The level of ultraviolet-B (UV-B) radiation on the Earth’s surface has increased due to depletion of the ozone layer. Here, we explored the effects of continuous wave He-Ne laser irradiation (632 nm, 5 mW·mm <sup>-2</sup>, 2 min·d <sup>-1</sup>) on proliferating-cell nuclear antigen (PCNA) damage repair function of wheat seedlings exposed to enhanced UV-B radiation (10.08 kJ ·m <sup>-2</sup>·d <sup>-1</sup>) at the early growth stages. Wheat seedlings were irradiated with enhanced UV-B, He-Ne laser treatment or a combination of the two. We explored the transcripts of PCNA in each treatment group using RT-PCR. In addition, total proteins were extracted from the 7-day-old wheat leaves, analyzed by SDS-PAGE and identified by western blot. The results showed that the transcription of PCNA was weakened following UV-B radiation compared to the control. However, when seedlings were subjected to elevated UV-B-damaging radiation followed by He-Ne laser irradiation, the expression of PCNA was signifi-cantly higher than UV-B radiation alone. These results suggest that He-Ne laser has an active role in repairing the UV-B damaging effects. In order to further investigate the function of PCNA, dynamic arrangements of PCNA in wheat root-tip cells were observed with confocal laser scanning microscopy (CLSM). The PCNA was marked fluorescent dimming and strength weakened in en-hanced UV-B radiation (UV-B) compared with the control group (CK) during processing. It shows that PCNA may be involved in the separation of chromosomes.
Plants as sessile organisms that require sunlight to grow and develop, are inevitably exposed to ultraviolet (UV) wavelengths (200 - 400 nm), which represent almost 7% of the electromagnetic radiation emitted from the sun [
Compared with chemical methods for plant UV-B protection, physical methods are more effective and beneficial. Low dose of laser irradiation can promote plant growth and development, physiological metabolism [
PCNA was originally identified in humans as an antigen in patients with systemic lupus erythaematosus and as a protein that was synthesized during the S-phase of the cell cycle. The presence of the protein in the nucleus of dividing cells led to the name PCNA. Subsequent studies demonstrated that genes that encode PCNA are present in all eukarya and that the proteins are essential for cell viability [
Preliminary work of our group found that enhanced UV-B could inhibit the cell mitosis frequency of wheat and cause chromosome aberration. The phenomenon of “partition-bundle division” is one of the distortion types. “Partition-bundle division” is also called multiple bundle division. It was found that during the anaphase to telophase of mitosis, chromosomes of the wheat root tip cells fell into “three bundles”, “four bundles”, “six bundles” after the UV-B radiation [
“ML7113” (Triticum aestivum) was supplied by wheat Research Institute of Shanxi Academy of Agricultural Sciences. The wheat seeds of fully germination and uniform size were selected, sterilized for 10 min with 1% NaClO, and then washed for 10 min with running water, and then cultured in Petri dishes with 30 seeds per dish, and each group of repeated three times at 25˚C and 60% relative humidity under a 8 h/16 h light/dark regime for 7 days. The materials were divided into four groups: control group(CK), UV-B radiation group (B), He-Ne laser irradiation group (L), compound treated group with UV-B radiation and He-Ne laser irradiation (BL). (The detailed methods were showed in
Enhanced UV-B radiation was provided by filtered Qin brand (Baoji Lamp Factory, China).The lamps were suspended above and perpendicular to the pots. The power intensity of UV-B radiation was 10.08 kJ∙m−2∙d−1 treatment germination of wheat, every day deal with 8 h, a total of 7 d processing.
High-power laser irradiation apparatus (Nanjing Laser Instrument; wavelength: 633 nm; power intensity: 5 mW・mm−2; beam diameter: 1.5 mm) were used for materials treatment. The wheat seedlings were exposed to He-Ne laser irradiation for 7 days with 2 min each day in the dark, to prevent the influence of stray lights.
Total RNA was extracted from approximately 100 mg of fresh wheat leaves with RNAiso Plus reagent (TaKaRa) according to the manufacturer’s instructions. Then, First-strand complementary cDNA was synthesized by reverse transcription of 5 µg total RNA using Prime Script RT-PCR Kit (TaKaRa) The following primers used in Semi-quantitative RT-PCR were PCNA1-F: 5’-TCGTGAGGATGCCTTCCAAT-3’; PCNA1-R: 5’-TTGCATCTTCCGGCTTGTCT-3’; PCNA2-F: 5’-ATGTCGCGTTGGTGTCTCTT-3’; PCNA2-R: 5’-AGTGTCGCTACCATCATCAGC-3; From cDNA to DNA Amplification conditions were carried out under the following conditions: 1 min of denaturation at 94˚C; 35cycles at 94˚C for 30 s, 56.5˚C for 30 s, and 72˚C for 30 s; followed by a 10 min extension at 72˚C. Amplified PCR products were separated on a 1% agarose gel containing 1.5% (w/v) ethidium bromide and visualized by a GIS Gelatum imaging system (Tanon, Shanghai, China). GAPDH was compared as a compared gene transcription: GAPDH-F: 5-T GGTTGATCTCGTTGTGCAGGTCTC-3’; GAPDH-F: 5’-GTCAGCCAAGTCAACAACTCTCTG-3’.
250 mg of the 7-day-old fresh wheat leaves of each group were taken and then were dissected with clean tools on ice and as quickly as possible to prevent degradation by proteases. Then we placed the tissue in eppendorf tubes and immersed the tubes in liquid nitrogen to snap freeze and kept samples on ice for immediate homogenization. 2000 µl lysis buffer (0.5 mM Calcium chloride, 0.5% NP-40, 0.5 mM β-Mercaptoethanol, 50 Mm Tris- Hcl pH 8.0, 0.2 μg/L Aprotinin and 1 mmol/LPMSF) were rapidly added into the tubes, and made the samples centrifuged for 20 min at 12,000 rpm at 4˚C in a micro centrifuge. Next, we removed the tubes from centrifuge and transferred the supernatant into fresh tubes. The supernatant was total protein samples. The last, total protein samples and the sample buffer (20% glycerol, 10% SDS, 10% β-Mercaptoethano, l50 mM Tris-Hcl) were mixed according to the proportion 1:1. Next the mixture in a boiling water bath for five minutes, after, the mixture cools, can be used for the Western blotting.
To determine protein level changes that we performed western blot analysis. Samples with same amount of pro-
Treatment | Light | Enhanced UV-B radiation | He-Ne laser irradiation | Dark culture |
---|---|---|---|---|
CK | 8 h/d | - | - | 16 h/d |
B | 8 h/d | 8 h/d | - | 16 h/d |
L | 8 h/d | - | 2 min/d | 16 h/d |
BL | 8 h/d | 8 h/d | 2 min/d | 16 h/d |
Annotations: Light/dark period of irradiation treatments. CK: control group; B: enhanced UV-B radiation alone; L: laser treatment; BL: combined UV-B and He-Ne radiation.
teins for Western blotting were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated at room temperature in blocking solution with 5% bovine serum albumin (BSA) for 2 h in phosphate- buffered saline (PBS,137mM Sodium chloride, 2.68 mM Potassium chloride, 8.04 mM Disodium hydrogen phosphate and 1.47 mM Potassium dihydrogen phosphate) containing 0.05% Tween-20. Afterwards, they were incubated with monoclonal mouse-anti-PCNA antibody (Abcam) attenuation 400-fold with TBST containing 1% Tween-20 and 3% BSA at 4˚C overnight after washed three times in TBST. Then the membrane was incubated with horseradish peroxidase conjugated anti-mouse IgG at room temperature for 2 h. Then, the Membrane was washed three times in TBST. Finally positive signals were visualized using eECL Western Blot Kit (CWBIO).
Quantity One can determination the contents of PCNA proteins. Then, SPSS software Analysis of the samples that arranged in completely randomized designs with four repeating groups. Statistical significance and ANOVA values are from four duplicate values. The last, origin software was used four repeating groups building a bar chart.
Root tips of wheat were fixed with paraformaldehyde for1 h, and cellulase and pectinase were used for enzymolysis to take out the cell wall for 2 h. Then, the root tip were used polyglutamic acid was fixed for the root tip did not move. Following, Then the root tip cells were incubated at 4˚C overnight with monoclonal mouse-anti- PCNA antibody (abcam) attenuation 300-fold with PBS containing 3% Tritonx-100, 5% DMSO and 2% BSA at 4˚C overnight. The FITC-conjugated goat anti-mouse IgG was used as secondary antibody (green) diluted 200-fold with PBS. 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to stain the cell nuclei (blue) at a concentration diluted 700-fold. Then, observed root-tip cells by confocal laser scanning microscopy (CLSM).
Specific primers against PCNA1 and PCNA2 respectively were carried out Semi-quantitative RT-PCR experiments. The PCR products the DNA were separated by electrophoresis on 1% agarose gels containing ethidium bromide 1.5% (w/v) to confirm its magnitude and verify the presence of a unique PCR product. The mRNA expression levels of each gene were measured using GAPDH as a reference (
Transcriptase (RT)-PCR product of PCNA1 and PCNA2 bands were analyzed. By densitometry using Quantity One 1-D gel image analysis softwar (Bio-Rad, Hercules, CA, United States). (
UV-B radiation on nucleinic acid metabolism of wheat.
At first, total proteins in wheat leaves with same amount of different groups for Western blotting were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proliferating cell nuclear antigen (PCNA) is a key component of the DNA replication machinery present in the nuclei of all dividing cells in eukaryotes. (RAD5a ubiquitin ligase is involved in ubiquitination of Arabidopsis thaliana proliferating cell nuclear antigen) Molecular weight is about 30 KD. Between 20.1 KD and 31 KD can clearly get a main band (
The contents of PCNA proteins were shown in (
The presence of the PCNA protein in the nucleus of dividing cells and synthesized during the S-phase of the cell cycle. PCNA play mainly role in S-phase, then in M-phase content gradually reduce of mitosis. But, PCNA still affect M-phase separation of chromosome in mitosis. To study the role of PCNA in mitosis, we examined by confocal laser Scanning microscopy the subcellular localization of PCNAs proteins at the stage of division. In this figure, chromatin and PCNAs are shown as blue and green respectively. From the figure we can see the distribution of PCNAs clearly during the cell mitosis. (Addition) We observed mitosis occurs abnormal and chromosome aberration. The PCNAs was marked fluorescent dimming, strength weakened in the UV-B are lower than the control group (CK) during processing. At the same time we found that PCNAs proteins were mainly located in the two poles of cell at the stage of division in control and this phenomenon was more obvious under UV-B condition (Addition) (
We all know proteins play an important role in various physiological processes. They are not only the important components of organisms, but also as biological enzymes in catalytic reaction. Due to the maximum absorption wavelength of proteins is just UV-B radiation wavelength range, the protein itself will be affected by UV-B radiation [
He-Ne laser impact on plant growth has received increasing attention, which has caused some preliminary studies. Studies have shown that Laser irradiation can change the enzymatic conformation, making activity of enzymes increased. But, low dose of laser irradiation can promote plant growth and development, physiological metabolism [
Recently, there are some studies of PCNA protein in plant resistance. This experiment based on different groups of PCNA proteins, then, extraction, identification. Illustrates the UV-B radiation reduces PCNA protein content, and after He-Ne laser processing its content will be restored. At the same time for each group of RT-PCR analysis comparison. Further to each set of chromosomes and PCNA protein in the FITC Mark after strong fluorescence degree analysis and comparison. We observed mitosis occurs abnormal and chromosome aberration. The PCNA was marked fluorescent dimming, strength weakened in the UV-B are lower than the control group (CK) during processing. Show that PCNA may be involved in the separation of chromosomes and the dynamic distribution of PCNA may have relationship with “partition-bundle division”.
In this article, we explored the effects of continuous wave He-Ne laser irradiation on PCNAs damage repair function of wheat seedlings exposed to enhanced UV-B radiation at the early growth stages. Altogether, our provide data evidenced by western blot and RT-PCR observation of PCNA protein and gene expression changed in wheat Seedlings. The results of LCSM showed that chromosomes were changed with PCNAs. At the same time, it preliminarily discussed some connections between the changes of chromosome and PCNAs under the enhanced UV-B, He-Ne laser irradiation. But it remained further researches about how PCNA protein regulated mitosis, and their quantitative relationship.
The work was supported by the National Nature Science Foundation of China (No. 30671061) and The Natural Science of Shanxi Province (No. 20081101, 2014011028-5).