Os01g0797600

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Annotated Information

Function

• OsERF3 (Os01g0797600), is AP2 domain-containing protein, it is found to be significantly up-regulated in both genotypes under cold stress.
• OsERF3 (Os01g0797600) belongs to class II ERFs with a conserved EAR-motif (DLNRPPP) at the C terminal (Nakano et al. 2006). In rice, OsERF3 participated in the resistance to disease and the herbivore-induced defense responses via mediating different hormone pathways (Luet al. 2011).We also found that OsERF3 negatively

affected ethylene production and drought tolerance in rice(Wan et al. 2011). However, little is known about whether its EAR motif is involved in the regulation of ethylene synthesis and drought response. Here, we generated transgenic rice overexpressing full-length (OE) and the mutation of OsERF3 with the A680/C substitution (mEAR), respectively. The phenotypic analyses suggested that mEAR lines showed better drought tolerance and more ethylene emission than those of OE lines and wild type (WT) plants. At the transcriptional level, the substitution of Leu/Ala disrupted the repression of ACO2 and ACS6 by OsERF3 in mEAR lines. Moreover, ACS2 was significantly up-regulated in mEAR lines compared with that in WT plants. Taken together, our results indicate that the EAR motif is required for OsERF3 to repress the expression of ethylene biosynthesis genes, deepening our understanding of the regulatory function of ERF proteins involved in ethylene biosynthesis and stress response.

• OsERF3 is a transcriptional repressor with an ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif (F/LDLNxxP), which transcriptionally represses the ethylene emission and drought tolerance in rice. However, its molecular mechanism to explore repression function remains unknown.
• OsERF3, an AP2 (APETALA 2 Gene)/ERF transcription factor, binds the LRK6 promoter at this new motif, which might cause differential expression of LRK6 in the 93-11/Nipponbare hybrid.The DNA binding ability ofOsERF3’s ERF/AP2 domain was analysed using the one-hybrid assay.

Expression

• The expression of OsERF3 was induced by drought, salt, ACC and ABA treatment. In addition, it showed a higher expression level in the root and sheath than that in the leaf.
• The full length of OsERF3 gene was obtained from the cDNA library using specific primers.The full length of OsERF3 gene was obtained from the cDNA library using specific primers. The mutation of EAR motif of OsERF3 (mEAR) was obtained by replacing the base of A680 with C (Fig. 1a) through PCR amplifications using specific primers (Table S1). Then, the full-length cDNA of OsERF3 and mEAR were inserted into pCAMBIA1307 modified from pCAMBIA1300, respectively. For the modification of pCAMBIA1307 vector (Fig. S1), we inserted a fragment including a CaMV 35S promoter and multiple cloning sites from vector pBluescript into the SstI and KpnI of pCAMBIA1300 vector. To generate the ERF3:GUS construct, the 1.0 kb promoter region upstream to start codon of OsERF3 was amplified using specific primers (OsERF3P-F and OsERF3P-R) (Table S1). Then, the fragment digested with EcoRI and NcoI was cloned into the multiple cloning sites of pCAMBIA1391Z vector. All of the transgenic rice plants were generated through Agrobacterium-mediated transformation. T1 seedlings were examined by PCR amplifications at the DNA and RNA levels with specific primers (Table S1); T2 and T3 seeds were screened by germinating with the selection of 50 ng/l hygromycin.

Fig. 1. Comparison of EAR motif and phylogenetic analysis of EAR motif containing ERF proteins from rice and Arabidopsis.

• The expression of OsERF3 was highly induced by ABA at 1 h, and reached peak at 2 h. Similarly, it was rapidly inducible by ACC at 0.5 h and peaked at 2 h, and then it decreased to normal level at 8 h (Fig. 2a). Moreover, OsERF3 was up-regulated by drought and peaked at 2 h. Similarly, OsERF3 was quickly induced by high salinity and peaked at 0.5 h. These results indicate that OsERF3 might be associated with ethylene- and ABArelated environmental stimuli in rice.the potential cis-elements withinthe 1 kb region (1,040 bp) upstream to the start codon(Table S2). There were more important elements involved in different hormones or stress signaling (including ethylene, ABA, Auxin and GA as well as dehydration, cold and light) and specific tissues. These data also support the molecular cues for the expression of OsERF3 in response to different hormones and stress treatments.
• As shown in Fig. 2b, the expression of OsERF3 in the root and sheath was two and three times higher than that in the leaf at the seedling phase, respectively. Moreover, the expression of GUS driven by the promoter of OsERF3 in transgenic rice was analyzed. Consistently, the GUS staining was observed in all parts of seedlings, but it showed more intense in sheath and root than that in leaf(Fig. 2c), further confirming its expression in seedlings.

Fig. 2.Expression of OsERF3 .

• The OsERF3 gene was predicted to encode a protein of 235 amino acids, with a calculated molecular weight of 24.3 kDa. OsERF3 is an AP2/ERF protein with an N-terminal domain containing one highly conserved ERF motif, and a C-terminus containing an y30 amino acid domain comprising a conserved ERF-associated amphiphilic repression(EAR) motif .

Mutation

• The physiological analysesransgenic of rice overexpressing full-length OsERF3 (OE) and its mutation of EAR motif with the A680/C substitution (mEAR), showed that mEAR lines showed better drought tolerance and more ethylene emission compared with those of OE lines and wild type plants.However, the repression of OsERF3 was eliminated in mEAR lines. Specifically, ACS2 was up-regulated in mEAR lines compared with that in OE lines andWTplants, suggesting that the Leu/Ala substitution

within the EAR motif resulted in loss of repression of OsERF3.theEARmotif is required for OsERF3 to transcriptionally regulate the ethylene synthesis and drought tolerance in rice, providing new insight to the roles of ethylene-response factor proteins in regulating ethylene biosynthesis and stress response.

• It has been reported that series stress-related genes (Luet al. 2011) and ethylene synthesis-related genes were regulated by OsERF3 in rice (Wan et al. 2011). In the present work, we focused on whether EAR motif is required for the repression of OsACS2, OsACS6, and OsACO2 by OsERF3 in rice. The expression level of these

genes in OE and mEAR lines was analyzed. As shown in Fig. 5, the expression level of ACO2, ACS2, and ACS6 was decreased in OE lines. Interestingly, the expression of ACS2 in mEAR lines was much higher than that in WT; the expression level of ACO2 and ACS6 in mEAR lines showed similar to that in WT. These results revealed that the mutation of Leu227/Ala within the EAR motif disrupted the repression function of OsERF3, implying that the EAR motif is essential for OsERF3 to repress the expression of ACO2, ACS2, and ACS6 in rice.

Fig. 4. Transcriptional repression of OsERF3 in the expression of ethylene biosynthesis-related genes via EAR motif.

• Plasmid rescue and cDNA sequencing identified three identical cDNA clones encoding an AP2/ERF protein, designated rice ERF3 (OsERF3). Yeast clones containing OsERF3–GAL4 consistently demonstrated strong growth on 5 mM 3-amino-1,2,4-triazole (3-AT) in repeated experiments. The OsERF3 cDNA sequence encoded a complete

open reading frame consisting of 708 nucleotides with no introns.

Evolution

Compared the drought tolerance among the transgenic lines (OE and mEAR) and wild type (WT) Nipponbare. As shown in Fig. 3a, most of the OE seedling leaves turned withered after the 2-week-old seedlings were exposed to successive drought by withholding water for 6 days, while WT and mEAR lines still grew well. When the seedlings of WT and OE lines exhibited serious stress symptom at the eighth day, mEAR lines showed mild stress symptom (Fig. 3a). After treated for 9 days, the resulting seedlings were recovered normal growth with watering for 7 days. The survival rates of seedlings in WT, OE lines, and mEAR lines were about 40, 19.5–28, and 43–49 %, respectively (Fig. 3b). Student’s t test analyses showed thatthe differences among WT and two OE lines were significant(P\0 05 or P\0 01). Although there was no significantdifference between WT and mEAR lines, the survival rate in mEAR lines was slight higher than that in WT plants. These findings suggest that the Leu227/Ala substitution results in the complete loss of negative regulation of drought tolerance by OsERF3. We also found that the OE lines showed drought sensitive at the flowering stage (Fig. S4). Taken together, our research demonstrates that that the EAR motif is necessary for OsERF3 to negatively regulate drought tolerance in rice.

Fig. 3. Negative regulation of OsERF3 in the drought tolerance through its EAR motif..

sequence analysis

The rice genome encodes for 163 putative AP2/ERF family proteins based on their conserved AP2 domain sequence (Sharoni et al. 2011). OsERF3 encodes 235 amino acids and belongs to class II ERF proteins. Sequence alignments showed that OsERF3 has a conserved AP2 DNA-binding domain and an EAR motif (DLNRPPP), which were highly similar to the other class II ERF genes from Arabidopsis and rice. Especially, the three amino acids (DLN) were highly conserved in all candidate proteins (Fig. 1b, Fig. S2), suggesting they might play important roles in EAR motif-containing proteins. Analyses with phylogenetic tree of the protein sequences showed that OsERF3 was grouped in a cluster with Os05g0497300, with identity of 67.2 %. These two proteins showed a close evolutionary relationship with Os04g0610400, AtERF4, AtERF8, and AtERF11 (Fig. 1c), coinciding with the recent report on the syntenic relationships of rice ERF proteins (Rashid et al. 2012). Of these candidate genes, AtERF11 (Li et al. 2011), AtERF3 (Ohta et al. 2001), AtERF4 (Yang et al. 2005), and AtERF7 (Song et al. 2005) were proved to function as transcriptional repressors involved in modulating hormone response and stress tolerance. These analyses imply that OsERF3 might be a transcriptional repressor and play roles in mediating stress response.

Labs working on this gene

• Institute of Crop Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing100081, China
• Shenzhen Institute of Breeding and Innovation, Chinese Academy of Agricultural Sciences, Shenzhen 518083, China
• Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
• National Key Facility of Crop Gene Resources and Genetic Improvement, Beijing 100081, China
• Rice Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China
• State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, 220 Handan Road, Shanghai 200433, China.

References

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Structured Information

Gene Name	Os01g0797600
Description	Ethylene responsive element binding factor3 (OsERF3)
Version	NM_001051054.1 GI:115440478 GeneID:4327621
Length	1090 bp
Definition	Oryza sativa Japonica Group Os01g0797600, 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 1:35522840..35523929
Sequence Coding Region	35522920..35523627
Expression	GEO Profiles:Os01g0797600
Genome Context	<gbrowseImage1> name=NC_008394:35522840..35523929 source=RiceChromosome01 preset=GeneLocation </gbrowseImage1>
Gene Structure	<gbrowseImage2> name=NC_008394:35522840..35523929 source=RiceChromosome01 preset=GeneLocation </gbrowseImage2>
Coding Sequence	<cdnaseq>atggcgcccagagcagctacggtggagaaggttgctgtggcgccacccaccgggcttggtcttggcgtcggcggaggtgtcggagccgggggtcctcactacaggggcgtccgcaagcgcccgtgggggcgttacgcagcggagatccgtgaccctgccaagaagagccgggtgtggctcggtacctacgacacggcagaggaggccgcccgcgcctacgacgccgccgctcgagagttccggggtgccaaggcaaaaacaaactttccgtttgcatcacagtcgatggtcggctgtggcggcagccccagcagcaatagcacggtagacaccggtggcggcggggttcagacgcctatgcgggccatgcctctgccgccgactctggacttggatttgttccaccgcgcggctgctgtgactgcagtcgccggcaccggcgttcgctttcctttcagaggatatcccgttgcacgtccagcaacgcatccttactttttctatgagcaggctgcagcggctgccgcagctgaggctggataccgtatgatgaagcttgcaccgccggtcaccgtggcggcggttgcacaaagtgactccgactcctcgtcggtggttgatctcgcgccgtcacctccagcggttacggcgaacaaggcggcagctttcgatctggatctgaaccggccgccgccggtagagaactag</cdnaseq>
Protein Sequence	<aaseq>MAPRAATVEKVAVAPPTGLGLGVGGGVGAGGPHYRGVRKRPWGR YAAEIRDPAKKSRVWLGTYDTAEEAARAYDAAAREFRGAKAKTNFPFASQSMVGCGGS PSSNSTVDTGGGGVQTPMRAMPLPPTLDLDLFHRAAAVTAVAGTGVRFPFRGYPVARP ATHPYFFYEQAAAAAAAEAGYRMMKLAPPVTVAAVAQSDSDSSSVVDLAPSPPAVTAN KAAAFDLDLNRPPPVEN</aaseq>
Gene Sequence	<dnaseqindica>81..788#cacacccaaacccaacctcccaaaacacccacccggtttaccagagatccgcgcccgccacttgtaaacctgctgcacccatggcgcccagagcagctacggtggagaaggttgctgtggcgccacccaccgggcttggtcttggcgtcggcggaggtgtcggagccgggggtcctcactacaggggcgtccgcaagcgcccgtgggggcgttacgcagcggagatccgtgaccctgccaagaagagccgggtgtggctcggtacctacgacacggcagaggaggccgcccgcgcctacgacgccgccgctcgagagttccggggtgccaaggcaaaaacaaactttccgtttgcatcacagtcgatggtcggctgtggcggcagccccagcagcaatagcacggtagacaccggtggcggcggggttcagacgcctatgcgggccatgcctctgccgccgactctggacttggatttgttccaccgcgcggctgctgtgactgcagtcgccggcaccggcgttcgctttcctttcagaggatatcccgttgcacgtccagcaacgcatccttactttttctatgagcaggctgcagcggctgccgcagctgaggctggataccgtatgatgaagcttgcaccgccggtcaccgtggcggcggttgcacaaagtgactccgactcctcgtcggtggttgatctcgcgccgtcacctccagcggttacggcgaacaaggcggcagctttcgatctggatctgaaccggccgccgccggtagagaactagctcaggatgggttagctgacgactttgtagtttctctcttattttcttctttgatggatatttctctccgatgttttggtcctctgtgtttttgtttagtagcctgtgagagacggaagagccttgtaaatagtttttctgccgagggcgaaattcatcttgggatctgttaattagaacagatcatgccggcgatgagatggactaaaccgtggagtgtatgtattcctttatattagtatgaagaaattattcagaaagtcacaaaaatatctgtgcacactgagtttgattgacgtttt</dnaseqindica>
External Link(s)	NCBI Gene:Os01g0797600, RefSeq:Os01g0797600