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Isolation of cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase gene promoters from Leucaena leucocephala, a leguminous tree species, and characterization of tissue-specific activity in transgenic tobacco

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Abstract

Promoter sequences of a 795 bp cinnamoyl CoA reductase (LlCCR) and 1,882 bp cinnamyl alcohol dehydrogenase (LlCAD) genes were isolated from Leucaena leucocephala, a leguminous tree species by genome walking, and analysed using bioinformatics tools. This revealed presence of cis-elements such as AC-boxes, XYLAT, WRKY, and MYB binding sites in addition to CAAT and TATA boxes. For functional characterization, each of LlCCR and LlCAD promoter sequences were fused to β-glucuronidase (GUS) reporter gene, immobilized into pBI101 plasmid, and introduced into tobacco via Agrobacterium tumefaciens strain LBA4404. Histochemical observations of transgenic lines indicated tissue-specific expression of GUS in the vascular tissues of leaves, stems, and roots. These results demonstrate that GUS expression driven by either LlCCR or LlCAD promoters were involved in lignifying tissues, and more specifically in differentiating xylem cells. This observed tissue-specific expression driven by either LlCCR or LlCAD promoters is sufficient for reducing the lignin content only in vascular tissues, thus overcoming the risks and challenges associated with down-regulation of lignin content in whole plants.

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Abbreviations

CCR:

Cinnamoyl CoA reductase

CAD:

Cinnamyl alcohol dehydrogenase

PAL:

Phenylalanine ammonia-lyase

C4H:

Cinnamate 4-hydroxylase

4CL:

4-Coumarate CoA ligase

F5H:

Ferulate 5-hydroxylase

CCoAOMT:

Caffeoyl-CoA O-methyltransferase

AEOMT:

Multi-functional O-methyltransferase

GUS:

β-Glucuronidase

MS:

Murashige and Skoog

NCBI:

National centre for biotechnology information

CBF:

C-repeat binding factor

DREB:

Dehydration-responsive element binding

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Acknowledgments

We gratefully acknowledge the financial assistance from the Council of Scientific and Industrial Research, New Delhi through CSIR-NMITLI project, University Grants Commission, New Delhi through CAS-UGC program and the Department of Science and Technology, New Delhi through DST-FIST program. Prashant and Sri Lakshmi Sunita also acknowledge University Grants Commission, New Delhi for the financial assistance through Dr. D. S. Kothari post-doctoral fellowship program.

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Authors and Affiliations

  1. Department of Genetics, Osmania University, Hyderabad, 500 007, India

    S. Prashant, M. Sri Lakshmi Sunita, V. L. Sirisha & P. B. Kavi Kishor

  2. Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, 500 007, India

    V. Vijaya Bhaskar

  3. Department of Botany, Telangana University, Nizamabad, 503 322, India

    A. Maruthi Rao

  4. Centre for Biotechnology, Institute of Science and Technology, Jawaharlal Nehru Technological University, Hyderabad, 500 085, India

    M. Lakshmi Narasu

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  1. S. Prashant
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Correspondence to P. B. Kavi Kishor.

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Prashant, S., Sri Lakshmi Sunita, M., Sirisha, V.L. et al. Isolation of cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase gene promoters from Leucaena leucocephala, a leguminous tree species, and characterization of tissue-specific activity in transgenic tobacco. Plant Cell Tiss Organ Cult 108, 421–436 (2012). https://doi.org/10.1007/s11240-011-0053-1

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