Os01g0884300

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Annotated Information

Function

 The OsNAC6 gene is a member of the NAC transcription factor gene family in rice. Expression of OsNAC6 is induced by abiotic stresses, including cold, drought and high salinity. OsNAC6 gene expression is also induced by wounding and blast disease. A transactivation assay using a yeast system demonstrated that OsNAC6 functions as a transcriptional activator, and transient localization studies with OsNAC6–sGFP fusion protein revealed its nuclear localization. Transgenic rice plants over-expressing OsNAC6 constitutively exhibited growth retardation and low reproductive yields. These transgenic rice plants showed an improved tolerance to dehydration and high-salt stresses, and also exhibited increased tolerance to blast disease. By utilizing stressinducible promoters, such as the OsNAC6 promoter, it is hoped that stress-inducible over-expression of OsNAC6 in rice can improve stress tolerance by suppressing the negative effects of OsNAC6 on growth under normal growth conditions. The results of microarray analysis revealed that many genes that are inducible by abiotic and biotic stresses were upregulated in rice plants over-expressing OsNAC6. A transient transactivation assay showed that OsNAC6 activates the expression of at least two genes, including a gene encoding peroxidase. Collectively, these results indicate that OsNAC6 functions as a transcriptional activator in response to abiotic and biotic stresses in plants. We conclude that OsNAC6 may serve as a useful biotechnological tool for the improvement of stress tolerance in various kinds of plants.

Expression

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 Expression of OsNAC6 is induced by both abiotic and biotic stresses
 The OsNAC6 gene (AB028185; AK068392; Os01 g0884300; ONAC048) encodes a protein of 303 amino acids containing the NAC domain in its N-terminal region (Figure 1a). The NAC domain contains predicted nuclear localization signals (NLS) at amino acids 71–83 and 107–123. RNA gel-blot and quantitative polymerase chain reaction (PCR) analyses showed that OsNAC6 was induced by dehydration, high salt (250 mM NaCl), cold (4�C), 100 lM ABA, 100 lM methyl

jasmonate (MeJA) (Figure 1b and Supplementary Figure S1) and wounding (Figure 1c). Induction of OsNAC6 was observed in leaves infected with the blast fungus Magnaporthe grisea Kyu89-246 (Figure 1d). We also examined the effects of stress-related chemicals on the expression of OsNAC6 in rice culture cells. Quantitative PCR analysis showed that OsNAC6 was moderately induced by hydrogen peroxide (H2O2) and weakly by the elictor N-acetylchitooligosaccharide (Figure 1e).

 In order to assess the effect of the promoter region on the expression of OsNAC6 under abiotic and biotic stresses in leaves and roots, we generated the transgenic rice plants containing 1.5 kb OsNAC6 promoter–GUS chimeric genes. Quantitative analysis of the OsNAC6 promoter–GUS transgenic rice plants showed that OsNAC6 was induced by dehydration, high salinity, cold, ABA, MeJA, hydrogen peroxide, wounding and blast disease (Figure 1f,g).

Sequences of various cis-acting elements involved in the response to abiotic stresses were identified in the 1.5 kb promoter region of OsNAC6 (Figure 1h). We found three ABA-responsive elements (ABREs; ACGTGG/TC) (Hattori et al., 2002), three recognition sites for MYB (MYBRSs; C/TAACNA/G) (Abe et al., 2003) and six recognition sites for MYC (MYCRSs; CANNTG) (Abe et al., 2003). The OsNAC6 promoter also includes some cis-acting elements involved in the reponse to biotic stresses, such as four W-boxes (TTGAC) (Eulgem et al., 2000) and four GCC boxes (GCCGCC) (Brown et al., 2003), which are known as recognition sites for WRKY and ERF transcription factors, respectively. Additionally, the OsNAC6 promoter has three

(a–c) Relative mRNA levels of OsNAC6 (a), AK104277 (b) and AK110725 (c) are shown.

Fourteen-day-old plants were used to isolate RNA. (i) Relative mRNA level of the genes in the untreated OsNAC6-OX rice plants compared with the mRNA level in the untreated vector control plants. (ii) Relative mRNA level of the genes in the 24 h DEX-treated OsNAC6-GR rice plants compared with the mRNA level in the 24 h DEX-treated vector control plants. (iii) Relative mRNA level of the genes in the POsNAC6-OsNAC6 and the PLIP9-OsNAC6 rice plants treated with 250 mM NaCl for 24 h compared with the mRNA levels in control plants exposed to the same saltstress treatment. (iv) Relative mRNA level of the genes in the stress-treated plants compared with the mRNA level in the untreated plants. Rice seedlings (Nipponbare) were grown hydroponically for 14 days and were then subjected to dry, high-salt (NaCl) and cold stresses for 24 h, and used to prepare total RNAs. (d) Activation of the promoter–GUS fusion gene by OsNAC6 using rice protoplasts. Rice protoplasts were transfected by the reporter plasmids using various sets of effector plasmids [vector containing ubiquitin promoter as a control (ubi-vector) and ubiquitin promoter–OsNAC6 (ubi-OsNAC6)] and reporter plasmids [the AK104277 promoter–GUS fusion (AK104277) and the AK110725 promoter–GUS fusion (AK110725)]. Co-transfection of a constitutively expressed luciferase (LUC) gene using the ubiquitin promoter allowed normalization of expression in independent experiments. Bars indicate the fold of the GUS activity compared with the reporter activity using the ubi-vector.

Evolution

Please input evolution information here. File:Gggg Figure 1. Structure and expression of OsNAC6 in rice. (a) Structure of the OsNAC6 protein. The NAC domain, NAC subdomains A–E, and the putative nuclear localization signal are shown. (b) Quantitative polymerase chain reaction (PCR) analysis of OsNAC6 expression under stress conditions and hormone treatments. Two-week-old rice plants grown hydroponically were dehydrated (dry), transferred to nutrient solution containing 250 mM NaCl, 100 lM ABA, 100 lM methyl jasmonate (MeJA), 100 lM salicylic acid (SA) or 100 lM ethephon, or transferred to and kept at 4�C (cold) for the indicated times. (c) Quantitative PCR analysis of OsNAC6 expression after wounding. The leaves of 2-week-old plants were wounded and kept on water-saturated filter paper for the indicated times. Relative mRNA levels for the wounded leaves (black) and undamaged leaves (white) are shown. (d) Quantitative PCR analysis of OsNAC6 in rice plants infected with blast disease. The leaves of 4-week-old plants were inoculated with rice blast fungus (Magnaporthe grisea). Relative mRNA levels for the infected leaves (black) and uninfected leaves (white) after the indicated times are shown. (e) Expression of OsNAC6 in rice cultured cells. Relative mRNA levels were analyzed using quantitative PCR. Cultured cells were grown in liquid medium containing 20 mM hydrogen peroxide (H2O2), 1 lg ml)1 N-acetylchitooligosaccharide elicitor (elicitor) or the liquid medium (medium) for the indicated times. (f) Quantitative analysis of OsNAC6 promoter–GUS transgenic rice plants under stress and hormone treatments. The 1516 bp region upstream of the start codon (ATG) was used to create rice plants containing the promoter–GUS gene. Stress and hormone treatments were performed as previously described. The plants were transferred from the basal nutrient solution to nutrient solution containing 20 mM H2O2 for hydrogen peroxide treatment. The GUS activities in the 24 h treated and untreated leaves or roots are shown. Relative GUS activities are shown compared with the GUS activity of the untreated leaves. (g) Quantitative analysis of OsNAC6 promoter–GUS transgenic rice plants infected with blast disease. The leaves of 4-week-old plants were inoculated with rice blast fungus. The GUS activities in the infected and uninfected leaves are shown. (h) Distribution of cis-acting elements in the promoter region of OsNAC6. DNA sequences similar to the stress-related cis-acting elements are indicated as follows: open circles, ABRE; closed circles, GCC box; closed inverted triangles, MYB recognition site; closed triangles, MYC recognition site; open diamonds, W-box; open inverted triangles, as1 motif; closed diamond, TATA.

Labs working on this gene

 Biological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki 305-8686, Japan,
 RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan,
 Plant Disease Resistance Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences (NIAS), Tsukuba, Ibaraki 305-8602, Japan,
 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology, Kawaguchi, Saitama 332-0012, Japan, and
 Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

References

 Abe, H., Urao, T., Ito, T., Seki, M., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell, 15, 63–78.
 Aguan, K., Sugawara, K., Suzuki, N. and Kusano, T. (1991) Isolation of genes for low-temperature-induced proteins in rice by a simple subtractive method. Plant Cell Physiol. 32, 1285–1289.
 Aida, M., Ishida, T., Fukaki, H., Fujisawa, H. and Tasaka, M. (1997) Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant Cell, 9, 841–857.

Becker, D. (1990) Binary vectors which allow the exchange of plant selectable markers and reporter genes. Nucleic Acids Res. 18, 203.

 Bray, E.A. (2004) Genes commonly regulated by water-deficit stress in Arabidopsis thaliana. J. Exp. Bot. 55, 2331–2341.
 Brown, R.L., Kazan, K., McGrath, K.C., Maclean, D.J. and Manners, J.M. (2003) A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis. Plant Physiol. 132, 1020–1032.
 Chiu, W.-L., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H. and Sheen, J. (1996) Engineered GFP as a vital reporter in plants. Curr. Biol. 6, 325–330.
 Christensen, A.H., Sharrock, R.A. and Quail, P.H. (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18, 675–689.
 Collinge, M. and Boller, T. (2001) Differential induction of two potato genes, Stprx2 and StNAC, in response to infection by Phytophthora infestans and to wounding. Plant Mol. Biol. 46, 521–529.
 Delessert, C., Kazan, K., Wilson, I.W., Van Der Straeten, D., Manners, J., Dennis, E.S. and Dolferus, R. (2005) The transcription

factor ATAF2 represses the expression of pathogenesis-related genes in Arabidopsis. Plant J. 43, 745–757.

 Dubouzet, J.G., Sakuma, Y., Ito, Y., Kasuga, M., Dubouzet, E.G., Miura, S., Seki, M., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2003) OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J. 33, 751–763.
 Eulgem, T., Rushton, P.J., Robatzek, S. and Somssich, I.E. (2000) The WRKY superfamily of plant transcription factors. Trends Plant

Sci. 5, 199–206.

 Fowler, S. and Thomashow, M.F. (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell, 14, 1675–1690.

Structured Information

Gene Name	Os01g0884300
Description	No apical meristem (NAM) protein domain containing protein
Version	NM_001051551.1 GI:115441472 GeneID:4325006
Length	2486 bp
Definition	Oryza sativa Japonica Group Os01g0884300, complete gene.
Source	Oryza sativa Japonica Group ORGANISM Oryza sativa Japonica Group Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP clade; Ehrhartoideae; Oryzeae; Oryza.
Chromosome	Chromosome 1
Location	Chromosome 1:40154843..40157328
Sequence Coding Region	40155348..40155818,40156680..40156954,40157054..40157219
Expression	GEO Profiles:Os01g0884300
Genome Context	<gbrowseImage1> name=NC_008394:40154843..40157328 source=RiceChromosome01 preset=GeneLocation </gbrowseImage1>
Gene Structure	<gbrowseImage2> name=NC_008394:40154843..40157328 source=RiceChromosome01 preset=GeneLocation </gbrowseImage2>
Coding Sequence	<cdnaseq>atgagcggcggtcaggacctgcagctgccgccggggttccggttccacccgacggacgaggagctggtgatgcactacctctgccgccgctgcgccggcctccccatcgccgtccccatcatcgccgagatcgacctctacaagttcgatccatggcagcttccccggatggcgctgtacggagagaaggagtggtacttcttctccccgcgagaccgcaagtacccgaacgggtcgcggccgaaccgcgccgccgggtcggggtactggaaggcgaccggcgccgacaagccggtgggctcgccgaagccggtggcgatcaagaaggccctcgtcttctacgccggcaaggcgcccaagggcgagaagaccaactggatcatgcacgagtaccgcctcgccgacgtcgaccgctccgcccgcaagaagaacagcctcaggttggatgattgggtgctgtgccggatttacaacaagaagggcgggctggagaagccgccggccgcggcggtggcggcggcggggatggtgagcagcggcggcggcgtccagaggaagccgatggtgggggtgaacgcggcggtgagctccccgccggagcagaagccggtggtggcggggccggcgttcccggacctggcggcgtactacgaccggccgtcggactcgatgccgcggctgcacgccgactcgagctgctcggagcaggtgctgtcgccggagttcgcgtgcgaggtgcagagccagcccaagatcagcgagtgggagcgcaccttcgccaccgtcgggcccatcaaccccgccgcctccatcctcgaccccgccggctccggcggcctcggcggcctcggcggcggcggcagcgaccccctcctccaggacatcctcatgtactggggcaagccattctag</cdnaseq>
Protein Sequence	<aaseq>MSGGQDLQLPPGFRFHPTDEELVMHYLCRRCAGLPIAVPIIAEI DLYKFDPWQLPRMALYGEKEWYFFSPRDRKYPNGSRPNRAAGSGYWKATGADKPVGSP KPVAIKKALVFYAGKAPKGEKTNWIMHEYRLADVDRSARKKNSLRLDDWVLCRIYNKK GGLEKPPAAAVAAAGMVSSGGGVQRKPMVGVNAAVSSPPEQKPVVAGPAFPDLAAYYD RPSDSMPRLHADSSCSEQVLSPEFACEVQSQPKISEWERTFATVGPINPAASILDPAG SGGLGGLGGGGSDPLLQDILMYWGKPF</aaseq>
Gene Sequence	<dnaseqindica>1511..1981#375..649#110..275#caagccctcctctcctcttcccaacactagtaggataaagccacagagagagcagtagtagtagcgagctcgccggagaacggacgatcaccggagaagggggagagagatgagcggcggtcaggacctgcagctgccgccggggttccggttccacccgacggacgaggagctggtgatgcactacctctgccgccgctgcgccggcctccccatcgccgtccccatcatcgccgagatcgacctctacaagttcgatccatggcagcttccccgtacgataatcctcctcctccatcctcccaatcatcaccaccatcaacgccgtcgtgaattgattgattgatttggtttgatttgttggtgttgtgtagggatggcgctgtacggagagaaggagtggtacttcttctccccgcgagaccgcaagtacccgaacgggtcgcggccgaaccgcgccgccgggtcggggtactggaaggcgaccggcgccgacaagccggtgggctcgccgaagccggtggcgatcaagaaggccctcgtcttctacgccggcaaggcgcccaagggcgagaagaccaactggatcatgcacgagtaccgcctcgccgacgtcgaccgctccgcccgcaagaagaacagcctcagggtaagcaaaaaccacacccaagattccatcactaaattcattactaaatctgtgttcatcgtgattattgattaatttagtcacctaattattcgcccaaaaccgcagctcgattcgaacagctggtggtacttctagatggatactactatttagatatttgatatatttattttgcaacttgtttaatcagctcatttcgctttcgaaatgaattgggaggataagcttagcgtggcccacggctttgggccgcagaaattaattggagacgttggctcatctcatctctagggccgcacctacgtggtgcaacttgcgcagccacgatcgaatcgttcgagcgtgaaacccattgccgtcaccacctcgcctcatccctttcagggaccaatcggtttttagccctacgcgcccctgcgatcgcgacgcccacgatagctaaatcccgaaagcaaataagcagtaatcggacagcgactcgaccgggattagttaaacaatggcttgattaattagatgctggaatttggagccttctgataagtttagggcctgtttggcacagctccagctccagcttcaccccttctggagctggagctcagccaaacagtttcggctccaccaaaacggggagtggagctgggtggagctctctcacaaaatgaactagagttgtggagttgggtttaggcagctccacaactccactccagactcaactcctggagttaaatttaggagttggagctgtaccaaacaggcccttagttttgcacttggtactttaatttttttttgagtgagtgtaaatttgtttctaaactttgtttatgaatttgttttgtattggtgcagttggatgattgggtgctgtgccggatttacaacaagaagggcgggctggagaagccgccggccgcggcggtggcggcggcggggatggtgagcagcggcggcggcgtccagaggaagccgatggtgggggtgaacgcggcggtgagctccccgccggagcagaagccggtggtggcggggccggcgttcccggacctggcggcgtactacgaccggccgtcggactcgatgccgcggctgcacgccgactcgagctgctcggagcaggtgctgtcgccggagttcgcgtgcgaggtgcagagccagcccaagatcagcgagtgggagcgcaccttcgccaccgtcgggcccatcaaccccgccgcctccatcctcgaccccgccggctccggcggcctcggcggcctcggcggcggcggcagcgaccccctcctccaggacatcctcatgtactggggcaagccattctagacgaccaaaaaaaaaaaaaaacaaccgcattggcagcaatggtgtcactgaacaccgtgcaggctagctagcttcatggccggtgaactttgactcaggcgagccgccggagttgactcaaagataattaaaagaagtgttttaagtggattggattggattagacagaggagatgaggactcgagaaaggcggcgatgagaccgtggttggggggaccctggcctggactgaacgacgacgaggcagcagcagaaagatggtgcaattgcatcgggtggcatgtcagtgtgtgtgtatagtggcatgtacatagtacatggtgattgattcggtatacagggggctagctttcctgtttctgtttcttcattggttaattattactcccattataaggtcttcttcagggttgctagcttaattaattaattaattagcccagtggttgaagtgtaagtcaaaattcatcaagtcagagactggaataatacaatacagtactg</dnaseqindica>
External Link(s)	NCBI Gene:Os01g0884300, RefSeq:Os01g0884300