Os06g0603000

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Annotated Information

Photoperiod-sensitive gene PHOTOPERIOD SENSITIVITY5 (SE5) encoding a heme oxygenase gene，is involved in phytochrome chromophore biosynthesis. It affects Rice's heading and maturity,and it's photoperiod sensitive. Short-day rice can promote early flowering.The photoperiodic sensitivity 5 (se5) mutant of rice, a short-day plant, has a very early flowering phenotype and is completely deficient in photoperiodic response.

Function

PHOTOPERIOD SENSITIVITY 5 ( SE5 ) was first presumed to encode a rice HO with high similarity to Arabidopsis HY1, although enzyme activity of SE5 was not confirmed (Izawa et al. 2000). The se5 mutant has a very early flowering phenotype under both short-day and long-day (LD) conditions, and is completely deficient in photoperiodic response (Izawa et al. 2000).Previous pharmacological results have shown that hematin, an HO-1 inducer, could protect wheat leaves from oxidative damage triggered by paraquat and Peroxide (Sa et al. 2007).In the present study,they showed that the SE5 protein was located in the chloroplast at least, and exhibited HO activity. Subsequently, RNAi knockdown of the rice SE5 gene results in loss of SE5 mRNA and SE5 protein. We also demonstrated that com-pared with wild-type, SE5 RNAi plants were more sus-ceptible to MV treatment. As expected in a se5 mutant (Izawa et al. 2000), there was a similar phenotype of early flowering under LD conditions. By contrast, the addition of exogenous CO aqueous solution partially rescued the cor-responding MV hypersensitivity. Transgenic Arabidopsis plants overexpressing SE5 and HY1 were generated and their corresponding tolerance phenotypes in the presence of MV were characterized. To investigate the physiological role of SE5 in rice, they obtained two knockdown transgenic lines by an RNAi approach (Fig. 3a). T-DNA insertion of the two T0 transgenic lines (RNAi-1 and RNAi-2) was confirmed by PCR-based analysis (Fig. 3b). Gene-specific RT-PCR showed that about 90 % of SE5 transcripts were specifically decreased by SE5 RNAi, whereas transcripts of OsHO2 were not affected in T2 progeny of transgenic plants (Fig. 3c). The protein levels of SE5 determined by western blotting displayed similar decreasing tendencies (Fig. 3d). The above results clearly indicate that only the transcripts and protein levels of SE5 were specifically reduced in SE5 RNAi transgenic plants. Subsequently, RNAi-1 and wild-type plants were selected for further analysis of the expression profiles of SE5 gene and corresponding protein levels in various tissues (Fig. 3e, f). Either SE5 transcripts or SE5 protein were highly expressed in stems and leaves in wild-type, but relatively less in roots and seeds. There was a higher level of SE5 protein in flowers compared to a low abundance of SE5 transcripts. However, the SE5 RNAi plants displayed decreased levels of both SE5 transcripts and SE5 protein. Additionally, except for relatively higher expression in root tissues, there was comparable expression of OsHO2 transcripts in either wild-type or RNAi-1 plants. In comparison with wild-type, the SE5 RNAi plants had weaker growth, with fewer stems and a yellowish color, when grown under LD conditions. For example, 50-day-old SE5 RNAi plants showed an apparent yellowish phenotype (Fig. 4a) and chlorophyll levels decreased to 47.9 and 54.4 % of the fifth and sixth leaves of wild-type, respectively, while corresponding chlorophyll a to b ratios were 14.5 and 14.0 % higher than those of wild-type plants (Fig. 4b).

Location and Expression

SE5 was localized to the chloroplast An 870-bp full length cDNA of SE5 was amplified by RT-PCR from rice seedling leaves. The coding region of SE5 encodes 289 amino acids with a calculated molecular weight of 31.9 kDa. This includes a 64-amino-acid transit peptide identified by the ChloroP algorithm (Emanuelsson et al. 1999; http://www.cbs.dtu.dk/​services/​ChloroP/​), suggesting a mature SE5 protein (mSE5; i.e. without the predicted transit peptide) of 25.6 kDa. To verify the subcellular localization of SE5, we constructed a vector that constitutively expressed SE5–GFP fusion protein using the CaMV 35S promoter. Then, the resulting construct and GFP control plasmid were transformed into Arabidopsis protoplasts, and the fluorescent signals were observed by a confocal laser scanning microscope. The green fluorescent signal of SE5-GFP fusion protein co-localized with the auto-fluorescence of chlorophylls in chloroplasts (Fig. 1), demonstrating that the fusion protein was efficiently targeted to chloroplasts. By contrast, the protoplast transformed with the empty GFP vector alone has green fluorescent signals in the cytosol and nucleus (Fig. 1). Additionally, non-transformed protoplast (control) for auto-fluorescence with the same acquisition parameters was shown.

To confirm that SE5 encodes a HO and to further characterize its properties, the recombinant His-tagged mSE5 protein was induced by IPTG. The recombinant protein was expressed as a soluble protein of 29.1 kDa, approximately corresponding to the molecular weight of the mSE5 protein (25.6 kDa) plus that of 6 × His-tag (0.7 kDa) and the translated vector sequence (2.6 kDa). After purification by Ni-affinity chromatography, it yielded a single band (Fig. 2a, lane 1), which was recognized by the polyclonal antiserum against mSE5 (Fig. 2a, lane 2).

In a subsequent test, they measured the HO activity of mSE5 by spectrophotometrically examining the conversion of heme to BV (Fig. 2b). Absorbance was monitored between 350 and 800 nm, with bound-heme showing strong absorbance at 405 nm and BV at 665 nm. Over a period of 20 min of incubation, the bound-heme peak decreased substantially, accompanied by a concomitant rise in the BV absorbance maxima (Fig. 2b). The reaction rate for the formation of BV was also determined by monitoring absorbance at 665 nm (Fig. 2c). Using the above data, we determined the kinetic constants for the HO reaction from a Lineweaver–Burk plot (Fig. 2c, insert). Under our experimental conditions, the V max value for the complete reaction was estimated as 62.5 nmol BV h−1 mg protein−1 with an apparent K m value for hemin of 2.9 μM. Furthermore, the rate of the mSE5 reaction increased to a peak value at pH 7.0 and declined thereafter (Fig. 2d, left). In contrast, mSE5 enzyme activity increased with rising temperature within 10–50 °C (Fig. 2d, right), comparable to values obtained from Arabidopsis HY1 (Muramoto et al. 2002, Gisk et al. 2010).

Investigation Proceeding

Plant heme oxygenase (HO) catalyzes the oxygenation of heme to biliverdin, carbon monoxide (CO), and free iron (Fe2+)—and Arabidopsis and rice (Oryza sativa) HOs are involved in light signaling. Here, we identified that the rice PHOTOPERIOD SENSITIVITY 5 (SE5) gene, which encoded a putative HO with high similarity to HO-1 from Arabidopsis (HY1), exhibited HO activity, and localized in the chloroplasts. Rice RNAi mutants silenced for SE5 were generated and displayed early flowering under long-day conditions, consistent with phenotypes of the null mutation in SE5 gene reported previously (se5 and s73). The herbicide methyl viologen (MV), which produces reactive oxygen species (ROS), was applied to determine whether SE5 regulates oxidative stress response. Compared with wild-type, SE5 RNAi transgenic plants aggravated seedling growth inhibition, chlorophyll loss and ROS overproduction, and decreased the transcripts of some representative antioxidative genes. By contrast, administration of exogenous CO partially rescued corresponding MV hypersensitivity in the SE5 RNAi plants. Alleviation of seed germination inhibition, chlorophyll loss and ROS overproduction, as well as the induction of antioxidant defense were further observed when SE5 or HY1 was overexpressed in transgenic Arabidopsis plants, indicating that SE5 may be useful for molecular breeding designed to improve plant tolerance to oxidative stress.

Labs working on this gene

1.College of Life Sciences, Cooperative Demonstration Laboratory of Centrifuge Technique, Nanjing Agricultural University, Nanjing, 210095, People’s Republic of China

2. Beckman Coulter Ltd. Co., Nanjing Agricultural University, Nanjing, 210095, People’s Republic of China

3. Jiangsu Province Key Laboratory for Plant Ex-situ Conservation, Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing, 210014, People’s Republic of China

4. Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, People’s Republic of China

5. Key Laboratory of Protection and Development Utilization of Tropical Crop Germplasm Resources, College of Horticulture and Landscape Architecture, Hainan University, Haikou, 570228, People’s Republic of China

6.Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0101, Japan

7.National Institute of Agrobiological Resources, Kannondai, Tsukuba, Ibaraki 305-8602, Japan

8.Research Institute for Advanced Science and Technology, University of Osaka Prefecture, Sakai, Osaka 599-8570, Japan

References

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Structured Information

Gene Name	Os06g0603000
Description	Similar to Heme oxygenase 1 (Fragment)
Version	NM_001064546.1 GI:115468823 GeneID:4341462
Length	4245 bp
Definition	Oryza sativa Japonica Group Os06g0603000, 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 6
Location	Chromosome 6:24730775..24735019
Sequence Coding Region	24730947..24731039,24731154..24731261,24731800..24732023,24734283..24734727
Expression	GEO Profiles:Os06g0603000
Genome Context	<gbrowseImage1> name=NC_008399:24730775..24735019 source=RiceChromosome06 preset=GeneLocation </gbrowseImage1>
Gene Structure	<gbrowseImage2> name=NC_008399:24730775..24735019 source=RiceChromosome06 preset=GeneLocation </gbrowseImage2>
Coding Sequence	<cdnaseq>atggcgcccgcggcagcgtcgctcacggcccccaacgcactcgcggcgacgtcgttgcccttcttgcacggaaggaagagcggcggcggcggcgtgtccgtgcacgccggggcgccttcgccttcgcgggcggtggcggtggtggcgcggaggttgtgggggagcgctagcagtagcaggaggatggtggtggcggcggcgacggcggcggagatggcgcccgcggcgagcggggaggaagggaagccgttcgtggaggagatgagggcggtggccatgcggctgcacaccaaggaccaggccaaggaaggggagaaggagccgcaggcgccgccggtggccaggtgggagccctccgtggacggctacctccgcttcctcgtcgacagcaagctcgtcttcgagacgctcgagaccatcgtcgaccgcgccgccgtcccctggtatgctgagttcaggaatactgggttggagagatcagaacaactgaagaaggatctggaatggttcaaggaacagggtcacacaattccagaaccatctgctcccggcactacatatgcttcctatctggaagagctggctgaaaaggactcccaagcttttatctgccatttctataatgtgtattttgctcatacggctggaggccgaatgattgggaagaaggtctctgagaatattctgaacaagaaggagctggagttctacaaatgggagggcaatctgtcccagctgctgcagaatgtccgcaacaagcttaacgaagtcgcctctagctggacccgggaggagaaggaccattgcctggatgaaacggagaagtcgttctcgtattctggagatctcctccgtcacatattcacctaa</cdnaseq>
Protein Sequence	<aaseq>MAPAAASLTAPNALAATSLPFLHGRKSGGGGVSVHAGAPSPSRA VAVVARRLWGSASSSRRMVVAAATAAEMAPAASGEEGKPFVEEMRAVAMRLHTKDQAK EGEKEPQAPPVARWEPSVDGYLRFLVDSKLVFETLETIVDRAAVPWYAEFRNTGLERS EQLKKDLEWFKEQGHTIPEPSAPGTTYASYLEELAEKDSQAFICHFYNVYFAHTAGGR MIGKKVSENILNKKELEFYKWEGNLSQLLQNVRNKLNEVASSWTREEKDHCLDETEKS FSYSGDLLRHIFT</aaseq>
Gene Sequence	<dnaseqindica>3981..4073#3759..3866#2997..3220#293..737#attcccggccattccaatccactcccaccaccaagcgcgagctcgattatttttttaatcgattttctccgatttaatccgtagaaaattccccactcctcctcctcctccaccgccgccgccgacgccgcactcctcactccgcagaagcaacaccaacgccgcactccaacgccaaccgcctccgcgcgcggcccgtcgccatcgtcgagcaccttcacgcttcctcctcccccgcacggaacggagacccctagcgccgctataagagggagagaggtgggcgggactcatggcgcccgcggcagcgtcgctcacggcccccaacgcactcgcggcgacgtcgttgcccttcttgcacggaaggaagagcggcggcggcggcgtgtccgtgcacgccggggcgccttcgccttcgcgggcggtggcggtggtggcgcggaggttgtgggggagcgctagcagtagcaggaggatggtggtggcggcggcgacggcggcggagatggcgcccgcggcgagcggggaggaagggaagccgttcgtggaggagatgagggcggtggccatgcggctgcacaccaaggaccaggccaaggaaggggagaaggagccgcaggcgccgccggtggccaggtgggagccctccgtggacggctacctccgcttcctcgtcgacagcaagctcgtcttcgagacgctcgagaccatcgtcgaccgcgccgccgtcccctggtgtgagtgatcggaaaagctcttcctcctccgtgctctactttggaccgcttggaatttttgtggatgacgtttttttttagaaaattttgaactgaaatgatatttgattggttcatacggcgacacccatgaaagttggaaatatttctcaccttctgttctagaatagtcaaatagtataattgtttagccagaaaaaaattggatgcacagctttttgtgaaggaaaatgtcaaattgaagttggatgtgccatatggtgggagtaggagcacttgcaaaccccctgttttgagtttggtttggatgcttgagaaaatatatgtaatccaagttaaggtattaaaagactttgagcctatgattatttcatgtcatcgaggaaattgcatcagctttggataagtcagctaccgatctgaaatttaaataaaaaaaatgtatatttccccttctgttacatgatatgctcttccactgaccataagaccagtgccatgtgcaggtggatccaatttaagtaacattcagcccacattttttaaaataaaacaaaggcatttctttgtcattgagaggcatccttgcctggcatgctgctagcaaaacatttggcaggcacagaatatgtttttgggagggtcttgataaaattgtttaggtgtttcgaaagtgttaaaacgctttactggcaacatttctttaagagtatcatcaaaatatcaatgtttgcctatatgtatcaagtaagaagtacaggaagccatgtgcatgtctatttgcagttggttggcacttggcggtataatcatctaaagtagtaccttactattggttcctattctatgggcaaacttgtatcgaccttctcgagagacatcctatattcctattcatggatgccttaaatcactaaaattttcattgtggctccctctgctgtttagaggtggcatccccagcacagttattttctgtatccacactcctgtattgcttcccttaatcatgtataagtcttgtattttggcattgtattgtttcgacacttggaattagaaatccttatattacattaccacttggcatgctatgtgcagaattacctcggtaggtttgtacttgcaaacagcttatgttggcaccatggtaccccaattttacatactcttgttctgttattctgctatggtctggtatgcaaataacacaataaacgttgcctccaattttggggggtgcacatcatgcagttattctgctagttgagttatgggcagcattttctttataacaggatttcttttgaaggaaatccttgagtctttataacagaatctttctggaatccgccttgtccttaacccttgagtctttgttacaccctttcatggacctacgttgtccgtcaatttgcctccactagctctctgtgtattggatggttggatgtgagaagcctctttcttattgatgattactttgcattttgcaaggaaaacatttcactagcaaagaaaattaaataatatataatgttaacaatgataccctttatatcttctagcttgcttgctgaagttttatggtatctggattgtctgcatttatctgtattggtgaaactctagatttttctatttgttttacatttgagttttagaacaacacgtagcttcgtgtttggctagttgaccagttccacttaagtacttctccgtttcatattatagtcgttttgattttgttgtagtcgaactttttaaaatttgaccaaatttgtagaaaaaatatagcaacatattcgacacaaaacaaacatattatccaaatatattaaatgttagttttaatgaaactaatttgatgttgtagatgttgctaaatttttctataaacttcatcaaacttctttaagtttgactaggaaaaaatcaaaacgacttataatatgaaatggagggattagtaaataattgagaagcaaataatgagtacctttattctactcctacataataccgtgtgcaatttgtttgcacctgaaatcttagcaggatagagaacttttgtagttttgtttatgctgtatggaagtatggaacaatgagtcataattacttgaacgtccacataaaatctgtaatattctttttagatggaaaattgaaactactataaaacagctagaagatttcttccatcaatatttgcaagaggctgcttattgacttcctatttatctgtatgtatttcagatgctgagttcaggaatactgggttggagagatcagaacaactgaagaaggatctggaatggttcaaggaacagggtcacacaattccagaaccatctgctcccggcactacatatgcttcctatctggaagagctggctgaaaaggactcccaagcttttatctgccatttctataatgtgtattttgctcatacggctggaggccgaatgattgggaagaaggtacagtttctgtcttttttaatgatgaattttttatatcattcaaaagctcactatcttttggtaacatagttcagaatggattcctataaatgcgtagaatattagctatcgaagcaacaaaactgacactaacagaaagtttgttctttgcttcattgcgctttcctgactgtatagtgtggacacttttgatgtgagatggtttcttttcttaggaatcagattgaccggttgctttggatgcttatattggtattttgttctaacaaaattatagagtacgctagtcgagataatgaaaatactacataaggcagcgagtgctttggcacgcagtcccttttaggatcctcttgccactactgttctcaagttgtgctgctcatagacgtcaaggtttttcaacccacctgacattattacaatgcggttttgtttgggcatatgaatagcagttttccatgcttgccacctcatttggttcaacttgtttgaatttcttaaacgtttaatcatgcccttgttatcatcttaggtctctgagaatattctgaacaagaaggagctggagttctacaaatgggagggcaatctgtcccagctgctgcagaatgtccgcaacaagcttaacgaagtcgcctctgtaagtccaggagctctatttgccacgccactatctcccccaattctttgatacgagcagtttgaaatgttgctaataaccaagctttaattgttccatttgctggccttgcagagctggacccgggaggagaaggaccattgcctggatgaaacggagaagtcgttctcgtattctggagatctcctccgtcacatattcacctaagcttaattgatcacggtgtctgctatctagtcttgataaacgctgtaaatactaaagttcacgtcagtcagacagactgttactggacactgggcagaggtcatttacttactcaaccagttggccttgcctcccatcaaattgccaaattctaataatccttcggtgtgcc</dnaseqindica>
External Link(s)	NCBI Gene:Os06g0603000, RefSeq:Os06g0603000