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Molecular Cloning and Characterization of a Cytokinin Dehydrogenase Gene from Upland Cotton (Gossypium hirsutum L.)

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Abstract

Cytokinins are plant hormones that play crucial roles in plant growth and development. Cytokinin dehydrogenase (CKX), regarded as a main negative regulator in cytokinin metabolism in plants, irreversibly degrades cytokinins into adenine/adenosine moiety. A CKX homologous gene, designated GhCKX, was cloned from upland cotton (Gossypium hirsutum L.). Transgenic tobacco plants over-expressing GhCKX showed a typical cytokinin-deficient phenotype, while CKX-silenced tobacco plants exhibited cytokinin over-producing phenotype. Tissue specifically enhancing the expression of GhCKX in the ovule epidermis of transgenic cotton led to a significant decrease of trans-zeatin and trans-zeatin riboside contents in the ovule. The decline of cytokinins resulted in a significant decrease in fiber initials on a single ovule. Our results indicate that GhCKX encodes a functional CKX, and cytokinins may be required for the initiation of cotton fiber cells.

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Abbreviations

CaMV35S:

Cauliflower mosaic virus 35S promoter

CKX:

Cytokinin dehydrogenase

dpa:

Day(s) post anthesis

EST:

Expressed sequence tag

FAD:

Flavin adenine dinucleotide

GA:

Gibberellin

BR:

Brassinosteriod

GUS:

β-glucuronidase

IAA:

Indole-3-acetic acid

Km:

Kanamycin

Cef:

Cefotaxime

As:

Acetosyringone

6-BA:

6-benzylaminopurine

NAA:

1-Naphthaleneacetic acid

IBA:

Indole-3-butyric acid

RT-PCR:

Real-time PCR

Z:

Trans-Zeatin

ZR:

Trans-Zeatin riboside

References

  • Arpat A, Waugh M, Sullivan JP, Gonzales M, Frisch D, Main D, Wood T, Leslie A, Wing R, Wilkins T (2004) Functional genomics of cell elongation in developing cotton fibers. Plant Mol Biol 54(6):911–929

    Article  PubMed  CAS  Google Scholar 

  • Ashikari M, Sakakibara H, Lin S, Yamamoto T, Takashi T, Nishimura A, Angeles ER, Qian Q, Kitano H, Matsuoka M (2005) Cytokinin oxidase regulates rice grain production. Science 309(5735):741–745

    Article  PubMed  CAS  Google Scholar 

  • Bartrina I, Otto E, Strnad M, Werner T, Schmulling T (2011) Cytokinin regulates the activity of reproductive meristems, flower organ size, ovule formation, and, thus, seed yield in Arabidopsis thaliana. Plant Cell 23(1):69–80

    Article  PubMed  CAS  Google Scholar 

  • Beasley C (1973) Hormonal regulation of growth in unfertilized cotton ovules. Science 179(4077):1003–1005

    Article  PubMed  CAS  Google Scholar 

  • Beasley C, Ting I (1973) The effects of plant growth substances on in vitro fiber development from fertilized cotton ovules. Am J Bot 60(2):130–139

    Article  CAS  Google Scholar 

  • Beasley C, Ting I (1974) Effects of plant growth substances on in vitro fiber development from unfertilized cotton ovules. Am J Bot 61(2):188–194

    Article  CAS  Google Scholar 

  • Burke JJ (2009) Cytokinin enhancement of cotton. The United States Patent No.US76634870 B1:1–13

  • Chen JG, Du XM, Zhou X, Zhao HY (1997) Levels of cytokinins in the ovules of cotton mutants with altered fiber development. J Plant Growth Regul 16(3):181–185

    Article  CAS  Google Scholar 

  • Dobra J, Motyka V, Dobrev P, Malbeck J, Prasil IT, Haisel D, Gaudinova A, Havlova M, Gubis J, Vankova R (2010) Comparison of hormonal responses to heat, drought and combined stress in tobacco plants with elevated proline content. J Plant Physiol 167(16):1360–1370

    Article  PubMed  CAS  Google Scholar 

  • Feng D-S, Wang H-G, Zhang X-S, Kong L-R, Tian J-C, Li X-F (2008) Using an inverse PCR method to clone the wheat cytokinin oxidase/dehydrogenase gene TaCKX1. Plant Mol Biol Report 26(3):143–155

    Article  CAS  Google Scholar 

  • Galuszka P, Frébortová J, Werner T, Yamada M, Strnad M, Schmülling T, Frébort I (2004) Cytokinin oxidase/dehydrogenase genes in barley and wheat. Eur J Biochem 271(20):3990–4002

    Article  PubMed  CAS  Google Scholar 

  • Gayle HD (1989) Fiber development in preanthesis cotton ovules. Physiol Plant 75(2):290–294

    Article  Google Scholar 

  • Gu R, Fu J, Guo S, Duan F, Wang Z, Mi G, Yuan L (2010) Comparative expression and phylogenetic analysis of maize cytokinin dehydrogenase/oxidase (CKX) gene family. J Plant Growth Regul 29:428–440

    Article  CAS  Google Scholar 

  • Houba-Herin N, Pethe C, d’Alayer J, Laloue M (1999) Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts. Plant J 17(6):615–626

    Article  PubMed  CAS  Google Scholar 

  • Ivanov Dobrev P, Kamínek M (2002) Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J Chrom 950(1–2):21–29

    Article  CAS  Google Scholar 

  • Ji SJ, Lu YC, Feng JX, Wei G, Li J, Shi YH, Fu Q, Liu D, Luo JC, Zhu YX (2003) Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array. Nucleic Acids Res 31(10):2534–2543

    Article  PubMed  CAS  Google Scholar 

  • Jones RJ, Schreiber BMN (1997) Role and function of cytokinin oxidase in plants. Plant Growth Regul 23(1):123–134

    Article  CAS  Google Scholar 

  • Kim H, Triplett B (2001) Cotton fiber growth in planta and in vitro. Models for plant cell elongation and cell wall biogenesis. Plant Physiol 127(4):1361–1366

    Article  PubMed  CAS  Google Scholar 

  • Konagaya K, Ando S, Kamachi S, Tsuda M, Tabei Y (2008) Efficient production of genetically engineered, male-sterile Arabidopsis thaliana using anther-specific promoters and genes derived from Brassica oleracea and B. rapa. Plant Cell Rep 27(11):1741–1754

    Article  PubMed  CAS  Google Scholar 

  • Kopecny D, Briozzo P, Popelkova H, Sebela M, Koncitikova R, Spichal L, Nisler J, Madzak C, Frebort I, Laloue M, Houba-Herin N (2010) Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study. Biochimie 92(8):1052–1062

    Article  PubMed  CAS  Google Scholar 

  • Kowalska M, Galuszka P, Frebortova J, Sebela M, Beres T, Hluska T, Smehilova M, Bilyeu KD, Frebort I (2010) Vacuolar and cytosolic cytokinin dehydrogenases of Arabidopsis thaliana heterologous expression, purification and properties. Phytochemistry 71(17–18):1970–1978

    Article  PubMed  CAS  Google Scholar 

  • Lee JJ, Hassan OSS, Gao W, Wei NE, Kohel RJ, Chen XY, Payton P, Sze SH, Stelly DM, Chen ZJ (2006) Developmental and gene expression analyses of a cotton naked seed mutant. Planta 223(3):418–432

    Article  PubMed  CAS  Google Scholar 

  • Lee JJ, Woodward AW, Chen ZJ (2007) Gene expression changes and early events in cotton fibre development. Ann Bot 100(7):1391–1401

    Article  PubMed  CAS  Google Scholar 

  • Li XB, Cai L, Cheng NH, Liu JW (2002) Molecular characterization of the cotton GhTUB1 gene that is preferentially expressed in fiber. Plant Physiol 130(2):666–674

    Article  PubMed  CAS  Google Scholar 

  • Luo M, Xiao Y, Li X, Lu X, Deng W, Li D, Hou L, Hu M, Li Y, Pei Y (2007) GhDET2, a steroid 5α-reductase, plays an important role in cotton fiber cell initiation and elongation. Plant J 51(3):419–430

    Article  PubMed  CAS  Google Scholar 

  • Luo K, Zhang G, Deng W, Luo F, Qiu K, Pei Y (2008) Functional characterization of a cotton late embryogenesis-abundant D113 gene promoter in transgenic tobacco. Plant Cell Rep 27(4):707–717

    Article  PubMed  CAS  Google Scholar 

  • Ma X, Feng D-S, Wang H-G, Li X-F, Kong L-R (2011) Cloning and expression analysis of wheat cytokinin oxidase/dehydrogenase gene TaCKX3. Plant Mol Biol Report 29:98–105

    Article  CAS  Google Scholar 

  • Malito E, Coda A, Bilyeu KD, Fraaije MW, Mattevi A (2004) Structures of Michaelis and product complexes of plant cytokinin dehydrogenase: implications for flavoenzyme catalysis. J Mol Biol 341(5):1237–1249

    Article  PubMed  CAS  Google Scholar 

  • Morris RO, Bilyeu KD, Laskey JG, Cheikh NN (1999) Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem Biophys Res Commun 255(2):328–333

    Article  PubMed  CAS  Google Scholar 

  • Pačes V, Werstiuk E, Hall R (1971) Conversion of N6-(Δ2-Isopentenyl)adenosine to adenosine by enzyme activity in tobacco tissue. Plant Physiol 48(6):775–778

    Article  PubMed  Google Scholar 

  • Pepper A, Morse A, Guinel F (2007) Abnormal root and nodule vasculature in R50 (sym16), a pea nodulation mutant which accumulates cytokinins. Ann Bot 99(4):765–774

    Article  PubMed  CAS  Google Scholar 

  • Pu L, Li Q, Fan X, Yang W, Xue Y (2008) The R2R3 MYB transcription factor GhMYB109 is required for cotton fiber development. Genetics 180(2):811–820

    Article  PubMed  CAS  Google Scholar 

  • Ryser U (2000) Cotton fiber initiation and histodifferentiation. In: Basra AS (ed) Cotton Fibers: Developmental Biology, Quality Improvement, and Textile Processing. Food Products Press, New York, pp 1–34

    Google Scholar 

  • Sawan ZM, Mohamed AA, Sakr RA, Tarrad AM (2000) Effect of kinetin concentration and methods of application on seed germination, yield components, yield and fiber properties of the Egyptian cotton (Gossypium barbadense). Environ Exp Bot 44(1):59–68

    Article  PubMed  CAS  Google Scholar 

  • Shi YH, Zhu SW, Mao XZ, Feng JX, Qin YM, Zhang L, Cheng J, Wei LP, Wang ZY, Zhu YX (2006) Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation. Plant Cell 18(3):651–664

    Article  PubMed  CAS  Google Scholar 

  • Tuskan G, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313(5793):1596–1604

    Article  PubMed  CAS  Google Scholar 

  • Wan CY, Wilkins TA (1994) A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Anal Biochem 223(1):7–12

    Article  PubMed  CAS  Google Scholar 

  • Werner T, Motyka V, Strnad M, Schmulling T (2001) Regulation of plant growth by cytokinin. Proc Natl Acad Sci 98(18):10487–10492

    Article  PubMed  CAS  Google Scholar 

  • Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, Schmulling T (2003) Cytokinin-deficient transgenic arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15(11):2532–2550

    Article  PubMed  CAS  Google Scholar 

  • Wu Y, Machado AC, White RG, Llewellyn DJ, Dennis ES (2006) Expression profiling identifies genes expressed early during lint fibre initiation in cotton. Plant Cell Physiol 47(1):107–127

    Article  PubMed  CAS  Google Scholar 

  • Xiao YH, Yin MH, Hou L, Pei Y (2006) Direct amplification of intron-containing hairpin RNA construct from genomic DNA. Biotechniques 41(5):548–552

    Article  PubMed  CAS  Google Scholar 

  • Yang S, Yu H, Goh C (2003) Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid. Plant Mol Biol 51(2):237–248

    Article  PubMed  CAS  Google Scholar 

  • Yang SS, Cheung F, Lee JJ, Ha M, Wei NE, Sze S-H, Stelly DM, Thaxton P, Triplett B, Town CD, Jeffrey Chen Z (2006) Accumulation of genome-specific transcripts, transcription factors and phytohormonal regulators during early stages of fiber cell development in allotetraploid cotton. Plant J 47(5):761–775

    Article  CAS  Google Scholar 

  • Yu XH, Zhu YQ, Chen XY, Xu ZH, Zhou BL, Chen S, Shen XL (2000) Alterations of root and fiber in transgenic cotton plants with chimeric ph/p-ipt gene expression. Acta Bot Sin 42(1):59–63

    Google Scholar 

  • Zalewski W, Galuszka P, Gasparis S, Orczyk W, Nadolska-Orczyk A (2010) Silencing of the HvCKX1 gene decreases the cytokinin oxidase/dehydrogenase level in barley and leads to higher plant productivity. J Exp Bot 61(6):1839–1851

    Article  PubMed  CAS  Google Scholar 

  • Zhang F, Liu X, Zuo K, Zhang J, Sun X, Tang K (2010a) Molecular cloning and characterization of a novel Gossypium barbadense L. RAD-like gene. Plant Mol Biol Report. doi:10.1007/s11105-010-0234-9

    Google Scholar 

  • Zhang J, Liu W, Yang X, Gao A, Li X, Wu X, Li L (2010b) Isolation and characterization of two putative cytokinin oxidase genes related to grain number per spike phenotype in wheat. Mol Biol Rep. doi:10.1007/s11033-010-0367-9

    Google Scholar 

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Acknowledgments

This work was supported by The Major State Basic Research Development Program of China (grant No 2010CB126000 to Y.P) and the National Natural Sciences Foundation of China (grant No. 30530490 to Y.P.).

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Authors and Affiliations

  1. Key Laboratory of Biotechnology and Crop Quality Improvement of Ministry of Agriculture of China, Biotechnology Research Center, Southwest University, Tiansheng Road 216, Beibei, Chongqing, 400716, People’s Republic of China

    Qi-wei Zeng, Shan Qin, Shui-qing Song, Mi Zhang, Yue-hua Xiao, Ming Luo, Lei Hou & Yan Pei

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  1. Qi-wei Zeng
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Correspondence to Yan Pei.

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Zeng, Qw., Qin, S., Song, Sq. et al. Molecular Cloning and Characterization of a Cytokinin Dehydrogenase Gene from Upland Cotton (Gossypium hirsutum L.). Plant Mol Biol Rep 30, 1–9 (2012). https://doi.org/10.1007/s11105-011-0308-3

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