Response of Arabidopsis Cohesin RAD21 to Cell Division after Enhanced UV-B Radiation

doi:10.11983/CBB20009

Abstract

Abstract: The effect of UV-B radiation on plants is reflected in multiple levels. DNA damage is caused by UV-B radiation, which leads to abnormal mitosis and ultimately affects plant growth and physiological and biochemical processes. RAD21.3 is a subunit of cohesin complex, which is involved in chromosome separation during mitosis. In this paper, Columbia-0 Arabidopsis thaliana and atrad21.3 were used as materials, and the control and UV-B treatment group were set up to analyze the root length, plant height, bolting time, physiological and biochemical parameters of WT, atrad21.3 and overexpressed transgenic plants. The mitosis of Arabidopsis root tip cells was observed by basic fuchsin staining, and the aberration rate was counted. After UV-B treatment of the WT and atrad21.3 mutants, it was found that the UV-B treated-WT and atrad21.3 had similar bolting time, plant height and various physiological and biochemical indexes. Through the construction of expression vector, the results showed that RAD21.3 was located in the nucleus. Further observation of mitosis revealed abnormal phenomena such as lagging chromosomes, chromosomes bridge, fragments chromosomes, etc. Statistics show that the aberration rate of the UV-B treated-WT is similar to atrad21.3, and the aberration rate of the UV-B treated-atrad21.3 increases. The above results indicate that RAD21.3 may respond to abnormal mitosis induced by UV-B radiation.

Key words: Arabidopsis thaliana, atrad21.3 mutant, UV-B radiation, chromosome, mitosis

Fangfang He,Huize Chen,Jinlin Feng,Lin Gao,Jiao Niu,Rong Han. Response of Arabidopsis Cohesin RAD21 to Cell Division after Enhanced UV-B Radiation[J]. Chinese Bulletin of Botany, 2020, 55(4): 407-420.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB20009

https://www.chinbullbotany.com/EN/Y2020/V55/I4/407

Figures/Tables 12

Figure 1 T-DNA insertions and identification of atrad21.3 (A) Gene structure and T-DNA insertions of atrad21.3; (B) Identification of DNA level in atrad21.3(M: Marker); (C) Transcriptional level of RAD21.3 gene in atrad21.3. WT: Wild type

Figure 2 Phenotypic analysis of wild type (WT) and atrad21.3 (A) Seed of WT; (B) Rosette leaf (day 12) of WT; (C) Bolting of WT; (D) Seed of atrad21.3; (E) Rosette leaf (day 12) of atrad21.3; (F) Bolting of atrad21.3; (G) Plant height of WT and atrad21.3. (A), (D) Bars=10 μm; (B), (C), (E)-(G) Bars=1.0 cm

Figure 3 Cloning of AtRAD21.3 (A) PCR products of AtRAD21.3 (1-6: Products); (B) Gel cutting recovery result of AtRAD21.3 (1, 2: Gel cutting recovery products); (C) Bacterial PCR result (1-4, 6: Positive single colony; 5: Negative control products); (D) Dual-restriction digestion of plasmid (1-4: Dual-restriction result). M: DNA marker

Figure 4 Transient expression of pSuper1300-RAD21.3-GFP in leaves of Nicotiana benthamiana (A) GFP signal of pSuper1300-RAD21.3-GFP; (B) DAPI staining of pSuper1300-RAD21.3-GFP in leaves of Nicotiana benthamiana; (C) Merged image of A and B. Bars=10 μm

Figure 5 Localization of pSuper1300-RAD21.3-GFP before and after UV-B treatment (Bars=10 μm)

Figure 6 Detection of UV-B radiation on AtRAD21 expres-sion quantity (A) Result of RT-PCR; (B) Gray value analysis. WT: Wild type. Different lowercase letters show significant differences (Duncan method, P<0.05).

Figure 7 The seed germination rate, root length, plant height and bolting time of Arabidopsis thaliana with UV-B treatment (A), (E) The seed germination rate of Arabidopsis thaliana with UV-B treatment; (B), (F) The root length of Arabidopsis thaliana seedling with UV-B treatment; (C), (H) The bolting time of Arabidopsis thaliana with UV-B treatment; (D), (G) The plant height of Arabidopsis thaliana with UV-B treatment. WT: Wild type. Different lowercase letters show significant differences (Duncan method, P<0.05).

Figure 8 The influence of physiological and biochemical in Arabidopsis thaliana with UV-B treatment (A) Effect of UV-B radiation on Arabidopsis thaliana malondialdehyde (MDA) concentration; (B) Effect of UV-B radiation on Arabidopsis thaliana superoxide dismutase (SOD) activity; (C) Effect of UV-B radiation on chlorophyll content in Arabidopsis thaliana; (D) Effect of UV-B radiation on content of soluble sugar of Arabidopsis thaliana. WT: Wild type. Different lowercase letters (or * ) show significant differences (Duncan method, P<0.05).

Figure 9 Relative expression of cell cycle related genes in Arabidopsis thaliana WT: Wild type. Different lowercase letters show significant differences (Duncan method, P<0.05).

Figure 10 Normal phase types of mitosis in root tip of Arabidopsis thaliana (A) Interphase; (B), (C) Prophase; (D) Metaphase; (E) Meta-anaphase; (F) Anaphase; (G) Telephase. Bars=20 μm

Figure 11 Different types of chromosome aberration in root tip of Arabidopsis thaliana (A) Unorientation at prophase (arrow); (B) Cofusion at prophase (arrow); (C) Fragments chromosomes (arrow); (D) Fragments chromosomes (arrow); (E) Asymmetric at anaphase (arrow); (F) Chromosome bridge (arrow); (G) Lagging chromosome (arrow); (H) Lagging chromosome (arrow). Bars=20 μm

Table1 Effects of the UV-B radiation on mitosis of Arabidopsis thaliana

Treatment	Total of observing cells	Total of dividing cells	Total of aberration cells	Percentage of dividing cells (%)	Percentage of chromosomal aberration (%)
WT CK	5390	195	8	3.6±0.202 a	0.148±0.102 c
WT UV-B	5376	166	25	3.08±0.258 c	0.465±0.056 b
atrad21.3 CK	6998	215	32	3.07±0.195 c	0.457±0.085 b
atrad21.3 UV-B	7044	194	45	2.75±0.182 d	0.638±0.105 a
35S:RAD21.3 CK	5603	186	7	3.3±0.132 ab	0.125±0.136 c
35S:RAD21.3 UV-B	5536	193	21	3.48±0.129 ab	0.379±0.125 bc

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