Effect of mcl-PHA synthesis in flax on plant mechanical properties and cell wall composition

Abstract

The high demand for new biomaterials makes synthesis of polyhydroxyalkanoates (PHA) in plants an interesting and desirable achievement. Production of polymers in plants is an example of application of biotechnology for improving the properties of plants, e.g. industrial properties, but it can also provide knowledge about plant physiology and metabolism. The subject of the present study was an industrially important plant: flax, Linum usitatissimum L., of a fibre cultivar (cv Nike). In the study the gene encoding PHA synthase from Pseudomonas aeruginosa, fused to a peroxisomal targeting signal, was expressed in flax plants with the aim of modifying the mechanical properties of plants. Medium-chain-length (mcl) hydroxy acids in flax plants from tissue cultures were detected by GC-FID and FTIR method. The introduced changes did not affect fatty acid content and composition in generated flax plants. Since mcl-PHA are known as elastomers, the mechanical properties of created plants were examined. Modified plants showed increases in the values of all measured parameters (except strain at break evaluated for one modified line). The largest increase was noted for tensile stiffness, which was 2- to 3-fold higher than in wild-type plants. The values estimated for another parameter, Young's modulus, was almost at the same level in generated flax plants, and they were about 2.7-fold higher when compared to unmodified plants. The created plants also exhibited up to about 2.4-fold higher tensile strength. The observed changes were accompanied by alterations in the expression of selected genes, related to cell wall metabolism in line with the highest expression of phaC1 gene. Biochemical data were confirmed by spectroscopic methods, which also revealed that crystallinity index values of cellulose in modified flax plants were increased in comparison to wild-type flax plants and correlated with biomechanical properties of plants.

Keywords: FTIR (Fourier transform infrared spectroscopy); Fatty acids; Flax; Mechanical properties; PHA (polyhydroxyalkanoates).

Fig. 1

a Schematic illustration of phaC1 cassette used for flax transformation. T-DNA contained: 35S CaMV- cauliflower mosaic virus promoter; phaC1, phaC1 synthase gene from P. aeruginosa; ICL-34 amino acids sequence of the Brassica napus isocitrate lyase targeting to peroxisomes; ocs, octopine synthase gene terminator; Pnos, nopaline synthase promoter; npt II, neomycin phosphotransferase gene; Tnos, nopaline synthase gene terminator. RB, LB: right and left border sequence of the T-DNA. b Selection of generated plants by PCR method. Top panel: Fragment of 792 bp of phaC1 gene from P. aeruginosa was amplified in PCR reaction on genomic DNA, isolated from plants. Integration of introduced gene was confirmed for 5 transgenic lines. Bottom panel: Fragment of 35S CaMV promoter (220 bp; first 5 lanes) and highly conserved region of chloroplast DNA (297 bp; last 5 lanes) were amplified with the use of specific primers. Genomic DNA was isolated from plants, cultivated in tissue cultures. pl, plasmid used for transformation (pART27-phaC1); wt, genomic DNA isolated from control, wild-type plants; different transgenic lines are numbered; m, marker 1 kb ladder. c Expression of phaC1 gene in selected plants (numbered 7 and 11). The total RNA was isolated from tissue-cultured flax plants and used for cDNA synthesis. Then semi-quantitative PCR reaction was carried out on the cDNA template. Quantitative data were normalized with expression of the actin gene, n = 3. d Determination of mcl-PHA isolated from tissue cultured flax plants, obtained after transformation with phaC1 gene. The level of PHA monomers was determined as described in the “Materials and methods” section by gas chromatography–flame ionization detection (GC-FID) method. The results derived from 3 samples analyzed per each line

Fig. 2

Fatty acids composition in green tissue of transgenic flax plants. The methyl esters of fatty acids were measured as described in the Materials and methods section. The statistical analysis was performed by Student’s t-test (*P < 0.05; **P < 0.01), n = 3

Fig. 3

Mechanical properties of flax stems determined in tensile tests for wild-type flax plants cv. Nike and two genetically engineered lines (#7 and 11). The parameters were estimated as described in the “Materials and methods” section. The statistical analysis was performed by Student’s t-test (*P < 0.05; **P < 0.01), n = 3

Fig. 4

Cellulose and lignin level in tested flax plants, obtained after transformation with phaC1 gene. Analysis was performed as described in the “Materials and methods” section. The level of cellulose and lignin was determined in plants derived from tissue cultures. The statistical analysis was performed by Student’s t-test (*P < 0.05; **P < 0.01), n = 3

Fig. 5

Spectroscopic analysis of analysed flax plants. a FTIR spectra of modified flax producing mcl-PHA (numbered as 7 and 11) and untransformed flax plants. b Raman spectra of the wt, 7 and 11 samples. c The integral intensity ratios of the IR bands at 1151/2920 cm⁻¹ (a), 1047/2920 cm⁻¹ (b), 988/2920 cm⁻¹ (c) and 890/2920 cm⁻¹ (d) for the control (wt), 7 and 11 samples. D. Crystallinity index (Icr) calculated for transgenic (#7, #11) and wild-type (wt) flax plants as the ratio between integral intensities of bands at 1380 cm⁻¹ and 2920 cm⁻¹