The study of the lignin biosynthesis and ways of its modification by genetic engineering methods are important in both scientific and practical terms. The mechanisms of its regulation have not yet been well studied, and no efficient methods of lignification decrease have been developed, which would not cause any undesirable attributes in the phenotype. We have used the method of synthesis of small interfering RNAs to the fourth exon of the 4-Coumarate: Coenzyme A Ligase gene in transgenic aspen plants, which resulted in the decreasing expression of the 4CL1 gene in the plants of the PtXIII4CL2c, PtXIII4CL4a, and PtXVI4CL9b lines by 72%, 45%, and 75%, accordingly. The 4CL1 gene expression inhibition was accompanied by the growth (height) speed change in greenhouse conditions. The height of cloned plants PtXIII4CL2c and PtXVI4CL9b was validly lower than the control values by 20% and 12%, accordingly. At the same time, the PtXIII4CL4a clone was higher than the control plants by 23%. The total lignin content in the wood of 6-month old cloned plants PtXIII4CL2c, PtXIII4CL4a, and PtXVI4CL9b decreased by 23%, 13%, and 16%, accordingly. We detected inverse correlation between the decrease in the lignin content and the development of the red-brown color of the wood. The correlation ratio was -0.87. Thus, the growth reduction was observed when the 4CL1 gene expression was inhibited by more than 70%, while the lignin content decrease and color change took place in all lines under study.

Anterola, A.M. & Lewis, N.G. (2002). Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity. Phytochemistry, 61(3), 221-294.

Blake, A.W., McCartney, L., Flint, J.E., Bolam, D.N., Boraston, A.B., Gilbert, H.J., Knox, J.P. (2006). Understanding the biological rationale for the diversity of cellulose-directed carbohydrate-binding modules in prokaryotic enzymes. J. Biol. Chem., 281: 29321-29329.

Chiang, V.L. & Funaoka, M. (1990). The dissolution and condensation reactions of guaiacyl and syringyl units in residual lignin during kraft delignification of sweetgum. Holzforschung, 44, 147- 155.

Gengshou, X. (2013). Repression of Lignin Synthesis in Rice by C4H and 4CL Using RNAi. International Journal of Bioscience, Biochemistry and Bioinformatics, 3(3), 226-228

Hu, W.J., Harding, S.A., Lung, J., Popko, J.L., Ralph, J., Stokke, D.D., Tsai, C.J. & Chiang, V.L. (1999). Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nature biotechnology, 17, 808- 812.

Hu, W.J., Kawaoka, A., Tsai, C.J., Lung, J., Osakabe, K., Ebinuma, H. & Chiang, V.L. (1998). Compartmentalized expression of two structurally and functionally distinct 4-coumarate: CoA ligase genes in aspen (Populus tremuloides). Plant Biology, 95, 5407- 5412.

Kajita, S., Hishiyama, S., Tomimura, Y., Katayama, Y. & Omori, S. (1997).  Structural characterization of modified lignin in transgenic tobacco plants in which the activity of 4-coumarate: coenzyme A ligase is depressed. Plant Physiol, 114, 871-879.

Kajita, S., Katayama, Y. & Omori, S.  (1996). Alterations in the biosynthesis of lignin in transgenic plants with chimeric genes for 4-coumarate: coenzyme A ligase. Plant Cell Physiol, 37, 957-965.

Lakin, G.F. (1990). Biometrics. Moscow: Vysshaya Shkola.

Leple, J.C., Dauwe, R., Morreel, K., Storme, V., Lapierre, C., Pollet, B., Naumann, A., Kang, K.Y., Kim, H., Ruel, K., Lefèbvre, A., Joseleau, J.P., Grima-Pettenati, J., De Rycke, R., Andersson-Gunnerås, S., Erban, A., Fehrle, I., Petit-Conil, M., Kopka, J., Polle, A., Messens, E., Sundberg, B., Mansfield, S.D., Ralph, J., Pilate & G., Boerjan, W.  (2007). Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure. Plant Cell, 19(11), 3669-91.

Li, L., Zhou, Y., Cheng, X., Sun, J., Marita, J.M., Ralph, J. & Chiang, V.L. (2003). Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. PNAS, 100, 4939-4944.

Livak, K.J. & Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)).  Methods, 25, 402-408.

Lundquist, K. (1992).  Methods in Lignin Chemistry. Berlin: Springer-Verlag.

Murashige, T. & Skoog, F.  (1962). A Revised Medium for Rapid Growth and Bioassays with Tobacco Cultures. Physiol. Plant, 15, 473-497.

Obolenskaya, A.V., Elnitskaya, Z.P. & Leonovich, A.A. (1991). Laboratory works on chemistry of wood and cellulose. Moscow: Ekologiya.

Pilate, G., Guiney, E., Holt, K., Petit-Conil, M., Lapierre, C., Leplé, J.C., Pollet, B., Mila, I., Webster, E.A., Marstorp, H.G. (2002). Field and pulping performances of transgenic trees with altered lignification. Nat Biotechnol, 20, 607-612.

Rogers, S. & Bendich, A. (1995). Extraction of Total Cellular DNA from Plants, Algae and Fungi. Dordrecht; Boston; London: Kluwer Academic Publishers.     

Sarkanen, K.V. & Hergert, H.L. (1971). Classification and distribution.  New York: Wiley-Interscience.

Shestibratov, K.A. Bulatova, I.V., Shadrina, T.E. & Miroshnikov, A.I. (2008). Genetic transformation of triploid aspen forms and drooping birch seed forms. Proceedings of the VIII International Conference of Young Scientists "Forests of Eurasia - the North Caucasus." Sochi.

Takahashi, M. (1976). Removal of lignin from partially delignified softwoods by soft rot- and white rot fungi. Wood Research, 61, 27-34.

Tian, X.M.,  Xie, J., Zhao, Y.L.,  Lu, H., Liu, S.C., Qu, L., Li, J.M., Gai, Y. & Jiang, X.N. (2013). Sense-, antisense- and RNAi-4CL1 regulate soluble phenolic acids, cell wall components and growth in transgenic Populus tomentosa Carr. Plant Physiology and Biochemistry, 65, 111-119.

Vanholme, R., Storme, V., Vanholme,V., Sundin,L., Christensen, J.-H., Goeminne,G., Halpin, C., Rohde, A., Morreel, K. & Boerjana, W.  (2012). A systems biology view of responses to lignin biosynthesis perturbations in Arabidopsis. The Plant Cell, 24, 3506-3529.

Voelker, S. L., Lachenbruch, B., Meinzer, F. C., Jourdes, M., Ki, C., Patten, A.M., Davin, L. B., Lewis, N. G., Tuskan, G. A., Gunter, L., Decker, S. R., Selig, M. J., Sykes, R., Himmel, M. E., Kitin, P., Shevchenko, O., Strauss, S.H.  (2010). Antisense Down-Regulation of 4CL Expression  Alters Lignification, Tree Growth, and Saccharification Potential of Field-Grown Poplar. Plant Physiology, 154, 874- 886.

Voelker, S.L., Lachenbruch, B., Meinzer, F.C., Kitin, P. & Strauss, S. (2011).Transgenic poplars with reduced lignin show impared xylem conductivity, growth efficiency and surval. Plant Cell Environ, 34, 655-68.

Wagner, A., Donaldson, L., Kim, H., Phillips, L., Flint, H., Steward, D., Torr, K., Koch, G., Schmitt, U. & Ralph, J.  (2009). Suppression of 4-Coumarate-CoA ligase in the coniferous gymnosperm Pinus radiata. Plant Physiol, 149, 370-383.