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Down-regulation of Leucaena leucocephala cinnamoyl CoA reductase (LlCCR) gene induces significant changes in phenotype, soluble phenolic pools and lignin in transgenic tobacco

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Abstract

cDNA and genomic clones of cinnamoyl CoA reductase measuring 1011 and 2992 bp were isolated from a leguminous pulpwood tree Leucaena leucocephala, named as LlCCR. The cDNA exhibited 80–85% homology both at the nucleotide and amino acid levels with other known sequences. The genomic sequence contained five exons and four introns. Sense and antisense constructs of LlCCR were introduced in tobacco plants to up and down-regulate this key enzyme of lignification. The primary transformants showed a good correlation between CCR transcript levels and its activity. Most of the CCR down-regulated lines displayed stunted growth and development, wrinkled leaves and delayed senescence. These lines accumulated unusual phenolics like ferulic and sinapic acids in cell wall. Histochemical staining suggested reduction in aldehyde units and increased syringyl over guaiacyl (S/G) ratio of lignin. Anatomical studies showed thin walled, elongated xylem fibres, collapsed vessels with drastic reduction of secondary xylem. The transmission electron microscopic studies revealed modification of ultrastructure and topochemical distribution of wall polysaccharides and lignin in the xylem fibres. CCR down-regulated lines showed increased thickness of secondary wall layers and poor lignification of S2 and S3 wall layers. The severely down-regulated line AS17 exhibited 24.7% reduction of Klason lignin with an increase of 15% holocellulose content. Contrarily, the CCR up-regulated lines exhibited robust growth, development and significant increase in lignin content. The altered lignin profiles observed in transgenic tobacco lines support a role for CCR down-regulation in improving wood properties of L. leucocephala exclusively used in the pulp and paper industry of India.

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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 University Grants Commission, New Delhi for financial assistance in the form of funds for Centre for Advanced Studies to the Department of Genetics, Osmania University.

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

    S. Prashant, M. Srilakshmi Sunita, Ranadheer K. Gupta, S. Anil Kumar & P. B. Kavi Kishor

  2. Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Anand, 388120, India

    S. Pramod & S. Rao Karumanchi

  3. Ajeet Seeds Ltd, 233 Chitegaon, Aurangabad, 431105, India

    S. K. Rawal

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  1. S. Prashant
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Fig. S1. Southern blot analysis of CCR sense and antisense transgenic tobacco plants showing gene integration using CCR gene specific probe. The DNA of sense, antisense transgenic plants and controls were digested with HindIII. Positive hybridization was observed at the 1011 bp region for each putative sense (A) and antisense (B) CCR transgenic plant samples except in controls. A - Lane1: Positive control; Lane2: Negative control; Lane3: S2; Lane4: S5; Lane11: S6; Lane12: S27 and Lane13: S9. B - Lane1: Positive control; Lane2: Negative control; Lane3: AS2; Lane4: AS3; Lane5: AS4; Lane5: AS12; Lane6: AS17

Fig. S2. RT-PCR analysis of CCR sense and antisense transgenic tobacco plants with CCR gene specific primers. Lane1: 1 kb ladder; Lane2: AS2; Lane3: AS3; Lane4: AS17; Lane5: AS4; Lane6: AS12; Lane7: Control; Lane8: S2; Lane9: S5; Lane10: S6; Lane11: S27; Lane12: S9. CCR antisense transgenic lines showed faint amplification as compared to sense CCR transgenics indicating up and down-regulation of CCR gene

Fig. S3 Phenotype of CCR sense and antisense transgenic lines. S5: CCR sense transgenic line; AS17: severely down-regulated CCR antisense transgenic line. C: Control

Fig. S4 Electron micrographs of stems of control and CCR sense and antisense transgenic tobacco lines. A and B Periodic acid–thiocarbohydrazide–silver proteinate (PATAg) staining. Control (A) showed less periodate reaction in the S1 and S3 layers. S2 layer showed relatively more carbohydrates. The secondary wall layers S1 and S2 in the antisense plants (B) showed intense staining indicating increased concentration of carbohydrates. C – E staining with KMnO4. Electron micrographs of fibre walls stained with KMnO4 showing lignin distribution pattern in control (C) and CCR antisense (D) and sense (E) plants. In control high lignin distribution was observed in CML, S1 and S3 layers of secondary wall. Antisense line showed lignin distribution limited to the middle lamellae and S1 layer. Sense line showed high lignin distribution in the S3 layer of secondary wall. CML: Cell corner middle lamellae; S1, S2, S3: Secondary cell wall layers. Scale bar = 0.5 micron

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Prashant, S., Srilakshmi Sunita, M., Pramod, S. et al. Down-regulation of Leucaena leucocephala cinnamoyl CoA reductase (LlCCR) gene induces significant changes in phenotype, soluble phenolic pools and lignin in transgenic tobacco. Plant Cell Rep 30, 2215–2231 (2011). https://doi.org/10.1007/s00299-011-1127-6

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