The influence of structural and functional characteristics of lipid transfer protein on the biological resistance of Pinus Sylvestris L.

Abstract

The dissertation presents the results of experimental studies of the structure and functions of the Scots pine lipid transfer protein gene (PsLTP1). New LTP gene sequences have been identified in the genome of Pinus sylvestris L. The ability of recombinant PsLTP1 to bind hydrophobic molecules in vitro and inhibit insect digestive enzymes has been investigated. The peculiarities of PsLTP1, PsLTP-B and PsLTP-D gene expression in pine seedlings in response to phytopathogenic fungal infection and under the influence of abiotic stressors, as well as after treatment with several growth stimulators, are described. The vital role of PsLTP in the defense mechanisms and involvement of these genes in response to biotic and abiotic stresses has been demonstrated. The correlations between the expression parameters of the PsLTP1, PsLTP-B and PsLTP-D genes in seedlings during heavy metal treatment and the dependence of these indicators on the contamination level of the growth conditions of the maternal organism were also clarified. The scientific and practical significance of the results lies in the possibility of using the properties of lipid transfer proteins genes for designing resistant to adverse environmental conditions genotypes of Scots pine and the cultivation of genetically improved planting material for forestry needs. The results of the research will also be used to update lecture courses on "Forest Phytopathology" on the harmfulness of phytopathogenic strains of microorganisms and the peculiarities of the infectious process under climate change, and "Plant Physiology" on the mechanisms of adaptation of conifers to environmental stressors. The study was conducted in the research laboratory of molecular genetic markers of wooden plants at the Forestry Department of the Ukrainian National Forestry University and the laboratory "Biotechnology and Analysis of Plant Preparations (VIO- 278)" of the Department of Biochemistry and Molecular Biology, University of Cordoba (Spain). LTP gene sequences were isolated from seven-day-old pine seedlings and deposited in the International GeneBank numbers JN980402, MN701087, MG679921, MG679922, MT424877 and MT424892. Additionally, 14 more sequences of Scots pine LTP were isolated from seed embryos, cloned and deposited in the GeneBank (MT424878, MT424879, MT424880, MT424881, MT424882, MT424883, MT424884, MT424885, MT424886, MT424887, MT424888, MT424889, MT424890 and MT424891). Moreover, five intron-containing Scots pine LTP sequences were cloned from genomic DNA; two of them were deposited in GenBank under numbers KY328829 and KY352340. In silico analysis of PsLTP sequences and sequences found in the National Center for Biotechnology Information (NCBI) databases showed the presence of 11 genes of lipid transfer proteins in the genome of Scots pine, thereby confirming the affiliation of Scots pine lipid transfer protein genes to the multigene family. Elucidation of the nucleotide sequence of the PsLTP1 gene and its amino acid sequence made it possible to model the 3D structure of the PsLTP1 molecule by analogy with the studied spatial models of other LTPs from angiosperm. The obtained structure is homologous to corn LTP by 45 % and consists of 4 α-helices and an unstructured C-terminal tail. The polypeptide chain structure contains eight conserved cysteines and the conserved DRQ and CKV domains required for binding to hydrophobic molecules. This cysteine skeleton is a characteristic feature of the family of lipid transfer proteins. Using polymerase chain reaction with reverse transcription, it was shown that PsLTP1 is constitutively expressed in Scots pine's vegetative organs (roots, hypocotyls, cotyledons of 8-day-old seedlings of Scots pine and in the bark, needles, vegetative buds and roots of 15-years old pine). It was found that lipid transfer protein genes PsLTP1, PsLTP-B and PsLTP-D constitutively express in the generative organs of Scots pine (macro- and microstrobiles, pollen). A significant amount of PsLTP transcripts are synthesized in embryos; however, we did not see any transcripts of PsLTP genes in the endosperm. The expression of PsLTP1, PsLTP-B and PsLTP-D genes differs in the generative organs and embryos. A significant amount of PsLTP1 transcripts was found in microstrobiles, while the transcription of both PsLTP-B and PsLTP-D genes was higher in embryos. Recombinant Scots pine lipid transfer protein (recPsLTP1) was isolated and purified in the heterologous expression system of E. coli. According to calculations, the molecular weight of purified recPsLTP1 is 11229,88 Da, and the value of the isoelectric point is 8,2, which correlates with the LTP of angiosperms. The ability of recPsLTP1 to bind to hydrophobic fluorescent probe (6,Ptoluidinylnaphthalene- 2-sulfonate (TNS)) in vitro has been investigated, indicating its functional activity. The value of the dissociation constant for the interaction of recPsLTP1 and ligand is in the micromolar range and is 9,6 μM, which indicates the involvement of LTP in low-affinity interactions. Based on this property, the ability of recPsLTP1 to bind to unsaturated fatty acids in vitro was demonstrated. Unsaturated fatty acids such as oleic, linoleic, linolenic and palmitoleic acids showed the highest rate of competing with TNS for binding sites in the hydrophobic cavity of recPsLTP1. Recombinant PsLTP1 showed the lowest affinity for saturated fatty acids with 18 atoms of carbon – stearic and palmitic fatty acids and did not interact with phospholipids. In addition to its ability to bind fatty acid molecules, recPsLTP1 could also bind the molecules of methyl jasmonic acid (MeJA) in vitro. MeJA is a derivative of linoleic acid and acts as a messenger in defense signaling during wounding and damage by necrotrophic pathogens. It has been experimentally confirmed that chimeric PsLTP1, which was translationally fused with green fluorescent protein (GFP), transiently expressed in the epithelial cells’ membrane of tobacco leaves. It has been shown that recPsLTP1 does not have the ability to inhibit the growth of mycelium of phytopathogenic fungi Fusarium solani (Mart.) Sacc., Alternaria alternata (Fr.) Keissl., Heterobasidion annosum (Fr.) Bref. and Phytophthora cinnamomi Rands and bacteria Escherichia coli, Pectobacterium carotovorum Waldie and Pseudomonas syringae Wan Hall. Investigating the ability of recPsLTP1 to inhibit the activity of insect digestive enzymes, it was found that recPsLTP1 can inhibit trypsin activity. It has been experimentally determined that 0,16 μM of recPsLTP1 is sufficient to reduce trypsin activity by 7,5 %, and at a concentration of 1,77 μM, trypsin activity decreased by 38 %. RecPsLTP1 showed high inhibitory activity against amylases of larvae of pine sawfly and May beetle. It appears that recPsLTP is much more effective in inhibiting the activity of pine sawfly amylases. The preparation 2,2 μM recPsLTP1 inhibits May beetle amylases by 17 %, while a decrease in the activity of pine sawfly amylases by 20 % is observed after the addition of 45 nM recPsLTP1. 450 nM recPsLTP1 inhibits the activity of pine sawfly amylases up to 43 %. It was demonstrated by a semiquantitative polymerase chain reaction that stress hormones such as jasmonic, salicylic and abscisic acids induce LTP gene expression in eight-day-old seedlings. As it turned out, the expression of the PsLTP1 and PsLTP-B genes is regulated by salicylic acid. In contrast, the regulation of the PsLTP-D gene is going by jasmonic acid and ABA-dependent pathways. It was also shown that after seedlings treatment with auxin and kinetin, the number of PsLTP-D transcripts was higher compared to PsLTP1 and PsLTP-B. The expression of PsLTP1, PsLTP-B and PsLTP-D genes has been shown to change during contact with Fusarium solani (Mart.) Sacc., Alternaria alternata (Fr.) Keissl., Heterobasidion annosum (Fr.) Bref. and Phytophthora cinnamomi Rands, which are pathogens of Scots pine. Chemibiotrophic microorganisms - F. solani and Ph. cinnamomi differently regulate the expression of PsLTP genes in the early stages of infection. The maximum number of transcripts of PsLTP genes was detected after 24 hours of contact with fusarium, while phytophthora significantly inhibited the expression of PsLTP genes, only the transcription of the PsLTP-B gene was 50 % higher compared to control values after six hours of contact with phytophthora. Another expression pattern of PsLTP1, PsLTP-B and PsLTP-D genes is observed in seedlings after contact with necrotrophic microorganisms: A. alternata and H. annosum. The highest levels of PsLTP genes transcription were reached after 48 hours of contact with A. alternata. PsLTP-D gene expression level was twice as high as PsLTP1 and PsLTP-B, and 4 times higher than control values. The PsLTP1 gene expression differed in time during infection with another representative of necrotrophic microorganisms – H. annosum; most PsLTP1 transcripts accumulated in the first two hours of contact with the pathogen and their values were twice as high as control values. It was found that the expression of PsLTP1, PsLTP-B and PsLTP-D genes also changes under the influence of abiotic stress conditions, such as salinity, extreme temperatures, drought, osmotic and oxidative stress, which indicates the participation of PsLTP genes in the mechanisms of response to abiotic stress. We noticed that salinity and low temperature positively regulate the expression of PsLTP genes, among which the PsLTP-D gene is susceptible to all studied stress conditions. The transcription level of the PsLTP-D gene is much higher while comparing to the control. It is shown that the growth conditions of the maternal organism affect the expression of PsLTP genes. It was demonstrated that the heavy metal ions in the salt complexes (copper, cadmium, cobalt, zinc, chromium) greatly affected the transcription levels of PsLTP1, PsLTP-B and PsLTP-D genes in eight-day seedlings. Copper and cobalt ions had the most stimulating effect on PsLTP-B and PsLTP-D gene expression, while the transcription of PsLTP1 gene after treatment of seedlings with solutions containing copper, chromium, cobalt, zinc and cadmium was slightly reduced. It was also demonstrated that growth conditions of the maternal organism have an effect on the transcription levels of PsLTP genes. Expression of PsLTP genes was higher in seedlings from the contaminated territory. From our research, we can conclude that Scots pine LTPs are promising candidates for use in the latest plant protection technologies and for the development of marker genes which can be used in marker assisted selection of drought and pestresistant pines. Increasing the trees' resistance to pests and microbial pathogens is one of the main goals in plant genetic engineering programs. Thus, the data obtained in the dissertation provide a basis for applying the knowledge about Scots pine LTPs in the development of specific molecular genetic approaches for the design and cultivation of genetically improved, biologically stable genotypes of Scots pine. Genetically modified trees with additional copies of Scots pine lipid transfer protein genes may be used for afforestation of unsuitable for maintaining areas due to drought or contamination with industrial wastes and heavy metals. Plant breeding for diseases and pests resistance will give the possibility to create buffer zones in order to reduce the distribution area of pathogens and plant-feeding insects and to afforest regions affected by these pests or by unfavorable growth conditions.