ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 5, с. 740-744
^_ЭКСПЕРИМЕНТАЛЬНЫЕ ^^^^^^^^^^^^^^
СТАТЬИ
УДК 581.1
THE EFFECTS OF ENHANCED UV-B RADIATION ON THE RCC1
IN WHEAT SOMATIC CELLS1 © 2015 X. L. Duan***, H. Z. Chen***, R. Han***
*School of Life Science, Shanxi Normal University, Linfen, China **Higher Education Key Laboratory of Plant Molecular and Environmental Stress Response (Shanxi Normal University)
in Shanxi Province, Linfen, China Received August 14, 2014
Regulator of Chromosome Condensation 1 (RCC1), a Guanine Nucleotide Exchange Factor (GEF) for the small Ran GTPase (Ran-GTP), is localized on chromatin in the nucleus where it mediates the generation of Ran-GTP. The production of Ran-GTP by RCC1 is critical for nuclear envelope formation, nucleocytoplas-mic transport, DNA replication, and chromosome-induced mitotic spindle assembly. Most of the previous studies on RCC1 were carried out in mammalian cells. Only few studies on RCC1 in plant cell have been reported. Here we present an investigation on the identification of RCC1 and its location in wheat (Triticum aestivum L.) somatic cells during mitosis using immunoblotting and immunofluorescence. It was postulated that RCC1 are richer in chromatin than the surrounding cytoplasm throughout mitosis. Compared with white light, enhanced UV-B radiation greatly reduced the content of RCC1 (immunoblotting) and its fluorescence intensity. When RCC1 was absent, laser scanning confocal microscope detected the abnormal chromosomes. The results indicated that the participation of RCC1 in cell cycle provided theoretical basics for revealing the mechanism of abnormal mitosis caused by enhanced UV-B radiation and guided practical significance for agricultural production.
Keywords: Triticum aestivum — somatic cell — mitosis — Regulator of Chromosome Condensation! (RCC1) — UV-B radiation
DOI: 10.7868/S0015330315050073
INTRODUCTION
The atmospheric ozone content has gradually been decreasing every year. Depletion of stratospheric ozone increases the solar ultraviolet radiation in the range of 290—320 nm (UV-B) that reaches the surface of the Earth [1]. Enhanced UV-B radiation directly affects the lives and development of organisms, leading to variations in morphological structure, physiological metabolism, genetic properties, and growth cycle of many animals and plants, and thereby further threatens human beings. Therefore, it is particularly essential to understand the influence of UV-B radiation and the mechanism by which it affects field crops production [2, 3].
Previous studies of UV-B effects on wheat plant have shown abnormal mitosis in somatic cells. Enhanced UV-B could inhibit mitosis frequency and also
1 This text was submitted by the authors in English.
Abbreviations: Ran-GTP — Ran-GTPase; RCC1 — Regulator of Chromosome Condensation 1.
Corresponding author: Rong Han. School of Life Science, Shanxi Normal University, Linfen, China; +86 0357-2058618; e-mail: hhwrsl@163.com
result in lagging chromosomes, chromosome bridges, free chromosomes, nuclear deformation, partition-bundle division, and other types of aberrations [4]. It was concluded that enhanced UV-B inhibited the DNA replication. The UV-B may also be associated with the chromosome movement pulled by mitotic spindle. However, it would require more investigation to fully understand the effects of UV-B and underlying mechanism.
RCC1 (Regulator of Chromosome Condensation 1) is a candidate enzyme associated with chromatin that could drive spindle assembly. This candidate is the guanine nucleotide exchange factor for Ran-GTPase (Ran-GTP) and regulates GTP binding and hydrolysis. Localized generation of a high Ran-GTP concentration by chromatin-associated RCC1 in condensed chromosomes has been hypothesized to be essential for mitotic spindle assembly, nucleocytoplasmic transport, and nuclear envelope formation. The phosphorylation of RCC1 in mitosis by Cdc2 kinase is necessary to generate Ran-GTP on mitotic chromosomes in mammalian cells activating a subset of mitotic motors and microtubule associated proteins (MAPs), which in turn are required for spindles assembly and chromosomes segregation [5, 6].
THE EFFECTS OF ENHANCED UV-B RADIATION ON THE RCC1
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Studies of cell extracts derived from Xenopus laevis eggs support that Ran-GTP is sufficient to promote the assembly of complete spindle-like structures and chromosome-induced spindle formation [7]. Mammalian RCC1 contains an NLS (nuclear location signal) at its N-terminus and is imported into the nucleus, where it interacts with chromatin via core histones H2A and H2B [8—11]. Fluorescent recovery after photobleaching (FRAP) experiments demonstrated a highly dynamic interaction between RCC1 fused to green fluorescent protein (GFP) with chromatin. Moreover, the methylated N-terminal tail of RCC1 stabilized its interaction with chromatin in live cells [12—15]. Temperature-sensitive (ts) mutants in the RCC1 gene of BHK cells might fail to maintain correct flow cell cycle. It may prematurely condense chromosomes and enter mitosis at the restrictive temperature without completed S-phase [16]. In tsBN2 cells, spindles form normally even at the nonpermis-sive temperatures [17, 18]. RanQ69L-GTP disrupts the RCC1-generated Ran-GTP gradient, while flattening the gradient eliminates spindle assembly around chromatin beads [19, 20]. These experiments demonstrated that a Ran-GTP gradient is required for chromatin-dependent spindle assembly in cell extracts from Xenopus eggs.
In this study, the wheat plants were regarded as test objects. We identified and researched the location of RCC1 in the mitotic cells using immunoblotting and immunofluorescence techniques. The results showed the effects of UV-B radiation on the location of RCC1 during mitosis in wheat somatic cells. The mechanism of abnormal mitosis by enhanced UV-B radiation has also been explained.
MATERIALS AND METHODS
Plant material. Wheat seeds (Triticum aestivum L., ML7113) used in this research were supplied by Wheat Research Institute of Shanxi Academy of Agricultural Sciences. The plump seeds of uniform size were selected and rinsed with running water. The seeds were cultured in clean dish paved three layers of wet gauze after budding, were and exposed to UV-B radiation (B) in a cycle of 8 h (light)/16 h (dark) for 7 days at 25°C. Control experiments (CK) were carried out using normal white light (table).
Nuclear proteins extraction. Wheat leaves (0.25 g) from groups CK and B were weighed, grinded with liquid nitrogen, and then put into the centrifuge tube. 1mL of ice-cold PBS buffer (Na2HPO4 - 1.1 g; NaCl - 4.25 g; NaH2PO4 - 0.1 g, pH 7.4; 500 mL), pH 7.9, was added to the sample; centrifuged at 6000 rpm for 5 min at 4°C, and all supernatant was removed. The centrifuge tube containing pellet was put on ice and 5x more buffer A (1 M Hepes, pH 7.9; 1 M KCl; 0.1 M EDTA; 0.1 M EGTA; 0.1 M DTT; 1 mM PMSF; 10% NP-40) was added to the tube.
Light/dark period of treatments
Treatment Light, h/day Dark, h/day
white light UV-B
CK 8 - 16
B - 8 16
CK — control, without UV-B radiation; B — UV-B radiation. The UV-B radiation intensity was 10.08 kJ/(m2-day).
(NP-40 is a commercially available detergent, which is nonyl phenoxypolyethoxylethanol.) The material was re-suspended in the buffer A and incubated on ice for 5 min, gently shaken occasionally, and was centrifuged at 3000 rpm for 5 min at 4°C. Supernatant was again re-suspended in equal volume of buffer B (1 M Hepes, pH 7.9; 5 M NaCl; 0.1 M EDTA; 0.1 M EGTA; 0.1 M DTT; 1 mM PMSF; 25% glycerin) and incubated on ice for 10 min, gently shaken occasionally, and then was centrifuged at 14 000 rpm for 5 min at 4°C. Collected supernatant was the nuclear proteins extraction.
Immunoblotting. The protein samples were separated on 12.5% one-dimensional SDS-PAGE. The amount of protein on the gel was quantified using Quantity One. For immunoblotting, the target band was excised from the gel and transferred onto PVDF membrane and probed with rabbit-anti RCC1 poly-clone antibody ("Shanghai Standard Biotech. Co., Ltd.", China) diluted at a ratio of 1 : 1000. The secondary anti-rabbit HRP conjugate was used in 1 : 1000 dilutions. The chemiluminescence signal was detected by film with ECL kit purchased from Bio-Rad Laboratories, USA.
Immunofluorescence. Wheat root tips (0.01 g) were fixed by 8% paraformaldehyde in PEM buffer (PIPES - 2.096 g; MgSO4 - 1.540 g; EGTA - 2.377 g; pH 6.9; 125 mL) in centrifuge tubes for 1 h and then were washed with PEM buffer (pH 6.8) three times. The plant material was digested in PEM with the addition of 2% cellulase and 1% pectinase at room temperature for 40 min and was washed with PEM buffer (pH 6.8) again. It was then treated with a mixture containing Triton X-100, DMSO, BSA, and PBS (pH 7.4) for 2 h. Samples were rinsed with PBS (pH 7.4) three times (10 min each time) and then incubated with primary antibody (1 : 100) overnight at 4°C in dark. After rinsing gently with PBS (pH 7.4) the secondary antibody (1 : 100) was used to incubate samples for 2 h at 37° C. Finally, the samples were washed with PBS (pH 7.4) for four times, and the nuclei were stained with fluorescent dye DAPI for 30 min. The samples were then kept on slides and sealed for visualization by confocal laser scanning microscope ("Olympus", Japan).
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DUAN h gp.
M
(a) CK
B
kD 97.466.2 ■
43.0 ■
31.0 ■
20.1 ■ 14.4
45 kD
(b)
CK B
45 kD
130 125
•s 120
s
•8 115
a
110
c
<D
5 105
100
95
(c)
2 6
00
2 4 2 4
CK
B
Fig. 1. RCC1 content in wheat somatic cells. a — RCC1 was separated from the nuclear proteins extraction using 12.5% SDS-PAGE. Every band showed different molecular weight and every lane showed treatment group. The arrow pointed general location for RCC1; b — immunoblotting was used for the preliminary analysis of RCC1 content; c — changes in RCC1 content were quantified using Quantity One. Numerals inside the columns — the
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