Protecting effect of He-Ne laser on winter wheat from UV-B radiation damage by analyzing proteomic changes in leaves


The ultraviolet-B (UV-B) radiation can affect gene expression of plant in growth and development. Winter wheat (Triticum aestivum), as a kind of economic crop cultured in the Northern China, is also damaged by present-day ultraviolet-B (UV-B) radiation. It was reported that He-Ne lasers could alleviate cell damage caused by UV-B radiation in plants. To get the proteomic changes of wheat alleviated by He-Ne lasers, we studied protein expression profiles of winter wheat (Jin Mai NO.79) leaves by running 2-DE (two-dimensional gel electrophoresis), which allowed the identification of some significantly different gel spots. The spots were further verified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, in which they were confirmed to be winter wheat proteins. The results showed: 1) the proteomic changes between the ultraviolet-B radiation group and normal light group were significantly different on sixth day; 2) The expression of some proteins such as catalase, DNA polymerase and reduced glutathione declines at the ultraviolet-B radiation group and increases at the UV-B + He-Ne laser group. This indicates UV-B could regulate genes encoding proteins in Winter wheat leaves and affect the growth of the Winter wheat. He-Ne lasers treatment could significantly alleviate UV-B-induced damage in wheat.

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Jia, Z. and Duan, J. (2013) Protecting effect of He-Ne laser on winter wheat from UV-B radiation damage by analyzing proteomic changes in leaves. Advances in Bioscience and Biotechnology, 4, 823-829. doi: 10.4236/abb.2013.48109.


With reduction of stratospheric ozone increasing ultraviolet-B (UV-B) radiation at the earth’s surface, UV-B radiation has been shown to be harmful to plants. An examination of more than 200 plant species reveals that roughly 20% are sensitive, 50% are mildly sensitive or tolerant and 30% are completely insensitive to UV-B radiation [1]. Winter wheat (Triticum aestivum), as a kind of economic crop cultured in the Northern China, is also affected by present-day ultraviolet-B (UV-B) radiation [2]. UV-B (280 - 320 nm) radiation is known to reduce the photosynthetic pigment and protein content of green leaves and inactivate molecular defense mechanisms. The UV-B radiation also affects gene expression for growth and development [3]. It was reported that He-Ne lasers could alleviate cell damage caused by UV-B radiation in plants. Studies have showed that He-Ne lasers play a positive role in accelerating plant growth and metabolism, and the suitable doses of He-Ne laser irradiation improved germination capacity of plant seeds, enzymatic activities and the concentration of chlorophyll. Previous studies have illustrated that a He-Ne laser pretreatment could protect plant cells from enhanced UV-B radiation [4]. Although abundant data concerning the influence of He-Ne lasers on plant have been obtained, to date, the proteomic change of He-Ne laser irradiation on wheat is not clear.

So, the aim of the present study is to investigate the proteomic change of wheat leaf alleviated by He-Ne lasers. For this purpose, we employed an instrument to emit He-Ne lasers (650 nm) that was transformed from the semiconductor laser to test the optical effect of laser protection on wheat damaged by UV-B irradiation.


2.1. Plant Materials and Growth Conditions

Winter wheat (Triticum aestivum, cv JinMai NO.79) seeds were obtained from Shanxi Wheat Research Institute of Agricultural Sciences. Seeds were selected for uniform size and sterilized for 10 min with 0.1% HgCl2 and were washed for 50 min by flowing water. The seeds were grown in Petri plates (diameter 18 cm) on cotton soaked with distilled water under continuous white fluorescent light at a fluence rate of 200 µmol/m2 at 25˚C without any external nutrient. Three replications of 30 pure seeds were used for each of the different treatment. The treatment groups were divided into normal light group (CK group), UV-B Radiation group (UV-B group) and He-Ne Radiation repairing group (UV-B + He-Ne laser group).

2.2. Establishment of Different Treatment Groups

When the seedlings began germination, Supplemental UV-B radiation was provided by filtered Tai brand (Shanxi, China) 30 W sunlamps. Lamps were suspended above. A semiconductor laser (wavelength 650 nm, power density 3.97 mW∙mm−2) was directly irradiated the wheat seeds for 2 min every day [5] (Table 1).

2.3. Preparation of Protein Extract [6]

At 12 day after treatment, the wheat leaf was collected. A portion (200 mg) of leaf was homogenized in 1 mL of lysis buffer containing 8 M urea, 2% NP-40, 0.8% ampholine (pH 3.5 to 10), 5% 2-mercapthoethanol and 5% Polyvinyl pyrrolidne-40, using a glass mortar and pestle on ice. The homogenates were centrifuged in a RA-50JS rotor (Kubota, Tokyo, Japan) for 5 min. The supernatant was centrifuged at 20,000 g for 5 min and subjected to electrophoresis.

2.4. 2-DE [7]

Prepared samples were separated by 2-DE in the first dimension by IEF (Isoelectro focusing) tube gels (Daiichi pure Chemicals, Tokyo, Japan) and in the second dimension by SDS-PAGE. An IEF tube gel of 11 cm length and 3 mm diameter was prepared. IEF gel solution consisted of 8 M urea, 3.5% acrylamide, 2% NP-40, 2% carrier ampholines (pH 3.5 - 10.0), 10% ammonium persulfate and TEMED. Electrophoresis was carried out at 200 V for 30 min, followed by 400 V for 16 h and 600 V for 1 h. After IEF, SDS-PAGE in the second dimension was performed using 15% polyacrylamide gels with 5% stacking gels. The gels were stained with CBB (Coomassie brilliant blue), and image analysis was performed. 2-DE images were synthesized and the position of individual proteins on gels was evaluated automatically with Image Master 2D Elite software (Amersham Biosciences, Uppsala, Sweden). The pI and Mr (molecular weight) of each protein was determined using 2-DE markers (Bio-Rad, Richmond, CA, USA).

Table 1. The establishment and procedure of different treatment groups.

2.5. Data Analysis

Different 2-DE gel spots were further verified by matrixassisted laser desorption ionization-time of flight mass spectrometry. The amino acid sequences obtained were compared with those of known proteins in the Swiss-Prot, PIR, GenPept and PDB databases with the Web-accessible search program FastA (http://www.dna.affre.go.jpl).

2.6. Identification of Rice Leaf Proteins by MALDI-TOF MS

The digestions were desalted by Poros R2 and eluted with 0.6 mL matrix solution consisted of CHCA (12 mg/mL) in 70% ACN with 0.1% TFA. The eluted solution was applied onto the target well, dried at room temperature and introduced into a Bruker AutoFlex MALDITOF MS. The mass spectrometer was operated under 19 kV accelerating voltage in the reflectron mode and a m/z range of 600 - 4000. The monoisotopic peptide masses obtained from MALDI-TOF MS were analyzed by m/z software, and interpreted with MASCOT (Matrix Science) against the NCBInr database and the nonredundant databases of the rice genome generated by the Beijing Genomics Institute. Some digestive products from 2-DE spots were carried out by LC-MS/MS using a LCQ Deca IT mass spectrometer (Thermo Finnigan, Ringoes, NJ, USA) for further confirmation of amino acid sequences. After capillary reverse phase HPLC, the separated peptides were subjected into IT MS with 3.2 kV spray voltage and 150˚C at the heated desolvation capillary. The m/z range from 400 - 2000 was scanned in 1.2 s, and the ions were detected with a high energy Conversion Dynode detector. The LC-MS/MS data were converted into DTA-format files which were further searched for proteins with MASCOT.


3.1. Protein Content of Wheat Leaf on Different Treatment Days

Figure 1 show levels of proteins with molecular weight of 100, 60, 40, 25 and 20 KDa polypeptides of wheat leaves after the UV-B on different treatment days. These alterations ranged in molecular weight from as low as 20 KDa to as high as 100 KDa. From the general picture of leaves proteins emerging from this work, one point is noteworthy, more protein alterations were scored in 66 KDa after enhanced UV-B radiation. The synthesis of 66 KDa polypeptides was decreased on the 6 and 9 treatment days. The synthesis of 21 - 31 KDa polypeptides was increased on the 6 and 9 treatment days. The total protein of content control group (CK) and experimental group(E) were significantly different on sixth day after enhanced UV-B radiation (Figure 2).

Conflicts of Interest

The authors declare no conflicts of interest.


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