The impact of the ccr1g mutation in A. thaliana was analyzed by a series of methods and approaches adapted to the characterization of the cell wall microstructure of individual cell types. LCM coupled with microanalysis has proven its efficiency in the investigation of the heterogeneous distribution of lignins and polysaccharides in A. thaliana stems. Moreover, it allowed the quantification of the effects of the mutation affecting different cell types. The results provide evidence that caution must be exerted in the interpretation of global analyses of plants which average the contribution of heterogeneous tissues. This new methodology appears to be of particular interest for genetically modified plants in which the impact of the mutation is tissue specific (Nakashima et al., 2008). Although some improvements need to be made in the tedious sample recovery, the results reported herein confirm the potential of such a microstrategy for cell wall investigation.
Taken together, our results demonstrate that the CCR1 mutation in A. thaliana has a strong impact on the assembly of the secondary walls of fibers and vessels. The defect in CCR activity, which slightly reduces the level of lignin content, clearly modifies the ultrastructural distribution of the G and S units, with a severe alteration of the assembly of the lignified secondary cell walls. TEM observations demonstrate that the overall result of the mutation is to impair the capacity of the lignified walls (ifs and vessels) to achieve the correct orientation of the CMF network in the S2 layer, as confirmed by the altered X-ray diffraction pattern. The defect in the orientation and packing of the CMFs in S2 seems to be the indirect consequence of lignin and, perhaps, hemicellulose modifications in quantity and structure. We found a correlation between the defect in lignin synthesis and the capacity of organization of CMFs. In this respect, our results substantiate genetic analysis documented by the use of linkage disequilibrium mapping (Thumma et al., 2005), which suggested an influence of the CCR gene on CMF angle orientation. They also correlate with a developmental study in wheat, showing that CCR contributes to stem strength support (Ma, 2007), a mechanical property which is closely related to the value of the microfibril angle (Evans & Ilic, 2001). As the perturbation of the microfibril angle denotes a disorder in microtubule orientation, the latter would also correlate with an alteration in cellulose synthesis. Such a change in general carbohydrate metabolism was observed in CCR-down-regulated transformants and was suggested to be associated with a stress response (Lepléet al., 2007). The topochemical investigation points to the particular participation of noncondensed lignin structures in CMF assembly in S2. It is remarkable that, in ccr1g, the capacity of the S1 layer to assemble properly is hardly affected, particularly in ifs, contrary to S2. Our results indicate that the absence of CCR1 influences the mode of coupling, leading to noncondensed units, whether they involve G units only or mixed SG units. Thioacidolysis shows that 80% of the lignin in the ccr1g mutant is of the condensed type, and immunolabeling completes these results in showing that this condensed moiety, which cannot be fully characterized by chemical analysis, is predominantly made up of G units. It is noteworthy that, despite their differences in tissue type and in lignin topochemistry, ifs and vessels in the CCR1-depleted mutant are affected in a similar manner, indicating that the implication of ccr1 is essentially directed to S2 assembly regardless of the tissue. Beyond the description of the precise impact of the ccr1g mutation on the wall structure of A. thaliana, the present results underscore the fundamental difference in the process of assembly of S1 and S2 in the lignified if wall. Thus, in A. thaliana, S1 can form in a coherent manner in spite of a minimum proportion of noncondensed S lignin units, whereas the participation of these units seems fundamental for S2 to organize a normal spatial pattern of its cellulose framework. This suggests that, in the process of lignified secondary wall thickening, the CCR1 gene is more specifically involved in the assembly of S2.
All of these results point to the role of noncondensed lignin structures in the interaction between cellulose and lignin, in which the orientation of the lignin rings is guided by the orientation of cellulose, shown by Raman spectroscopy (Houtman & Atalla, 1995) and further demonstrated by molecular modeling (Besombes & Mazeau, 2005). Reminiscent of the inhibition of cellulose synthesis causing the abnormal deposition of lignin in zinnia cells (Taylor et al., 1992), we witness here, with the ccr1g mutant, the reverse situation, in which the alteration of lignin synthesis impedes cellulose organization. Together, our observations support the view that, during secondary wall assembly, the synthesis of one component mediates the patterning of the others (Taylor et al., 1992).