, physiology, and the related gene transcription. Thus, we used A. thaliana as the model plant to study the effects of exogenous AsA on plants at the physiological, biochemical, and genetic levels.
MATERIALS AND METHODS
Plant materials, root length analysis, and water content measurements. Arabidopsis thaliana (ecotype Columbia (Col)) seeds were sterilized and germinated on agar plates with MS supplemented with 30 g/L sucrose for 1 week, and the seedlings were transplanted to Metromix 360 soil. Triplicate cultures were prepared for each treatment. The plants were grown in a controlled chamber maintained at a temperature of 25 ± 0.5°C with a 12-h photoperiod and a light intensity of160 mmol photons/(m2 s) (provided by fluorescent bulbs). AsA (2 or 8 mM) was sprayed on the leaves twice every day at a given time (9:00 a.m. and 4:00 p.m.). The samples were collected on the 5th and 10th days to analyze the effect of the AsA on the plant growth, including the root length, fresh weight, and water content (WC).
Chlorophyll, proline, and MDA contents. After 5 and 10 days of treatment, approximately 0.5 g of leaf tissue was collected and homogenized with N,N-dim-ethylformamide, according to the report of Inskeep and Bloom [9], to analyze the Chl a, Chl b, and total Chl in the plant tissue. The proline was measured according to Claussen [10], and the measurement of MDA was performed according to our previous report [11].
Superoxide radical and hydrogen peroxide stain -ing. The H2O2 assay consisted of visualization using 3,3'-diaminobenzidine (DAB) staining, according to the method of Thordal-Christensen et al. [12]. DAB is rapidly absorbed by plant tissues and is polymerized locally in the presence of H2O2 to yield a visible brown
color. The O2- was detected using nitroblue tetrazolium (NBT), producing a blue color by the precipitation of NBT according to the method of Rao and Davis [13].
Enzyme extraction and analysis. The A. thaliana plantlets were ground with 20 mM phosphate buffer (pH 7.4) in an ice bath, and the homogenate was cen-trifuged to obtain a crude extract for assaying the enzyme activities. The activities of SOD, POD, CAT, GPX, and APX were determined according to our previous report [14]. The protein concentration was determined using a protein quantification kit ("Dojindo", Japan).
RNA extraction, reverse transcription, and realtime PCR analysis. In this study, we selected the fol-
lowing antioxidant enzyme genes to analyze the effect of exogenous AsA on the antioxidant system: three Cu/Zn-SOD genes (CSD1-3); one Mn-SOD gene (MSD1); three Fe-SOD genes (FSD1-3); one CAT gene (CAT); five ascorbate peroxidase genes (APX1-5), and eight glutathione peroxidase genes (GPX1-8). The nomenclature of antioxidant genes was according to our previous report [14]. We also analyzed several aquaporins: SIP1;1, SIP1;2, TIP1;1, PIP 1;2, PIP 2;1, and PIP 2;2. For performing the real-time PCR, the RNA of A. thaliana was extracted using RNAiso Plus, according to the manufacturer's instructions ("TaKaRa", China). The RNA was reverse transcribed into cDNA using a reverse transcriptase kit ("Toyobo", Japan), and the cDNA was used as the template to perform the realtime PCR analysis using the Eppendorf Mastercycler® ep Realplex4 ("Wesseling-Berzdorf", Germany). The primer pairs for each are listed in table 1.
Data analysis. The statistical analyses were performed using the StatView 5.0 program. When ANOVA indicated a significant effect, the differences among the means were determined using the Tukey test at the 0.05 or 0.01 probability level.
RESULTS
Effects of exogenous AsA on plant growth, fresh weight, and water content
We observed a growth inhibition after the A. thaliana seedlings were sprayed with 2 and 8 mM AsA, especially at the 8 mM concentration. We measured the seedling fresh weights and found that they were approximately 78.6 and 64.3% of the control after the 5-day treatment with 2 and 8 mM AsA, respectively, and approximately 81.3 and 62.5% of the control after the 10-day treatment, respectively. As a measure of leaf development, we assessed the lengths and widths of the leaves and observed reduction in these parameters after AsA treatment, with values less than 70% of the control after 10 days of 8 mM AsA treatment (fig. 1). In addition to the change in the fresh weight and size of the leaves, we found that the leaves appeared slightly wilted. Therefore, we assayed the water content in the seedlings and found the WC in the control and 2 and 8 mM AsA-treated groups was approximately 93.6, 93.3, and 92.9% after 5 days oftreat-ment, respectively, and was approximately 93.1, 92.6, and 92.1% after 10 days of treatment, respectively. The data analysis showed that the water loss was significant after AsA treatment.
Effects of exogenous AsA on the contents of chlorophyll, protein, proline, and MDA
The change in the morphology was caused by changes in the physiology and biochemistry. In this study, we measured the chlorophyll, protein, proline, and MDA content
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