OsSUI1 deficiency do not affect the growth and development of rice, and the development of the floral organs is normal
Previous research has indicated that the expression of OsSUI1 gene is induced by high and low temperatures, but OsSUI1 overexpression transgenic plants resulted in floral organ malformation and vegetative sterility, making it unable to perform abiotic stress experiments. To reveal the biological role of OsSUI1 in rice growth and development, loss-of-function mutants of OsSUI1 were generated through CRISPR/Cas9 gene-editing system in the rice variety 9522. We obtained 17 regenerated seedlings, all of which were positive plants. We analyzed the mutations in the OsSUI1 gene, two lines with different types of loss-of-function mutations, ossui1-1 and ossui1-2, were obtained. In ossui1-1, the OsSUI1 gene exhibits a 2 bp deletion, while ossui1-2 shows a 1 bp insertion at the editing site. Despite the mutation types being dissimilar, both lead to the loss of the SUI1 structural domain of the OsSUI1 gene. (Fig. 1a). The ossui1 mutant plants were planted in the experimental field alongside wild-type plants with routine pest and disease management. Through phenotypic analysis and investigation of major agronomic traits in rice, it was found that the ossui1 had normal development of floral organs and there were no significant differences between the mutant and the wild-type plants in terms of number of spikelets per panicle, seed setting rate, panical length(Fig. 1b-g). All subsequent experiments referring to ossui1 mutants will be conducted using the ossui1-1 line.
RNA-seq analysis reveals the involvement of gene in abiotic stress response in rice
As our previously report, the OsSUI1 gene is predominantly expressed in floral organs(Song et al. 2018). To investigate the differences in global gene expression across different genetic backgrounds, RNA-seq analysis was performed using the wild-type plants (WT), OsSUI1 over-expression plants (OE), and the ossui1 mutants. Although the ossui1 mutant exhibited growth and development essentially identical to that of the WT plants, transcriptional data revealed that the 548 differentially expressed genes were discovered, of which 150 genes (27.4%) were up-regulated, and 398 genes (72.6%) were down-regulated in the mutant line (Fig. 2a, Table S1). In the ossui1 mutant, the expression of OsDREB1B was notably reduced (log2FoldChange=-2.49). Previous studies have demonstrated that OsDREB1A and OsDREB1B expression in rice are triggered by cold stress, and that OsDREB1B is involved in the response to cold stress. Additionally, over-expression of OsDREB1B plants in both tobacco and Arabidopsis has been shown to enhance tolerance to both low and high temperatures(Qin et al. 2007; Gutha et al. 2008). Under drought stress, RCN1/OsABCG5 takes part in ABA accumulation within guard cells and may even be responsible for ABA import, which is crucial for stomatal closure(Matsuda et al. 2016). The expression level of RCN1 was found to be down-regulated significantly in the ossui1 mutant (log2FoldChange=-1.78) according to the RNA-seq data.
RNA sequencing data showed that a total of 987 significantly differentially expressed genes were identified in overexpressed plants compared to wild-type plants, of which 689 genes (69.9%) were up-regulated, and 298 genes (30.1%) were down-regulated (Fig. 2b, Table S2). The qSE3 encodes the protein OsHAK21, which promotes the uptake of K+ and Na+ ions, represses the accumulation of reactive oxygen species, and ultimately enhances salt tolerance during seed germination (He et al. 2019). Our data showed that the expression level of qSE3 gene was up-regulated 11.44-fold in the OsSUI1-OE lines. HAN1 functions as a negative cold tolerance regulator (Mao et al. 2019) and was found to be down-regulated (log2FoldChange = -3.36) in the OsSUI1-OE lines. The OsGS1;1/OsGS2 co-overexpressing transgenic plants showed considerably enhanced photosynthetic and agronomic performance under drought and salinity stress imposed during the reproductive stage (Singh et al. 2013). In our data, the expression level of OsGS2 in the OsSUI1-OE lines was significantly higher than that in WT (log2FoldChange = 1.24).
Several differentially expressed genes were discovered in both datasets, which have previously been documented to be linked to abiotic stresses. This mainly consists of members from MYB, bZIP, WRKY, ERF/DREB, and NAC gene families, in addition to genes directly associated with drought, salt, and cold stress, such as LOC_Os03g55540, LOC_Os08g23110, LOC_Os01g65500, LOC_Os01g72530, LOC_Os04g51160, LOC_Os02g01590 (Zhang et al. 2019; Li et al. 2021; Golldack et al. 2003; Golldack et al. 2003; Diédhiou et al. 2006; Xu et al. 2011; Saika et al. 2002; Baillo et al. 2019). Components that were also involved were a few genes that participate in the ABA pathway, including LOC_Os05g38290 and LOC_Os11g05110 (Zong et al. 2016; Zhang et al. 2012), as well as genes belonging to the calcium-dependent protein kinase group, like LOC_Os12g03970, LOC_Os01g55440, LOC_Os05g11790 (Wang et al. 2011; Kanwar et al. 2014;) (Fig. 2c-d).
According to the results of GO enrichment and significantly differentially expressed gene analyses, there were significant differences in the expression levels of several stress-related genes in both ossui1 mutant and overexpressing plants compared to wild-type plants. Among them, the mutant group was mainly enriched in oxide metabolism (GO:0016684, GO:0004601, GO:0042744, GO:0042743) or cellular response to hydrogen peroxide (GO:0042542, GO:1901700, GO:0070301, GO:0034599, GO: 0016209), it is worth noting that there are 3 items related to reactive oxygen species (GO:0000302, GO:0034614, GO:0072593) and there are also items related to thermal response (GO:0009408, GO:0034605) in Fig. 3a (Table S3). The overexpression group was enriched in the regulation of enzyme activity (GO:0005088, GO:0004553, GO:0004857, GO:0005976, GO:0015291, GO:0015299, GO:0015979), glucose metabolism (GO:0005089, GO:0005618, GO:000957, GO:0016020) and ion transport activity (GO:0009765, GO:0015035), among others(Fig. 3b, Table S4). There were 43 significant GO items shared between the overexpression group samples and the mutant group samples, of which 16 were directly or indirectly associated with plant tolerance, mainly related to reactive oxygen metabolism, metal ion binding, defence response and terpene synthase activity.
The RNA-seq data indicated that the OsSUI1 gene was strongly associated with abiotic stress response in rice, and to verify this conclusion even further, we detected a number of genes for abiotic stress response in both mutant and the WT plants using qRT-PCR. The six DREB family genes examined, except for OsDREB1D, the other five, OsDREB1B, OsDREB1C, OsDREB1G, OsDREB1H, OsDREB41, showed a significant down-regulation of the expression levels in the mutant leaves as compared to the wild-type plants. And in the spikes of the ossui1 mutant, except for OsDREB1G and OsDREB1H, the other four genes also showed significant down-regulation. According to the RNA-seq results, SNAC1, RCN1, HAN1, OsWRKY24, qSE3 were the other five genes we detected. The results showed that the negative regulator of stress response, qSE3, was significantly up-regulated in the mutant, while the other four genes were significantly down-regulated. The results of qRT-PCR were consistent with the results of RNA-Seq, which further suggests the involvement of the OsSUI1 gene in the response of rice to abiotic stress.
OsSUI1 deficiency increases the sensitivity to cold, heat and salt stress
To investigate the significance of the OsSUI1 gene in rice's response to abiotic stresses, we exposed ossui1 mutants to cold, heat and salt stress treatments in the seedling stage. Results revealed that after cold treatment (4℃, 3 days), the survival rates of ossui1 and wild type were 11.1% and 55.6%, respectively. Upon heat treatment (42℃, 4 days), the survival rates of ossui1 and wild type were 2.9% and 44.0%, respectively. Upon salt treatment (0.6% NaCl, 7 days), the survival rates of ossui1 and wild type were 6.3% and 60.4%, respectively (Fig. 5). These results together support the view that OsSUI1 plays a key role in the response of rice to abiotic stress.
Sequence polymorphism analysis of the OsSUI1 gene
It was suggested that abundant genetic diversity would tremendously benefit crop improvement (Joshi et al. 2023), DNA polymorphisms are ubiquitous genetic variations among individuals and include single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and other larger rearrangements (Suehiro et al. 2013). In order to analyze the polymorphism of OsSUI1 gene sequences and the presence of haplotypes in different rice varieties, we analyzed in detail the coding sequences and the variants present in the regulation of the OsSUI1 gene on the basis of a public sequencing database(RFGB: https://www.rmbreeding.cn/Index/) (Wang et al. 2020). Based on the SNP profile of the coding region, we identified six haplotypes for the OsSUI1 gene(Fig. 6a), of which, Hap1, Hap3 and Hap5 encode proteins containing 241 amino acids, Hap2, Hap4 encode proteins containing 243 amino acids, and Hap6 encodes a 171 amino acid protein due to a deletion of the G base at position Chr04:31958170, which directly leads to the premature termination of the coding product(Fig. 6b). Our analysis revealed that the Hap6, which contains a base deletion, exists only in japonica varieties(Fig. 6c).
In order to analyze the subcellular localization of these proteins, we constructed vectors for fusion expression with GFP proteins respectively, and transiently expressed them in tobacco leaf cells, and the results showed that OsSUI1243-GFP and OsSUI1241-GFP had signals only in the nucleus, whereas the signals of OsSUI1173-GFP were distributed in the nucleus, and in the cell membrane(Fig. 7). We do not yet know what this association of hap6, and the truncated protein OsSUI1173, with japonica means, and we need more data and in-depth studies to resolve the relationship.
Polymorphism in the promoter region can alter gene expression and lead to biological changes. To gain insight into the transcriptional differences of the OsSUI1 gene in different rice varieties, we blast the promoter sequence of the OsSUI1 gene by the database the Rice Resource Center (http://ricerc.sicau.edu.cn/) (Qin et al. 2021) with 133 varieties. Three promoter types have been identified, the initial type is the regular type represented by 9311 and Nipponbare, there are a total of 69 materials with this promoter type, and the CDS haplotypes of these materials contains Hap4 and Hap6. The second type is the transposon insertion type, as exemplified by ShuHui498 (R498), which exhibits a 4542 bp transposon at Chr4:31958931 (MSU7.0). This type includes a total of seven materials, and all the CDS regions of these seven materials are of the Hap2 haplotype. The ZhenShan97 represents the 29 bp insertion type with a non-contiguous 29 bp insertion located at Chr4:31959010 (MSU7.0) within a 40 bp range. A total of 40 samples are classified as this type, and their coding DNA sequences all contain two haplotypes, Hap1, Hap3 and Hap5 (Fig. 8a).
The effect of transposons on host gene expression and function is related to their insertion site, which may lead to gene silencing when inserted into the promoter sequence of a gene. To study the effect of this transposon on the OsSUI1 gene, we analyzed in detail the transposon. The full-length sequence of this transposon is 4542 bp, and we named it TE-4542. The sequence has typical reverse repeats (IR) at both ends and is 244 bp in length, with no predicted coding gene sequences found between IR sequence(Fig. 8a). The insertion position of TE-4542 was located 315 bp upstream of the ATG of the OsSUI1 gene, but we showed no significant changes in the expression level and pattern of the OsSUI1 gene in rice varieties with different promoter type by expression assay within 2 h heat treatment(Fig. 8b). We need more data and studies to analyze the effect of transposon insertion on the transcriptional regulation of the OsSUI1 gene.