Fatty acid desaturase gene transcription at Solanum tuberosum L. cold adaptation

The problem of the survival of plants under low temperatures becomes more relevant in the light of global climate change and the growing needs of the population. In this regard, studies of the impact of hypothermia on plants are not only fundamental, but also applied. Potatoes are an important food crop, ranking fourth in the world in terms of growing. The actual yield of potatoes is significantly lower than its potential productivity, and one of the limiting factors is the lack of resistance of many modern varieties to spring frosts. Decoding of the potato genome made it possible to use advances in molecular biology to study the role of individual genes and identify key proteins that can increase resistance to low temperature. It is widely known that under the action of low temperatures, there is a phase transition of membrane lipids, which is accompanied by a decrease in membrane fluidity and loss of their barrier properties and, as a result, by inactivation of enzymes. In response to changes in physical properties of membranes, cells activate protection systems, among which an important role is played by the cold-induced increase in the degree of unsaturation of fatty acids of membrane lipids. Therefore, one of the main goals of adaptation is the stabilization of membranes, for example, due to the work of enzymes, fatty acid desaturase (encoded by genes FAD), catalyzing the conversion of saturated fatty acids (FA) into unsaturated. Among all plant cell membranes, chloroplast membranes play a special role in the formation of plant resistance to low temperatures, since it is in chloroplasts that photosynthesis, the main source of energy necessary for the restructuring of metabolism during the adaptation period, takes place. The aim of the research was to study the role of chloroplast localized ∆9-, ∆12 - and ω3(∆15)-desaturases in adaptive transformations of the fatty acid composition of chloroplast membranes when forming potato plant cold resistance during hardening. The object of the study was potato plants (Solanum tuberosum L., cultivar Jubilee Zhukov), 3 weeks of age, grown in soil culture at a temperature of 22°C, illumination of 100 μmol∕(m² c) and 16-h photoperiod. Hardening of plants was carried out in the climatic chamber KBW-240 “Binder” (Germany) under 16-h photoperiod and illumination of 100 μmol∕(m²c) at a temperature of 3°C for 7 days. Controls were nonhardened plants. To assess the effectiveness of adaptation, whole plants were frozen at a temperature of 2°C for 18 hours in the climatic chamber MIR-153 “Sanyo” (Japan), and then transferred to the growing conditions to determine survival. The following genes of FA desaturases were selected for the study: SAD (encodes one of the soluble ∆9-ACP-), FAD6 (encodes membrane-bound acyl-lipid ∆12-), FAD7 (encodes membranebound acyl-lipid Δ15(ω3)-desaturase). Protein products of these genes are localized in chloroplasts. Total RNA from leaves was isolated using Spectrum Plant Total RNA Kit “Sigma” (USA). The reverse transcription reaction was performed using a set of reagents and the Protocol MMLV RT Kit “Eurogen” (Russia). The resulting cDNA was used for real-time PCR (q-PCR) using the amplifier CFX96 Touch Real-Time PCR Detection System “Bio-Rad” (USA), using a set of reagents qPCRmix-HS SYBR kit “Eurogen” (Russia). The relative transcript content was calculated by calculating the normalized expression (ΔΔC_T). Primers for the genes of FA desaturases were selected using the database NCBI and Internet resource Primer3Plus. Intact chloroplasts were isolated by centrifugation in a percol step gradient. Chloroplast lipids were methylated by boiling in a mixture of CH₃OH and CH₃COCl. The obtained LC methyl esters were analyzed by GL-MS using Agilent 7890A GC (USA). The experiments were conducted in 5-6 biological replicates and 3-4 analytical ones. Statistical data processing was performed using the program SigmaPlot 11. The data are presented as means and their standard errors. We showed that the hardened potato plants survived after -2°C for 18 h, which indicates the successful hardening of S. tuberosum, Jubilee Zhukov cultivar (See Fig. 1). Among the studied genes ∆9-, Δ12- and ω3-desaturases of chloroplasts, a short-term (after 2 h of adaptation) increase in the transcripts of the FAD6 gene encoding acyllipid Δ12-desaturase was found. The relative content of FAD7 gene transcripts encoding ω3-desaturase remained stable and maintained at the level of control. The character of SAD gene expression encoding ∆9-ACP desaturase differed from the others: the relative transcript content decreased during adaptation (See Fig. 2). It should be noted that potatoes have 13 soluble ∆9-ACP-desaturase genes forming the first double bond, whose proteins are localized in the stroma of chloroplasts. Perhaps, the studied gene is not cold-inducible. The total percentage of polyunsaturated fatty acids (PUFA) of lipids in chloroplasts of potato was high and constitutive in non-hardened plants accounted for almost 90% of the total content of all FA (See Table). Probably, therefore, in the process of adaptation there was no noticeable increase in the relative content of the transcripts of the studied genes Δ12- and ω3-desaturases of chloroplasts. During the period of low-temperature hardening, the part of PUFA, and especially α-linolenic acid, was maintained at a high level; there was an increase in the content of palmitic acid, which may indicate an increase in the intensity of synthesis of FA de novo. In addition, an increase in the content of C_{16: 1}^∆7 acid was observed, which is also important for adaptation. It is known that the fluidity of membranes with decreasing temperature is determined not only by the content of FA with a larger number of double bonds, but also by the content of FA with a smaller number of carbon atoms. Maintaining a high amount of PUFA helped to maintain the thylakoid membranes of chloroplasts in a functional state during the hardening process, which, in turn, allowed the potato plants to realize other processes of adaptation. The paper contains 2 Figures, 1 Table and 25 References.

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