Introduction

During the geographical distribution and production of crops in temperate and high-altitude environments, low temperature stress often constitutes one of the major limiting factors (Zhu et al. 2007). Rice (Oryza sativa L.) is one of the most important staple crops worldwide, feeding around half of the world population (Wing et al. 2018). However, among the major cereal crops, rice is one of the most sensitive to cold stress (Zhang et al. 2014). Therefore, improving cold tolerance in rice has always been a primary goal in both breeding and genetic research. Based on the genetic diversity of different rice cultivars, many QTLs have been identified including qLTG3-1, Ctb1, COLD1, CTB4a and bZIP73 (Fujino et al. 2008; Saito et al. 2010; Ma et al. 2015; Schläppi et al. 2017; Zhang et al. 2017; Liu et al. 2018).

Low temperature signals, a response by plants to cold climates, can be perceived through the changes in membrane fluidity, molecular conformation, and metabolite concentration (Chinnusamy et al. 2007; Arshad et al. 2017). Based on the studies in the model plant Arabidopsis, a major low temperature response signaling pathway has been identified, including the transcriptional regulation cascades CBFs (C-repeat/dehydration-responsive element binding factors) (Medina et al. 2011). CBFs can bind to the C-repeat/dehydration-responsive element (CRT/DRE), a common cis-element in cold-responsive genes (Wang et al. 2008). Many key components have been identified in the signaling cascade upstream of CBFs, including transcription factors ICE1 (Inducer of CBF Expression 1), ICE2, CAMTAs, and MYB15 (Chinnusamy et al. 2003; Agarwal et al. 2006; Doherty et al. 2009; Fursova et al. 2009). Meanwhile, the key transcription factor ICE1 is also under regulation at post-transcriptional levels by SIZ1, HOS1 and SnRK2.6/OST1 (Dong et al. 2006; Miura et al. 2007; Ding et al. 2015).

As a gaseous plant hormone, ethylene plays important roles in plant growth, development, and responses to biotic and abiotic stresses (Yang and Hoffman 1984; Morgan and Drew 1997; Alexander and Grierson 2002; Anderson et al. 2004; Dubois et al. 2018). Ethylene induces the accumulation of antifreeze proteins (AFPs) in winter rye (Secale cereale) and the expression of LeCBF1 in tomato (Lycopersicon esculentum) (Yu et al. 2001; Zhao et al. 2009). It has been shown that increasing the ethylene synthesis can promote the chilling stress resistance in tomato (Ciardi et al. 1997; Zhang and Huang 2010). In addition, genes involved in ethylene signaling could be induced rapidly under low temperature in Arabidopsis (Fowler and Thomashow 2002).

The major components in the ethylene signaling pathway have been identified in Arabidopsis. Membrane localized (especially ER-localized) receptors can sense ethylene and negatively regulate the ethylene signaling pathway. In Arabidopsis, ethylene receptors can be classified into two subfamilies, subfamily I (AtETR1 and AtERS1) and subfamily II (AtETR2, AtERS2 and AtEIN4) (Light et al. 2016). Five ethylene receptor genes have been cloned in rice (Yau et al. 2004). It has been reported that OsETR2, one ethylene receptor can negatively regulate ethylene sensitivity and the transition from the vegetative to the reproductive stage (Wuriyanghan et al. 2009). In addition, OsETR2 also functions in starch metabolism (Wuriyanghan et al. 2009). The differentially localized OsERS1 and OsETR2 are involved in submergence tolerance with differential functions (Yu et al. 2017). In addition, ethylene receptor genes can also respond to various abiotic stresses at transcriptional level (Zhang et al. 2001). However, the function of ethylene receptors in response to low temperature stress remains elusive. In this study, we identified that the transgenic rice seedling overexpressing OsETR4 (OsETR4-OE lines) showed higher cold resistance than wild types. Transcriptomic analysis revealed significant differences in cold response between OsETR4-OE and WT.

Materials and Methods

Generation of OsETR4 overexpression transgenic rice

The full-length CDS of OsETR4 was cloned and fused to overexpressing vector driven by maize Ubiquitin promoter to generated pUbi::OsETR4 construct. Agrobacterium-mediated transformation was adopted to produce transgenic lines in the Kitaake (Oryza sativa L. sspp. japonica) calli (Hiei et al. 1994). The primers used were given in Table 1.

Growth conditions and cold tolerance analysis

Seeds of OsETR4-OE transgenic rice and WT were incubated in distilled water under 30°C for two days. Then germinated seeds with uniform plumule length were planted in pots with nutrient soil and then incubated in growth chamber of 12-h light (28°C, 80Lx)/12-h dark (25°C) photoperiod. For cold treatment, the seedlings of 3-leaf stage were transferred to a growth chamber of 8°C 12-h light/12-dark for two weeks and then recovered at 28°C for one week. Then the seedling survival rates were counted. Three independent replicates with 100 seedlings of each line were performed at one time. For RNA-seq analysis, rice seedlings were sampled as control first, and the rest were transferred to 8°C for 3 h and sampled as cold treatment.

RNA extraction, cDNA preparation and RNA-seq analysis

Total RNA was extracted using the RNA extraction kit (TianGen, Beijing). The following construction of cDNA library and sequencing were conducted by Novelbio (Shanghai). Three independent samples were collected for RNA extraction and Next-generation sequencing by ABI IonProton (2 × 101 bp). Raw sequence data were obtained, and the sequence reads were submitted to NCBI SRA database under accession number PRJNA524199. FASTQ_Quality_Filter tool was used to generate clean data for further analysis. The reads were mapped to the rice reference genome (MSU RefGen_v7) using a spliced aligner Tophat. The software Cufflinks was adopted to generate transcript fragments (Trapnell et al. 2010). Cuffdiff program was used for the differential expression analysis between four samples (Trapnell et al. 2013).

Quantitative real-time PCR

The cDNA was synthesized using One-step gDNA Removal and cDNA Synthesis Super Mix (TRansScript, Beijing). qRT-PCR was performed with SYBR Green mix (TOYOBO, Japan) using Chromo4 real-time PCR detection system (BIO-RAD, CFX96). The rice Actin1 gene was used as the internal control. The primers used for qRT-PCR was given in Table 1.

Results

OsETR4 is involved in cold stress response in rice

The expression of OsETR4 was significantly up-regulated in OsETR4-overexpression (OE) lines compared with Kitaake (wild-type) under the normal growth conditions (Fig. 1A). After cold treatment, seedlings of the OsETR4 overexpression lines showed a significantly higher survival rates compared to Kitaake seedlings (Fig. 1B, D). The seedling survival rates of two OsETR4-OE lines ETR4-1 and ETR4-2 were 45 and 47% respectively after low temperature treatment, and only 18% of the Kitaake (Fig. 1B).

Transcriptomic analysis of the OsETR4-OE and WT lines

Table 1: The primers used in this study

Fig. 1: Overexpression of OsETR4 increased cold tolerance of rice seedlings

(A) Expression level of OsETR4 in wild type (WT) and OsETR4-OE lines (ETR4-1 and ETR4-2) by qRT-PCR. (B) The seedling survival rates of the OsETR4-OE lines and WT (Kitaake). (C, D) Representative phenotype of OsETR4-OE lines and WT before (C) and after (D) cold treatment. Bar = 10 cm

To further investigate the functions of OsETR4 in the cold stress responses in rice, we performed RNA-seq analysis of the OsETR4-OE1 and WT before and after cold treatment. Three independent samples were collected for RNA extraction and Next-generation sequencing by ABI IonProton. A total of 192,044,538 reads (93.3%) were mapped to the rice genome. The detailed mapped reads of each samples were listed in Table 2. Cluster analysis of the 12 samples was performed with R software Cluster package. The samples before and after cold treatment were separately clustered, indicating significant transcriptome changes after cold treatment (Fig. 2A). In addition, after cold treatment, overexpressed line (ETR4) and wildtype (Kitaake) were separated clearly. Principle component analysis (PCA) was also performed, and the result was in consistent with the cluster analysis (Fig. 2B). Therefore, the transcriptomic responses of OsETR4-OE1 and WT were different and also consistent with the different seedlings’ survival rates. In conclusion, the transcriptome data showed high quality results and could be used for subsequent analysis.

Transcriptomic responses to cold treatment of WT and OsETR4-OE

We identified a total of 33958 annotated transcripts in the four treatments by Cufflinks (Trapnell et al. 2010). A total of 25844 genes (76%) were represented in all treatments. Before low temperature stress, 28987 (85.3%) and 28880 (85.0%) of the genes were expressed in wildtype (Kitaake) and ETR4 overexpressed lines respectively. After low temperature stress, 88.1% (29915) and 86.9% (29501) were expressed in wildtype (Kitaake) and ETR4 overexpressed lines, respectively (Fig. 3).

Differential expression analysis of WT and OsETR4-OE

Table 2: RNA-seq quality control

Fig. 2: Cluster (A) and PCA (B) diagram of 12 rice RNA-seq samples

WTck and WTcold indicate samples of WT before and after cold treatment. ETR4ck and ETR4cold indicate samples of OsETR4-OE1 before and after cold treatment. Numbers indicate independent biological replicates

Fig. 3: Profile of gene expression of WT and OsETR4-OE line before and after low temperature treatment

Fig. 4: Profile of differentially expressed genes (DEGs)

(A) The number of DEGs in the four groups of comparison, i.e., WTck_WTcold, WTck_ETR4ck, WTcold_ETR4cold and ETR4ck_ETR4cold. The number of up-regulated DEGs and down-regulated DEGs were also shown. (B) Venn diagram illustrating the relationship between the four groups of DEGs

To further explore the differential transcriptomic responses between WT and OsETR4-OE, the differential expressed genes (DEGs) of four groups were analyzed: WTck_WTcold, WTck_ETR4ck, WTcold_ETR4cold, and ETR4ck_ETR4cold. We used the fold change ≥ 2 and FDR value ≤ 0.05 as a screening criterion. A total of 4199 genes were differentially expressed in WT before and after cold treatment, among which 2399 genes were up-regulated and 1800 were down-regulated after cold treatment (Fig. 4A). In the OsETR4-OE line, 2550 genes were up-regulated and 2654 were down-regulated after cold treatment. Before cold treatment, it was found that 91 DEGs between WT and OsETR4-OE. Among those, 75 genes had higher expression levels in OsETR4-OE and 16 genes had higher expression levels in WT. After cold treatment, 875 genes were differentially expressed between WT and OsETR4-OE: the expression level of 331 genes was higher in OsETR4-OE and 544 genes were higher in WT.

A Venn diagram including the four different comparisons was drawn in order to elucidate the relationships of the four groups of DEGs (Fig. 4B). The numbers in the different parts of the diagram indicate the DEGs specifically shared between different comparison groups. In addition, some parts of genes (I, II, III, and IV) possessed specific biological meanings and Gene Ontology (GO) analysis of these genes using AgriGO was performed (Tian et al. 2017).

Gene Ontology analysis of differentially responsive DEGs

Part I represents DEGs that were only differentially expressed between WT and OsETR4-OE after cold treatment (WTcold_ETR4cold). This group of genes showed little response to cold treatment in WT and OsETR4-OE and showed no significant difference before cold treatment between WT and OsETR4-OE. However, after cold treatment, these genes showed significant difference between WT and OsETR4-OE. A total of 258 genes were identified. The GO terms related to protein phosphorylation were significantly enriched in the biological process (P) (Fig. 5A). GO terms related to ATP binding and kinase activity were significantly enriched in the molecular function (F). Three GO terms were significantly enriched in the cellular component (C), i.e., “cell wall”, “external encapsulating structure” and “membrane”.

Part II represents DEGs specifically shared between WTcold_ETR4cold and ETR4ck_ETR4cold. This group of genes showed significant difference between WT and OsETR4-OE after cold treatment and also respond to the cold treatment in the OsETR4-OE. A total of 400 genes were identified and GO analysis were also performed. The GO terms related to “oxidation reduction”, “carbohydrate metabolic process”, “lipid metabolic process” and “protein phosphorylation” were significantly enriched in the biological process (Fig. 5B). In the molecular function, GO terms related to “oxidoreductase activity”, “hydrolase activity” and “protein kinase activity” were significantly enriched, consistent with the biological process identified (Fig. 5C). Three GO terms were significantly enriched, including “cell wall”, “apoplast” and “external encapsulating structure”.

Specific DEGs shared between WTck_ETR4ck, WTcold_ETR4cold and ETR4ck_ETR4cold

In part III, a total of 7 genes were identified as DEGs specifically shared between WTck_ETR4ck, WTcold_ETR4cold and ETR4ck_ETR4cold. This group of genes showed no significant response to cold treatment in the WT, but responded to cold treatment in OsETR4-OE and showed significant difference between WT and OsETR4-OE before and after cold treatment. Therefore, the OsETR4 overexpression may have changed the cold responses of rice seedlings (Table 3). After cold treatment, 3 genes showed induced expression and 4 genes showed repressed expression in OsETR4-OE. LOC_Os11g17954 may be involved in glutathione-mediated detoxification. LOC_Os03g60000 and LOC_Os02g06290 encode proteins involved in the mitochondrial functions. LOC_Os06g06490 encodes a U-box domain-containing protein kinase and may be involved in stress response. LOC_Os12g33194 belongs to the trichome birefringence-like gene family and its homologues in Arabidopsis have been shown to be involved in the synthesis and deposition of secondary wall cellulose (Bischoff et al. 2010). LOC_Os04g09604 encodes an O-methyltransferase and its homologue in Arabidopsis has been shown to be involved in lignin biosynthetic process (Goujon et al. 2003).

Common freezing responsive genes with different expression levels

Part IV represents DEGs shared between all four groups of comparison. Besides being cold responsive both in WT Table 3: Differentially expressed genes in part III of Fig. 4B

Fig. 5: Gene Ontology analysis of differentially expressed genes

(A) GO analysis of the 258 DEGs. (B) Significantly enriched GO terms belonging to biological process of 400 DEGs. (C) Significantly enriched GO terms belonging to molecular function of 400 DEGs

and OsETR4-OE, these 6 genes were also differentially expressed between WT and OsETR4-OE, both before and after cold treatment. This group of genes was cold-responsive in both WT and OsETR4-OE, but they were differentially expressed between WT and OsETR4-OE before and after cold treatment (Table 4). Therefore, the different expression level of these genes may contribute to the different seedling survival rates.

Table 4: Differentially expressed genes in part IV of Fig. 4B

Fig. 6: Expression levels of ethylene-related genes

(A) The expression profile of 45 ethylene-related genes in WT and OsETR4-OE before and after cold treatment. (B) Validation of the expression levels of 6 genes at 0 h and 3 h after cold treatment by qRT-PCR. Values are means± SD (n = 3). **: P < 0.01, *: P < 0.05

All 6 genes are repressed after cold treatment in WT and OsETR4-OE. Among these, the expression levels of 2 genes were consistently higher in WT than OsETR4-OE. LOC_Os01g09700 is a member of 1-aminocyclopropane-1-carboxylate synthase (ACS) gene family, also known as OsACS5. LOC_Os01g23710 encodes a NAC family transcription factor and may participate in signal transduction in the cold response. The other 4 genes all showed higher expression levels in the OsETR4-OE than WT, indicating that the high expression of these genes may contribute to the cold tolerance of OsETR4-OE. LOC_Os04g17660 encodes an arsenate reductase, OsHAC1;2 and overexpression of OsHAC1;2 can enhance the tolerance to arsenate (Shi et al. 2016). No reference literatures were found in the cases of LOC_Os07g44250 and LOC_Os08g03020.

Different expression patterns of ethylene signaling related genes

Table 5: Expression information of ethylene related genes

To further investigate the role of ethylene signaling in the cold response of rice seedlings, the expression profile of 45 genes involved in the rice ethylene biosynthesis and signaling were analyzed. Many ethylene-related genes were differentially expressed, either between WT and ETR4-OE, or between control and cold treatment (Fig. 6A). The detailed expression value and gene information were listed in Table 5. LOC_Os05g05680, also known as OsACO5, was both repressed in WT and ETR4-OE after cold treatment, consistent with the OsACS5 identified in Part IV of Fig. 4b. The expression of OsACO1, OsACO3, and OsACS1 were upregulated after cold treatment both in WT and ETR4-OE. OsACO2 was induced by cold treatment in WT but repressed in ETR4-OE. The expression of the other members of OsACS and OsACO family showed no significant differences. Most of the ethylene response factors investigated here were upregulated both upregulated in WT and ETR4-OE after cold treatment, including LOC_Os09g11480 and LOC_Os09g11460. LOC_Os09g11480 and LOC_Os09g11460 are also known as Sub1B and Sub1C and participate in the submergence response of rice (Xu et al. 2006). Two ethylene response factor gene, OsWR1 and OsWR2, both showed opposite response patterns between WT and ETR4-OE, which were induced in WT but repressed in ETR4-OE. Therefore, the ethylene signaling was different between WT and ETR4-OE under cold treatment, which may contribute to the differences in cold tolerance. The expression of 6 ethylene-related genes was further validated using qRT-PCR and the expression patterns were in agreement with our RNA-seq results (Fig. 6B).

Discussion

In Arabidopsis, genes involved in ethylene signaling can be rapidly up-regulated after exposure to low temperatures (Fowler and Thomashow 2002). In addition, it has been reported that ethylene synthesis is positively associated with cold tolerance in tomato plants (Ciardi et al. 1997; Zhang and Huang 2010). Therefore, ethylene has been shown to have positive effects on plant cold tolerance. However, ethylene receptors usually play negative roles in the ethylene response pathway. OsACS5, which participates in the ethylene synthesis, can be induced abiotic and biotic stress (Straeten et al. 2001; Iwai et al. 2006; Hu et al. 2011). Among 6 common freezing responsive genes with different expression levels, OsACS5 was repressed both in WT and OsETR4-OE after cold stress. More importantly, the expression level of OsACS5 was consistently lower in OsETR4-OE than WT. Two ethylene response factor genes, OsWR1 and OsWR2 can be induced by drought and their over-expression can enhance the drought tolerance in rice (Wang et al. 2012; Zhou et al. 2014). Interestingly, OsWR1 and OsWR2 both showed opposite response patterns between WT and OsETR4-OE. Therefore, the differential ethylene signaling response between WT and OsETR4-OE under cold treatment may contribute to their different cold tolerance.

In addition, OsACO5, responsible for encoding an aminocyclopropane-1-carboxylate oxidase which is involved in ethylene biosynthesis (Iwai et al. 2006), was both repressed in WT and ETR4-OE after cold treatment. Most of the ethylene response factors investigated were both upregulated in WT and ETR4-OE after cold treatment, indicating that cold stress indeed induced the ethylene signaling response in rice.

Although OsETR4 also expresses in young seedlings and anthers of rice, its expression level is quite low (Yau et al. 2004), which is also in agreement with our RNA-seq results. Among the five reported ethylene receptors in rice, OsERS1 had the highest expression level and was constitutively expressed in different tissues, while OsETR2 and OsERS2 had lower expression level (Yau et al. 2004). The expression of OsETR2 and OsERS1 can be induced by exogenous stimulus such as IAA or ethylene while OsERS2 was repressed (Yau et al. 2004). In our RNA-seq results, ethylene receptors barely respond to the cold treatment both in WT and OsETR4-OE, suggesting that ethylene receptors may not respond to cold stress in rice and the OsETR4-OE had no effects on the other ethylene receptors.

The ethylene receptors also have kinase activities: AtETR1 has histidine kinase activity, whereas the others mainly have Ser/Thr kinase activities (Gamble et al. 1998; Xie et al. 2003; Moussatche and Klee 2004). The Gene Ontology term “protein phosphorylation” is significantly enriched in DEGs that are only differentially expressed between WT and OsETR4-OE after cold treatment. This means that after cold treatment, genes involved in protein phosphorylation are differentially expressed between WT and OsETR4-OE and these genes may contribute to the cold tolerance of OsETR4-OE. Overexpressing OsETR4 protein may help the rice seedlings in response to cold treatment through enhancing its kinase activities. However, the detailed protein activity assay needs to be further studied.

Conclusion

Overexpression of an ethylene receptor OsETR4 could increase the seedling survival rates of rice under cold stress. Transcriptomic analysis by RNA-sequencing (RNA-seq) showed significant difference in OsETR4-OE line compared to wild type (WT) in response to cold. The Gene Ontology term “protein phosphorylation” is significantly enriched in DEGs that are only differentially expressed between WT and OsETR4-OE after cold treatment, indicating that the enhanced kinase activities by OsETR4 overexpression may contribute to its cold tolerance. In addition, genes involved in the rice ethylene biosynthesis and signaling were differentially expressed, either between WT and ETR4-OE, or between control and cold treatment. Therefore, genetic engineering of ethylene receptor OsETR4 could contribute to the cold climate tolerance of rice in the future.

Acknowledgements

We acknowledge financial support from the National Key Research and Development Plan of China (Grant Nos.2016YFD0300502), the National Natural Science Foundation of China (31701396), International S&T Cooperation Program of Jilin provincial science and Technology Department (20160414024GH), China Postdoctoral Science Foundation (2018M631878) and partly supported by the open funds of the State Key Laboratory of Crop Genetics and Germplasm Enhancement (ZW201701).

Author Contributions

XD and WJ designed the experiments; ZL, MW, KH, XY, HZ, ZM, SJ, LG, LD and XZ performed the experiments; ZL analyzed the RNA-seq data, WJ and ZL wrote the manuscript, XD, TW and MB modified the manuscript; all the authors agreed on the final submission and posted no conflicting interest.

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