Spectroscopic characterization of genetically modified flax fibers

Accepted Manuscript Spectroscopic Characterization of Genetically Modified Flax Fibers L. Dymińska, A. Gągor, J. Hanuza, A. Kulma, M. Preisner, M. Żuk, M. Szatkowski, J. Szopa PII: DOI: Reference:

S0022-2860(14)00609-7 http://dx.doi.org/10.1016/j.molstruc.2014.06.013 MOLSTR 20694

To appear in:

Journal of Molecular Structure

Received Date: Revised Date: Accepted Date:

21 March 2014 4 June 2014 4 June 2014

Please cite this article as: L. Dymińska, A. Gągor, J. Hanuza, A. Kulma, M. Preisner, M. Żuk, M. Szatkowski, J. Szopa, Spectroscopic Characterization of Genetically Modified Flax Fibers, Journal of Molecular Structure (2014), doi: http://dx.doi.org/10.1016/j.molstruc.2014.06.013

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Spectroscopic Characterization of Genetically Modified Flax Fibers

L. Dymińska a,*, A. Gągor b, J. Hanuza a,b, A. Kulma c, d, M. Preisner c, d, M. Żuk c, e, M. Szatkowski c, d, J. Szopa c,d,e a

Department of Bioorganic Chemistry, Institute of Chemistry and Food Technology, Faculty of Engineering and Economics, Wrocław University of Economics, Komandorska 118/120, 50-345 Wrocław, Poland

b

Institute of Low Temperatures and Structure Research, Polish Academy of Sciences, Okólna 2, 50-422 Wrocław, Poland

c

Faculty of Biotechnology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland

d

The Wroclaw Research Centre EIT+ Sp. z o.o., ul. Stabłowicka 147, 54-066 Wroclaw, Poland

e

Linum Fundation, Stabłowicka 147/149, 54-066 Wroclaw, Poland

AUTHOR INFORMATION Corresponding Author * Tel: +48 71 3680299. Fax: +48 71 3680292. E-mail: [email protected]

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ABSTRACT The principal goal of this paper is an analysis of flax fibre composition. Natural and genetically modified flax fibres derived from transgenic flax have been analysed. Development of genetic engineering enables to improve the quality of fibres. Three transgenic plant lines with different modifications were generated based on fibrous flax plants as the origin. These are plants with: silenced cinnamyl alcohol dehydrogenase (CAD) gene; overexpression of polygalacturonase (PGI); and expression of three genes construct containing β-ketothiolase (phb A), acetoacetyl-CoA reductase (phb B), and poly-3hydroxybutyric acid synthase (phb C). Flax fibres have been studied by FT-IR spectroscopy. The integral intensities of the IR bands have been used for estimation of the chemical content of the normal and transgenic flaxes. The spectroscopic data were compared to those obtained from chemical analysis of flax fibres. X-ray studies have been used to characterize the changes of the crystalline structure of the flax cellulose fibres.

Keywords: FT-IR, flax fibres, structure, cellulose, lignin, pectin

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1. INTRODUCTION Several methods have been proposed for GMO identification and quantification. These are enzyme-linked immunosorbant assay analyses based on protein information [1], Western blot [2], immunoassay TRAIT test [3], and polymerase chain reaction analyses based on DNA information [4,5]. In recent years, rapid and inexpensive analyses have been developed. They include near-infrared (NIR) [6] and Fourier-transform (FT-IR) spectroscopies for the discrimination of genomics DNA from different genotypes [7]. The former method is often coupled with photoacoustic (PAS) cell in the studies of plants [8] and foods [9]. FT-IR-PAS method has been recognized as a rapid and nondestructive technique that allows to distinguish between transgenic and non-transgenic plants. This method is particularly effective when combined with canonical discriminant analyses [10,11]. The above described methods have been applied to several products derived from genetically modified plants, e.g. soybean seeds [10], tobacco [12,13], rice [14], and aspen [15]. In the present research we wanted to apply another method that allows to characterize differences between natural and GMO organisms. This method applies the deconvolution of the spectral contour into Lorentzian components and comparison of their integral intensities for the transgenic and non-transgenic flax fibers. Flax (Linum usitatissimum) is an annual plant cultivated in temperate climate, particularly in Central Europe. Products obtained from this plant are useful in industry, being a valuable source of oil and fibers. Development of genetic engineering enables to improve the quality of these products. The genetical flax modification enhances the quality of flax fibers, oil properties, elevation of antioxidant level and creation of pathogen-resistant plants. These modifications made flax more useful and precious source for a broad range of products applicable in industry. The transgenic approach was used to give overexpression of fungal pectinase enzymes in flax PGI 11. The introduced modifications resulted in a pectin content

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decrease and thus an improved retting efficiency [16]. The reduction in the lignin content in CAD 27 plants resulted in an improvement in the elastic properties of flax plants; transgenic plants showed increased mechanical properties in comparison to nontransformants [17]. Significant modification in stem mechanical properties was accompanied by the PHB (poly-3hydroxybutyric acid) accumulation in growing cells of fibers in the transgenic plants (M50) [18]. Properties of flax fibre depend strongly on the growing conditions and the applied fibre processing technique [19]. Among the methods used in the studies of these materials, FT-Raman and FT-IR spectroscopies were found to be very suitable to detect the major chemical components of the flax stems in vivo [20]. These techniques provide information on the molecular changes of the flax fibres caused by ageing [21], mechanical processing [22], and chemical treatment [23]. Many papers have been published on the vibrational spectra of the flax fibres and the components isolated from this plant [12,23-28]. The application of these methods allowed to recognise several important problems of the flax chemistry. For instance, they gave the information on polarisation behaviour of the IR bands for oriented cellulose fibres [28], structural changes of flax fibres in chemical treatment [23,27], the fibre content of flax stems, strain induced shifts of the Raman bands of natural cellulose fibres [29,30], effects of the enzymatic retting of flax stems [26], and the role of the hydrogen bonds in the different packing of the celluloses I and II [31]. These investigations resulted also in finding of the diagnostic way to differentiate two categories of the native celluloses, i.e. algal and bacterial celluloses IA, and higher plant celluloses IB [32]. The biochemical, mechanical, and structural properties of flax stems and fibres, derived from field grown genetically modified (GM) flax, were studied in our previous papers [33-36]. In the present work the results of FT-IR studies of native (NIKE) and transgenic flax

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(M50, CAD 27, PGI 11) fibres have been reported. The chemical content and structure of the fibers have been analyzed on the basis of the IR spectra. X-ray diffraction method has been used to compare the crystalline structures of the studied flax cellulose fibres.

2. EXPERIMENTAL SECTION 2.1.Plant Material Three types of transgenic plant were generated based on fibrous flax plants as the origin. Cinnamyl alcohol dehydrogenase gene repression [17], fungal polygalacturonase overexpression [16], and PHB synthesising [18] transgenic flax plants were the source of fibres. All modified plant types were generated using an Agrobacterium method [18,37-40] with the constructs described in Table 1. To avoid doubts that not all of the CAD isoforms were silenced, a conservative fragment of 480 nt was chosen to be used in the transformation. It has over 90% identity with a large number of CAD sequences from angiosperms. Briefly, cotyledons of 6-day-old seedlings were infected with an A. thumefaciens strain containing a dedicated binary plasmid introduced by electroporation. After the transfer of cotyledons to callus induction medium and subsequently to shoot induction medium, the explants were collected and grown on root-inducing medium. The pre-selection and further selection of modified plants were carried out by means of PCR and Northern blot analysis as described previously [16-18]. From each transformation, one line that showed the expected effect of the introduced modification and normal phenotype was chosen for the field trial as a source of fibre to be used throughout this study. Table 1 Flax plants from all transgenic lines and the control were cultivated in the field in the vicinity of Wrocław (in the 2010 season). After harvesting 4-month-old plants, the straw was

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retted for 3 weeks using the dew method and then processed to obtain the fibre used for further analysis.

2.2. Cellulose Content Analysis Cellulose amount was measured with a colorimetric method employing anthrone reagent. To remove all contaminants for further analyzing, the samples were incubated for 1 h at 100 °C in a mixture of HNO3 and AcOH (1:8 v/v). After this the samples were centrifuged, the supernatant discarded, the pellet washed twice with distilled water and then dissolved in 67% H2SO4 (v/v). Cooled anthrone reagent was added to mixed samples. The cellulose content was measured spectrophotometrically at 620 nm [41].

2.3. Lignin Content Measurement Total lignin content was measured by the 'acetyl-bromide' method. Briefly, dried and ground into powder tissue samples were heated for 1 h at 65 °C and next filtrated through GF/A filters washed several times with different organic solvents. The samples prepared in such a way were dried for 12 h and then, after adding 25% acetyl-bromide in AcOH, they were incubated for 2 h at 50 °C and further dissolved in 10 ml of 2N NaOH mixed with 12 ml AcOH. After incubating the samples for at least 12 hours at room temperature lignin content was measured spectrophotometrically at 280 nm. The results were given as an equivalent of coniferyl alcohol, for which calibration curve had been made [42].

2.4. Determination of Pectin Content The measurements were conducted in three steps. Initially, the contamination from tissues was removed by extracting the samples in the following way, with 96% EtOH at 100 °C, 80% EtOH at 80 °C, chloroform:methanol solution (1:1 v/v) and then acetone at room

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temperature. After drying, the samples were hydrolyzed in an ice bath with concentrated H2SO4. Dilluted with water and centrifuged, the supernatant containing pectin was collected to new tubes and further the amount of pectin was determined spectrophotometrically by biphenyl method. The hydrolyzate was supported in turn with 4M sulfamic acid potasium sulfonate solution, pH=1.6; Na2B4O7 in H2SO4, than incubated for 20 min at 100 °C. Finally, m-hydroxybiphenyl was added to measure absorption at 525 nm. The results were given as an equivalent of glucuronic acid [43].

2.5. IR Studies The IR spectra at room temperature were measured in the spectral range 50–4000 cm-1 using a FT-IR Biorad 575C spectrometer (made in the USA) with 2 cm-1 resolution. The samples were prepared in the KBr pellet. All spectroscopic measurements were performed for the pulvered samples obtained by milling of the dried flax fibres in Retsch mill the ZM 200 model. The mathematical processing of the measured spectra was performed using the computer programm ORIGIN 7.5. Lorentzian distribution function was used for the data fitting. The fitting parameter χ2 was of order 10-6. Due to short time of measurements the moisture of the studied samples remained unchanged during the spectra acquisition. It means that the residual water was not present in the studied samples.

2.6. X-ray Diffraction Powder diffraction data were collected on X'Pert PRO X-ray diffractometer with PIXcel ultra- fast line detector and Soller slits for Cu Kαradiation. The measurements were performed in transmission mode in the 10-60 2Θ range. All samples contained unintentionally

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introduced contamination that was not identified and it gave an intensive sharp peak around 22° and 35°.

2.7. Statistical Analysis The data (±SD) were obtained from 3 to 5 samples per line and are presented as the averages of independent replicates ± standard deviations. Statistical analyses were performed using Statistica 7 software (Statsoft, USA). The significance of the differences between the means was determined using the Student’s t test.

3. RESULTS AND DISCUSSION 3.1. Biochemical Analysis Plant cell wall composition is based on a biopolymer built mainly of cellulose and lignin. This net is encrusted in polysaccharides, proteins and low-molecular weight phenolics. Although composition of a plant cell wall is quite well described, still very little is known about origins and mutual interactions of cellulose and other polymers while a cell wall is synthesized. The biosynthesis and assembling these polymers into the structure that we call the cell wall are under the control of a complex network of several hundred genes [44,45]. Up- or downregulating the expression of different cell wall genes provokes modifications in the composition/structure of the plant cell wall. Therefore, lignin and pectin have naturally been one of the first targets for the cell wall engineering in flax. We expected that the introduced modifications would lead to changes in polymer levels in fibre. The results of the analysis of polymers composition of fibres isolated from the control and transgenic plants are presented in Table 2. Table 2

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Indeed, biochemical analysis of flax fibre from CAD27 and PGI11 plants showed a decrease in the level of lignin and pectin content, respectively. However, the most interesting results were obtained from measurements of cellulose. In both cases, in lignin- and pectin-low plants, the fibres showed an increase in the cellulose content and the highest rise (30%) was in the case of the fibre from the PGI11 plant. Thus, it may suggest that for improved fibre quality, the pectin level reduction appears to be the most effective. It is reasonable to note that the pectin decrease also affected the lignin content (7% decrease) in the fibre. Since pectin and cellulose are polymers of sugars, it may suggest that the excess of sugar monomers upon pectin down-regulation is redirected to cellulose synthesis. However, in the case of lignin reduction, this hypothesis can be countered by the fact that lignin belongs to a completely different pathway. It can thus be suggested that perhaps both spatial and substrate availability determine the level of cellulose in fibre upon lignin or pectin reduction. The obtained results clearly show that, in fact, each modification caused direct and indirect changes in the fibre composition. Research made so far in this field indicates that the synthesis of lignin and cellulose are coupled to each other [46]. Even though this link is obvious, the molecular background still remains unclear. Earlier research examining transgenic aspen with silenced 4-coumarate coenzyme A ligase (4CL), catalyzing a generation of activated phenolic precursors, indicated that a modification of the lignin biosynthesis pathway causes an increase in the cellulose level. Obtained plants showed a strong decrease in lignin level (up to 45%) while at the same time about 15% increase in cellulose level was observed [47]. Other data from plants with an under-expression the cinnamyl alcohol dehydrogenase (CAD), caffeoyl-CoA O-methyl tranferase (CCoAOMT) or hydroxycinnamoyl CoA transferase (HCT) gene, which encode the enzyme catalysing different steps in the biosynthesis of lignin monomers, have shown that lignin content in several plant species is reduced [48-51].

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Another aspect of the integrity of plant cell wall is incorporation of the low-molecular weight phenolics such as cinnamylaldehydes into lignin and also in the cellulose net, which is reported to happen when the lignin synthesis pathway is perturbed. Arabidopsis mutants with strongly silenced CAD gene had about 40% lowered lignin level and expressed weaker phenotype. The condensation of the lignin biopolymer was significantly affected by the huge decrease in a number of β-O-4 linkages and by the incorporation of sinapyl and coniferyl aldehyde into lignin's structure [49,52-54]. Engineering pectin level in plants is also a subject of numerous investigations. Blocking the expression of ACC oxidase, the enzyme involved in the ethylene biosynthetic pathway, or overexpression of an ethylene-responsive transcription factor gene increased accumulation or biosynthesis of pectin, respectively [55,56]. Furthermore, expression of the fungal pectinases (polygalacturonase, rhamnogalacturonase), a polygalacturonase-inhibiting protein, a pectin methylesterase inhibitor gene or plant treatments with pathogen, resulted in the dramatic changes in pectin content [16,57-59]. Analyses of transgenic flax plants showed that polygalacturonase (PGI) overexpression was associated with reductions (8–18%) in stem pectin content, as well as with slight modifications in the structure of the pectin polymer [18]. Additionally, alfalfa expression of UDP-glucose dehydrogenase, the key enzyme in biosynthesis of uronic acids, resulted in an increase in the rhamnose content accompanied by a slightly higher level of xylose and a higher lignin level measured by the Klason method. No significant increase in glucose, galactose and uronic acids levels was observed in the cell wall residues [60]. Hypothetically, maintaining the proper ratio of biopolymers can result in a defined number of accessible binding sites on the cellulose polymer, the space available and/or defined and limited accessibility of substrates for biopolymers synthesis.

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In this aspect, another suitable approach is the introduction of external polymer in the plant stem. This may be a tool to verify the hypothesis whether the cell wall biopolymers’ aggregate is a closed structure or it has an open structure and incorporating a new polymer does not diminish existing biopolymers amount and chemical reactivity. Analysis of the fibre from M50 plants (the fibre contained 50 µg of PHB per 1g DW), showed a slight decrease (4%) in the cellulose content and a reasonable reduction (14%) in the lignin level. Biochemical analysis of fibres from transgenic plants revealed an increase in cellulose content upon lignin and pectin reduction. An exception was the fibres with external PHB, where the cellulose level was slightly decreased. This allowed us to draw a conclusion that flax fibre composition is not an open structure; the newly introduced polymer exists at the expense of the ligninocellulosic polymer amount and structure. The changes in the polymer composition and structure were continued and confirmed by FTIR and X-ray analysis.

3.2. Vibrational Data FT-IR spectroscopy has been found to be very suitable to detect the major chemical components of flax fibres. Figure 1 shows the IR spectra of the fibres isolated from the control and transgenic plants. The main contours are similar to those reported in literature for other flax stems [23-26]. The IR spectra of natural and genetically modified flax fibres consist of the bands characteristic mainly for cellulose [23,26-27,61-65]. Lorentzian distributions of some contours allow to compare the integral intensities of the weak bands corresponding to lignin and pectin were evaluated by their subtraction from the base line of the cellulose spectrum. Figure 1

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The spectra of flax fibres were shifted to a common point. The intensity of the 2920 cm-1 band can be chosen as a standard. This band corresponds to the νas(CH2). The previous results showed that the intensity of the corresponding band remained unchanged for different studied samples [66]. This approach allows quantitative comparison of the band intensities in the spectra of the flax fibres both from control and transgenic plants. The broad absorption band at 3400 cm-1 corresponds to the stretching ν(OH) mode of the free hydroxyl groups and those involved in the intra- and inter-molecular hydrogen bonds [63]. The shape of this band is nearly the same for the all studied samples (i.e. the control and transgenic fibres) but the bands differ in terms of their absorption intensity. The overall intensity of the all components for the investigated samples follows the direction IM50 > INIKE ≥ IPGI11 > ICAD27 (Figure 1). The changes in the intensity of the 3400 cm-1 band components for the control and transgenic fibres probably resulted from different conformations of the intramolecular and intermolecular hydrogen bonds O-H⋅⋅⋅O of the glucopyranose system. Such changes are expected when different rotary isomers appear in the skeleton of the cellulose polymer, since they differ in the strength and orientation of the hydrogen bond. This leads to a disordered arrangement of the pyranoid rings in the cellulose polymers in fibres from transgenic flax. It should be pointed out that opposite effect was observed for the fibres from transgenic flax producing polyhydroxybutyrate (M50). In this case the intensity of the 3400 cm-1 band components was significantly higher than that of the control NIKE fibres. The IR absorbtion bands in the OH stretching range were deconvoluted into six Lorentzian components. The peak positions and integral intensities of these bands are summarized in Table 3. These bands are related to the vibrations of hydrogen bonded OH groups: the intramolecular hydrogen bond 2-OH⋅⋅⋅O-6 and 3-OH⋅⋅⋅O-5, the intermolecular hydrogen bond 6-OH⋅⋅⋅O-3' and to the OH stretching vibration [61-63,65].

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Table 3 The component in the 3455–3410 cm-1 range corresponds to the intramolecular hydrogen bond 2-OH⋅⋅⋅O-6 in cellulose. The band in the 3375–3340 cm-1 range corresponds to the intramolecular hydrogen bond 3-OH⋅⋅⋅O-5 in cellulose. The next band, which appears at about 3310-3230 cm-1 corresponds to intermolecular hydrogen bond 6-OH⋅⋅⋅O-3' in cellulose. It can be seen that the wavenumbers of peak position of PGI11 and CAD27 are higher than those of NIKE. Furhermore, it can be seen that the integral intensity of the bands corresponding to the intramolecular hydrogen bond in PGI11 is higher than that in NIKE. This means that the number of hydrogen bonds in PGI11 fibres is higher than that in natural fibre. But the integral intensities of the all components for CAD27 are lower than those for NIKE. The decrease of integral intensity of the band assigned to the intra- and intermolecular hydrogen bond for CAD27 indicates that the cellulose molecular chains are more flexible. Two additional components at 3482 and 3400 cm-1 appear for the transgenic fibre M50. It suggests forming additional hydrogen bonds in this transgenic fibres. The band at above 3500 cm-1 corresponding to the stretching ν(OH) mode of the free hydroxyl groups disappears for the transgenic fibre M50. It confirms that the free OH groups take part in additional hydrogen bonds. The main aim of significant modification in stem mechanical properties was to accumulate PHB in growing cell of fibres in the transgenic plants (M50), and the broad absorption band at about 3400 cm-1 shows that cellulose polymers of M50 are more highly bound than those of control NIKE. It should be noted that presented above discussion on the role of the hydrogen bonds in the structure of the flax fibres has taken into account the possibility of the presence of the residual water in the studied materials. Therefore, they were dried before the spectroscopic measurements and checked after the studies. It was stated that the residual water was not present in the studied by us samples.

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The contours observed in the IR spectra of the flax fibres in the ranges 1500–1800, 850–1500 cm-1 and 400–850 cm-1 are typical for the flax cellulose consisting of some amounts of lignins and pectins (Figure 1). The bands from these multiplets can be assigned to the vibrations: δas(CH3,CH2) at 1429 cm-1, δs(CH3,CH2) at 1372 cm-1, δ(CH) at 1319 and 1336 cm-1, ν(C-C) and ν(C-O) in the range from 1200 to 1300 cm-1, δ(φ-OH) at 1163 cm-1, νas(C-OC) in the range 1000–1110 cm-1, γ(CH) in the range of 850–1000 cm-1, and δ(θ) in the range from 500 to 720 cm-1[67,68]. Bands at 1429 and 1318 cm-1 correspond to the δas(CH2, CH3) and δ(CH) + δ(OH) vibrations, respectively. The comparison of the integral intensities of these bands gives the following relationships: for 1429 cm-1 band IPGI11 > INIKE > ICAD27 > IM50 and for 1318 cm-1 band IPGI11 > INIKE > ICAD27 > IM50. The weak band at 898 cm-1 corresponds to vibrations of cellulose. The integral intensity of this band fulfil the relations: IPGI11 > INIKE > IM50 > ICAD27 showing that the PGI11 fibres exhibit greater content of cellulose (Figure 2). The difference between the integral intensities of the NIKE and PGI11 is 23.4%. The integral intensity for this band is almost the same for the NIKE, M50, CAD27 (6.8-14.6%). Data above suggest that the fibre from PGI11 exhibit the highest content of cellulse compared to control fibre, while fibres from M50 and CAD27 revealed lower amount of cellulose. Figure 2 The important difference between the control and transgenic fibres appeared in the two ranges at about 1200 cm-1 and from 950 to 1160 cm-1. The former corresponds to the in-plane stretching vibrations of pyranoid rings coupled with the in-plane bending of the OH···O bonds and the latter to the νas(C-O-C) vibrations. The comparison of the integral intensities of the δ(OH···O) band at about 1200 cm-1 for NIKE and M50 gives the following relationships: INIKE < IM50 (43% difference). The integral intensitiy are practically very close for the NIKE and PGI11 flax fibres: INIKE ≅IPGI11 ( 2.2%

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difference) (Figure 3). The integral intensitiy of this component for CAD27 is lower than this for NIKE: INIKE >ICAD27 (37% difference). A broad band at about 600 cm-1 is observed for the all studied samples. It corresponds to the out-of-plane bending γ(OH⋅⋅⋅O) modes of the hydroxyls engaged in the O-H⋅⋅⋅O HB [63]. The integral intensities of the band at 665 cm-1 corresponding to the γ(OH⋅⋅⋅O) vibration also change. For the NIKE, M50 and CAD27 samples the relative intensities of this band reflect the same trend as in the case of the δ(OH) modes discussed above: INIKE < IM50, but the difference is very small 3.3%; INIKE > ICAD27 (difference is 23.2%). This band exhibits a different trend in integral intensity for NIKE and PGI11: INIKE < IPGI11 (23.4% difference). Figure 3 The bands at 1158 and 990 cm-1 originate from the ν(COC) vibrations of β-1,4glycosidic bond of cellulose chains. The integral intensities analyzed for these bands for NIKE, M50, CAD27 and PGI11 show the following trend: for 1158 cm-1 band IM50 > IPGI11 > INIKE > ICAD27 and for 990 cm-1 band IM50 > INIKE > IPGI11 > ICAD27 (Figure 4). These data suggest that the M50 contains more C-O-C bridges than the control NIKE does, because this bridge exists not only in cellulose polymers but also in PHB. Figure 4 The data from IR study lead to the conclusion: cellulose polymer of the transgenic fibres M50 are longer and more highly bound than those from the control NIKE; the transgenic fibres CAD27 are shorter and more loosely bound than those from the control NIKE; the fibres PGI11 are shorter and more highly bound than those from the control NIKE. Pectins constitute a heterogenous group of polysaccharides rich in D-galacturonic acid. They are components of practically all fruit and vegetables, and their content in a plant can be determined using several physicochemical methods. They are composed of galacturonants consisting of un-branched α1-4 linked residues interspread with rhamnose and 15

distinctive branched rhamnogalacturons. The degree of methylesterification is the factor that characterize the pectins. The structures of pectins affect their physicochemical properties and among various methods, FT-IR and Raman spectroscopies are excellent tools for such studies. The vibrational spectra of natural pectins and their derivatives have been recorded by several authors [69-79]. A few problems were discussed on the basis of IR methods, for instance: a content of free carboxyls, carboxylate anions, methyl esters, acetyls, amides, uronic acids, and sugars in pectins containing media, as well as the degree of their methylesterification [79]. On the other hand, Raman spectroscopy has been applied in structural investigations of polysaccharides [74,78-81]. It has been commonly accepted that the most diagnostic ranges that characterize the pectin state are following: 2850 – 3500, 1400 – 1800 and 830 – 900 cm1

. In the IR spectra the intense and broad ν(O-H⋅⋅⋅O) band is observed at about 3400 cm-1. For

H-pectins a very strong IR band that corresponds to the ν(C=O) vibrations of COOH unit appears at about 1760 cm-1. For pectin salts the respective νas and νs(COO) bands are observed at 1630 and 1420 cm-1 [79]. In the range 830–900 cm-1 a very strong and sharp Raman band at about 850 cm-1 for H-pectins and at about 860 cm-1 for pectin salts is expected. In IR spectra this band is weak and often overlapped by a stronger out-of-plane γ(OH) band [7173]. On the other hand, the bands at 825–860 and 880–900 cm-1 have been assigned to the equatorial anomeric H(α-anomers and α-glycosides) and axial anomeric H(β-anomers and βglycosides), respectively [24-27,63,82-84]. It has commonly been accepted that the most diagnostic spectral range that characterize the pectin constituents of fibres is 1600–1800 cm-1 range of IR spectra. The bands in this range of IR spectra for the control and transgenic flax fibres could be deconvoluted into three Lorentzian components. The component at about 1737 cm-1 corresponds to the νas(COO) vibrations of the unconjugated carboxyl group of pectins. The band at about 1650 cm-1 corresponds to the νas(COO) vibration of the conjugated carboxyl group [85]. The third band, 16

which appears at about 1609 cm-1, corresponds to the νs(COO) vibrations of the carboxyl group present in pectins. The integral intensities analyzed for these bands for the NIKE, M50, CAD27 and PGI11 show the following trend: for the 1737 cm-1 band IM50 > INIKE ≥ IPGI11 > ICAD27; for the 1655 cm-1 band IM50 > INIKE > IPGI11 ≥ ICAD27 and for the 1602 cm-1 band IM50 > INIKE > ICAD27 > IPGI11 (Figure 5). These data suggest that the fibres from PGI11 contain lower amount of pectin in comparison with the NIKE fibres. This agrees with the aim of genetic modification for PGI11. For the M50 flax fibres the multiplet in the 1600–1800 cm-1 range corresponds to the ν(COO) vibrations of the carboxyl groups present not only in pectins but also in the PHB compacted between the flax fibres. For that reason dependence IM50 > INIKE does not mean higher amount of pectins in the M50 flax fibres, but it gives information on the carboxyl group content in this sample. Figure 5 Lignins apart from cellulose, hemicellulose and pectins, are next polymeric composites that occur in plants. FT-IR spectroscopy is a useful technique for determination of the lignins content in pulp and plant [85-90]. The vibrational properties of lignins derived from different plants have been defined in details and widely used for their characterization. Their IR spectra exhibit a very broad band at 3400–3500 cm-1 corresponding to the hydroxyl groups in phenolic and aliphatic units, as well as a multiplet of bands in the range 2800–2950 cm-1 arising from the ν(CH) of aromatic metoxyl, methyl and methylene groups of side chains. The ν(C=O) and ν(COO) bands are observed in the range 1700–1730 cm-1 for un-conjugated units with the shoulder at about 1680 cm-1 that corresponds to the conjugated carbonylcarboxyl system. Aromatic skeleton vibrations appear at about 1600, 1520 and 1430 cm-1. Another band, observed at about 1460 cm-1, has been attributed to the coupled δ(CH) + νφ (φring) vibration. Lignin samples exhibit several weaker bands in the range below 1400 cm-1 originating from the phenolic OH and aliphatic CH3 and CH2 groups (1360–1380 cm-1), ν(C17

C) + ν(C-O) + δ(C=O) coupled vibrations (1200–1300 cm-1), ν(φ) (φ-ring) vibration (1325 cm-1) aromatic δ(CH) + δ(C-O) (1000–1040 cm-1) as well as γ(CH) (900–920 cm-1) [85,87-90]. Reviewed above data can be applied in the discussion of the results obtained by us for the natural and transgenic flax fibers. The IR bands in the 1300 – 1200 cm-1 range may be used for identification of the changes of lignin content in the natural and genetically modified flax fibres. It can be deconvoluted into four Lorentzian components. The components at about 1264 cm-1 and about 1247 cm-1 correspond to lignin vibrations [63]. The integral intensities of these bands for the NIKE, M50, CAD27 and PGI11 fulfil the relation: INIKE ≥ IPGI11 > ICAD27 >IM50 (Figure 6). The integral intensities are practically very close for the NIKE and PGI11 flax fibres. These data show that concentration of the lignins is almost the same for the NIKE, and PGI11 flax fibres. The fibres from M50 contain lower amount of lignin in comparison with the NIKE fibres. The same dependence can be observed in chemical analysis. The data presented in Figure 5 prove that the integral intensity of the bands at 1515 and 1462 cm-1 follow the trend INIKE ≅ IPGI11 > IM50 > ICAD27. Such a sequence agrees with the results of the biochemical analysis: all transgenic fibres showed reduction in lignin content and this was the highest in case of CAD27 flax and the lowest for PGI11 flax. These data suggest that the fibres from CAD27 contain lower amount of lignin in comparison with the NIKE fibres. This agrees with the aim of genetic modification for CAD27. Figure 6 3.3. X-ray Diffraction Cellulose is a linear homopolymer composed of b-D-glucopyranose units which are linked by 1,4-glicosidic bonds. The chains aggregate into microfibrils which form either ordered, crystalline or less ordered, amorphous regions. Microfibrils aggregate to fibrils and further to cellulose fibres. The presence of three different hydroxyl groups with different

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polarities in one glucopyranose unit introduces strong intermolecular and intramolecular hydrogen bonds that organize packing of microfibrils. Despite the simple molecular structure the crystalline cellulose crystalizes in several polymorphic forms adopting monoclinic P1121 symmetry (Iβ, II, IIII allomorphs) or triclinic P1 space group (Iα, IVI or IVII allomorphs) [91,92]. The crystal structure of all forms consists of polymer chains propagating along c direction. The chains are organized into sheets via hydrogen bond interactions. The layout and bonding between the sheets are different for all allomorphs. There are two native cellulose forms: Iα and Iβ. Iαis mostly found in bacterial cellulose and in cell walls of some algae whereas Iβ rich specimens are most common for cotton and higher plants. Because of the fact that investigated samples are flax fibres the X-ray diffractograms are indexed using Iβpolymorph patterns, see Figure 7. Figure 7 and 8 The average size of crystallites in [200] direction calculated from the Scherrer formula for the all samples range from 5-6 nm and is slightly larger than crystallites in native cellulose that are typically 2–5 nm in diameter [93]. Better crystallinity of the samples in comparison to native cellulose is also confirmed by well resolved diffraction peaks. For the all samples the (200) peak is shifted towards lower diffraction angles comparing to the standard patterns. The displacement is due to the expansion of the crystal lattice which is observed for nanocrystalline compounds. The fact that (200) peak is the most affected is related to the weak character of the interactions present in [200] direction. Cellulose chains are bonded in [200] direction only by weak electrostatic bonds and van der Vaals forces. In other directions, where the forces are stronger, the lattice expansion is not so distinct. Introduction of different genes does not change drastically the spatial organization of the cellulose chains. There is no evidence of conversion to other polymorphic forms. The most noticeable differences in diffraction patterns of genetically modified samples concern

19

the intensity ratio between individual peaks. The long range order between (102) lattice planes is established due to the hydrogen bonds within different cellulose chains whereas in [200] direction only weak electrostatic forces are present. In the all modified samples 200 peaks are much more intense in comparison to 102 peaks, see Figure 8. It means either that for modified flax the degree of ordering between (102) lattice planes is lower in transgenic flax (hydrogen bonds are broken) or the long range order in [200] direction is higher in comparison to the control NIKE sample. Taking into account that the intensity of 200 in comparison to 110 peaks in the modified samples is twice as high as in the control NIKE. The second scenario is more probable.

4. Conclusions The results of the chemical analyses and comparison of the IR spectra of the native and transgenic flax fibers allow to draw the following conclusions: •

FT-IR spectroscopy is a very suitable method to detect the major chemical components of flax fibers and to characterize the molecular changes and amount of components in flax fibers produced by genetic modifications.

•

The natural and transgenic flax fibers differ in the conformation of their cellulose skeletons due to the greater tendency to rotation of the pyranoid rings in the transgenic flax. They differ in strength and orientation of the hydrogen bonds joining the cellulose chains.

•

The wavenumbers and intensity of the IR bands corresponding to the intramolecular hydrogen bonds in the transgenic PGI11 flax are clearly higher than those of the natural NIKE flax. It means that the number of HBs in genetically modified flax is higher. The opposite effect occur when the CAD27 and NIKE flax are compared, and cellulose chains in the CAD27 are more flexible.

20

•

The cellulose chains in the transgenic M50 flax are joined by additional hydrogen bonds that couple the fibers stronger in it in comparison with the natural NIKE flax.

•

The amount of C-O-C bridges in the M50 flax is higher than that of the natural NIKE flax what is the result of formation of additional bridges of such a type between the PHB polymers the content of which is higher in the transgenic M50 flax.

•

Cellulose chains of the transgenic M50 fibers are longer and bound stronger than those in the control NIKE, whereas transgenic fibers in the CAD27 are shorter and bound more loosely than those in the control NIKE; and the PGI11 fibers are shorter and bound stronger than those in the control NIKE.

•

The amount of pectins is higher in natural flax, their content is lower in the PGI11 than in the natural NIKE flax. It means that for the PGI11 transgenic flax fibers the concentration of pectin decreases despite the aim of genetic modification.

•

The amount of lignins in the studied NIKE and PGI11 flax fibers is comparable, although higher than in the CAD27; it means that the aim of the genetic modification was reached.

•

The spatial organization of the cellulose chains, i.e. their ordering and crystallinity, is higher in the transgenic flax than in the natural NIKE flax.

21

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27

List of figures: Figure 1. IR spectra of NIKE (a), PGI11 (b), CAD27 (c), and M50 (d) fibres. Figure 2. Differences in the integral intensities of the bands at 1429 cm-1 (a), 1318 cm-1 (b) and 898 cm-1 (c) for the NIKE, M50, CAD27, and PGI11 fibres. Figure 3. Differences in the integral intensities of the bands at 1200 cm-1 (a) and 665 cm-1 (b) for the NIKE, M50, CAD27, and PGI11 fibres. Figure 4. Differences in the integral intensities of the bands at 1158 cm-1 (a) and 990 cm-1 (b) for the NIKE, M50, CAD27, and PGI11 fibres. Figure 5. Differences in the integral intensities of the bands at 1737 cm-1 (a), 1655 cm-1 (b), 1602 cm-1 (c) for the NIKE, M50, CAD27, and PGI11 fibres. Figure 6. Differences in the integral intensities of the bands at 1515 cm-1 (a), 1462 cm-1 (b), 1325 cm-1 (c), 1264 cm-1 (d), and 1247 cm-1 (e) for the NIKE, M50, CAD27, and PGI11 fibres. Figure 7. XRD diagram of genetically modified cellulose. The most intensive reflections are marked with Miller indices. The patterns are indexed for cellulose Iβ. The impurity phase is marked with asterisk. Figure 8. The intensity ratio of the 102 / 200 diffraction peaks.

List of tables: Table 1. The summarised scheme of the modifications including introduced gene and the kind of a genetic manipulation, effect of the modification and the construct content. Table 2. Biochemical composition of the cell wall fibre from control (NIKE) and transgenic lines. Table 3. Wavenumbers (ν) and integral intensity (A) of the Lorentzian components derived for the 3000-3700 cm-1 range for the natural (NIKE) and transgenic flax fibres.

28

(a) Absorbance

Absorbance

(a) (b) (c)

(b) (c) (d)

(d) 4000

3800

3600

3400

3200

3000

-1

2800

2600

1800

1750

Wavenumber / cm

Absorbance

Absorbance

(a) (b) (c) (d) 1500

1700

1650

1600

-1

1550

1500

Wavenumber / cm

(a) (b) (c) (d)

1400

1300

1200

1100

1000

-1

Wavenumber / cm

900

850

800

750

700

650

600

550

-1

500

450

400

Wavenumber / cm

29

Integral intensity

60

40

(b) (a)

20

(c) 0

Nike

M50

CAD27

PGI11

30

Integral intensity

20

15

10

(b) (a)

5

0

Nike

M50

CAD27

PGI11

31

100 80

Integral intensity

60 40

(a)

18 16 14 12

(b)

10 8

Nike

M50

CAD27

PGI11

32

Integral intensity

80

(b)

60

40

(c) 20

(a)

15 10 5

Nike

M50

CAD27

PGI11

33

40

Integral intensity

30

(a)

20

(b) 10 5

(d)

4

(e) (c)

3 2 1 0

Nike

M50

CAD27

PGI11

34

*

200

M50 CAD33

102

NIKE

1-10

7

110

I N T E N S I T Y [arb. units]

PGI11

14

* * *

0 10

20

30

2 theta (Cu Kα )

35

Intensity 102/200

0 .7 0 .6 0 .5 0 .4

0.3

0.2

N IK E

P G I11

C A D 27

M 50

36

Table 1 The summarised scheme of the modifications including introduced gene and the kind of a genetic manipulation, effect of the modification and the construct content. Origin flax variety

Line

Gene introduced into plasmid and its source Cinnamyl alcohol deydrogenase, Linum usitatissimum sp.

Modification

Effect in plant

The construct content

L.usitatissi mum L., fibrous cultivar Nike

CAD27

Silencing CAD gene using RNAi method.

Lower lignin level [17]

PGI11

Polygalacturon ase I, Aspergillus aculeatus

Expressing fungal gene encoding polygalactouronase responsible for pectin degradation.

Reduced amount of pectin [16]

M50

β-ketothiolase (phb A), acetoacetylCoA reductase (phb B), and PHB synthase (phb C), Ralstonia eutropha

Expressing bacterial genes responsible for producing polyhydroxybutyrate.

Producing PHB in plant's tissues [18]

•The binary plasmid pHellsgate2-CAD: pHellsgate2 (EMBL: AJ311874); 506 bp fragment from flax; •CAD gene (EMBL: DQ487210); •promoter 35S CaMV and terminator OCS • The pBinAR vector (Frish et al. 1995); • the cDNAs for PGI I from Aspergillus aculeatus (GenBank: AF054893); • promoter 35S CaMV and terminator OCS • The binary vector pBinARHygABC14-3-3; • the phb B and phb C genes under a 35S CaMV promoter; the phb A gene under 14-3-3 promoter

37

Table 2 Biochemical composition of the cell wall fibre from control (NIKE) and transgenic lines. PLANT TYPE

Cellulose [mg/g DW]

Lignin [mg/g DW]

Pectin [mg/g DW]

CONTROL

641 +/- 9.1

26.6 +/- 0.9

37.2 +/- 2.8

CAD27

710** +/- 9.8

21.0** +/- 1.23

36.0^ +/- 2.3

PGI11

865** +/- 36

24.8 +/- 0.6

24.8** +/- 3.5

M50

615* +/- 1.2

22.9^ +/- 0.40

38.8^ +/- 3.1

The methods used to determine all components are described in the experimental section. The test of significance was performed using the Student’s t-test (* P < 0.05, ** P < 0.001, ^ P<0.1).

38

Table 3 Wavenumbers (ν) and integral intensity (A) of the Lorentzian components derived for the 3000-3700 cm-1 range for the natural (NIKE) and transgenic flax fibres. NIKE ν 3603 3537

A 21 114

PGI11 ν 3605 3540

A 16 64

CAD27 A ν 3605 15 3538 55

3445

176

3451

205

3459

140

3350 3259 3166

273 313 591

3355 3277 3222

295 98 359

3352 3262 3201

199 132 438

M50 ν

A

3482 3440 3400 3349 3290 3238

78 114 180 300 188 73

39

CAD27

PGI11 CAD27 M50

M50 4000

3500

3000 1800

1600 -1 1400

Wavenumber / cm

665

990

1264 1247 1200 1158

NIKE

Absorbance

NIKE PGI11

1318

1462 1429 1515

1655 1602

1737

Absorbance

3537 3445 3350 3259

Graphical abstract

1300 1200 1100 1000

900

800

-1

700

600

500

Wavenumber / cm

40

Highlights

•

The content of bio-chemicals in GM and natural flax fibres has been compared.

•

FT-IR spectra of fibres have been reported and analysed.

•

XRD studies have been used to characterize the cellulose fibres.

41