Comparison of Differentially Expressed Genes Involved in Drought Response between Two Elite Rice Varieties

Dear Editor, Drought is the most devastating factor limiting the productivity and geographical distribution of rice (Oryza sativa). Several drought-tolerant varieties have been developed from drought-tolerant germplasm. These varieties make it imperative to clarity the mechanism of drought responses at the genomic level, which will provide a foundation for future breeding and genetic engineering efforts (Zhou, 2007Zhou J. et al.Global genome expression analysis of rice in response to drought and high-salinity stresses in shoot, flag leaf, and panicle.Plant Mol. Biol. 2007; 63: 591-608Crossref PubMed Scopus (244) Google Scholar). Yangdao6 (YD6) was used to breed many elite varieties in China, which was developed from Yangdao2 (YD2) through a series of crosses (Supplemental Figure 1). Here, we verified that YD6 seedlings showed better drought tolerance than YD2, Yangdao4 (YD4), and 3021 seedlings (Figure 1A). After drought treatment for 8 d, over 81.9% of YD2 plants were rolled, which is about three times that of YD6 plants. Recovering normal growth with water, the survival rates of YD6 and YD2 seedlings were 92.2% and 65.6%, respectively. Drought invariably causes oxidative damage to cells due to excessive generation of reactive oxygen species (ROS) in crops (Selote et al., 2004Selote D.S. Bharti S. Khanna-Chopra R. Drought acclimation reduces O2*- accumulation and lipid peroxidation in wheat seedlings.Biochem. Biophys. Res. Commun. 2004; 314: 724-729Crossref PubMed Scopus (68) Google Scholar; Moumeni, 2011Moumeni A. et al.Comparative analysis of root transcriptome profiles of two pairs of drought-tolerant and susceptible rice near-isogenic lines under different drought stress.BMC Plant Biol. 2011; 11: 174Crossref PubMed Scopus (115) Google Scholar). In rice, drought-tolerant varieties showed lower H2O2 level and enhanced antioxidative enzyme activity compared to drought-sensitive varieties (Guo et al., 2006Guo Z. Ou W. Lu S. Zhong Q. Differential responses of antioxidative system to chilling and drought in four rice cultivars differing in sensitivity.Plant Physiol. Biochem.: PPB/Societe francaise de physiologie vegetale. 2006; 44: 828-836Crossref PubMed Scopus (265) Google Scholar; Rabello, 2008Rabello A.R. et al.Identification of drought-responsive genes in roots of upland rice (Oryza sativa L).BMC Genom. 2008; 9: 485Crossref PubMed Scopus (97) Google Scholar). Here, we found that the SOD and CAT activities in YD6 were higher than those in YD2 under normal conditions. When exposed to drought, the increasing extents of SOD and CAT in YD6 were more significant than those in YD2 (Supplemental Figure 2A and 2B). Then, we compared the accumulation of superoxide between two varieties using nitroblue tetrazolium (NBT) staining. Without stress treatment, both YD6 and YD2 plants showed slight NBT staining. After drought treatment, the level of NBT staining in YD2 seedlings was more intense than that in YD6 seedlings (Figure 1B), suggesting that YD2 plants accumulated much higher superoxide than YD6 plants. These results suggested that YD6 showed better drought tolerance associated with higher antioxidant enzyme activity and less ROS accumulation compared to YD2. Understanding the molecular basis of plant performance under drought will be helpful for the development of drought-adapted crops. In rice, many stress-responsive genes, encoding various antioxidant enzymes, oxido-reductases, kinases, and detoxification proteins, were rapidly induced by drought and contributed to tuning the balance of ROS metabolism in the drought-tolerant cultivars (Gorantla, 2007Gorantla M. et al.Identification of stress-responsive genes in an indica rice (Oryza sativa L.) using ESTs generated from drought-stressed seedlings.J. Exp. Bot. 2007; 58: 253-265Crossref PubMed Scopus (120) Google Scholar; Degenkolbe, 2009Degenkolbe T. et al.Expression profiling of rice cultivars differing in their tolerance to long-term drought stress.Plant Mol. Biol. 2009; 69: 133-153Crossref PubMed Scopus (202) Google Scholar; Yang et al., 2010Yang S. Vanderbeld B. Wan J. Huang Y. Narrowing down the targets: towards successful genetic engineering of drought-tolerant crops.Mol. Plant. 2010; 3: 469-490Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Moreover, many differentially expressed genes under drought lead to alternative physiological and phenotypic responses among varieties (Lenka et al., 2011Lenka S.K. Katiyar A. Chinnusamy V. Bansal K.C. Comparative analysis of drought-responsive transcriptome in Indica rice genotypes with contrasting drought tolerance.Plant Biotechnol. J. 2011; 9: 315-327Crossref PubMed Scopus (209) Google Scholar; Moumeni, 2011Moumeni A. et al.Comparative analysis of root transcriptome profiles of two pairs of drought-tolerant and susceptible rice near-isogenic lines under different drought stress.BMC Plant Biol. 2011; 11: 174Crossref PubMed Scopus (115) Google Scholar). Here, we identified numerous differentially expressed genes between YD2 and YD6 seedlings (Supplemental Table 1). For examples, CuZnSOD, CAT-A, GSR-1, CYP71A1, CYP72A1, CYP72A5, and CYP86A1 were notably induced by drought in YD6, but mildly or not altered in YD2 (Figure 1C). Their up-regulated expression might be conducive to the higher antioxidant enzyme activity and lower ROS accumulation in YD6 compared to those in YD2. Instead, Rboh H was extremely up-regulated by drought in YD2, but down-regulated in YD6 (Figure 1C). Rboh proteins are key regulators of various cellular responses to biotic and abiotic stresses by mediating the generation of ROS in rice (Wong, 2007Wong H.L. et al.Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension.Plant Cell. 2007; 19: 4022-4034Crossref PubMed Scopus (336) Google Scholar). The expression status of Rboh H might be correlated with the different ROS accumulation in YD6 and YD2 seedlings under drought. Moreover, we identified several preferentially expressed genes in YD6 or YD2. APN2-1, GST26, CYPM, CYP-2, STPK-4, and AP4-S1 preferentially expressed only in YD6 (Figure 1D), which encode important function proteins including oxidoreductases, metabolism enzymes, and kinase, respectively. However, 3-HCD, RBCSC, and CYP86A1 were detectable only in YD2. Importantly, 3-HCD encodes a multifunctional peroxisomal isozyme involved in the beta-oxidation of fatty acids associated with generating H2O2 (Arent et al., 2010Arent S. Christensen C.E. Pye V.E. Norgaard A. Henriksen A. The multifunctional protein in peroxisomal beta-oxidation: structure and substrate specificity of the Arabidopsis thaliana protein MFP2.J. Biolog. Chem. 2010; 285: 24066-24077Crossref PubMed Scopus (30) Google Scholar). Its high expression in YD2 might be related to more production of H2O2 under drought compared to that in YD6. Thus, we considered that the transcriptional divergence might be responsible for the different physiological tolerance to drought between YD2 and YD6 seedlings. DNA sequence polymorphisms commonly occur in both coding and promoter regions, which cause transcriptional difference in specific genes and contribute to phenotypic adaptation among different rice varieties (Han and Xue, 2003Han B. Xue Y. Genome-wide intraspecific DNA-sequence variations in rice.Curr. Opin. Plant Biol. 2003; 6: 134-138Crossref PubMed Scopus (88) Google Scholar; Miyashita et al., 2005Miyashita N.T. Yoshida K. Ishii T. DNA variation in the metallothionein genes in wild rice Oryza rufipogon: relationship between DNA sequence polymorphism, codon bias and gene expression.Genes Genet. Systems. 2005; 80: 173-183Crossref PubMed Scopus (7) Google Scholar; Liu et al., 2010Liu L. Zhou Y. Szczerba M.W. Li X. Lin Y. Identification and application of a rice senescence-associated promoter.Plant Physiol. 2010; 153: 1239-1249Crossref PubMed Scopus (38) Google Scholar). We analyzed the DNA polymorphisms of several candidate genes among YD2, YD4, YD6, and 3021. As shown in Supplemental Figure 3, 3-HCD was just amplified from YD2. This result is consistent with its preferential expression in YD2. Similarly, APN2-1 could be amplified from 3021 and YD6, but not from YD4 or YD2. For the four clustered genes (GST26, AP4-S1, N/A-1, and N/A-2), three of them except AP4-S1 were only amplified from YD4 and YD6. For AP4-S1, we found that there were many SNPs and INDELs (insertions and deletions of DNA fragments) within this gene in YD2 and 3021 compared with those in YD6 and YD4 (Supplemental Figure 4). These data implied that YD6 inherited some specific genes from YD4 or 3021, and reflected their exclusive expression status in YD6 against that in YD2. Promoters are considered to be more ‘evolvable’’ than coding regions and play crucial roles in transcriptional regulation. We compared the promoter sequences of RBCSC, CYP86A1, CuZnSOD, CYP72A1, CAT-A, and CYPM among four varieties through PCR amplification (Supplemental Figure 5A) and further sequence analyses. We verified that a 3497-bp fragment was inserted in –321 bp of RBCSC in YD6 and 3021 (Supplemental Figure 6), but not in YD2 or YD4 (Supplemental Figure 5A), suggesting that RBCSC in YD6 originated from 3021, and the insertion disrupted its normal expression in YD6 against that in YD2. Moreover, we confirmed that many SNPs and INDELs existed in the promoters of CAT-A, CYP72A1, CYP86A1, and CYPM among four varieties (Supplemental Figures 5B and 7). Based on the DNA polymorphisms, we inferred that CAT-A and CuZnSOD in YD6 were derived from YD4, while CYP72A1, CYP86A1, and CYPM were inherited from 3021. Importantly, some SNP and INDELs resulted in the changes of numerous putative elements within the promoters of specific genes among four varieties (Supplemental Table 2), which were reported to be involved in controlling the expression of many stress-inducible genes. The changes of many elements might lead to the differential expression of corresponding genes between YD6 and YD2. YD6 was developed from YD2 undergoing a series of breeding processes (Supplemental Figure 1). In this research, we confirmed numerous differentially expressed genes in YD6 against those in YD2 at the genomic DNA and transcriptional levels. The breeding processes might cause the following alterations: (1) some genes, like 3-HCD, in YD2 were lost in YD6; (2) many genes in YD6 including CuZnSOD, CAT-T, APN2-1, N/A-1, N/A-2, GST26, and AP4-S1 were inherited from YD4 or 3021, which did not exist in YD2; (3) InDels or SNPs existed in specific genes such as RBCSC and CYP86A1 in YD2 against that in YD6. Taken together, we proposed that the genetic diversity due to breeding processes gave rise to many differentially expressed genes and led to the different physiological responses to drought between YD2 and YD6. Moreover, these SNPs and INDELs also provided molecular genetic markers to accelerate the breeding progress using molecular marker-assisted methods. Supplementary Data are available at Molecular Plant Online. This work was supported by grants from the National Natural Science Foundation of China (Grant Nos 30971542 and 30730060).