Distribution and Chemical Composition of Lignin in Secondary Xylem of Cactaceae.

Cactaceae family has heterogeneity in the accumulation of lignocellulose due to the diversity of shapes and anatomy of the wood. Most studies focus on fibrous and dimorphic species; but the non-fibrous species are poorly studied. The aims of this work were to analyze the syringyl/guaiacyl ratio of lignin and its distribution in secondary xylem, especially in non-fibrous species. The syringyl/guaiacyl (S/G) ratio was quantified from 34 species of cacti by nitrobenzene oxidation of free-extractive wood. The distribution of lignocellulose in wood sections stained with safranin O/fast green was determined with epifluorescence microscopy. The S/G ratio was heterogeneous; most of the non-fibrous species had a higher percentage of syringyl, while the fibrous ones accumulate guaiacyl. Fluorescence emission showed that vessel elements and wide-band tracheids had similar tonalities. It is hypothesized that the presence of a higher percentage of syringyl in most cacti is part of the defense mechanism against pathogens, which together with the succulence of the stem represent adaptations that contribute to survival in their hostile environments.

Lignin Analysis by HPLC and FTIR: Spectra Deconvolution and S/G Ratio Determination.

Fourier transform infrared spectroscopy (FTIR) is a simple nondestructive technique that allows the user to obtain quick and accurate information about the structure of the constituents of wood. Spectra deconvolution is a computational technique, complementary to FTIR analysis, which improves the resolution of overlapped or unobserved bands in the raw spectra. High performance liquid chromatography (HPLC) is an analytical technique useful to determine the ratio of the lignin monomers obtained by the alkaline nitrobenzene oxidation method. Furthermore, lignin content has been commonly determined by wet chemical methods; Klason lignin determination is a quick and accessible method. Here, we detail the procedures for chemical analysis of the wood lignin using these techniques. Additionally, the deconvolution process of FTIR spectra for the determination of the S/G ratio, in lignin isolated by this or other methods, is explained in detail.

Structural Characterization of Lignin in Four Cacti Wood: Implications of Lignification in the Growth Form and Succulence.

Wood lignin composition strongly depends on anatomical features and it has been used as a marker for characterizing major plant groups. Wood heterogeneity in Cactaceae is involved in evolutionary and adaptive processes within this group; moreover, it is highly correlated to the species growth form. Here we studied the lignin structure from different types of woods in four Cactaceae species with different stem morphologies (Pereskia lychnidiflora, tree/fibrous wood; Opuntia streptacantha and Pilosocereus chrysacanthus, tree/succulent fibrous wood; Ferocactus hamatacanthus, cylindrical stem/dimorphic wood) in order to determine their relationship with the wood anatomy in an evolutionary-adaptive context. Dioxane lignin was isolated and analyzed by pyrolysis coupled with gas chromatography and mass spectrometry (Py-GC/MS), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). The main linkages are the ß-O-4' ether (67-85%), the ß-ß' resinol (10-26%) and the ß-5' and α-O-4' linkages of the phenylcoumaran structures (≤7%). Spirodienone structures have a considerable abundance (5%) in the dimorphic wood of F. hamatacanthus. In addition, low contents (≤3%) of α,ß-diaryl ether, α-oxidized ß-O-4' ether and dibenzodioxocin structures were found. The sinapyl- and coniferyl acetates are not part of the wood lignin in any of the studied species. The low (≤5%) γ-acetylation in the F. hamatacanthus and P. chrysacanthus wood lignin is here interpreted as an evidence of a high specialization of the wood elements in the conduction/storage of water. The lignin of the studied Cactaceae is composed predominantly of guaiacyl and syringyl units (S/G: 0.9-16.4). High abundance of syringyl units (62-94%) in three of the four species is considered as a defense mechanism against oxidative agents, it is a very conspicuous trait in the most succulent species with dimorphic wood. Furthermore, it is also associated with ferulates and the herein called γ-acetylated guaiacyl-syringaresinol complexes acting as nucleation sites for lignification and as cross-links between lignin and carbohydrates at the wide-band tracheid-fiber junctions.

Lignin Analysis by HPLC and FTIR.

Fourier transform infrared spectroscopy (FTIR) is a simple non-destructive technique which allows the user to obtain quick and accurate information about the structure of the constituents of wood. High performance liquid chromatography (HPLC) is an analytical technique useful to determine the ratio of the lignin monomers obtained by the alkaline nitrobenzene oxidation method. Furthermore, lignin content has been commonly determined by wet chemical methods; Klason lignin determination is a quick and accessible method. Here, we detail the procedures for chemical analysis of the wood lignin using these techniques.

Expression of the KNOTTED HOMEOBOX Genes in the Cactaceae Cambial Zone Suggests Their Involvement in Wood Development.

The vascular cambium is a lateral meristem that produces secondary xylem (i.e., wood) and phloem. Different Cactaceae species develop different types of secondary xylem; however, little is known about the mechanisms underlying wood formation in the Cactaceae. The KNOTTED HOMEOBOX (KNOX) gene family encodes transcription factors that regulate plant development. The role of class I KNOX genes in the regulation of the shoot apical meristem, inflorescence architecture, and secondary growth is established in a few model species, while the functions of class II KNOX genes are less well understood, although the Arabidopsis thaliana class II KNOX protein KNAT7 is known to regulate secondary cell wall biosynthesis. To explore the involvement of the KNOX genes in the enormous variability of wood in Cactaceae, we identified orthologous genes expressed in species with fibrous (Pereskia lychnidiflora and Pilosocereus alensis), non-fibrous (Ariocarpus retusus), and dimorphic (Ferocactus pilosus) wood. Both class I and class II KNOX genes were expressed in the cactus cambial zone, including one or two class I paralogs of KNAT1, as well as one or two class II paralogs of KNAT3-KNAT4-KNAT5. While the KNOX gene SHOOTMERISTEMLESS (STM) and its ortholog ARK1 are expressed during secondary growth in the Arabidopsis and Populus stem, respectively, we did not find STM orthologs in the Cactaceae cambial zone, which suggests possible differences in the vascular cambium genetic regulatory network in these species. Importantly, while two class II KNOX paralogs from the KNAT7 clade were expressed in the cambial zone of A. retusus and F. pilosus, we did not detect KNAT7 ortholog expression in the cambial zone of P. lychnidiflora. Differences in the transcriptional repressor activity of secondary cell wall biosynthesis by the KNAT7 orthologs could therefore explain the differences in wood development in the cactus species.

Wood chemical composition in species of Cactaceae: the relationship between lignification and stem morphology.

In Cactaceae, wood anatomy is related to stem morphology in terms of the conferred support. In species of cacti with dimorphic wood, a unique process occurs in which the cambium stops producing wide-band tracheids (WBTs) and produces fibers; this is associated with the aging of individuals and increases in size. Stem support and lignification have only been studied in fibrous tree-like species, and studies in species with WBTs or dimorphic wood are lacking. In this study, we approach this process with a chemical focus, emphasizing the role of wood lignification. We hypothesized that the degree of wood lignification in Cactaceae increases with height of the species and that its chemical composition varies with wood anatomy. To test this, we studied the chemical composition (cellulose, hemicellulose, and lignin content) in 13 species (2 WBTs wood, 3 dimorphic, and 8 fibrous) with contrasting growth forms. We also analyzed lignification in dimorphic and fibrous species to determine the chemical features of WBTs and fibers and their relationship with stem support. The lignin contents were characterized by Fourier transform infrared spectroscopy and high performance liquid chromatography. We found that 11 species have a higher percentage (>35%) of lignin in their wood than other angiosperms or gymnosperms. The lignin chemical composition in fibrous species is similar to that of other dicots, but it is markedly heterogeneous in non-fibrous species where WBTs are abundant. The lignification in WBTs is associated with the resistance to high water pressure within cells rather than the contribution to mechanical support. Dimorphic wood species are usually richer in syringyl lignin, and tree-like species with lignified rays have more guaiacyl lignin. The results suggest that wood anatomy and lignin distribution play an important role in the chemical composition of wood, and further research is needed at the cellular level.