SaRCC1, a Regulator of Chromosome Condensation 1 (RCC1) Family Protein Gene from Spartina alterniflora, Negatively Regulates Salinity Stress Tolerance in Transgenic Arabidopsis

Abstract

A regulator of chromosome condensation 1 (RCC1) family protein has been functionally characterized to be involved in various cellular processes. In this study, one RCC1 gene named SaRCC1 was cloned from the full-length cDNA library of Spartinaalterniflora. The open reading frame (ORF) of SaRCC1 was 1440 bp, and it encoded 479 amino acids with a calculated molecular mass of 51.65 kDa. Multiple amino acid sequence alignments showed that SaRCC1 had high identity with other plant RCC1s, and the phylogenetic analysis indicated that SaRCC1 had a closer affinity to Zea mays RCC1 family protein (ZmRCC1). SaRCC1 gene was induced under salt stress conditions, and its encoded protein was located in peroxisome. In order to further investigate the function of SaRCC1, transgenic Arabidopsis plants ectopically both sense-overexpressing and antisense-overexpressing SaRCC1 were generated. SaRCC1-overexpressing lines exhibited an increased salt and ABA hypersensitivity and reduced resistance to salinity stress. On the other hand, the transcripts of some stress-responsive genes in the SaRCC1 transgenic plants were affected in response to salinity stress. Our results provide evidence for the involvement of SaRCC1, negatively regulating salt stress responses by affecting stress-related gene expression in Arabidopsis.

Keywords: Spartina alterniflora; UVR8; regulator of chromosome condensation 1; salinity stress.

Figure 1

The SaRCC1 protein structure, sequence alignments, and phylogenetic relationships of SaRCC1 and other plant RCC1 family proteins. (A) Computational analyses of SaRCC1 protein. Expasy online software (http://www.prosite.expasy.org/scanprosite, accessed on 6 May 2022) predicted that Arabidopsis RCC1 family protein AtRCC1-2 (At3g02300) contains seven RCC1 repeats, whereas SaRCC1 protein contains six predicted RCC1 repeats. (B) Amino acid alignment analysis of identity between AtRCC1-2 and SaRCC1 protein. Arrows and box in different colors mark RCC1 repeat domains. (C) The phylogenetic relationships of SaRCC1 and its closely related RCC1 family proteins in other plants. The phylogenetic tree was constructed with MEGA version 5.0 software, and numbers on branches indicate the percentage of bootstrap analysis supporting the grouping of each branch. SaRCC1 is highlighted in red.

Figure 2

Expression pattern of SaRCC1 gene under salinity stress. S. alterniflora seedlings were subjected to different concentrations of NaCl, and the leaves were then collected for SaRCC1 gene expression analysis at the indicated time points. SaTubulin was used as the reference gene. The asterisks over the bars indicate statistical significance (p < 0.05) at each treatment time.

Figure 3

Localization of SaRCC1–eGFP fusion protein. eGFP alone (A) or SaRCC1–eGFP fusion protein (B) expressed under the control of the CaMV 35S promoter in Arabidopsis protoplasts. (C) Arabidopsis protoplasts co-expressing SaRCC1–eGFP and AtHMGB1–mcherry protein. (D) Arabidopsis protoplasts co-expressing SaRCC1–eGFP and AtPEX7–mcherry protein. The pictures were photographed under the green channel (eGFP fluorescence), red channel (mCherry fluorescence), combination of the green and red channels, and bright channel. Chlorophyll represents chlorophyll autofluorescence. Scale bar = 10 μm.

Figure 4

Phenotypes (A) and hypocotyl length (B) of WT and SaRCC1 transgenic seedlings grown under white- and UV-B-light conditions for 5 days. Scale = 0.5 cm. Different letters indicate significant differences (p < 0.05) according Duncan’s multiple test.

Figure 5

Analysis of salinity tolerance in Arabidopsis wild-type (WT) and SaRCC1-overexpressing and antisense-overexpressing lines. The phenotype (A), germination rate (B), and root length (C) of wild-type (WT) and SaRCC1 overexpressing and antisense-overexpressing Arabidopsis on 1/2 MS medium supplemented with or without 150 mM NaCl for 7 d. Different letters indicate significant differences (p < 0.05) according Duncan’s multiple test.

Figure 6

Phenotypes of wild-type (WT) and SaRCC1 transgenic Arabidopsis lines under abscisic acid (ABA) treatment. (A) Seedling growth of WT and SaRCC1 transgenic Arabidopsis lines on 1/2 MS medium supplemented with or without 0.5 μM of ABA after incubation for 7 days. (B) Green cotyledon rate of WT and SaRCC1 transgenic Arabidopsis lines. Different letters indicate significant differences (p < 0.05) according Duncan’s multiple test.

Figure 7

Expression levels of stress-responsive genes in Arabidopsis wild-type (WT) and transgenic plants under salinity stress, ABA treatment, and osmotic stress conditions. Seven-day-old seedlings were subjected to 100 mM NaCl, 5 μM ABA, or 300 mM mannitol, respectively, for 24 h, before determining the expression levels of RD22, RD29A, RD29B, COR15A, COR47, and P5CS1 by qRT-PCR. Actin2 was used as an internal reference gene. The asterisks over the bars indicate the significant differences between the wild-type and SaRCC1 transgenic Arabidopsis lines.