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. 2017 Apr 17;12(5):e1316442. doi: 10.1080/15592324.2017.1316442

The rice transcription factors OsICE confer enhanced cold tolerance in transgenic Arabidopsis

Cuiyun Deng a,*, Haiyan Ye b,*, Meng Fan b, Tongliang Pu a, Jianbin Yan b,c,✉
PMCID: PMC5501220  PMID: 28414264

ABSTRACT

Cold stress is one of the major constraints for crop yield. Plants have in turn evolved highly sophisticated mechanisms involving altered physiologic and biochemical processes to cope with the cold stress. Previous studies have revealed that the INDUCER OF CBF EXPRESSION 1 (ICE1), a basic helix-loop-helix (bHLH) transcription factor, directly binds and activates the expression of C-Repeat Binding Factor/Dehydration-Responsive-Element-Binding protein (CBF/DREB1) to regulate the cold-response pathway in Arabidopsis thaliana. However, the function of AtICE1 orthologues in rice is largely unknown. Here we identified that OsICE1 and OsICE2 in rice shared highly conserved amino acid sequence with AtICE1 in Arabidopsis. Overexpression of OsICE1 and OsICE2 in Arabidopsis significantly enhanced the cold tolerance of Arabidopsis seedlings and improved the expression of cold-response genes. Furthermore, we showed that both OsICE1 and OsICE2 physically interact with OsMYBS3, a single DNA-binding repeat MYB transcription factor that is essential for cold adaptation in rice, suggesting that OsICE1/OsICE2 and OsMYBS3 probably act through specific signal transduction mechanisms to coordinate cold tolerance in rice. These results demonstrated that the 2 OsICEs are orthologues of AtICE1 and play positive regulators in activation of cold-response genes to regulate the cold tolerance.

KEYWORDS: ICE1, cold stress, positive regulator, transcription factors

Plants frequently encounter a wide array of environmental stresses, including drought, cold, salinity, UV radiation, ozone, and other abiotic stresses, which seriously affect plant growth, development and crop yield. Among the various abiotic stresses, cold (chilling or low temperature) is one of the most significant limitations to crop productivity.1,2,3 To ensure normal growth and development, plant itself has evolved diverse stress adaptive mechanisms to survive and adapt to the cold stress,4 such as improvement the membrane stabilization, synthesis of specific solutes and adjustment in enzyme activity, leading to the acquisition of cold tolerance.2,3,4,5

It has been extensively studied that the transcription factors known as C-repeat binding factors (CBFs) or dehydration-responsive-element-binding proteins (DREBs) can bind to CRT/DRE cis elements to activate the expression of the downstream cold-regulated (COR) genes, resulting in conferring enhanced cold tolerance in Arabidopsis.6,7 Recently, studies show that INDUCER OF CBF EXPRESSION (ICE1),8 a MYC-like basic helix-loop-helix (bHLH) transcription factor,9 is an upstream transcription factor that directly binds to the CBF/DREB1 genes promoter regions, so that enhances the expression of DREB/CBF to improve the plant cold tolerance.10,11,12 In addition, studies reported that OsMYBS3, a single DNA-binding repeat MYB transcription factor, plays a critical role in cold adaptation in rice.13 Under the cold stress, the expression of OsMYBS3 gene was induced to high level16 which further activated several cold-response genes, leading to enhance stress responses and cold tolerance in rice.14,15,16

Rice (Oryza sativa L.) is a warm-season crop that is especially sensitive to cold stress. Cold tolerance plays a critical role in determining rice development and biomass yield, so that studying on cold-response pathways will help inform genetic improvement of rice to increase biomass production. Although OsICE1/OsICE2 have high similarity in sequence with formerly reported AtICE1 (AT3G26744) and AtICE2 (AT1G12860) from Arabidopsis,19,20,23 it is still largely unknown about their function in regulation of rice cold tolerance. In this study, we investigate whether OsICE1 (LOC_Os11g32100) and OsICE2 (LOC_Os01g70310) share conserved function through analysis of amino acid sequence similarity between OsICE1/OsICE2 and AtICE1/AtICE2. As shown in Fig. 1A, the amino acids from 347 site to 407 site are highly conserved in all 4 proteins, indicating that they might have the similar functions in regulation of cold tolerance (Fig. 1A). Phylogenetic analysis reveals that OsICE1 is clustered closer with AtICE1 and AtICE2 (Fig. 2B). Moreover, analysis of amino acid similarity and identity showed that AtICE1 are 45.9% similar to OsICE1, and 48.5% similar to OsICE2 (Fig. 1C), suggesting that they have highly similar amino acids. Taken together, these results suggest that OsICE1/OsICE2 in rice share highly conserved amino acid features with the AtICE1/AtICE2 in Arabidopsis.

Figure 1.

Figure 1.

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Bioinformatics analysis of ICE1 and ICE2 proteins in rice and Arabidopsis. (A) Amino acid sequence alignment of OsICE1/OsICE2 with AtICE1/AtICE2. The majority of ICE1/2 proteins is indicated by arrowhead on top of the amino acids. The red bar is represent the similarity. (B) Phylogenetic analysis of Arabidopsis AtICE1/2 and rice OsICE1/2 proteins. (C) Percentage of amino acid similarity and identity among rice OsICE1/2 and Arabidopsis AtICE1/2.

Figure 2.

Figure 2.

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Overexpression of OsICE1/2 in Arabidopsis enhances cold tolerance. (A) RT-PCR analysis of OsICE1/OsICE2 transcripts in overexpression transgenic Arabidopsis lines. PCR amplification of ACTIN2 was used as a control. (B) Freezing phenotypes of 12-d-old seedlings grown on MS medium after exposure to indicated freezing temperatures. The seedlings were treated at -6°C for 1 h, and followed by recovery at 22°C for 4 d. Experiments were performed 3 times with similar results. (C) The Survival rates of 12-d-old seedlings grown on MS medium after exposure to indicated freezing temperatures. The ein3 mutant plant is used as the control. Error bars show SD from 3 replicates. (D) Expression of cold-related gene COR15A, COR47 and RD29A in the overexpression plants under cold stress. 12-d-old MS-grown plants were treated at -6°C for the indicated time periods. ACTIN8 was used as the internal control. Error bars show SD from 3 independent experiments.

Recently, studies demonstrated that overexpression of AtICE1 in wild-type plants activates expression of the CBF genes through directly binding to the MYC recognition sequences in the CBF promoter under the cold stress, resulting in enhanced cold tolerance of Arabidopsis seedlings.1,12 To investigate whether OsICE1 and OsICE2 have similar function in regulation of cold tolerance as the AtICE1, we generated the OsICE1/OsICE2-overexpression plants (OsICE1/OsICE2-OE) under Arabidopsis wild-type (WT) background. To examine the overexpression level of OsICE1 and OsICE2 in the Arabidopsis transgenic lines, we performed the RT-PCR analysis to detect the transcripts of OsICE1 and OsICE2 (Fig. 2A) and 4 individual lines (OsICE1-OE1, OsICE1-OE2, OsICE2-OE7 and OsICE2-OE9) were chosen for further experiments. To investigate the cold tolerance of OsICE1/OsICE2-OE plants, we analyzed the phenotypes and survival rates of the OsICE1/OsICE2-overexpression plants in response to freezing stress. The ein3 mutant was used as a control, which was reported to positively regulate the cold tolerance.21 As shown in Fig. 2C, the survival rates of the OsICE1/OsICE2-overexpression plants were higher than the wild-type plants (Fig. 2C): the survival rates of OsICE1-OE1 is about 60%, OsICE1-OE2 is about 76%, OsICE2-OE7 is about 64%, OsICE2-OE9 is about 74%, and the survival rates of the positive control ein3 mutant is about 70%, which are all higher than that of wild-type plants, suggesting that OsICE1/OsICE2 positively regulate plant cold tolerance in Arabidopsis. We further examined the expression level of cold-response genes (including RESPONSIVE TO DESSICATION29A (RD29A), COLD REGULATED 15A (COR15A), and COLD REGULATED (COR47)) under cold stress by qRT-PCR. As shown in Fig. 2D, transcripts of these target genes were significantly upregulated in OsICE1/OsICE2-overexpression plants compared with wild-type plants under cold stress (Fig. 2D). Taken together, these results indicate that both OsICE1 and OsICE2 share conserved function with AtICE1, which positively regulate cold-response genes and improve cold tolerance in Arabidopsis.

Previous studies showed that OsMYBS3 regulates many cold-response marker genes in rice to enhance plant tolerance to cold stress.1,12,17,18 To investigate whether OsICE1/OsICE2 and OsMYBS3 have correlation in plant cold-stress responses, we performed the yeast-two hybrid (Y2H) to detect the interaction between OsICE1/OsICE2 and OsMYBS3. Y2H assay showed that both the OsICE1 and OsICE2 interact with OsMYBS3 (Fig. 3), suggesting that OsICE1/OsICE2 probably regulate cold tolerance by MYBS3-mediated cold-response signaling pathway.

Figure 3.

Figure 3.

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OsICE1/OsICE2 directly interact with OsMYBS3. Interaction was indicated by the ability of cells to grow on synthetic dropout medium lacking Leu, Trp, His, and Ade. The GAL4 activation domain expressed by pGADT7 (shown as AD) was used as negative controls.

AtICE1 has been previously reported to function as a key transcription factor in regulation of plant cold tolerance.1,12,17,18 Here we report the identification and characterization of OsICE1 and OsICE2 in rice that activate cold-stress response genes and positively regulate plant cold tolerance. Most recently, JA was established as a critical signal in cold-stress response in Arabidopsis. JA may regulates CBF/DREB1 expression by activating ICE transcription factors which acts as an important upstream signal to activate the ICE-CBF/DREB1 signaling pathway and positively regulates Arabidopsis cold tolerance.22 In this study, we revealed that OsICE1/OsICE2 in rice shared conserved function with AtICE1 in regulation of cold-stress response. The further investigation of JA-regulated OsICE1/OsICE2 signaling cascade will help to provide more insights into the function of JA's role in cold-tolerance in rice.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by grants from the Ministry of Science and Technology (2016YFA0500501), the National Science Foundation of China (31421001).

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