Comparison of Differentially Expressed Genes

Molecular Plant ? Volume 5 ? Number 6 ? Pages 1403–1405 ? November 2012LETTER TO THE EDITOR Comparison of Differentially Expressed Genes Involved in Drought Response between Two Elite Rice Varieties

Dear Editor,

Drought is the most devastating factor limiting the pro-ductivity and geographical distribution of rice (Oryza sativa). Several drought-tolerant varieties have been developed from drought-tolerant germplasm. These varieties make it imper-ative to clarity the mechanism of drought responses at the genomic level, which will provide a foundation for future breeding and genetic engineering efforts (Zhou et al., 2007). 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 veri-fied that YD6seedlings 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., 2004; Moumeni et al., 2011). In rice, drought-tolerant varieties showed lower H2O2level and enhanced antioxidative enzyme activity compared to drought-sensitive varieties (Guo et al., 2006; Rabello et al., 2008). Here, we found that the SOD and CAT activities in YD6were higher than those in YD2under normal condi-tions. When exposed to drought, the increasing extents of SOD and CAT in YD6were 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 treat-ment, both YD6and YD2plants showed slight NBT stain-ing. After drought treatment, the level of NBT staining in YD2 seedlings was more intense than that in YD6 seedlings (Figure 1B), suggesting that YD2plants accumulated much higher superoxide than YD6 plants. These results suggested that YD6showed better drought tolerance associated with higher antioxidant enzyme activity and less ROS accumula-tion 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 et al., 2007; Degenkolbe et al., 2009; Yang et al., 2010). Moreover, many differentially expressed genes under drought lead to alternative physiological and phenotypic responses among varieties (Lenka et al., 2011; Moumeni et al., 2011). 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 CYP86A1were 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 et al., 2007). 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 oxi-doreductases, metabolism enzymes, and kinase, respectively. However, 3-HCD, RBCSC, and CYP86A1 were detectable only in YD2. Importantly, 3-HCD encodes a multifunctional peroxi-somal isozyme involved in the beta-oxidation of fatty acids associated with generating H2O2 (Arent et al., 2010). Its high expression in YD2might be related to more production of H2O2 under drought compared to that in YD6. Thus, we con-sidered that the transcriptional divergence might be respon-sible for the different physiological tolerance to drought between YD2 and YD6 seedlings.

? The Author 2012. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.

doi:10.1093/mp/sss053, Advance Access publication 8 August 2012 Received 22 March 2012; accepted 3 April 2012

1404Letter to the Editor

DNA sequence polymorphisms commonly occur in both coding and promoter regions, which cause transcriptional dif-ference in specific genes and contribute to phenotypic adap-tation among different rice varieties (Han and Xue, 2003; Miyashita et al., 2005; Liu et al., 2010). 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, APN2-1could 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 YD2and 3021compared with those in YD6and 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 cod-ing 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 fur-ther 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), sug-gesting that RBCSC in YD6 originated from 3021, and the inser-tion 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

Figure 1.Analyses of the Physiological and Molecular Difference in Response to Drought between YD2 and YD6 Seedlings.

(A) Comparisons of drought tolerance of YD2, YD4, YD6, and 3021 seedlings. Control: 2-week-old rice seedlings growing under normal conditions. Drought: 2-week-old rice seedlings subjected to successive withdrawing water for 8 d.

(B) Detection of superoxide by NBT staining in YD2 and YD6 seedlings under normal condition (Control) and drought treatment (Drought).

(C) Determination of the differentially expressed genes in 1-week-old plants under drought. YD2 and YD6: plants growing under normal condi-tions; YD2-D and YD6-D: plants subjected to drought.

(D) Confirmation of the preferentially expressed genes in YD6 or YD2. Transcript levels are referred to that of actin in the same samples. Error bars are based on three replicates.

Letter to the Editor 1405

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.

YD6was developed from YD2undergoing 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 transcrip-tional levels. The breeding processes might cause the follow-ing alterations: (1) some genes, like 3-HCD, in YD2 were lost in YD6; (2) many genes in YD6including CuZnSOD, CAT-T, APN2-1, N/A-1, N/A-2, GST26, and AP4-S1were 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 YD2and YD6. Moreover, these SNPs and INDELs also provided molecular genetic markers to accelerate the breeding progress using molecular marker-assisted methods. SUPPLEMENTARY DATA

Supplementary Data are available at Molecular Plant Online.

FUNDING

This work was supported by grants from the National Natural Science Foundation of China (Grant Nos 30971542 and 30730060). ACKNOwleDGMeNTS

We thank Dr Xiqin Fu (China National Hybrid Rice Center, Changsha, Hunan) for providing the rice seeds. No conflict of interest declared.

Hai-wen Zhang a,2, Xiao-wu Pan a,b,2, Yong-Chao li b, li-Yun wan a, Xiao-Xiang li b

and Rong-Feng Huang a,1

a Biotechnology Research Institute, Chinese Academy of Agricultural

Sciences, Beijing 100081, China

b Rice Research Institute, Hunan Academy of Agricultural Sciences,

Changsha 410125, China 1 To whom correspondence should be addressed. E-mail rfhuang@https://www.360docs.net/doc/fa6890549.html,, fax: +86-10-82106143.

2 These authors contributed equally to this work.ReFeReNCeS

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