Sugar-cane cell anatomy and topochemical distribution of aromatic components
Rind and pith regions were excised from sugar cane internodes, cut into transverse sections 1 μm thick, and examined by microscopy. Using toluidine blue staining, we found that the sugar-cane internodes had the typical vascular bundles (vessels and surrounding fibers) and sucrose-storing parenchyma cells (Figure 1). The vascular bundles were more abundant in the rind, whereas parenchyma cells predominated in the pith region, consistent with the literature on sugar-cane anatomy [15] and other grasses as revised recently [9]. These cell types are well described in the literature, and are characterized by various lengths, diameters and wall thicknesses [15, 16].
UMSP was used to record UV spectra from spots 1 μm2 in size located in the secondary wall (S2) of the three main cell types (Figure 2). The highest UV absorbance was measured in the S2 of vessels from rind and pith, followed by fibers and parenchyma. UV spectra of fiber and vessel S2 walls had defined bands near 278 nm and 315 nm (Figure 2). The band at 278 nm is produced by the aromatic rings in guaiacyl lignin, whereas the strong band at 315 nm is typical of hydroxycinnamic acids linked to the lignin and/or the arabino-methylglucurono-xylan backbones often found in grasses [9, 18, 20]. The spectra from the S2 of parenchyma cell walls had the lowest absorbance values, especially in the pith region. The band at 278 nm was not resolved in these spectra, but a pronounced band appeared at 315 nm, which is consistent with the predominance of hydroxycinnamic acids esterified to arabino-methylglucurono-xylans [9, 21].
Selected areas of the sugar-cane tissues were further scanned at constant wavelengths of 278 and 315 nm with a spatial resolution of 0.25 μm. The signals of the UV-spectrophotometer were converted into digitized images (APAMOS software; Zeiss, Jena, Germany). Colored micrographs for selected samples are illustrated in Figure 3 (see Methods). The scanned micrographs of fiber tissues from the rind and the pith had similar absorbance levels, with the most intense absorbance seen in the cell corners (CC) and the compound middle lamella (CML). Frequency histograms from each image were evaluated to calculate average absorbance values [22]. In the rind and pith fibers, the average absorbance values at 278 nm were 0.40 and 0.39 (including all cell-wall layers), respectively, whereas, those of the parenchyma tissue were significantly lower at 0.31 and 0.18, respectively. Comparing the results obtained from the spectral point measurements and scanning analyses for the individual cell types and layers, both datasets had very good congruence (compare Figure 2 and Figure 3).
Excised rind from untreated sugar cane contained 19% lignin, 30% hemicelluloses and 44% cellulose, whereas the pith fraction had corresponding values of 12%, 24% and 53%. Considering that parenchyma cells predominate in the pith region, these data are in close agreement with those of He and Terashima [17, 18], who reported lower lignin content in the parenchyma compared with the vascular bundle tissues. Notably, part of the lignin measured by the Klason procedure actually corresponds to the hydroxycinnamic acids that condense with lignin during the analytical acid treatment [23].
Treatment of the rind and pith samples with aqueous acetic acid/chlorite caused selective removal of lignin and hydroxycinnamic acids. Total lignin contents decreased rapidly, from 19% to 7% after 4 hours of treatment for rind samples, and from 12% to 7% after 2 hours of treatment for pith samples. Selected portions of the treated sugar-cane tissues were also examined by the UV spectroscopic techniques. The lignin (and accessory aromatic compounds) originally deposited in the fiber cell walls of the rind tissue mainly resisted a delignification process of 1 hour, whereas the pith fiber tissue was rapidly delignified (Figure 3a-d). In fact, the rind fibers required prolonged treatment (4 hours) for significant removal of the aromatic components. By contrast, the cell walls of the rind parenchyma tissue displayed a distinct decrease in UV absorbance values after 1 hour of treatment with aqueous acetic acid/chlorite (Figure 3e,g). The highest level of delignification was found in the cell walls of the pith parenchyma tissue (Figures 3f,h); after a treatment of 1 hour, none absorbance values were quantified using the highly-sensitive UMSP technique. The studied vessel cell walls were characterized by heterogeneous delignification (measured at 278 nm, representing guaiacyl lignin), but the absorbance at 315 nm decreased significantly for both rind and pith cells (data not shown).
UV spectra obtained from the S2 of fibers, vessels and parenchyma cells of the aqueous acetic acid/chlorite-treated samples are shown in Figure 4 and Figure 5. Treatment time was extended up to 4 hours, but in general, the removal of lignin and hydroxycinnamic acids was fully completed after 2 hours. After this time, the remaining lignin (detected by the topochemical analysis) was located in the compound middle lamella and cell corners. Evaluation of the S2 UV spectra of all cell types revealed that the hydroxycinnamic acids were the first to be removed, because the 315 nm band decreased in intensity more rapidly than did the bands at 278 nm (Figure 4, Figure 5). In pith cells, removal of hydroxycinnamic acids was even more pronounced (Figure 5).
Enzymatic hydrolysis of rind and pith samples
The untreated rind and pith samples and the aqueous acetic acid/chlorite-treated samples were digested with a mixture of cellulolytic enzymes. Beside cellulose, xylan present in the bagasse (fibre) samples was also hydrolyzed by the enzymatic cocktail (Figure 6a-d). The xylan hydrolysis was a result of the xylanase and β-xylosidase activities present in the commercial enzyme preparations [24–26]. For rind samples, the xylan conversion to xylose was similar to that seen for cellulose hydrolysis, but for pith samples, it was significantly lower than the cellulose hydrolysis (Figure 6b,d), indicating that the hemicellulose remaining in the pith fraction after the delignification was more recalcitrant to enzymatic attack.
Cellulose from untreated pith samples was promptly hydrolyzed to glucose, reaching 63% conversion after 72 hours of hydrolysis, whereas untreated rind samples had only 20% conversion, indicating that the less lignified parenchyma cells, which mainly occurred in the pith region, were significantly less recalcitrant than the fibers and vessels predominating in the rind region. This is consistent with previous work showing that parenchyma cells from maize stems at various states of maturation were preferentially degraded by rumen biota [27]. Hansen et al. [28] also reported that parenchyma cells surrounding the pith cavity lining of thermally pretreated wheat straw were promptly hydrolyzed by a mixture of cellulolytic enzymes. Akin revised the histochemical findings for degradation of various tissues by cellulolytic systems, and confirmed that cells with lower levels of lignin are the first to be hydrolyzed in several plant species [9].
The UMSP data showed that parenchyma cells in the pith region are characterized by the lowest UV-absorbance values, mostly related to hydroxycinnamic acids, because the measured S2 spectrum had its peak only at 315 nm. Taken together, these results suggest that the action of the cellulolytic enzymes was not restrained by the aromatics occurring in the pith parenchyma. In addition, aqueous acetic acid/chlorite treatment of pith did not enhance the cellulose or xylan conversion, corroborating the notion that the recalcitrance of this fraction does not depend on the presence of aromatics only. It is likely that the limited hydrolysis of xylan to xylose also reflects the hindrance to cellulase action, as the hemicelluloses are known to encapsulate cellulose fibrils in the cell walls [3]. More recently, Qing and Wyman [25] found that xylo-oligomers accumulating during enzymatic treatment of xylans can also inhibit cellulolytic enzymes. In our study, we found that partially delignified fiber and vessels remained in the pith fraction, and might also account for the maximum level of cellulose conversion of around 63-66% (Figure 6b). Conversely, treatment of the rind cells with aqueous acetic acid/chlorite, which led to significant removal of hydroxycinnamic acids and lignin (Figure 4), resulted in significant enhancement of cellulose and xylan conversion by the commercial cellulases (Figure 6a).
There was an inverse correlation between cellulose conversion levels and lignin contents or absorbance data for the various tissue types in rind (Figure 7). Absorbance data at 280 nm and 315 nm (Figure 4, Figure 5) were used as an estimate of lignin and hydroxycinnamic acid contents, respectively, in each cell type. The r2 values for data correlation by linear models were relatively high (Figures 7b,c). An exception was the data for rind parenchyma at 280 nm, which gave an r2 value of only 0.655. For the total lignin detected in the rind fraction, the correlation with cellulose conversion was also high (r2 = 0.83), reflecting the weighted average of absorbances at 280 nm of each individual cell type. The inverse correlation seen for the lignin content or the absorbance assigned to aromatics with the levels of cellulose conversion is in close agreement with data reported in previous studies by Grabber et al. [29] for mature and immature alfalfa fibers, Jung and Casler [27] for maize stems at various stages of maturation, Chen and Dixon [30] for transgenic alfalfa, and Lee et al. [31] for selectively delignified wood. With sugar-cane bagasse, we also found previously that a mild chemo-thermo-mechanical pretreatment led to enhanced cellulose conversion by cellulases, which was due to lignin removal during the pretreatment [26]. In this study, these correlations were not noticeable for pith samples, because the untreated fraction was promptly hydrolyzed by the cellulases to a cellulose conversion level of 63-66%, and treatment with acetic acid/chlorite did not enhance this conversion efficiency.