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Molecular characterization of Myb transcription factor ( TaMyb ) gene that is expressed in response to hypoxic condition in wheat ( Triticum aestivum L.) roots. Tong Geon Lee 1 , Cheol Seong Jang 1 , Jae Yoon Kim 1 , Jae Han Park 1 , Dae Yeon Kim 1 , Dong Sub Kim 2 , and Yong Weon Seo 1
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Molecular characterization of Myb transcription factor (TaMyb) gene that is expressed in response to hypoxic condition in wheat (Triticum aestivum L.) roots Tong Geon Lee1, Cheol Seong Jang1, Jae Yoon Kim1, Jae Han Park1, Dae Yeon Kim1, Dong Sub Kim2, and Yong Weon Seo1 (1) Division of Biotechnology and Genetic Engineering, Korea University, Anam-Dong, Seongbuk-Gu, Seoul 136-713, Republic of Korea (2) Department of Radiation Plant Breeding and Genetics, Korea Atomic Energy Research Institute, Yuseong-Gu, Daejeon 305-600, Republic of Korea B A Introduction.………………………………………... • Oxygen deficiency associated with waterlogging in the field is one of the major problems in the crop growing areas. The appropriate signaling pathway under oxygen deficiency may help a successful adaptation to change in O2 concentration.……….. • In a previous study, we analyzed 1,344 clones from the cDNA library of hypoxia-stressed wheat roots and registered 1,274 ESTs in the GenBank database (accession nos. DN828708 - DN829789 and DN948989 - DN949180). Several clones including a clone (accession no. DN828996) that shared high homology with a Myb transcription factor gene exhibited various expressions in roots under hypoxia.…..…………………………………….. • We have analyzed and report our results for the tissue specific transcription of the Myb transcription factor gene in wheat roots under oxygen deficiency treatment and abiotic stresses. T C Time (d) 0 1 2 4 5 0 1 2 4 5 TaMyb1 PDC pH O2 concentration (mol m-3) rRNA Time (d) 0 1 2 4 0.011 DOC (mol m-3): TaMyb1 0.037 0.011 0.042 0.033 rRNA E light DOC 0 12 18 21 (h) L N D 0 1 2 4 Time (d) Time (days) C TaMyb1 rRNA rRNA N D DOC (mol m-3): 0.047 0.016 rRNA Fig. 3. Oxygen deficiency treatment and Northern blot hybridization of TaMyb1. (A) Changes in O2 concentration and pH in the solution as treatment time progresses. O2 is presented as a measure of the concentrations of dissolved O2 (mol m-3). The values are means of three replicates ±s.e. (B) Northern blot hybridization of the TaMyb1 and PDC under oxygen deficiency treatment. (C) Northern blot hybridization of the TaMyb1 under oxygen deficient condition combined with dark treatment. (D) Northern blot hybridization of the TaMyb1 under oxygen deficiency, darkness, together with cutting treatment in the roots. (E) Northern blot hybridization of the TaMyb1 under dark condition (normoxia, DOC: > 0.06 mol m-3) in the roots. h, hour(s); d, day(s); C, control; T, treatment; D, dark conditions; L, light conditions; N, normoxia; DOC, dissolved oxygen concentration; PDC, pyruvate decarboxylase. ……………………………………………………………………………………………………………….. TaMyb1 Myb1 MybHv1 Zm38 R2R3 Myb protein Arabidopsis TaMyb1 Myb1 MybHv1 Zm38 R2R3 Myb protein Arabidopsis TaMyb1 Myb1 MybHv1 Zm38 R2R3 Myb protein Arabidopsis TaMyb1 Myb1 MybHv1 Zm38 R2R3 Myb protein Arabidopsis TaMyb1 Myb1 MybHv1 Zm38 R2R3 Myb protein Arabidopsis --------MGRSPCCEKAHTNKGAWTKEEDDRLTAYIKAHGEGCWRSLPKAAGLLRCGKS --------MGRSPCCEKAHTNKGAWTKEEDDRLTAYIKAHGEGCWRSLPKAAGLLRCGKS --------MGRSPCCEKAHTNKGAWTKEEDDRLTAYIKAHGEGCWRSLPKAAGLLRCGKS --------MGRSPCCEKAHTNRGAWTKEEDERLVAYIRAHGEGCWRSLPKAAGLLRCGKS --------MGRSPCCEKEHTNKGAWTKEEDERLVAYIRAHGEGCWRSLPKAAGLLRCGKS MEDYERINSNSPTHEEDSDVRKGPWTEEEDAILVNFVSIHGDARWNHIARSSGLKRTGKS CRLRWINYLRPDLKRGNFSDEEDELIIKLHSLLGNKWSLIAGRLPGRTDNEIKNYWNTHI CRLRWINYLRPDLKRGNFSDEEDELIIKLHSLLGNKWSLIAGRLPGRTDNEIKNYWNTHI CRLRWINYLRPDLKRGNFSHEEDELIIKLHSLLGNKWSLIAGRLPGRTDNEIKNYWNTHI CRLRWINYLRPDLKRGNFTADEDDLIVKLHSLLGNKWSLIAARLPGRTDNEIKNYWNTHV CRLRWINYLRPDLKRGNFTADEDDLIIKLHSLLGNKWSLIAARLPGRTDNEIKNYWNTHI CRLRWLNYLRPDVRRGNITLEEQFMILKLHSLWGNRWSKIAQYLPGRTDNEIKNYWRTRV RRKLTSRGIDPVTHRAINSDHAASNITISFEAAQ---RDDKGAVFRRDAEPTKVAAAAAA RRKLTSRGIDPVTHRAINSDHAASNITISFETAQ---RDDKGAVFRRDAEPTKVAAAAAA RRKLTSRGIDPVTHRAINSDHAASNITISFESAQ---RDDKGAVFRRDAEPAKAAAAAAA RRKLLGRGIDPVTHRPIAADAVTVTTVSFQPSPS-------------------------- RRKLLGRGIDPVTHRPVNAAAATISFHPQPPP---------------------------- QKQAKHLRCDVNSNLFKETMRNVWMPRLVERINAQSLPTTCEQVESMITDPSQPVNEPSP ITHVDHHHRS--NPHHQMEWGQGKPLKCPDLNLDLCISPPS-----HEDPMVDTKPVXKR ITHVDHHHHHRSNPLHQMEWGQGKPLKCPDLNLDLCISPPS-----HEDPMVDTKPVVKR ISHHVDHHHR---SNPQLDWGQGKPLKCPDLNLDLCISPPI-----HEDPMVDTKPVVKR ---------AAAAAAAEAEATAAKAPRCPDLNLDLCISPPCQQQEEEEVDLKPSAAVVKR -------------TTKEEQLILSKPPKCPDLNLDLCISPPSCQEEDDDYEAKPAMIVRAP VEPG---------FVQFSQNHHQQFVPATELSATSSNSPAETFSDVRGGVVNGSGYDPSG EA-----------VVGLCFSCSMGLPRSADCSAAAFMGL------------------- EA-----------VVGLCFSCSMGLPRSADCKCSSFMGL------------------- EAGVGV------GVVGLCFSCSMGLPRSSDCKCSSFMGL------------------- EVLLGGRGHGHGHGGALCFGCSLGVQKGAPGCSCSSSNGHRCLGLRGGMLDFRGLKMK ELQR--------RRGGLCFGCSLGLQKECKCS-------------------------- QTGFG---------EFNDWGCVGGDNMWTDEESFWFLQDQFCPDTTSYSYN------- 52 52 52 52 52 60 112 112 112 112 112 120 169 169 169 146 144 180 222 224 221 197 191 231 250 252 254 255 215 273 T C Time (h) 0 18 24 36 48 0 18 24 36 48 MX A D PX E PC ABA C MX PEG D P NaCl PX Fig. 4. In situ hybridization of the TaMyb1 mRNA in radicle of control and hypoxia-stressed wheat. Radicles from the control (A) and anaerobic stressed (B, C, and D) plants were collected under hypoxia (DOC: 0.011 mol m-3). Solid arrowheads indicate a high positive signal. Magnifications are ×400 (A and B) and ×1000 (C and D). AE, aerenchyma; C, cortex; D, epidermis; E, endodermis; MX, metaxylem; P, phloem; PC, pericycle; PX, protoxylem. Bars = 100 ㎛. …... MX B C AE MX E Citric acid PC AE E rRNA PC D Fig. 5. Northern blot hybridization of TaMyb1 in seminal roots treated with citric acid (10 mM), NaCl (250 mM), polyethylene glycol (PEG, 25%) or ABA (100 µM). h, hours; C, control; T, treatment. ………………………………………… AE Fig. 1. Amino acid sequence alignment of TaMyb1 with those of other plant Myb transcription factors. Accession numbers: TaMyb1 (wheat, DN828996), transcription factor Myb1 (wheat, AAT37167), MybHv1 (barley, CAA50224), Myb-related protein Zm38 (maize, P20025), typical P-type R2R3 Myb protein (rice, AAL84628), AtMYB2 (Arabidopsis, BAA03534).…………………………………………………………... Results and Discussion.…………………………………………………………………………….………… • A clone (accession no. DN828996) contained a 750-bp open reading frame (ORF) encoding for the putative Myb transcription factor with 250 amino acids. This clone was designated as TaMyb1 (Triticum aestivumMyb transcription factor 1).…………………………………………………………………………….. • Presence of TaMyb1 on the 3BL was confirmed by using Chinese Spring aneuploid accessions including nullisomic-tetrasomic and ditelosomic lines.….............……………………………………………... • Dramatic increases in the transcripts of a TaMyb1 occurred under hypoxia. The transcriptional expression of TaMyb1 was continued until approximate anoxia, being enhanced by light under hypoxia, but little expression during anoxia could be shown by Northern blot hybridization. Under normoxic condition, the presence or absence of light did not affect expression of TaMyb1.……………………….... • In situ hybridization of the hypoxic stressed radicle showed that TaMyb1 expression was occurred in the epidermis, endodermis, and cortex which was in contact with the endodermis...……………………… • TaMyb1 transcription levels in roots gradually increased as the result of treatment with NaCl.……….. • The results of our Northern blot analysis and tissue specific hybridization revealed that TaMyb1 expression was strongly related to the oxygen concentration in root environment and the responses to the abiotic stresses in the wheat roots. ………………………………………………………………………… N7D-T7A N5B-T5A N7A-T7B N6D-T6B N2B-T2D N6B-T6D N2D-T2A N3D-T3B N5A-T5D N5D-T5B Dt3BL N7B-T7A N1B-T1A N1D-T1B N3B-T3D N3D-T3A N1A-T1B N6A-T6D N4A-T4B N3A-T3D M Fig. 2. Chromosomal localization of TaMyb1 in aneuploid lines of Chinese Spring. ………………………………………………… TaMyb1 Acknowledgement………………………………………… This work was financially supported by the grant from the Proton Engineering Frontier Project, KAERI, Republic of Korea. This work was partially supported by a grant (20050301-034-432-006-01-00) from BioGreen 21 Program, Rural Development Administration, Republic of Korea.…………………………………………………………. * This study has been accepted in Physiologia Plantarum (PPL-2006-00042.R2).
