Response of alternative splice isoforms of OsRad9 gene from Oryza sativa to environmental stress

Plant material, growth condition, and treatments

Seeds of rice (Oryza sativa L.) Zhonghua 11 and male sterile line m393LA were grown in a natural environment on a farmland in Chendu City (Sichuan Province, China). Samples of root, stem, leaf, abortion, unicellular pollen, bicellular pollen, and tricellular pollen were collected and stored at −80°C. The isolation and purification of developmental pollen were conducted according to the method described by Peng et al. [15].

Stress treatments included genotoxic stress, UV radiation, antibiotic agents such as bleomycin and mitomycin C (MMC), and environmental stresses such as drought, salt (NaCl), CdCl₂, and HgCl₂ stress. Ten-day-old seedlings were exposed to UV rays at 240 lm for 1, 2, 5, or 10 h. The wavelength of UV rays was 253.7 nm (wavelength region, 220–300 nm), which is mostly used in biological studies. Antibiotic stress assays were performed as previously described by Heitzeberg et al. [14]. Approximately 1 cm-tall seedlings grown in darkness were incubated in MS medium containing 1 μg/mL bleomycin or 40 μg/mL MMC. For environmental stress treatments, 1-month-old seedlings were treated with 0.2 M NaCl, 0.5 M CdCl₂, and 0.5 M HgCl₂, respectively, whereas drought stress was induced by natural drying instead of watering the solution.

To study the expression pattern of Rad9 response to auxin in the context of rice callus formation, rice callus induction assays were conducted as described by Luo et al. [16]. After surface sterilizing with 75% ethanol and 1.5% sodium hypochlorite and then washing three times with sterile water, seeds were inoculated on MS medium supplemented with different auxin sources [2,4-D, Indole-3-acetic acid (IAA), and Indole-3-Butytric acid (IBA)] and subsequently cultured in darkness. Auxin concentrations were 0.5, 1.0, 2.0, and 5.0 mg/L.

All harvested materials were immediately frozen in liquid nitrogen and then stored at −80°C for RNA preparation. At least three independent replicates of each experiment were performed.

Amplification and sequence analysis of OsRad9 gene

Total RNA of rice samples was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription of pooled RNA was carried out with oligodeoxythymidine using the PrimeScript™ RT Reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. PCR primers (Table 1) were designed based on the sequence from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/, LOC_Os03g22450). PCR reaction was conducted as described previously by Wang et al. [17].

After identifying on 1% agarose gels, PCR products were cloned into the pMD19-T vector (Takara, Dalian, China) and sequenced. Sequence similarities were examined with the GenBank/EMBL database using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The amino acid sequences of cloned cDNA fragments were deduced and protein sequences were aligned using the program DNAMAN 6.0 (http://www.lynnon.com). Phylogenetic relationship was analyzed by multiple alignments of Rad9 proteins using the MEGA 4.1 program and visualized by iTOL (http://itol.embl.de/) [18].

Location assays of OsRad9-GFP-Fusion proteins

Transient expression assays in onion epidermal cells were carried out by Agrobacterium mediated transformation. cDNAs of OsRad9.1 and OsRad9.2 were linked to the 5′ end of gfp gene and then inserted to multiple cloning sites of pBI221 vector to make fusion gene expression vectors. Agrobacterium transformation was conducted as described by Li et al. [19]. A small piece (~1 cm²) of onion epidermis was torn off under sterile conditions and infected with Agrobacterium suspension for 20 min. After bacterial liquids were absorbed with filter paper, the flake was placed on MS medium and cocultured for 24 h at 25°C and 16 h light/8 h darkness. After cleaning the bacteria with sterile liquid MS medium, cell layers were mounted in water and examined by epifluorescence microscopy (Olympus SZX2-ILLB, Japan) with a GFP2 filter (480 nm excitation filter/510 nm barrier filter).

Real-time quantitative PCR analysis of OsRad9 mRNA

Total RNA was extracted using the method described above. Approximately 0.5 μg of total RNA from each pool was reverse transcribed to cDNA with oligodeoxythymidine in a volume of 10 μL following the PrimeScript™ RT Reagent Kit protocol.

The synthesized cDNA was diluted to 100 μL with sterile water and used as template in real-time quantitative PCR (qRT-PCR). Gene-specific primers (Table 1) were designed using Primer 5.0 software (PREMIER Biosoft International, Palo Alto, CA, USA) to amplify the specific cDNA fragment of two isoforms, OsRad9.1 and OsRad9.2. The two isoforms had same reverse primer but different forward primes (Table 1). As showed in Figure 1, the forward primer of isoform OsRad9.1 (Rq1-F) includes 15 nt of the end of Exon 8 and 6 nt of the front of Exon 9. The forward primer of isoform OsRad9.2 also includes the same 6 nt of the front of Exon 9, but the other 15 nt of the primer is from the end of Exon 6. All qRT–PCRs were performed using SYBR® Premix Ex Taq™II (TaKaRa, Dalian, China) and a Bio-Rad CFX96 Real-time PCR machine. Cycling parameters for OsRad9 were 95°C for 10 s initially, followed by 40 cycles, each comprising 95°C for 10 s, 60°C for 20 s and 72°C for 20 s, then 3 min at 72°C. Os-actin PCR conditions were 95°C for 10 s initially, followed by 40 cycles, each comprising 95°C for10 s, 63°C for 20 s and 72°C for 20 s, then 3 min at 72°C.

Figure 1:

Exon-structure diagram of the two mRNA isoforms of OsRad9. The location of the primers is shown in the box. Rq1-F is located beteewn exon8 and 9 junctions, Rq2-F is located beteewn exon6 and 9 junctionsis, Rq-R is located downstream of the exon9.

Isolation and sequence analysis of OsRad9

According to the rice genome data (http://rice.plantbiology.msu.edu/), OsRad9 gene was located on the long arm of chromosome 3 (Loc_Os03g22450) and contained nine exons forming an ORF of 1344 nucleotides coding for a putative protein of 447 aa (Figure 2A). The putative protein of OsRad9 was homologous to ARF75 (AJ441077) of Arabidopsis thaliana in Genbank. However, we further found that the naming of AtARF75 was based on experimental evidence, no additional details recorded (http://www.ncbi.nlm.nih.gov/protein/CAD29645.1). And with no auxin response elements (AuxREs), the putative ARF75 do not in the ARF family of both rice and Arabidopsis thaliana [20]. When the putative amino acids of OsRad9 were used for BLASTP, two records (EEC75240.1 and ABF95895.1) were found in GenBank, and both of them had completed Rad9 domain (Figure 2B). Hence, our research focus was transferred to Rad9 gene. Sequence analysis revealed that OsRad9 protein shared conserved domains and that the difference between the two records mainly occurred in the C-terminal portion of OsRad9 (Figure 2A and B). These analyses indicated that EEC75240.1 and ABF95895.1 were products alternatively spliced from OsRad9 transcripts. For confirmation, we conducted reverse-transcriptase PCR (RT-PCR) analysis using total RNA from rice callus. As expected, two bands could be amplified (Figure 2C) and designated as OsRad9.1 and OsRad9.2, corresponding to EEC75240.1 and ABF95895.1, respectively. Further cloning, sequencing, and sequence comparison showed that OsRad9 had very high similarity to Rad9 from other species, especially in the N-terminal region of the encoded peptides. Some examples were as follows: 24.54% similarity to hRad9, 20.55% to SpRad9, and the closest homolog in the PCNA family that charged for the Rad9–Hus1–Rad1 clamb complex. Thus, they can all act as DNA damage sensors and are loaded around DNA [21], [22].

Figure 2:

OsRad9 gene structure and RT-PCR analysis. (A) Schematic depiction of the genomic structure of OsRad9 and alternative splicing of the two transcript isoforms. Line above shows the genomic contig from genome database. The exons and introns are represented by boxes and lines, respectively. (B) BLASTP analysis of amino acid sequences of OsRad9.1 and OsRad9.2 in the GenBank. The polyline denotes alternative splicing region. The hatched box indicates a Rad9 domain. (C) Detection of alternative transcripts of OsRad9 by RT-PCR. Lane M, DNA molecular weight marker (bp); lane 1, PCR amplification from rice callus cDNA; lane 2 (OsRad9.1), lane 3 (OsRad9.2).

C-terminal analysis using the web-based server KinasePhos [23] with default parameters showed that OsRad9.1 contained 21 phosphorylation sites (amino acids 301–447), whereas OsRad9.2 only had 12 phosphorylation sites (amino acids 301–384) with exon7 and exon8 missing (Figure 3).

Figure 3:

Multiple alignment of Rad9 proteins from several species. The sequences for alignment from the published sequences of Rad9s, SpRad9 (CAA46693.1) from Schizosaccharomyces pombe, AtRad9 (NP_001030644.1) from Arabidopsis thaliana and hRad9 (AAH14848.1) from Homo sapiens. Dark shadow amino acids indicate identity and light shadow amino acids indicate chemically similar amino acids. The continuous underline denotes the Rad9 region. The C-terminal region is enclosed by the red box. The asterisk denotes potential phosphorylation sites.

Phylogenetic analysis showed that Rad9 had obvious differentiation between animals and plants, with OsRad9 transcript isoforms closely related to ARF75 of monocot maize; plant Rad9 homologs were divided into two main groups, dicotyledons and monocotyledons (Figure 4).

Figure 4:

Phylogenetic tree of Rad9 protein sequences from different species. The OsRad9 proteins were shown continuous underline in picture.

Subcellular localization of OsRad9

Rad9 proteins are reportedly localized to the cell nucleus [24], [25], [26]; thus, information on the subcellular localization of these proteins can help elucidate the functional roles that proteins play in cells. To analyze the localization patterns of OsRad9.1 and OsRad9.2, we linked them to GFP for fusion proteins, and transient expression conducted in onion epidermal cells showed that OsRad9.1 and OsRad9.2 protein signals appeared only in the nucleus (Figure 5). This finding indicated that both OsRad9.1 and OsRad9.2 functioned in the nucleus and that genome DNA was their main target.

Figure 5:

Subcellular localization of the two OsRad9 transcript isoforms. (A) Schematic depiction of the GFP, OsRad9.1::GFP and OsRad9.2::GFP fusion constructs. The constitutive 35S CaMV promoter was used to express these fusion proteins in onion cells. (B) Subcellular localization of OsRad9.1::GFP and OsRad9.2::GFP. The fluorescent images of the GFP fusion proteins are shown in the left panels and the bright images of the GFP fusion proteins are shown in the right panels.

Spatial expression patterns of OsRad9 transcript isoforms

The expression analysis of OsRad9 based on RT-PCR and qRT-PCR showed that the two OsRad9 transcript isoforms had different expression patterns (Figure 6). OsRad9.1 was expressed predominantly in root, stem, leaf, especially in callus (Figure 6A and C).

Figure 6:

Spatial expression patterns of two OsRad9 isoforms by RT-PCR (A) and qRT-PCR (C) in different tissues: leaf, root, stem, callus, abortion pollen, unicellular pollen, bicellular pollen and tricellular pollen, (B) Pollen grains were isolated from the tricellular stage of the male sterile line m393LA and Zhonghua 11 line, respectively, and examined by Dye 4′-6-diamidino-2-phenylindole (DAPI) staining, arrows denote the nucleus, bars: 20 μm.

Meanwhile, OsRad9.2 expression was much higher in pollen than somatic tissues (Figure 6A and C). In pollen development, the expression of OsRad9.1 sharply increased from unicellular pollen to bicellular pollen, and then maintained a high expression level, consistent with the fact that pollen was more sensitive to environmental factors [27], [28], whereas OsRad9.2 continued its high-level expression over OsRad9.1 (Figure 6A and C). If pollen (such as in the male sterile line m393LA) was aborted in the unicellular stage (Figure 6B), the expression pattern of Rad9 was similar to that in roots instead of that in normally developed pollen (Figure 6A and C). These results suggested that the two OsRad9 transcript isoforms played different roles in different developmental tissues.

Responses of OsRad9 transcript isoforms to DNA genotoxic stresses

In response to DNA-damaging agents (UV, bleomycin, and MMC), OsRad9 expression was promoted by UV, bleomycin, and MMC (Figure 7) similar to hRad9 [26]. However, OsRad9.1 expression increased faster with prolonged treatment time of genotoxicity compared with OsRad9.2. For example, expression of the former reached eight times over after 10 h of UV irradiation, whereas the response of OsRad9.2 to UV irradiation was not sensitive (Figure 7A).

Figure 7:

Transcript accumulation of OsRad9 transcript isoforms in rice leaves in response to DNA genotoxic stresses as determined by qRT–PCR. Rice seedlings were treated with UV (A), bleomycin(z) and MMC(Mc) (B) at the indicated times.

Induction of OsRad9 transcript isoforms by environmental stresses

To assess the responses of OsRad9 to environmental stresses, the expression of OsRad9.1 and OsRad9.2 was detected under stress conditions such as drought, salt, and heavy metals (CdCl₂ and HgCl₂). Results indicated that OsRad9.1 and OsRad9.2 expression was upregulated in all treatments (Figure 8), but the expression of OsRad9.1 was much higher than that of OsRad9.2.

Figure 8:

Transcript accumulation of OsRad9 transcript isoforms in rice leaves in response to environmental stresses as determined by qRT–PCR. Rice seedlings were treated with drought (A), 0.2 M NaCl (B), 0.5 M CdCl₂ (C) and 0.5 M HgCl₂ (D) at the indicated times.

Expression patterns of OsRad9 transcript isoforms response to auxin in the context of rice callus formation

ARF75 is recorded in the GenBank database; thus, OsRad9 expression can be affected by the plant hormone auxin. For confirmation, we investigated the expression patterns of OsRad9 transcript isoforms in the context of rice callus formation. Results showed that 2,4-D stimulated Rad9 expression and that the expression trend of OsRad9.1 was similar to that of OsRad9.2, i.e. the expression level increased with prolonged time (Figure 9A). However, the increase rate was much higher than that of OsRad9.2. At the optimum concentration 2 mg/L of 2,4-D, the expression level of OsRad9.1 was 13 times (26/2) that of OsRad9.2 (Figure 9A).

Figure 9:

Expression patterns of OsRad9 transcript isoforms response to auxin in the context of rice callus formation by qRT-PCR. (A) The effect of 2 mg/L 2,4-Dichlorophenoxyacetic acid (2, 4-D) on OsRad9 transcript isoforms induction in the callus formation processes at different time points. (B) The effect of different auxins (2.0 mg/L 2, 4-D, IAA and IBA) on OsRad9 transcript isoforms induction in calli at indicated times.

OsRad9.1 displayed different response patterns elicited by different species of growth hormones (Figure 9B). Short treatment times (24 h) with 2,4-D showed the strongest effect in promoting OsRad9 expression, whereas IAA showed the weakest. However, long-term treatment (14 days) with IAA intensively stimulated OsRad9.1 expression rather than the opposite. As to OsRad9.2, the expression difference was not significant between IAA and 2,4-D (Figure 9B).