Four Cold Responsive Genes That Show Consistent Differential Expression between the Japonica and Indica subspecies of rice

Rice (Oryza sativa L.) is the most important staple crop that feeds nearly half of the world’s population. Because originated in tropical or subtropical areas, rice is sensitive to low temperatures throughout its entire life cycle. Cold stress can inhibit photosynthesis by reducing the chlorophyll concentration, inducing the accumulation of Reactive Oxygen Species (ROS) and by causing severe damage to various cellular components including membrane lipids, structural proteins and enzymes [1-3]. Low temperatures have the potential to negatively affect the growth and development of rice plants during any developmental stage, from germination to grain filling, resulting in large losses in rice production [4].

Plants have developed several strategies to respond to low temperatures that are known collectively as ‘cold acclimation’ [5]. Numerous studies of cold acclimation have been reported in recent years, and they showed that many pathways participate in the cold-response process [6,7]. The Dehydration-Responsive Element Binding protein (DREB1)/C-repeat binding factor (CBF)-dependent cold-responsive pathway is the most well-known among all the pathways [8]. Another pathway with a central role in the rice response to cold stress is the MYBS3-dependent pathway. Interestingly, DREB1A responds early and transiently, while MYBS3 responds relatively slowly to cold stress in rice, suggesting that theMYBS3-dependent pathway is important for long-term adaptation to persistent cold stress [9]. In addition to these two pathways, the plant hormone systems play an indispensable role in cold stress tolerance [10]. For example, Jasmonate (JA) positively regulates cold tolerance in plants because it can positively modulate the CBF-dependent or –independent pathway, leading to accumulation of cryoprotective compounds such as polyamine, glutathione and anthocyanins [11]. DELLA proteins, the key components of the Gibberellic Acid (GA)-signaling pathway, are involved in the CBF1-mediated cold stress response [12]. The cross-talk between Cytokinin (CK) and Abscisic Acid (ABA) are also important in cold stress signaling. Components of CK signaling, Histidine Kinases (AHKs) and the cold-inducible A-type ARRs, play negative regulatory roles in cold stress response via inhibition of the ABA response [13]. Components of Brassinosteroid (BR) and ethylene signaling were also found to modulate freezing tolerance in Arabidopsis plants [14,15].

There are two major subspecies of rice, Oryza sativa ssp. Indica and Oryza sativa ssp. japonica. Generally, japonica rice varieties are more tolerant to low temperatures than varieties of indica rice [16]. Therefore, cold tolerance in indica rice can be improved by introducing genes from japonica rice through molecular breeding. Recent advances in next generation sequencing technology provide a powerful strategy for discovering new genes associated with cold tolerance in rice [17-21]. In this study, we combined transcriptome sequencing with genome re-sequencing to identify the Cold-Responsive-genes (CORs) that are differentially-expressed between the two rice subspecies. Using this approach, we found four COR genes that are differentially-expressed between indica and japonica varieties. Moreover, these genes show important natural variation between the two subspecies which predicted to result in changes of cold responsive cis-elements. Therefore, these CORs may be useful for improving cold tolerance in rice through molecular breeding.

Global mRNA expression profiles in rice seedlings in response to cold stress

 Figure S1: Phenotypes of the seedlings of the indica and japonica rice cultivars before (A) and after exposure at 4°C for 4 days (B) and 10oC for 5 days(C).Teqing and 02428 are indicated with red arrows). Graphical representation of the relative survival rates of 7 indica and 6 japonica seedlings following exposure to cold stress at 4°C for 4 days (D) or10°C for 5 days (E). Mean values with one or two asterisks were found to be significantly different by Student's t test (*P≤0.05; **P≤0.01; n≥6).

Table 1: Numbers of sequence tags in the 10 transcriptome libraries from rice cultivars Teqing and 02428 exposed to low temperatures.

Identification of COR genes in the indica and japonica rice cultivars

 

Figure 1: (A) Hierarchical cluster analysis of gene expression based on the log ratio fold-change data. I0 and J0 indicate the control samples of Teqing (I) and 02428 (J) before cold treatments, respectively. The number before the letter (I or J) indicates the temperature, the number after the letter indicates the hours of exposure. For example, 10I24 means the Teqing sample exposed in 10oC for 24 hours. (B) Number of up- and down-regulated Cold-Responsive genes (CORs) from Teqing and 02428 rice seedlings exposed to cold (4°C and 10°C) for 3 and 24hr.

Gene ontology and pathway enrichment analysis of the rice CORs

 Figure 2: Gene Ontology (GO) analysis of COR genes in the “molecular function” (A), “biological process” (B) and “cell component” (C) GO classes in Teqing and 02428 at 4°C and 10°C for 3hr and 24hr. The y-axis and x-axis indicate the percentage of genes in a GO category and the names of the clusters, respectively. The numbers before the letter (I or J) indicate the temperatures, the numbers after the letter (I or J) indicate the hours of exposure, J and I mean Teqing and 02428, respectively. For example, ‘CORs in 4I3’ means differentially expressed genes in Teqing between before (0hr) and after (3hr) cold treatment (4I3 vs I0, |log2Ratio| ?1 and FDR ≤ 0.001).

 

Figure 3: KEGG pathway analysis of CORs in Teqing and 02428.
BG refers to all the genes in the background. ‘CORs in 4I3’ means differentially expressed genes in Teqing between before (0hr) and after (3hr) cold treatment (4I3 vs I0, |log2Ratio| ≥1 and FDR ≤ 0.001). By analogy, the numbers before the letter (I or J) indicate the temperatures, the numbers after the letter (I or J) indicate the hours of exposure; J and I mean Teqing and 02428, respectively.* indicates significantly enriched KEGG pathways with correct P value ≤0.05.

Identification of consistent differentially-expressed COR genes in multiple rice cultivars

 Figure 4: Venn diagrams showing the shared COR genes in Teqing and 02428 after exposure to cold treatments. (A) 563 COR genes respond to cold in both cultivars at 4°C for 3 hours; (B) 3361 COR genes respond to cold in both cultivars at 4°C for 24 hours; (C) 716 COR genes respond to cold in both cultivars at 10°C for 3 hours; (D) 3219 COR genes respond to cold in both cultivars at 10°C for 24 hours (CORs only in Teqing are shown in blue circle with the number written in white, CORs only in 02428 are shown in yellow circle with the number written in black, CORs shared by both cultivars are shown in center part of the Venn diagrams with the number written in white). (E) among all the common CORs we get from picture A, B, C and D, 171 COR genes respond to cold in both cultivars in all these cold treatments (Words above the pictures indicate the source of CORs. For example, common CORs in 4°C/3hr means CORs shared by Teqing and 02428, when they are treated in 4°C for 3 hours).

 Figure 5: qRT-PCR analysis of 13 selected COR genes identified by RNA-seq in leaves of Teqing and 02428 plants before (0hr) and after 3 h or 24 h of cold treatment at 4°C or 10°C. The data of expression levels were separately compared with the Teqing sample before cold treatment (0hr). OsActin was used as an internal standard (Student’s t test, *P≤0.05, **P≤0.01, n=3).

To determine whether the observed differences in expression for the five genes are associated with cold tolerance in other cultivars of the two subspecies, we included an additional five cold tolerant japonica rice cultivars (Nipponbare, Ta Hung Ku, Taibei309, Baber, Zhonghua 11) and five cold sensitive indica rice cultivars (9311, Lal-Aman, Pao-Tou-Hung, Ai-Chiao-Hong, Guan-Yin-Tsan) to the study. Details of the cold responses of these 10cultivars are shown in figure S1. All of the japonica rice cultivars showed significantly stronger cold tolerance than the indica cultivars, as expected. We then we assayed the expression levels of the five genes (OsABCB5, OsPHIL2, OsDREB1A, OsCDPK7 and OsREM4.1) in the 10 cultivars. Among these five genes, the expression profiles of OsABCB5, OsPHIL2, OsDREB1A and OsREM4. 1still showed clear distinctions between the indica and japonica subspecies, while OsCDPK7 did not show the indica-japonica difference in the multi-cultivar comparison (Figure 6). In the initial comparison between Teqing and 02428, OsABCB5, OsPHIL2 and OsDREB1Awere expressed at higher levels in Teqing than in 02428; similarly, in the comparison of the five cultivar pairs, the expression of the three genes was also higher in indica than in japonica. OsREM4.1 showed higher expression levels in the japonica rice cultivars, confirming the results from the comparison of Teqing and 02428. After cold treatment, expression of OsABCB5, OsPHIL2, OsDREB1A and OsREM4. 1 was significantly higher than in the control, and the expression of OsPHIL2 was up-regulated >400-fold after 4°C treatment in the indica cultivars.

 

Figure 6: Relative expression levels determined by qRT-qPCR of the OsABCB5 (A), OsREM4.1 (B), OsPHIL2 (C), OsDREB1A (D) and OsCDPK7 (E) genes in leaves of 6 indica and 6 japonica plants before (0hr) and after 3 h or 24 h of cold (4°C or 10°C) treatment. Values are the averages of three samples ± SE. Error bars indicate the standard deviation. Mean values with one asterisk were found to be significantly different by Student’s t test (*P≤0.05; NS, not significant; n=3).

Analysis of natural variation in these COR genes in multiple rice cultivars

 Figure 7: Schematic diagrams showing the position of SNP/Indels in the2.0-kb promoter regions and Coding Regions (CDS) of the OsABCB5 (A), OsPHIL2 (B), OsDREB1A (C) and OsREM4.1(D) genes. The phylogenetic tree was generated by MEGA5.0 software using neighbor joining method, with the segment length of interior branches indicating bootstrap values (1000 replications). The scale shows nucleotide substitutions per site. In gene schematic diagrams, gray bars indicate exons, and black lines indicate introns, the red vertical lines indicate the SNPs in coding regions while the black vertical lines indicate the SNPs in promoter regions. In the left table, Aus, Indica, Japonica and MIX represent the ecotypes of cultivated rice, such as aus, indica, japonica and mixture of other ecotypes, and the numbers in each block show the number of varieties. In the right table, the position of TSS is 0, negative and positive numbers indicate the positions of polymorphic sites in the promoter and coding region, respectively.

 Based on nucleotide polymorphisms, we could divide the sequences of the 575 lines into four haplotypes for OsABCB5; among these, HapII was the most represented in japonica lines, and Hap I was the most represented in indica lines. There were 12 SNP/Indel variations identified in the OsABCB5 promoter region between indica and japonica lines. Of these, four were predicted to be located within low temperature responsive cis-elements. For example, Hap II and Hap I differed in the nucleotides located at positions -642 G/A, -1633 A/G, -1638 G/A, and -1808 G/A upstream of the start codon; these four SNPs were related to theMYBST1, WRKY71OS/ARR1AT, WBBOXPCWRKY1 and LTRECOREATCOR15motifs, respectively, that are predicted to function in the cold response.

The japonica subspecies of Oryza sativa has adapted to a temperate climate, while the indica subspecies has adapted to tropical and subtropical environments. As a result, japonica rice cultivars are generally more tolerant to cold stress than indica rice cultivars (Figure S1). In our study, we used comparative transcriptome analysis on two rice cultivars to identify a group of COR genes in these two cultivars. From the CORs, we further identified four key genes, OsABCB5, OsPHIL2, OsDREB1A and OsREM4.1, that showed ubiquitous and distinctive activity in indica and japonica cultivars in an integrative study using KEGG and GO annotations, Venn diagram analysis, qRT-PCR, and natural variation analysis.

The DREB1/CBF pathway is a well-known cold-responsive pathway in plants [23]. The pathway includes the CBF1, CBF2 and CBF3 genes, which are also known as DREB1B, DREB1C and DREB1A, respectively, in Arabidopsis [24]. However, not all three transcription factors act as positive regulators in the CBF/DREB1 pathway. In Arabidopsis, freezing tolerance of the cbf2 mutant is enhanced by up-regulating the expression of CBF1/DREB1B and CBF3/DREB1A, which means that CBF2/DREB1C plays a negative role in the cold response [25]. OsDREB1A has been reported previously to be induced by cold stress [26,27]. Consistent with these results, we found that the expression of OsDREB1Aincreased after cold treatment. Lourenco et al., have claimed that in OsHOS1 knockdown plants, the reduced expression of OsHOS1 promoted the accumulation of OsICE1 protein and resulted in the up-regulation of OsDREB1A. However, the transgenic plants did not show increased cold tolerance [28]. In our study, the expression level of OsDREB1A was higher in indica rice compared to japonica rice. Therefore, we speculate that OsDREB1Amay play a negative role in cold signaling. In our natural variation analysis, we discovered a SNP related to MYB in the promoter region of OsDREB1Ain HapI (japonica). Interestingly, a MYB-like transcription factor has been reported to be involved in the cold regulation of CBF genes, and MYB15 over expression reduced the expression of CBF genes under cold treatment [29]. Thus, the MYB negative regulation of OsDREB1A may result in the stronger cold tolerance observed in japonica rice cultivars.

Among the four genes we screened, OsREM4.1 is related to the plant hormone ABA, which is known as a cold stress-responsive hormone. Two SNP variations within ABA responsive or MYB elements were located within the promoter region of OsREM4.1. Interestingly, OsREM4.1 was previously characterized and found to be up-regulated by ABA signaling; the OsREM4.1 protein can bind to OsSERK1 and inhibit the formation and activation of the OsBRI1-OsSERK1 receptor complex, which is crucial for BR signaling [30]. In Arabidopsis thaliana, application of exogenous ABA can enhance cold tolerance and induce the expression of several COR genes, including RAB18, RD29A and KIN2 [31,32]. Several studies showed that plants treated with BR grew better at low temperature compared to optimal conditions [33]. In addition, Li et al., described two key components of BR signaling, BIN2 and BZR1, regulate plant freezing tolerance; BIN2 and its homologs play negative roles, while BZR1 plays a positive role in the cold response [14]. In our study, OsREM4.1hada higher level of expression in japonica rice, so we predicted that OsREM4.1 may play a positive role in regulating cold tolerance via crosstalk between the ABA and BR pathways.

Another gene we found that is associated with the BR pathway is OsPHIL2, which encodes a Phosphate-Induced protein (PHI). Interestingly, two BR-response genes in Arabidopsis, EXORDIUM (EXO) and EXORDIUM-LIKE1 (EXL1), were previously reported to have the same PHI conserved region [34]. Expression of the EXO gene is up-regulated by BR and EXL1 has a similar expression pattern to EXO in Arabidopsis in that it can be induced by BR [35,36]. We therefore predicted that OsPHIL2 may be similarly connected to BR signaling pathway. Considering that OsPHIL2 is expressed at higher levels in indica cultivars when exposed to low temperature, OsPHIL2 may negatively regulate cold stress via BR signaling.

ABC transporters constitute one of the largest protein families, and are present in organisms ranging from bacteria to humans [37]. In plants, ABC proteins were originally identified as transporters involved in the final detoxification process [38]. OsABCB5 is predicted to encode an MDR-like ABC transporter. There are several studies that have reported that MDR-like ABC transporters are involved in the auxin signaling pathway. Noh et al., showed that two MDR-like genes, At MDR1 and AtPGP1, are required for auxin transport and auxin-mediated development in Arabidopsis. Also, mutation of two related MDR-like genes (MDR1 and PGP1) resulted in reduced polar auxin transport [39]. Another study found that the Arabidopsis MDR-like ABC transporter4 (AtPGP4) is involved in auxin-mediated root development [40]. Although the role of auxin in cold stress remains unclear, there are reports that potentially link cold stress to auxin by showing that cold stress inhibits the inflorescence gravity response in Arabidopsis thaliana [41,42]. An early study demonstrated that temperature affects the speed of exogenous auxin transport in a variety of plant species [43]. Considering that auxin is at the center of hormonal crosstalk, auxin may mediate the response to cold by interacting with other hormones [44]. These results indicate that OsABCB5may play a crucial role in the cold response in rice seedlings by transporting auxin.

Based on the expression profiles and predicted pathways of the four key genes involved in the cold response in rice, we can conduct further functional studies to determine the biological and biochemical functions of the proteins encoded by these genes.

Plant materials and Evaluation of Phenotypes

RNA isolation

Library construction and Illumina cDNA sequencing

Identification of COR genes

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of gene expression

Table S1: Sequences of the oligonucleotide primers used in the qRT-PCR assays.

Haplotype analysis and SNP discovery of the four key COR genes

DM conceived and designed the experiments. XH and DM performed the experiments. XH and YT analyzed the data, and XH wrote the manuscript.

We thank Dr. Caiyan Chen for his help in conceiving the project. This work was supported by grants from the National Natural Science Foundation of China (31101211, 31371603).

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