Os01g0263300

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Annotated Information

Function

OsPOX1is the only flowerpreferential and cold-inducible peroxidase gene in rice.The cDNA encodes a putative class III peroxidase of 335 amino acids with 77–98% identity with rice peroxidases and namedOsPOX1OsPOX1is coldinducible in floral-organ preferential manner. A promoter region of OsPOX1was isolated, and the transgenic rice plants ofGUSgene driven by the OsPOX1 promoter was investigated under cold-stressed and unstressed conditions.

Expression

  To understand the regulation ofOsPOX1expression, a 1.8 kb

promoter region ofOsPOX1was isolated and fused toβ-glucuronidase(GUS) reporter gene. Transgenic rice plants expressing POsPOX1-GUSshowed minimal GUS activity in both shoots and roots at the vegetative stage. In the flowers at early young microspore stage, GUS activity was detected in the veins and anthers. Interestingly, at the later vacuolated pollen stage, the GUS activity was highly induced by cold stress, suggesting that OsPOX1is a

flower-preferential cold-responsive gene in ric.

Genomic structure ofOsPOX1on chromosome 1. ATGand arrow represent the start codon and the direction of translation, respectively. OsPOX1coding regions are shown inblack boxeswith introns aswhite boxes. Barindicates the position of 0.5 kb ddOs319 fragment on OsPOX1gene..

 the expression ofOsPOX1in the seedling was not changed by various stress treatment as shown:

The nucleotide and deduced amino acid sequences of OsPOX1. The positions of nucleotides are shown on the left and the positions of amino acids on theright. Asteriskindicates the translation termination site. The forward and reverse primers used in the nested PCR areunderlined..

The deduced OsPOX1 protein was compared with two peroxidases of rice, Rice-PrxRP and Rice-POX (Ito et al. 2000) and peroxidases of Arabidopsis(AtPOX; Capelli et al. 1996), Asparagus (AoPOX; accession no. BAA94962), tobacco (NtPOX; accession no. AAK52085), and soybean (GmPOX; Yi and Hwang 1998) using the ClustalW program (Thompson et al. 1994). The results of comparison are displayed using GeneDoc (Nicholas et al.1997). The lines indicate N-terminal signal peptide, a distal heme-binding domain (I), a central conserved domain (II), and a proximal heme-binding domain (III), respectively. Arrowheads and arrows indicate eight conserved Cys residues which form a disulfide bond network and three His residues involved in the acid/base catalysis and hemestabilization (Welinder 1992). Identical amino acids are highlighted in black. Identical amino acids in six or five sequences are shown ingray.

Evolution

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Labs working on this gene

   Plant Samples and Bacterial Strains

Field-grown rice plants (Oryza sativacv. Dongjin) were treated as described by Kim et al. (2007). After cold-stress treatment, plant samples were immediately frozen in liquid nitrogen and stored at −70°C. For GUS assay, various tissues at different stages were immediately immersed into the X-Glu solution. Two Escherichia colistrains, XL-1 Blue MRF', and

   MC1000 were used as hosts for molecular cloning. The f1 helper phage, R408, was used for excision of the pBluescript phagemid from the λUNI-Zap XR vector (Stratagene, USA).
   Isolation of the Full-Length Sequence of OsPOX1

For obtaining the full-length cDNA of ddOs319, reverse transcriptase-mediated PCR (RT-PCR) was performed using specific primers designed based on ddOs319 sequence and its corresponding genomic sequence in the NCBI database. Total RNA was isolated as described by Koh et al. (2007), and cDNA templates were synthesized using 5μg of total RNAs from the cold-treated flowers,0.5 mM dNTP, 0.1μM of a specific primer (POX-A1, 5′-TAG AAC AAC CAA ATG AAC TC-3′), and 200 U of SuperScript™II reverse transcriptase (Invitrogen, USA).RT-PCR was performed in 50μl solution containing a 2-μl aliquot of the cDNA reaction, 0.2μM of specific primers (POX-S1, 5′-AGT GCA GTG CAG TGC AGT GC-3′;POX-A1), 0.2 mM dNTPs, and 5 units of Ex-Taq DNA polymerase (TakaRa, Japan). The reaction included an initial 3 min denaturation at 94°C, followed by 30 cycles of PCR (94°C, 1 min; 54°C, 1 min 30 sec; 72°C, 1 min 30 sec), and a final 10 min at 72°C. Oneμl of the primary PCR products were used for a nested PCR reaction containing 0.2μM of specific primers (POX-S2, 5′-GCG AAG GTT TAC TTG GCG AC-3′; POX-A2, 5′-TGA ACT CAT TTT ATA TAT TG-3′), 0.2 mM dNTPs, and 5 units of Ex-Taq DNA polymerase. The reaction included an initial 3-min denaturation at 94°C, followed by 30 cycles of PCR (94°C, 1 min; 47°C, 1 min 30 sec; 72°C, 1 min 30 sec), and a final 10 min at 72°C.

   Northern Blot Analysis

Ten μg of total RNA was denatured and resolved by electrophoresis through 1.3% agarose gel, blotted onto anylon membrane, and then hybridized at 42°C with 32

Plabeled OsPOX1probe for 16 to 24 h (Sambrook et al.1989). The membrane was washed in 0.1× SSC, 0.1% SDS at room temperature, and then exposed to either Hyperfilm™MP film (Amersham, UK) or a phosphoimage plate of BAS 1500 (Fuji, Japan).

Northern analysis ofOsPOX1in response to various stress. a Northern analysis ofOsPOX1in response to cold stress (12°C for 4 days) at reproductive stage. Total RNA (10 μg) isolated from unstressed leaves (L) or flowers (F) and cold-treated leaves (CL)or flowers (CF) was separated on agarose gel, blotted onto a nylon membrane, and hybridized with radio-labeledOsPOX1cDNA. EtBrstained rRNA bands are shown inlower part of thepanel. bNorthern analysis ofOsPOX1in response to cold stress (12°C for 4 days) at reproductive stage. Total RNA (10μg) isolated from seedlings treated with various stress of cold (4°C), physical wounding, ABA (100μM), drought, and salt (250 mM) was separated on agarose gel, blotted onto a nylon membrane, and hybridized with radio-labeledOsPOX1cDNA

   Isolation of the OsPOX1Promoter

For obtaining the promoter region ofOsPOX1, PCR was performed with genomic DNA using specific primers designed based on its genome sequence in the NCBI database. The primary PCR was in 50 μl solution containing 0.1 μg of rice genomic DNA, 0.2 μMof specific primers (POX1-P5, 5′-GAT CTA CAT CAG AAC AAG G-3′; POX1-P7, 5′- AGC ATC AAC AGG CAA CC-3′), 0.2 mM dNTPs, and 2.5 units of Ex-Taq DNA polymerase (TakaRa, Japan). The reaction included an initial 5-min denaturation at 94°C, followed by 30 cycles of PCR (94°C, 1 min; 53°C, 1 min 30 sec; 72°C, 2 min 10 sec), and a final 10 min at 72°C. Aliquots (1μl) from 20-fold dilutions of the primary PCR products were applied directly to nested PCR reaction (50μl) containing 0.2 μM of specific primers (POX1-P6, 5′-GTT CTA GAG CTA TAC ACG-3′; POX1-P8, 5′-AAG GTA CCG GAA GTC GCC AAG-3′), 0.2 mM dNTPs, and 2.5 units of Ex-Taq DNA polymerase. The reaction included an initial 5-min denaturation at 94°C, followed by four cycles of PCR (94°C,1 min; 42°C, 1 min 30 sec; 72°C, 2 min 10 sec), and then by 26 cycles of PCR (94°C, 1 min; 59°C, 1 min 30 sec; 72°C,2 min 10 sec) and a final 10 min at 72°C.

OsPOX1promoter sequence, a schematic diagram of POsPOX1-GUSconstruct, and histochemical localization of GUS activity in the POsPOX1-GUStransgenic plants. a The nucleotide sequence of ca. 1.8 kb ofOsPOX1promoter region. The positions of nucleotides are shown on theleft. Putative CAAT, TATA boxes, and start codon are underlined. bChimeric gene construct ofGUSunder theOsPOX1 promoter. TheOsPOX1promoter region was fused to theGUSgene of the binary vector, generating pSK228 and introduced into rice genome byAgrobacterium-mediated cocultivation method.RB, right border of T-DNA; POsPOX1, OsPOX1promoter; GUS, β-glucronidase gene; TNOS, nopaline synthase terminator; P35S, CaMV 35S promoter; hph, hygromycin phosphotransferase gene; 7’, termination region of Transcript 7 of pTiA6;LB, left border of T-DNA.

  DNA Sequence Analysis

Both strands of the PCR-amplified promoter as well as cDNA were sequenced by the dideoxynucleotide chain termination method (Sanger et al. 1977) with an ABIPRISM Autosequencer (Perkin-Elmer, USA). The sequences were then compared with those in the NCBI database,using the BLAST program (Altschul et al.1990), and the nucleotide/amino acid sequences were analyzed using softwares, ClustalX (Thompson et al.1997) and GeneDoc (Nicholas et al. 1997).

   Construction of Rice Transformation Vector,POsPOX1-GUS(uidA)

The nested PCR product ofOsPOX1promoter was purified with PCR clean Up-M™kit (Viogene, USA) and digested withXbaI and KpnI. The 1.8 kb of OsPOX1promoter region was ligated into a promoter-less plant transformation binary vector containing GUSreporter gene, generating pSK228. The vector was transferred into Agrobacterium tumefaciens strain LBA4404 by the freeze–thaw method(An et al. 1988).

   Rice Transformation

Rice was transformed via theAgrobacterium-mediated cocultivation method, as previously described (Hiei et al.1994; Lee et al. 1999). All the transgenic plants were produced on a 40-mg L−1 hygromycin B-containing medium and were transferred to the greenhouse afterward.To determine whether the plants were truly transgenic, a GUSportion was amplified by PCR using primers (GUS5′,5′-CTA CAC CAC GCC GAA CAC CT-3′; GUS3′,5′-CAG GCA CAG CAC ATC AAA GA-3′). After harvesting seeds, 15 seeds from each line were grown in paddy field for harvesting seeds. The seeds of T2 generation weregerminated in Petri dish containing SDW for 3 days, and then the hygromycin B media (200 mg L−1) was added for 4 days. From the plates ~10 hygromycin resistant plants were transferred into Magenta-box with non-hygromycin media for further growth of ca. 1 week. After then, the seedling plants were transplanted to greenhouse for the GUS assay.

   GUS Assay

GUS assay was performed according to Jefferson et al.(1987). Various tissues of transgenic plants at 5–10 days before heading were incubated in 100 mM sodium

phosphate buffer (pH 7.0) containing 0.1% (w/v)5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 10 mM EDTA, 0.5% (v/v) Triton X-100, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 20% methanol at 37°C in the dark for 10 to 13 h. To allow a better penetration of substrate, vacuum was applied to samples in the X-Gluc solution for 2 min. After staining, the tissues were cleared using ethanol at 50°C several times and observed under a microscope (Leica, Germany) using dark-field illumination.

cGerminating seed under normal growth condition.dGerminating seed exposed to cold stress (4°C for 6 h). e Seedling of ca. 10 days old under normal growth condition.f Flower at early young microspore stage under normal growth condition.gFlower at vacuolated pollen stage under normal growth condition.hFlower at vacuolated pollen stage exposed to cold stress (12°C for 4 days). Transverse section at the position of upper barofhgivesi andi’(enlargement). Transverse section at the position of middle bar ofhgivesj andj’ (enlargement). Transverse section at the position oflower bar ofhgiveskandk’(enlargement). Bar: c~h1 mm;i, j, k0.5 mm;i’,j’,k’ 0.2 mm

References

1. Sung-Hyun Kim;Hong-Seok Choi;Young-Chan Cho;Seong-Ryong Kim

 Cold-Responsive Regulation of a Flower-Preferential Class III Peroxidase Gene, OsPOX1, in Rice (Oryza sativa L.)
 Journal of Plant Biology, 2011, 55(2): 123-131

2. Sung-Hyun Kim;Ji-Youn Kim;Soo-Jin Kim;Kyung-Sook An;Gynheung An;Seong-Ryong Kim

 Isolation of cold stress-responsive genes in the reproductive organs, and characterization of the OsLti6b gene from rice (Oryza sativa L.)
 Plant Cell Reports, 2007, 26(7): 1097-1110