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Cloning, characterization and impact of up- and down-regulating subabul cinnamyl alcohol dehydrogenase (CAD) gene on plant growth and lignin profiles in transgenic tobacco

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Abstract

Both cDNA including 5′UTR and 3′UTR and genomic clones of cinnamyl alcohol dehydrogenase (CAD) were isolated and characterized from a pulp-yielding leguminous tree Leucaena leucocephala (LlCAD1). The deduced amino acid sequence shared high identity with orthologous sequences of Acacia mangium × Acacia auriculiformis (83%), Medicago sativa (83%), Nicotiana tabaccum (83%) and Aralia cordata (81%). Full length cDNA contained 78 bases of 5′UTR and 283 bases of 3′UTR, while the genomic clone contained 5 exons and 4 introns. Western blot analysis revealed elevated expression of LlCAD1 in seedling roots and shoots compared to leaves. Sense and antisense CAD tobacco transgenics showed increased and reduced CAD activity accompanied by a change in monomeric lignin composition. Histochemical staining of lignin in down-regulated plants suggested an increase in aldehyde units and a decrease in S/G ratio. Down-regulation of CAD resulted in accumulation of syringic, ferulic, p-coumaric and sinapic acids compared to untransformed controls. These observations were validated by anatomical studies of down-regulated transgenic stems which showed thin walled, elongated phloem and xylem fibres, accompanied by a reduction in the density of vessel elements and amount of secondary xylem when compared to untransformed plants. Furthermore, Klason lignin analysis of CAD antisense transgenics showed 7–32% reduced lignin and normal phenotype as compared to untransformed plants. Such a reduction was not noticed in up-regulated transgenics. These results demonstrate a unique opportunity to explore the significant role that down-regulation of CAD gene plays in reducing lignin content thereby offering potential benefits to the pulp and paper industry.

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Acknowledgments

This work was supported by a grant from the Council of Scientific and Industrial Research (CSIR-NMITLI), New Delhi, India and we gratefully acknowledge the financial assistance. We are also thankful to the UGC, New Delhi for financial assistance in the form of Centre for Advanced Studies to the Department of Genetics, and DBT-OU-ISLARE program to the Osmania University.

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Authors and Affiliations

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

    V. L. Sirisha, S. Prashant, D. Ranadheer Kumar, N. Jalaja, P. Hima Kumari, P. Maheshwari Rao & P. B. Kavi Kishor

  2. Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Anand, 388 120, Gujarat, India

    S. Pramod, Preeti Mishra & S. Rao Karumanchi

  3. Department of Botany, Osmania University, Hyderabad, 500 007, India

    S. Nageswara Rao

  4. National Chemical Laboratory, Pune, 411 008, India

    B. M. Khan

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  1. V. L. Sirisha
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Correspondence to P. B. Kavi Kishor.

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Sirisha, V.L., Prashant, S., Ranadheer Kumar, D. et al. Cloning, characterization and impact of up- and down-regulating subabul cinnamyl alcohol dehydrogenase (CAD) gene on plant growth and lignin profiles in transgenic tobacco. Plant Growth Regul 66, 239–253 (2012). https://doi.org/10.1007/s10725-011-9647-1

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