Characterization of Nicotiana tabacum plants expressing hybrid genes of cyanobacterial Δ9 or Δ12 acyl-lipid desaturases and thermostable lichenaseby I. M. Gerasymenko, L. A. Sakhno, T. N. Kyrpa, A. M. Ostapchuk, T. A. Hadjiev, I. V. Goldenkova-Pavlova, Y. V. Sheludko

Russ J Plant Physiol


Plant Science


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Society of Automotive Engineers of

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ISSN 10214437, Russian Journal of Plant Physiology, 2015, Vol. 62, No. 3, pp. 283–291. © Pleiades Publishing, Ltd., 2015.

Original Russian Text © I.M. Gerasymenko, L.A. Sakhno, T.N. Kyrpa, A.M. Ostapchuk, T.A. Hadjiev, I.V. GoldenkovaPavlova, Y.V. Sheludko, 2015, published in Fiziologiya Rastenii, 2015, Vol. 62, No. 3, pp. 307–316. 283 1 INTRODUCTION

Desaturation of fatty acids (FA) is an essential reaction determining physical properties of membrane glycerolipids. Changes in level of unsaturated FAs in membrane lipids are an important part of plant adap tive response to abiotic and biotic factors, mainly due to stabilization of integrity and function of mem braneassociated protein complexes end electron transport chains under stress conditions [1].

Two groups of enzymes take part in FA desatura tion in plant cells: acyl–acyl carrier protein (ACP) desaturases and acyl–lipid desaturases [2]. Acyl–ACP Δ9 desaturases function in plastids catalyzing the first double bond formation in saturated FAs (C16:0 and

C18:0). Further desaturation of monounsaturated FA 1 Published in the author’s version. occurs in plastid or ER membranes by means of mem branebound acyl–lipid desaturases accepting etheri fied FAs in glycerolipids as substrates. This leads to formation of di and triunsaturated FAs with double bonds in Δ12 and Δ12,15 positions. Additionally to enzymes realizing these most common reactions, sev eral desaturases introducing double bonds at Δ4, Δ6 and other positions have been described [3].

In cyanobacteria, membranebound acyl–lipid desaturases are responsible for formation of unsatur ated FA. Four proteins DesC, DesA, DesB, and DesD introduce double bonds at the Δ9, Δ12, Δ15 and Δ6 positions, respectively. In the first reaction DesC converts stearic acid (C18:0) to oleic acid (C18:1 Δ9).

Next step includes formation of linoleic acid (LA;

C18:2 Δ9,12) by DesA desaturase. Further conversion may introduce double bonds at Δ15 and/or Δ6 posi tions [2].

To clarify the role of acyl–lipid desaturases in molecular mechanisms of plant adaptive response, we need data about changes in FA composition in mem


Characterization of Nicotiana tabacum Plants Expressing Hybrid

Genes of Cyanobacterial Δ9 or Δ12 Acyl–lipid Desaturases and Thermostable Lichenase1

I. M. Gerasymenkoa, L. A. Sakhnoa, T. N. Kyrpaa, A. M. Ostapchukb, T. A. Hadjievc,

I. V. GoldenkovaPavlovac, Y. V. Sheludkoa aInstitute of Cell Biology and Genetic Engineering National Academy of Science of Ukraine, Kyiv bZabolotny Institute of Microbiology and Virology National Academy of Science of Ukraine, Kyiv; fax: +38 (044) 5267104; email: cTimiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia; fax: 007 (499) 9778018; email:

Received October 15, 2014

Abstract—We established transgenic lines of Nicotiana tabacum expressing hybrid genes of Synechocystis sp.

PCC 6803 Δ12 (desA) acyl–lipid desaturase and Synechococcus vulcanus Δ9 (desC) acyl–lipid desaturase with or without sequence coding for transit peptide of Rubisco small subunit of Arabidopsis thaliana under control of a constitutive promoter. Reliable increase of linoleic acid portion (C18:2; Δ9,12) accompanied with decrease of αlinolenic acid (C18:3; Δ9,12,15) relative amount was detected for plants expressing hybrid desA::licBM3 gene. No reliable changes were detected in fatty acid profiles and unsaturation index of plants transformed with Δ9 desaturase gene desC::licBM3 lacking signals of intracellular targeting while expression of this gene with Arabidopsis thaliana Rubisco small subunit transit peptide sequence caused growth of C18:3 αlinolenic acid part simultaneously with reduction of C18:2 linoleic acid part, as well as increase of unsat uration index. No changes in relative amount of Δ9monounsaturated fatty acids were observed in any of studied lines. All plants expressing desaturase genes exhibited enhanced levels of superoxide dismutase (SOD) activity after cold treatment in contrast to control lines with suppressed SOD activity after cold treatment.

Keywords: Nicotiana tabacum, Synechocystis sp. PCC 6803, Synechococcus vulcanus, Clostridium thermocel lum, acyl–lipid desaturases, lichenase, fatty acids, transgenic plants

DOI: 10.1134/S1021443715030073

Abbreviations: ACP—acyl carrier protein; CoA—coenzyme A;

FA—fatty acid; GFP⎯green fluorescent protein; RTP⎯transit peptide of Rubisco small subunit; SOD⎯superoxide dismutase;

TSP⎯total soluble protein. 284


GERASYMENKO et al. brane lipids depending on subcellular localization of desaturases. Although several publications describe plant sequences homologous to Δ9 acyl–lipid desatu rases of cyanobacteria [4, 5], little information exists on correlation of their intracellular localization and function. It is still not clear if the nonplastid desatu ration of FAs with Δ9 desaturase is possible. To answer this question, it is necessary to compare plants expressing heterologous Δ9 desaturase localized in chloroplasts or cytosol. This can be achieved by trans fer and expression of heterologous Δ9 desaturase gene with or without chloroplast localization signal. It is noteworthy that additional information about func tions of desaturases in plant cell and their influence on

FA composition of membrane lipids can be gained by expression of a heterologous Δ12 desaturase gene.

Although heterologous expression of some desaturase genes in plants has been described earlier (e.g., [6, 7]), these experiments were carried out with different plant species that complicates the estimation of contribu tion of a given desaturase in change of FA composition in membrane lipids.

In the present work we describe the results of expression of two heterologous acyl–lipid desaturase genes whose protein products are localized in different cell compartments (chloroplasts and cytosol) in model plant Nicotiana tabacum, as well as comparative anal ysis of FA composition of membrane lipids and esti mation of plant antioxidant system response to low temperature treatment. In order to estimate the level of heterologous protein accumulation we fused the target gene sequences in one reading frame with the sequence of thermostable lichenase reporter gene from Clostridium thermocellum. As it was proved ear lier, such hybrid system allows for efficient monitoring of recombinant product accumulation and retains, in most cases, the function of the target protein unchanged [8, 9].