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FTIR spectroscopy monitoring of cell wall modifications during the habituation of bean (Phaseolus vulgaris L.) callus cultures to dichlobenil
Plant Science 167 (2004) 1273–1281 www.elsevier.com/locate/plantsci
FTIR spectroscopy monitoring of cell wall modifications during the habituation of bean (Phaseolus vulgaris L.) callus cultures to dichlobenil Ana Alonso-Simo´n, Antonio E. Encina, Pene´lope Garcı´a-Angulo, ´ lvarez, Jose´ L. Acebes* Jesu´s M. A Departamento de Biologı´a Vegetal. A´rea de Fisiologı´a Vegetal, Universidad de Leo´n, E-24071, Leo´n, Spain Received 18 May 2004; received in revised form 25 June 2004; accepted 26 June 2004 Available online 23 July 2004
Abstract The habituation of bean calluses to dichlobenil results from the acquisition of a modified cell wall, with an enhancement in pectins and a decrease in cellulose and hemicelluloses. In this work, Fourier transform infrared (FTIR) spectroscopy in conjunction with a set of multivariate analyses and other statistical tools, such as principal component analyses and Student’s t-test applied to clusters from a dendrogram, were used to monitor the modifications occurring in the cell walls of bean callus cultures due to a habituation program to dichlobenil. Forty samples of calluses, differing in the dichlobenil concentration at which they were growing and in the number of subcultures in a given concentration of dichlobenil, corresponding to two habituation experiments, were analyzed. Multivariate analyses of the spectra showed that the type and the extent of cell wall modifications depended on the concentration of the inhibitor in the culture medium and the time that the callus had been present at a given concentration of the inhibitor, and the analyses distinguished among three groups of calluses with different levels of habituation to dichlobenil: (i) non-habituated and habituated to low dichlobenil concentrations, (ii) habituated to intermediate concentrations (up to 4 mM), and (iii) habituated to high concentrations (4–12 mM). A slight modification of cell walls was only detected after 13 subcultures in 0.5 mM dichlobenil. In the presence of a higher concentration of dichlobenil, the content of cellulose was clearly reduced while that of pectins was increased. We conclude that FTIR spectroscopy associated with a set of statistical tools is a powerful method for analyzing in muro–and more rapidly–the changes in polysaccharides related to dichlobenil habituation, and that it could be used in the future to identify cell wall changes related to habituation to other herbicides or stress factors. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Cellulose; Cell wall; Dichlobenil; FTIR; Herbicide habituation; Pectin
1. Introduction Habituation of cell cultures to cellulose biosynthesis inhibitors, such as isoxaben or dichlobenil, is a useful method for assaying the plasticity of plant cell wall composition and structure [1]. Thus, cell cultures of several Abbreviations: FTIR spectroscopy, Fourier transform infrared spectroscopy; Tnm, callus growing in n mM dichlobenil during m subcultures; NTm, callus subcultured m times in the absence of dichlobenil; PCA, principal components analysis; PC, principal component . * Corresponding author. Tel.: +3487291482; fax: +3487291479. E-mail address: [email protected] (J.L. Acebes).
dicots have been habituated to dichlobenil. Tomato [2], tobacco [3–6], and bean [7] cell suspensions, and bean calluses [8] showed reduced levels of cellulose and hemicelluloses, and an enhancement of pectins. Similar modifications have been described in cell cultures habituated to isoxaben, such as bean calluses [9] and tobacco BY-2 cell suspensions [1], although the habituation of both cultures to isoxaben was quicker and displayed several minor differences regarding habituation to dichlobenil. The above chemical and structural changes were mainly characterized by cell wall fractionation of isolated cell walls, followed by gas chromatography of the derivatized
0168-9452/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2004.06.025
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sugars in each fraction. These analytical methods are timeconsuming, require relatively large amounts of sample, and more importantly the solvents and conditions required to extract and solubilize the polysaccharides from the cell wall can produce undesirable reactions. In contrast, Fourier transform infrared (FTIR) spectroscopy is a powerful and rapid assay for cell wall components and putative cross-links in that it non-destructively identifies polymers and functional groups and is able to provide abundant information about their in muro organization. Moreover, FTIR spectroscopy requires only small amounts of sample [10–11]. Despite the improvement brought to the procedures of cell wall analysis, the application of FTIR methodology may still be limited by the large amount of data and overlapping band components. The development of computer systems and the use of statistical tools have enabled easier processing of the information obtained from the spectra, thus increasing the interest and usefulness of spectroscopic techniques. Principal components analysis (PCA) is a method that reduces the dimensionality of the data from the several hundred data points of a spectral set to a fewer number of dimensions, and it is valid for gaining an exploratory analysis of the bulk of data in order to detect internal groupings [12]. The variability in each spectrum in relation to the mean of the population can then be represented as a smaller set of values (axes) termed principal components (PC). It is possible to derive a spectrum related to the PC score (named a PC loading) that represents an independent source of spectral variability with respect to all the data. The analysis of loading factors can be used to identify the molecular factors that underlie the grouping or the discrimination of the original data [13]. Principal component analysis (PCA) associated with FTIR spectroscopy has proved to be an important tool for the rapid identification and classification of mutants with altered cell wall compositions [13–14] and to characterize the modifications appearing in several of these mutants [15–17] or in transgenic plants with altered cell walls [18]. FTIR spectroscopy has also been used to characterize the modifications of cell walls in cell lines habituated to cellulose biosynthesis inhibitors [7–9,19]. The changes associated with the habituation of cultures to a cellulose biosynthesis inhibitor have been analyzed using high-concentrations of herbicide and long-term habituation periods. However, some short-term changes related to habituation have also been observed [1], although they have not been fully characterized. Accordingly, monitoring of the effects of this group of herbicides on cell wall composition along the habituation procedure would be useful to identify short-term habituation events, as compared to long-term events. The present work reports the monitoring of the cell wall changes in bean callus cultures through a procedure of habituation to dichlobenil using FTIR spectroscopy and a set of multivariate analyses and other statistical tools.
2. Material and methods 2.1. Callus culture and habituation to dichlobenil Calluses were obtained from bean leaves as previously described [8]. Leaves from 10-day-old bean seedlings were cultured aseptically at 27 8C for 30 days on Murashige and Skoog medium [20] solidified with 8 g l 1 agar, containing 30 g l 1 sucrose and 10 mM 2,4-dichlorofenoxyacetic acid (2,4-D). Calluses were removed from the explants and routinely subcultured for 30-day periods on an identical medium. Dichlobenil (>98% pure) from Fluka was dissolved in ethanol. The ethanol concentration never exceeded 0.2% (v/v), and it was checked that this concentration did not affect callus growth. Two habituation experiments developed under the same conditions but were carried out in two different periods. Callus pieces weighing 0.5–0.7 g (fresh weight) were transferred to a dichlobenil concentration equal to its I50 value (0.5 mM [8]) and subsequently subcultured in increasing concentrations of the herbicide. At least four subcultures of 30 days each were made between each increase in dichlobenil concentration. Aliquots of these calluses were taken at the end of each subculture and frozen at 20 8C until use. Habituated calluses growing at different dichlobenil concentrations are referred to as Tnm calluses, where ‘‘n’’ represents the mM concentration of dichlobenil, and ‘‘m’’ the number of subcultures in that concentration. Samples from calluses corresponding to experiment 1 are marked by* (Tnm). 2.2. Preparation of cell walls Calluses were homogenized in liquid nitrogen with a mortar and pestle, washed twice with cold 100 mM potassium phosphate buffer, pH 7.0, and treated with VI a-amylase from hog pancreas (Sigma Co.) for 4 h at 37 8C. The suspension was centrifuged and the pellet was washed with distilled water (3). The resulting pellet was washed with acetone (3), methanol:chloroform (1:1; v/v) (3), diethylether (2) and air-dried [21]. 2.3. FTIR spectroscopy and multivariate analysis Tablets for FTIR spectroscopy were prepared in a GrasebySpecac Press, using cell walls (2 mg) mixed with KBr (1:100 p/p). Spectra were obtained on a Perkin-Elmer instrument at a resolution of 1 cm 1. A window between 900 and 1800 cm 1, which contains information of characteristic polysaccharides, was selected in order to monitor cell wall structure modifications. All spectra were normalized and baseline-corrected with Perkin-Elmer IR Data management software. Then data were exported to Microsoft Excel 2000 and all spectra were area-normalized. Citrus polygalacturonic acid (sodium salt) (Sigma Co.), arabinogalactan
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(Sigma Co.), isolated xyloglucan from bean hemicelluloses [7], and fibrilar cellulose CF-11 (Whatman) exhaustively washed with 35% KOH, were used as standards. The samples of standard compounds were treated exactly in the same way. Cluster analysis was performed using the Ward method, and the Pearson coefficient was selected as distance measurement. Principal component analysis (PCA) was performed using a maximum of five principal components. Student’s t-test was calculated at the P = 0.95 significance threshold. All analyses were carried out using the Statistica software package.
calluses corresponding to two habituation experiments were analyzed (Fig. 1). The FTIR spectra showed a higher variability for the fingerprint (900–1200 cm 1) and 1600–1750 cm 1 regions. The fingerprint profile of spectra from cell walls of nonhabituated calluses resembled that of hemicellulosic polysaccharides. In contrast, the same region of the spectra in calluses habituated to high concentrations of dichlobenil showed peaks characteristic of commercial pectin. To elucidate differences among these spectra, a set of multivariate analyses was performed.
2.4. Cellulose analysis
3.2. Cluster analysis
Cellulose was quantified in crude cell walls by the Updegraff method [22], using the hydrolytic conditions described by Saeman [23] and quantifying the glucose released by the anthrone method [24].
The dendrogram corresponding to the cell wall spectra from all types of calluses displayed three main branches, designated A, B and C (Fig. 2). The spectra within the upper branch (A) were mainly those from non-habituated calluses or calluses habituated to 0.5 mM dichlobenil for up to seven subcultures. The central branch (B) was separated by 0.21 units from branch A and was constituted by spectra corresponding to cell walls of habituated calluses ranging from 0.5 mM dichlobenil, with more than 13 subcultures (T0.513), to 4 mM for their first subculture (T41). The lower branch (C), separated by 0.29 units from the former branches, was integrated by the spectra of calluses grown on the highest concentrations of dichlobenil (4–12 mM). Thus, three groups of calluses could be established, corresponding to non-habituation plus a low-level of habituation (A), an intermediate-level of habituation (B) and a high-level of habituation (C).
3. Results 3.1. FTIR spectra A habituation protocol was implemented in order to monitor the changes that occur in cell walls, when bean calluses are cultured in the presence of dichlobenil. Bean calluses were cultured for different numbers of subcultures at dichlobenil concentrations ranging from 0.5 mM—the I50 for bean calluses [8]—to 12 mM. Forty representative FTIR cell wall spectra from non-habituated and habituated
Fig. 1. Area normalized and baseline-corrected FTIR spectra of cell walls obtained from 40 bean calluses by the dichlobenil habituation procedure. Nonhabituated calluses (NT) and calluses habituated to different dichlobenil concentrations (T) were used (see Fig. 2). Representative spectra from non-habituated calluses and calluses habituated to high dichlobenil concentration are marked: (–) NT88; (- - -) T1274.
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Fig. 2. Dendrogram obtained by cluster analysis of non-habituated calluses and calluses habituated to different dichlobenil concentrations based on their FTIR spectra. NTm, non-habituated calluses growing during m subcultures; Tnm, habituated calluses growing in n mM dichlobenil during m subcultures. Asterisks reflect calluses corresponding to experiment 1. A–C are the clusters discussed in the text.
Each main branch was split into two sub-branches, named 1 and 2, separated by about 0.04 units in each case. In clusters A and B, the sub-branches (1 and 2) grouped cell cultures in accordance with their habituation level (A1 < A2 and B1 < B2). However, the two sub-branches of cluster C contained separately spectra from experiment 1 and 2, respectively. Within a subgroup, the distances among spectra were shorter than 0.03 units. 3.3. Principal components analysis Principal components 2 and 3 (PC2 and PC3) could be used to separate the above three groups of spectra (Fig. 3). There was a gradient from the negative side of both principal components, occupied by all NT and T0.52 calluses (cluster A), to the positive side of both PCs, which contained all the spectra from calluses with a higher level of habituation (cluster C). When the loadings for PC2 and PC3 were analyzed (Fig. 4), several wave numbers appeared as negative peaks regarding both PCs. Several negative peaks were characteristic of cellulose. Thus, the peak at 988 cm 1 was assigned to a bond shared by cello–triose, –tetraose, and –pentose [25]; the
doublet at 1036 and 1056 cm 1 was attributed to C–O–C and C–C bonds of the cellulose sugar rings [26], 1120 cm 1 to the C–O and C–C vibrations of cellulose [26]; 1164 cm 1 to the glycosidic C–O–C vibration of cellulose [26], and 1312 and 1372 cm 1 to a CH2 stretch of cellulose [26]. These results reflect a higher content of cellulose in both non-habituated and short-term-habituated calluses with respect to the content of long-term-habituated calluses. Attending to the wave numbers, which appeared as positive peaks for both PC2 and PC3, it was observed that these were assigned to pectins: 950 cm 1 was attributed to CO bending [27]; 1004 and 1096 cm 1 were assigned to C– O and C–C stretching, and 1140 cm 1 was assigned to asymmetric stretching of the glycosidic link [10]. Thus, calluses with a higher habituation level were enriched with pectins. PC3, but not PC2, also showed positive peaks at 1408 and 1604 cm 1, assigned to carboxylate group of uronic acids, whereas the 1715–1720 cm 1 area, assigned to ester linkages [12], appeared as negative. Since only calluses with the intermediate level of habituation were allocated at the positive side of PC3 but not PC2, their pectins seem to have a lower degree of methyl-esterification.
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Fig. 3. Principal component analysis of callus spectra. A plot of the second and third PCs is represented based on the FTIR spectra of non-habituated calluses and calluses habituated to different dichlobenil concentrations. A–C are the clusters discussed in the text.
3.4. Interpretation of the spectral differences among the three groups of calluses In order to appreciate differences among calluses with low, intermediate or high level of habituation, the average spectra from the three clusters observed in Fig. 2 were obtained (Fig. 5). The internal differences in clusters A and B were higher in the 990–1150 cm 1 and 1500–1630 cm 1 regions, whereas in cluster C, nearly all wave numbers were affected. Cluster A had a fingerprint region with more pronounced peaks than the other two clusters, and less marked peaks in the carboxylate (1420–1600 cm 1) regions. However, in habituated calluses (cluster C), more prominent peaks at
950, 1010, and 1150 cm 1 were observed, and the 1040– 1060 cm 1 area was split into two peaks. A shift in some peaks was noted, when cluster A was compared to clusters B and C: from 1620 to 1600 cm 1, and from 1440 to 1420 cm 1. A flattening of the 1500–1600 cm 1 area was also observed from cluster A to cluster C. Two Student’s t-tests were used to determine the significance of the difference between average values for each individual wave number of the spectrum, one of them comparing clusters A + B and C, in order to analyze differences related to the habituation to high levels of the herbicide (Fig. 6A), and the other comparing clusters A and B, in order to assay differences occurring during habituation to intermediate levels of dichlobenil (Fig. 6B).
Fig. 4. Loadings for PC2 (—) and PC3 (- - -) corresponding to Fig. 3.
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Fig. 6. Representation of the t-value from Student’s t-test for the comparison between clusters. Student’s t-test values between clusters in Fig. 2 (y-axis) were plotted as a function for the wave numbers (x-axis). Horizontal lines refer to the P = 0.95 significance threshold. (A) Clusters A + B compared to cluster C, showing changes during habituation to high dichlobenil concentrations; (B) cluster A compared to cluster B, reflecting changes during habituation to low dichlobenil concentrations.
Compared to high-habituated calluses, the most important differences in the second Student’s t-test (cluster A versus cluster B, Fig. 6B), were: Fig. 5. Plot of mean and standard deviation for each wave number corresponding to clusters A–C in the dendrogram shown in Fig. 2.
When clusters A + B were assayed against cluster C (Fig. 6A), among the strong positive and sharp peaks corresponding to t-values (higher in the non-habituated and low- and intermediate-habituated calluses than in the highhabituated ones), a series of prominent peaks were seen at wave numbers assigned to several cellulose linkages (988, 1040, 1060, 1172, 1316 and 1376 cm 1), xyloglucan (1128 cm 1) [28], and esterified pectins (1260 cm 1) [29], whilst the highest significant negative t-values were located at wave number intervals associated with free carboxylic acid groups of pectins: 1420 cm 1, which appeared displaced to 1436 cm 1, and 1600 cm 1, flattened between 1592 and 1628 cm 1 interval. A similar shift from 1420 cm 1 towards higher wave numbers has previously been reported in a t-test carried out on cellulose deficient mutants [14]. Other pectin wave numbers appeared as negative peaks (956 cm 1, corresponding to galactose, and 1008, 1100 and 1148 cm 1, assigned to galacturonic acid) between contiguous characteristic peaks of cellulose, but in the nonsignificant zone.
a) Most peaks characteristics of cellulose were positive but less prominent (988–1164 cm 1) or appeared at the negative (1316 cm 1) or in the non-significant (1376 cm 1) zones of Student’s t-values. This fact shows that, at initial stages of dichlobenil habituation, cell wall cellulose content was not as depleted as that with highhabituated calluses. b) The negative peaks at 1008–1100 cm 1, characteristic of pectins, were also more attenuated. These results indicate that calluses with low levels of habituation show lesspectin modification than high-habituated calluses. c) Some ranges, as those located between 900 and 980 cm 1 and between 1180 and 1360 cm 1 showed negative Student’s t-values. d) The peaks at 1476 and 1736 cm 1 were shifted towards 1456 and 1716–1690 cm 1, respectively. 3.5. Cellulose content of calluses cell wall As expected, the increase in dichlobenil concentration during the habituation procedure caused a reduction in the cellulose content (Fig. 7). Regarding experiment 1, with only few exceptions, the three clusters from the dendrogram grouped calluses with different cellulose contents among them: the upper branch, with cellulose levels higher than
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Fig. 7. Cellulose contents in crude cell walls of non-habituated calluses and calluses habituated to different dichlobenil concentrations. Asterisks reflect calluses corresponding to experiment 1.
16% of the cell wall, the central branch, with cellulose contents between 15 and 10%, and the lower branch, with cellulose contents below 10%. However, no differences between sub-branches 1 and 2 within a cluster could be observed. At a same level of habituation, calluses from experiment 1 showed higher cellulose contents as compared with those of experiment 2. However, at dichlobenil concentrations higher than 8 mM, this effect was not significant. In relation to low habituation levels, the cellulose content in the cell wall from calluses habituated to 0.5 mM dichlobenil in the initial subcultures was similar to that of nonhabituated calluses. As the culture in 0.5 mM dichlobenil progressed, the cellulose content underwent an important reduction. Interestingly, after 84 subcultures, the cellulose content increased again, and was even higher than that of the non-habituated cultures. Similarly, at initial stages of habituation, experiment 1 calluses showed also a higher cellulose content than non-habituated counterparts.
4. Discussion Tolerance to dichlobenil in bean calluses and cell suspensions [7–8] and in cell suspensions of tomato [2] and tobacco [3–6] largely results from the ability of these cells to modify the composition and structure of their cell walls. The altered cell walls have reduced amounts of cellulose and hemicelluloses, mainly xyloglucan, and enhanced levels of pectins. The above cell wall modifications have previously been assayed with great precision using cell wall fractionation of isolated cell walls followed by sugar analysis of each fraction, but the method is too cumbersome, and can therefore only be applied to a limited number of samples. In the present report, we have demonstrated that FTIR is an efficient and rapid method for identifying a broad range of
the structural modifications in cell walls that appear as a consequence of habituation to dichlobenil. Sabba et al. [4] distinguished between the effects caused by short-term treatment to dichlobenil (3–24 h) and longterm habituation (months to years). Our study, carried out throughout the habituation procedure, establishes that the type and extent of cell wall modifications depend on two important features: the concentration of the inhibitor in the culture medium, and the time that the calluses have been present in a given concentration of the inhibitor. During habituation to dichlobenil, no appreciable changes in the cell wall spectra were detected when the calluses were cultured for less than 7 subcultures in 0.5 mM dichlobenil, indicating that a lag period would be necessary until cell wall modifications arise. However, after 13 subcultures in 0.5 mM dichlobenil, the spectra of the cell walls underwent several changes, including an attenuation of the peaks related to cellulose. Cell wall modifications became more evident between the spectra from calluses that were grown in a low concentration of dichlobenil (low-habituation level) and in an intermediate concentration of dichlobenil (intermediate-habituation level), and between these and long-term habituated calluses. In the two habituation experiments, although the range of intermediate dichlobenil concentrations in which main cell wall modifications were observed and the extent of these modifications were different, further increases in the dichlobenil concentration did not seem to affect cell wall composition and long-term habituated calluses were similar in both experiments. The spectra from calluses that were growing in the same dichlobenil concentration for a low number of subcultures resembled those they came from, but after several subcultures they modified their cell wall composition, showing more pectins and less cellulose. In a range of low dichlobenil concentrations (i.e. 0.5–1 mM), the calluses showed a tendency to revert to their original cellulose
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content, and in some cases even to be higher (i.e. in T0.584 from experiment 2 or in T0.51 from experiment 1), as the number of subcultures increased. This reversal of certain callus features as habituation proceeded suggests the existence of a second mechanism of tolerance, such as the degradation or compartmentalisation of the herbicide in addition to a modified cell wall. In accordance with this, we have previously reported that dehabituated bean cell suspensions in which dichlobenil had been suppressed from the culture medium maintained a high-tolerance towards this herbicide [7]. In addition, we have recently observed that dichlobenil-habituated calluses show a high-tolerance to other cellulose inhibitors, such as isoxaben or flupoxam (Garcı´a-Angulo, personal communication). There was a strong correlation between the assignment of a callus to a group in the dendrogram and the cellulose content of the callus. In the Student’s t-test plot (Fig. 6A), six peaks characteristics of cellulose were observed. By analyzing cell wall mutants, Mouille et al. [14] found changes in the same wave numbers related to cellulose and predicted that these could be used to follow cellulose contents in different developmental contexts or in different environmental conditions. Our results seem to confirm their prognosis. We have previously reported that the cell walls of dichlobenil-habituated bean cell suspensions accompany their decrease in cellulose with a higher proportion of noncrystalline cellulose [7]. It would be interesting to check whether some of the FTIR features associated with cellulose display any differences, i.e in some peak intensities, during habituation in order to monitor differences in cellulose crystallinity. Previous analyses of habituated bean cultures showed that they had a high-proportion of pectins, and that these pectins had a lower degree of esterification [8]. Also, recent immunocytochemical analyses of these cultures seem to corroborate these observations (Garcı´a-Angulo and Willats, personal communication). The results reported here confirm this idea since PCA analysis of our data reflects a PC3 loading profile with peaks of pectin groups, indicating that their cell walls were becoming enriched in pectin throughout the habituation process. Moreover, tomato cells habituated to dichlobenil also show an FTIR pattern with enhanced free carboxylate peaks [19]. Three types of esters may be present in the wall: free methyl esters of carboxylic groups of pectin, other saturatedalkyl esters that may cross-link pectin molecules, and phenolic esters [29]. The broadness and irregularity of the peak at 1700–1740 cm 1 in the Student’s t-test and PC loadings plots pointed to the coexistence of several ester groups and/or the presence of variations in environment among the ester groups [30]. The multiple wave numbers corresponding to ester bonds suggest that different carboxylic ester linkages were present in the habituated calluses or that the reduced cellulose content caused changes in the chemical features of subsets of pectic polysaccharides. When bean calluses were continuously subcultured at high
concentrations of dichlobenil, the peak at about 1700 was shifted to 1736 cm 1, indicating a possible shift from pectinsaturated esters to phenolic or other saturated-alkyl esters. Further observations in callus cultures habituated to highdichlobenil concentrations, such as browning and the typical autofluorescence attributed to phenolic esters (data not shown), pointed to a higher proportion of phenolics in their cell walls. The fact that the reduction of cellulose in cell walls is accompanied by a higher proportion of pectins is confirmed by several other reports, such as those carried out with Arabidopsis hypocotyls treated with dichlobenil [11,14], or with cell wall mutants such as prc1, kob1, rsw1, rsw2, Kor1-1, pom1, etc. [14], or those reports referring to the cellulose synthase gene silenced by virus induction [31]. In addition, impaired cellulose biosynthesis may be accompanied by an ectopic accumulation of lignin, as in eli1 mutants [32]. Also, a reduction in the amount of cellulose has recently been reported to be associated with a down-regulation of an aexpansin gene in Petunia hybrida [18]. Our results are in agreement with most results and also highlight the parallelism between the decrease in cellulose and the increase in pectin during the habituation of bean cells to dichlobenil. Here, we also demonstrate that principal component analysis of FTIR spectra and the data from Student’s t-test applied to clusters from a dendrogram constructed using the ward clustering algorithm contribute with complementary information and allow us to establish the different contributions of different components during habituation to dichlobenil. Thus, it may be concluded that multivariate analysis together with chemical characterisation of cell walls is a valid tool for monitoring and analyzing the changes occurring in cell wall composition and architecture associated with habituation to cellulose biosynthesis inhibitors, and could in the future be used for the identification of cell wall changes related to other stress conditions, including habituation to other herbicides.
Acknowledgements This work was supported by grants from DGICYT (BFI2002-03253) and Junta de Castilla y Leo´ n (LE 17/04) and predoctoral grants to A.A.-S. and P.G.-A. from the University of Leo´ n and the Spanish Science and Technology Ministry respectively. We thank N. Skinner for correcting the English version of the manuscript.
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FTIR spectroscopy monitoring of cell wall modifications during the habituation of bean (Phaseolus vulgaris L.) callus cultures to dichlobenil Ana Alonso-Simo´n, Antonio E. Encina, Pene´lope Garcı´a-Angulo, ´ lvarez, Jose´ L. Acebes* Jesu´s M. A Departamento de Biologı´a Vegetal. A´rea de Fisiologı´a Vegetal, Universidad de Leo´n, E-24071, Leo´n, Spain Received 18 May 2004; received in revised form 25 June 2004; accepted 26 June 2004 Available online 23 July 2004
Abstract The habituation of bean calluses to dichlobenil results from the acquisition of a modified cell wall, with an enhancement in pectins and a decrease in cellulose and hemicelluloses. In this work, Fourier transform infrared (FTIR) spectroscopy in conjunction with a set of multivariate analyses and other statistical tools, such as principal component analyses and Student’s t-test applied to clusters from a dendrogram, were used to monitor the modifications occurring in the cell walls of bean callus cultures due to a habituation program to dichlobenil. Forty samples of calluses, differing in the dichlobenil concentration at which they were growing and in the number of subcultures in a given concentration of dichlobenil, corresponding to two habituation experiments, were analyzed. Multivariate analyses of the spectra showed that the type and the extent of cell wall modifications depended on the concentration of the inhibitor in the culture medium and the time that the callus had been present at a given concentration of the inhibitor, and the analyses distinguished among three groups of calluses with different levels of habituation to dichlobenil: (i) non-habituated and habituated to low dichlobenil concentrations, (ii) habituated to intermediate concentrations (up to 4 mM), and (iii) habituated to high concentrations (4–12 mM). A slight modification of cell walls was only detected after 13 subcultures in 0.5 mM dichlobenil. In the presence of a higher concentration of dichlobenil, the content of cellulose was clearly reduced while that of pectins was increased. We conclude that FTIR spectroscopy associated with a set of statistical tools is a powerful method for analyzing in muro–and more rapidly–the changes in polysaccharides related to dichlobenil habituation, and that it could be used in the future to identify cell wall changes related to habituation to other herbicides or stress factors. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Cellulose; Cell wall; Dichlobenil; FTIR; Herbicide habituation; Pectin
1. Introduction Habituation of cell cultures to cellulose biosynthesis inhibitors, such as isoxaben or dichlobenil, is a useful method for assaying the plasticity of plant cell wall composition and structure [1]. Thus, cell cultures of several Abbreviations: FTIR spectroscopy, Fourier transform infrared spectroscopy; Tnm, callus growing in n mM dichlobenil during m subcultures; NTm, callus subcultured m times in the absence of dichlobenil; PCA, principal components analysis; PC, principal component . * Corresponding author. Tel.: +3487291482; fax: +3487291479. E-mail address: [email protected] (J.L. Acebes).
dicots have been habituated to dichlobenil. Tomato [2], tobacco [3–6], and bean [7] cell suspensions, and bean calluses [8] showed reduced levels of cellulose and hemicelluloses, and an enhancement of pectins. Similar modifications have been described in cell cultures habituated to isoxaben, such as bean calluses [9] and tobacco BY-2 cell suspensions [1], although the habituation of both cultures to isoxaben was quicker and displayed several minor differences regarding habituation to dichlobenil. The above chemical and structural changes were mainly characterized by cell wall fractionation of isolated cell walls, followed by gas chromatography of the derivatized
0168-9452/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2004.06.025
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sugars in each fraction. These analytical methods are timeconsuming, require relatively large amounts of sample, and more importantly the solvents and conditions required to extract and solubilize the polysaccharides from the cell wall can produce undesirable reactions. In contrast, Fourier transform infrared (FTIR) spectroscopy is a powerful and rapid assay for cell wall components and putative cross-links in that it non-destructively identifies polymers and functional groups and is able to provide abundant information about their in muro organization. Moreover, FTIR spectroscopy requires only small amounts of sample [10–11]. Despite the improvement brought to the procedures of cell wall analysis, the application of FTIR methodology may still be limited by the large amount of data and overlapping band components. The development of computer systems and the use of statistical tools have enabled easier processing of the information obtained from the spectra, thus increasing the interest and usefulness of spectroscopic techniques. Principal components analysis (PCA) is a method that reduces the dimensionality of the data from the several hundred data points of a spectral set to a fewer number of dimensions, and it is valid for gaining an exploratory analysis of the bulk of data in order to detect internal groupings [12]. The variability in each spectrum in relation to the mean of the population can then be represented as a smaller set of values (axes) termed principal components (PC). It is possible to derive a spectrum related to the PC score (named a PC loading) that represents an independent source of spectral variability with respect to all the data. The analysis of loading factors can be used to identify the molecular factors that underlie the grouping or the discrimination of the original data [13]. Principal component analysis (PCA) associated with FTIR spectroscopy has proved to be an important tool for the rapid identification and classification of mutants with altered cell wall compositions [13–14] and to characterize the modifications appearing in several of these mutants [15–17] or in transgenic plants with altered cell walls [18]. FTIR spectroscopy has also been used to characterize the modifications of cell walls in cell lines habituated to cellulose biosynthesis inhibitors [7–9,19]. The changes associated with the habituation of cultures to a cellulose biosynthesis inhibitor have been analyzed using high-concentrations of herbicide and long-term habituation periods. However, some short-term changes related to habituation have also been observed [1], although they have not been fully characterized. Accordingly, monitoring of the effects of this group of herbicides on cell wall composition along the habituation procedure would be useful to identify short-term habituation events, as compared to long-term events. The present work reports the monitoring of the cell wall changes in bean callus cultures through a procedure of habituation to dichlobenil using FTIR spectroscopy and a set of multivariate analyses and other statistical tools.
2. Material and methods 2.1. Callus culture and habituation to dichlobenil Calluses were obtained from bean leaves as previously described [8]. Leaves from 10-day-old bean seedlings were cultured aseptically at 27 8C for 30 days on Murashige and Skoog medium [20] solidified with 8 g l 1 agar, containing 30 g l 1 sucrose and 10 mM 2,4-dichlorofenoxyacetic acid (2,4-D). Calluses were removed from the explants and routinely subcultured for 30-day periods on an identical medium. Dichlobenil (>98% pure) from Fluka was dissolved in ethanol. The ethanol concentration never exceeded 0.2% (v/v), and it was checked that this concentration did not affect callus growth. Two habituation experiments developed under the same conditions but were carried out in two different periods. Callus pieces weighing 0.5–0.7 g (fresh weight) were transferred to a dichlobenil concentration equal to its I50 value (0.5 mM [8]) and subsequently subcultured in increasing concentrations of the herbicide. At least four subcultures of 30 days each were made between each increase in dichlobenil concentration. Aliquots of these calluses were taken at the end of each subculture and frozen at 20 8C until use. Habituated calluses growing at different dichlobenil concentrations are referred to as Tnm calluses, where ‘‘n’’ represents the mM concentration of dichlobenil, and ‘‘m’’ the number of subcultures in that concentration. Samples from calluses corresponding to experiment 1 are marked by* (Tnm). 2.2. Preparation of cell walls Calluses were homogenized in liquid nitrogen with a mortar and pestle, washed twice with cold 100 mM potassium phosphate buffer, pH 7.0, and treated with VI a-amylase from hog pancreas (Sigma Co.) for 4 h at 37 8C. The suspension was centrifuged and the pellet was washed with distilled water (3). The resulting pellet was washed with acetone (3), methanol:chloroform (1:1; v/v) (3), diethylether (2) and air-dried [21]. 2.3. FTIR spectroscopy and multivariate analysis Tablets for FTIR spectroscopy were prepared in a GrasebySpecac Press, using cell walls (2 mg) mixed with KBr (1:100 p/p). Spectra were obtained on a Perkin-Elmer instrument at a resolution of 1 cm 1. A window between 900 and 1800 cm 1, which contains information of characteristic polysaccharides, was selected in order to monitor cell wall structure modifications. All spectra were normalized and baseline-corrected with Perkin-Elmer IR Data management software. Then data were exported to Microsoft Excel 2000 and all spectra were area-normalized. Citrus polygalacturonic acid (sodium salt) (Sigma Co.), arabinogalactan
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(Sigma Co.), isolated xyloglucan from bean hemicelluloses [7], and fibrilar cellulose CF-11 (Whatman) exhaustively washed with 35% KOH, were used as standards. The samples of standard compounds were treated exactly in the same way. Cluster analysis was performed using the Ward method, and the Pearson coefficient was selected as distance measurement. Principal component analysis (PCA) was performed using a maximum of five principal components. Student’s t-test was calculated at the P = 0.95 significance threshold. All analyses were carried out using the Statistica software package.
calluses corresponding to two habituation experiments were analyzed (Fig. 1). The FTIR spectra showed a higher variability for the fingerprint (900–1200 cm 1) and 1600–1750 cm 1 regions. The fingerprint profile of spectra from cell walls of nonhabituated calluses resembled that of hemicellulosic polysaccharides. In contrast, the same region of the spectra in calluses habituated to high concentrations of dichlobenil showed peaks characteristic of commercial pectin. To elucidate differences among these spectra, a set of multivariate analyses was performed.
2.4. Cellulose analysis
3.2. Cluster analysis
Cellulose was quantified in crude cell walls by the Updegraff method [22], using the hydrolytic conditions described by Saeman [23] and quantifying the glucose released by the anthrone method [24].
The dendrogram corresponding to the cell wall spectra from all types of calluses displayed three main branches, designated A, B and C (Fig. 2). The spectra within the upper branch (A) were mainly those from non-habituated calluses or calluses habituated to 0.5 mM dichlobenil for up to seven subcultures. The central branch (B) was separated by 0.21 units from branch A and was constituted by spectra corresponding to cell walls of habituated calluses ranging from 0.5 mM dichlobenil, with more than 13 subcultures (T0.513), to 4 mM for their first subculture (T41). The lower branch (C), separated by 0.29 units from the former branches, was integrated by the spectra of calluses grown on the highest concentrations of dichlobenil (4–12 mM). Thus, three groups of calluses could be established, corresponding to non-habituation plus a low-level of habituation (A), an intermediate-level of habituation (B) and a high-level of habituation (C).
3. Results 3.1. FTIR spectra A habituation protocol was implemented in order to monitor the changes that occur in cell walls, when bean calluses are cultured in the presence of dichlobenil. Bean calluses were cultured for different numbers of subcultures at dichlobenil concentrations ranging from 0.5 mM—the I50 for bean calluses [8]—to 12 mM. Forty representative FTIR cell wall spectra from non-habituated and habituated
Fig. 1. Area normalized and baseline-corrected FTIR spectra of cell walls obtained from 40 bean calluses by the dichlobenil habituation procedure. Nonhabituated calluses (NT) and calluses habituated to different dichlobenil concentrations (T) were used (see Fig. 2). Representative spectra from non-habituated calluses and calluses habituated to high dichlobenil concentration are marked: (–) NT88; (- - -) T1274.
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Fig. 2. Dendrogram obtained by cluster analysis of non-habituated calluses and calluses habituated to different dichlobenil concentrations based on their FTIR spectra. NTm, non-habituated calluses growing during m subcultures; Tnm, habituated calluses growing in n mM dichlobenil during m subcultures. Asterisks reflect calluses corresponding to experiment 1. A–C are the clusters discussed in the text.
Each main branch was split into two sub-branches, named 1 and 2, separated by about 0.04 units in each case. In clusters A and B, the sub-branches (1 and 2) grouped cell cultures in accordance with their habituation level (A1 < A2 and B1 < B2). However, the two sub-branches of cluster C contained separately spectra from experiment 1 and 2, respectively. Within a subgroup, the distances among spectra were shorter than 0.03 units. 3.3. Principal components analysis Principal components 2 and 3 (PC2 and PC3) could be used to separate the above three groups of spectra (Fig. 3). There was a gradient from the negative side of both principal components, occupied by all NT and T0.52 calluses (cluster A), to the positive side of both PCs, which contained all the spectra from calluses with a higher level of habituation (cluster C). When the loadings for PC2 and PC3 were analyzed (Fig. 4), several wave numbers appeared as negative peaks regarding both PCs. Several negative peaks were characteristic of cellulose. Thus, the peak at 988 cm 1 was assigned to a bond shared by cello–triose, –tetraose, and –pentose [25]; the
doublet at 1036 and 1056 cm 1 was attributed to C–O–C and C–C bonds of the cellulose sugar rings [26], 1120 cm 1 to the C–O and C–C vibrations of cellulose [26]; 1164 cm 1 to the glycosidic C–O–C vibration of cellulose [26], and 1312 and 1372 cm 1 to a CH2 stretch of cellulose [26]. These results reflect a higher content of cellulose in both non-habituated and short-term-habituated calluses with respect to the content of long-term-habituated calluses. Attending to the wave numbers, which appeared as positive peaks for both PC2 and PC3, it was observed that these were assigned to pectins: 950 cm 1 was attributed to CO bending [27]; 1004 and 1096 cm 1 were assigned to C– O and C–C stretching, and 1140 cm 1 was assigned to asymmetric stretching of the glycosidic link [10]. Thus, calluses with a higher habituation level were enriched with pectins. PC3, but not PC2, also showed positive peaks at 1408 and 1604 cm 1, assigned to carboxylate group of uronic acids, whereas the 1715–1720 cm 1 area, assigned to ester linkages [12], appeared as negative. Since only calluses with the intermediate level of habituation were allocated at the positive side of PC3 but not PC2, their pectins seem to have a lower degree of methyl-esterification.
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Fig. 3. Principal component analysis of callus spectra. A plot of the second and third PCs is represented based on the FTIR spectra of non-habituated calluses and calluses habituated to different dichlobenil concentrations. A–C are the clusters discussed in the text.
3.4. Interpretation of the spectral differences among the three groups of calluses In order to appreciate differences among calluses with low, intermediate or high level of habituation, the average spectra from the three clusters observed in Fig. 2 were obtained (Fig. 5). The internal differences in clusters A and B were higher in the 990–1150 cm 1 and 1500–1630 cm 1 regions, whereas in cluster C, nearly all wave numbers were affected. Cluster A had a fingerprint region with more pronounced peaks than the other two clusters, and less marked peaks in the carboxylate (1420–1600 cm 1) regions. However, in habituated calluses (cluster C), more prominent peaks at
950, 1010, and 1150 cm 1 were observed, and the 1040– 1060 cm 1 area was split into two peaks. A shift in some peaks was noted, when cluster A was compared to clusters B and C: from 1620 to 1600 cm 1, and from 1440 to 1420 cm 1. A flattening of the 1500–1600 cm 1 area was also observed from cluster A to cluster C. Two Student’s t-tests were used to determine the significance of the difference between average values for each individual wave number of the spectrum, one of them comparing clusters A + B and C, in order to analyze differences related to the habituation to high levels of the herbicide (Fig. 6A), and the other comparing clusters A and B, in order to assay differences occurring during habituation to intermediate levels of dichlobenil (Fig. 6B).
Fig. 4. Loadings for PC2 (—) and PC3 (- - -) corresponding to Fig. 3.
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Fig. 6. Representation of the t-value from Student’s t-test for the comparison between clusters. Student’s t-test values between clusters in Fig. 2 (y-axis) were plotted as a function for the wave numbers (x-axis). Horizontal lines refer to the P = 0.95 significance threshold. (A) Clusters A + B compared to cluster C, showing changes during habituation to high dichlobenil concentrations; (B) cluster A compared to cluster B, reflecting changes during habituation to low dichlobenil concentrations.
Compared to high-habituated calluses, the most important differences in the second Student’s t-test (cluster A versus cluster B, Fig. 6B), were: Fig. 5. Plot of mean and standard deviation for each wave number corresponding to clusters A–C in the dendrogram shown in Fig. 2.
When clusters A + B were assayed against cluster C (Fig. 6A), among the strong positive and sharp peaks corresponding to t-values (higher in the non-habituated and low- and intermediate-habituated calluses than in the highhabituated ones), a series of prominent peaks were seen at wave numbers assigned to several cellulose linkages (988, 1040, 1060, 1172, 1316 and 1376 cm 1), xyloglucan (1128 cm 1) [28], and esterified pectins (1260 cm 1) [29], whilst the highest significant negative t-values were located at wave number intervals associated with free carboxylic acid groups of pectins: 1420 cm 1, which appeared displaced to 1436 cm 1, and 1600 cm 1, flattened between 1592 and 1628 cm 1 interval. A similar shift from 1420 cm 1 towards higher wave numbers has previously been reported in a t-test carried out on cellulose deficient mutants [14]. Other pectin wave numbers appeared as negative peaks (956 cm 1, corresponding to galactose, and 1008, 1100 and 1148 cm 1, assigned to galacturonic acid) between contiguous characteristic peaks of cellulose, but in the nonsignificant zone.
a) Most peaks characteristics of cellulose were positive but less prominent (988–1164 cm 1) or appeared at the negative (1316 cm 1) or in the non-significant (1376 cm 1) zones of Student’s t-values. This fact shows that, at initial stages of dichlobenil habituation, cell wall cellulose content was not as depleted as that with highhabituated calluses. b) The negative peaks at 1008–1100 cm 1, characteristic of pectins, were also more attenuated. These results indicate that calluses with low levels of habituation show lesspectin modification than high-habituated calluses. c) Some ranges, as those located between 900 and 980 cm 1 and between 1180 and 1360 cm 1 showed negative Student’s t-values. d) The peaks at 1476 and 1736 cm 1 were shifted towards 1456 and 1716–1690 cm 1, respectively. 3.5. Cellulose content of calluses cell wall As expected, the increase in dichlobenil concentration during the habituation procedure caused a reduction in the cellulose content (Fig. 7). Regarding experiment 1, with only few exceptions, the three clusters from the dendrogram grouped calluses with different cellulose contents among them: the upper branch, with cellulose levels higher than
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Fig. 7. Cellulose contents in crude cell walls of non-habituated calluses and calluses habituated to different dichlobenil concentrations. Asterisks reflect calluses corresponding to experiment 1.
16% of the cell wall, the central branch, with cellulose contents between 15 and 10%, and the lower branch, with cellulose contents below 10%. However, no differences between sub-branches 1 and 2 within a cluster could be observed. At a same level of habituation, calluses from experiment 1 showed higher cellulose contents as compared with those of experiment 2. However, at dichlobenil concentrations higher than 8 mM, this effect was not significant. In relation to low habituation levels, the cellulose content in the cell wall from calluses habituated to 0.5 mM dichlobenil in the initial subcultures was similar to that of nonhabituated calluses. As the culture in 0.5 mM dichlobenil progressed, the cellulose content underwent an important reduction. Interestingly, after 84 subcultures, the cellulose content increased again, and was even higher than that of the non-habituated cultures. Similarly, at initial stages of habituation, experiment 1 calluses showed also a higher cellulose content than non-habituated counterparts.
4. Discussion Tolerance to dichlobenil in bean calluses and cell suspensions [7–8] and in cell suspensions of tomato [2] and tobacco [3–6] largely results from the ability of these cells to modify the composition and structure of their cell walls. The altered cell walls have reduced amounts of cellulose and hemicelluloses, mainly xyloglucan, and enhanced levels of pectins. The above cell wall modifications have previously been assayed with great precision using cell wall fractionation of isolated cell walls followed by sugar analysis of each fraction, but the method is too cumbersome, and can therefore only be applied to a limited number of samples. In the present report, we have demonstrated that FTIR is an efficient and rapid method for identifying a broad range of
the structural modifications in cell walls that appear as a consequence of habituation to dichlobenil. Sabba et al. [4] distinguished between the effects caused by short-term treatment to dichlobenil (3–24 h) and longterm habituation (months to years). Our study, carried out throughout the habituation procedure, establishes that the type and extent of cell wall modifications depend on two important features: the concentration of the inhibitor in the culture medium, and the time that the calluses have been present in a given concentration of the inhibitor. During habituation to dichlobenil, no appreciable changes in the cell wall spectra were detected when the calluses were cultured for less than 7 subcultures in 0.5 mM dichlobenil, indicating that a lag period would be necessary until cell wall modifications arise. However, after 13 subcultures in 0.5 mM dichlobenil, the spectra of the cell walls underwent several changes, including an attenuation of the peaks related to cellulose. Cell wall modifications became more evident between the spectra from calluses that were grown in a low concentration of dichlobenil (low-habituation level) and in an intermediate concentration of dichlobenil (intermediate-habituation level), and between these and long-term habituated calluses. In the two habituation experiments, although the range of intermediate dichlobenil concentrations in which main cell wall modifications were observed and the extent of these modifications were different, further increases in the dichlobenil concentration did not seem to affect cell wall composition and long-term habituated calluses were similar in both experiments. The spectra from calluses that were growing in the same dichlobenil concentration for a low number of subcultures resembled those they came from, but after several subcultures they modified their cell wall composition, showing more pectins and less cellulose. In a range of low dichlobenil concentrations (i.e. 0.5–1 mM), the calluses showed a tendency to revert to their original cellulose
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content, and in some cases even to be higher (i.e. in T0.584 from experiment 2 or in T0.51 from experiment 1), as the number of subcultures increased. This reversal of certain callus features as habituation proceeded suggests the existence of a second mechanism of tolerance, such as the degradation or compartmentalisation of the herbicide in addition to a modified cell wall. In accordance with this, we have previously reported that dehabituated bean cell suspensions in which dichlobenil had been suppressed from the culture medium maintained a high-tolerance towards this herbicide [7]. In addition, we have recently observed that dichlobenil-habituated calluses show a high-tolerance to other cellulose inhibitors, such as isoxaben or flupoxam (Garcı´a-Angulo, personal communication). There was a strong correlation between the assignment of a callus to a group in the dendrogram and the cellulose content of the callus. In the Student’s t-test plot (Fig. 6A), six peaks characteristics of cellulose were observed. By analyzing cell wall mutants, Mouille et al. [14] found changes in the same wave numbers related to cellulose and predicted that these could be used to follow cellulose contents in different developmental contexts or in different environmental conditions. Our results seem to confirm their prognosis. We have previously reported that the cell walls of dichlobenil-habituated bean cell suspensions accompany their decrease in cellulose with a higher proportion of noncrystalline cellulose [7]. It would be interesting to check whether some of the FTIR features associated with cellulose display any differences, i.e in some peak intensities, during habituation in order to monitor differences in cellulose crystallinity. Previous analyses of habituated bean cultures showed that they had a high-proportion of pectins, and that these pectins had a lower degree of esterification [8]. Also, recent immunocytochemical analyses of these cultures seem to corroborate these observations (Garcı´a-Angulo and Willats, personal communication). The results reported here confirm this idea since PCA analysis of our data reflects a PC3 loading profile with peaks of pectin groups, indicating that their cell walls were becoming enriched in pectin throughout the habituation process. Moreover, tomato cells habituated to dichlobenil also show an FTIR pattern with enhanced free carboxylate peaks [19]. Three types of esters may be present in the wall: free methyl esters of carboxylic groups of pectin, other saturatedalkyl esters that may cross-link pectin molecules, and phenolic esters [29]. The broadness and irregularity of the peak at 1700–1740 cm 1 in the Student’s t-test and PC loadings plots pointed to the coexistence of several ester groups and/or the presence of variations in environment among the ester groups [30]. The multiple wave numbers corresponding to ester bonds suggest that different carboxylic ester linkages were present in the habituated calluses or that the reduced cellulose content caused changes in the chemical features of subsets of pectic polysaccharides. When bean calluses were continuously subcultured at high
concentrations of dichlobenil, the peak at about 1700 was shifted to 1736 cm 1, indicating a possible shift from pectinsaturated esters to phenolic or other saturated-alkyl esters. Further observations in callus cultures habituated to highdichlobenil concentrations, such as browning and the typical autofluorescence attributed to phenolic esters (data not shown), pointed to a higher proportion of phenolics in their cell walls. The fact that the reduction of cellulose in cell walls is accompanied by a higher proportion of pectins is confirmed by several other reports, such as those carried out with Arabidopsis hypocotyls treated with dichlobenil [11,14], or with cell wall mutants such as prc1, kob1, rsw1, rsw2, Kor1-1, pom1, etc. [14], or those reports referring to the cellulose synthase gene silenced by virus induction [31]. In addition, impaired cellulose biosynthesis may be accompanied by an ectopic accumulation of lignin, as in eli1 mutants [32]. Also, a reduction in the amount of cellulose has recently been reported to be associated with a down-regulation of an aexpansin gene in Petunia hybrida [18]. Our results are in agreement with most results and also highlight the parallelism between the decrease in cellulose and the increase in pectin during the habituation of bean cells to dichlobenil. Here, we also demonstrate that principal component analysis of FTIR spectra and the data from Student’s t-test applied to clusters from a dendrogram constructed using the ward clustering algorithm contribute with complementary information and allow us to establish the different contributions of different components during habituation to dichlobenil. Thus, it may be concluded that multivariate analysis together with chemical characterisation of cell walls is a valid tool for monitoring and analyzing the changes occurring in cell wall composition and architecture associated with habituation to cellulose biosynthesis inhibitors, and could in the future be used for the identification of cell wall changes related to other stress conditions, including habituation to other herbicides.
Acknowledgements This work was supported by grants from DGICYT (BFI2002-03253) and Junta de Castilla y Leo´ n (LE 17/04) and predoctoral grants to A.A.-S. and P.G.-A. from the University of Leo´ n and the Spanish Science and Technology Ministry respectively. We thank N. Skinner for correcting the English version of the manuscript.
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