Lignification in the flax stem: evidence for an unusual lignin in bast fibers

In the context of our research on cell wall formation and maturation in flax (Linum usitatissimum L) bast fibers, we (1) confirmed the presence of lignin in bast fibers and (2) quantified and characterized the chemical nature of this lignin at two developmental stages. Histochemical methods (Weisner and Maüle reagents and KMnO₄-staining) indicating the presence of lignin in bast fibers at the light and electron microscope levels were confirmed by chemical analyses (acetyl bromide). In general, the lignin content in flax bast fibers varied between 1.5% and 4.2% of the dry cell wall residues (CWRs) as compared to values varying between 23.7% and 31.4% in flax xylem tissues. Immunological and chemical analyses (thioacidolysis and nitrobenzene oxidation) indicated that both flax xylem- and bast fiber-lignins were rich in guaiacyl (G) units with S/G values inferior to 0.5. In bast fibers, the highly sensitive immunological probes allowed the detection of condensed guaiacyl-type (G) lignins in the middle lamella, cell wall junctions, and in the S1 layer of the secondary wall. In addition, lower quantities of mixed guaiacyl–syringyl (GS) lignins could be detected throughout the secondary cell wall. Chemical analyses suggested that flax bast-fiber lignin is more condensed than the corresponding xylem lignin. In addition, H units represented up to 25% of the monomers released from bast-fiber lignin as opposed to a value of 1% for the corresponding xylem tissue. Such an observation indicates that the structure of flax bast-fiber lignin is significantly different from that of the more typical woody plant lignin, thereby suggesting that flax bast fibers represent an interesting system for studying an unusual lignification process.     

Neighboring Parenchyma Cells Contribute to Arabidopsis Xylem Lignification,while Lignification of Interfascicular Fibers Is Cell Autonomous

Lignin is a critical structural component of plants, providing vascular integrity and mechanical strength. Lignin precursors (monolignols) must be exported to the extracellular matrix where random oxidative coupling produces a complex lignin polymer. The objectives of this study were twofold: to determine the timing of lignification with respect to programmed cell death and to test if nonlignifying xylary parenchyma cells can contribute to the lignification of tracheary elements and fibers. This study demonstrates that lignin deposition is not exclusively a postmortem event, but also occurs prior to programmed cell death. Radiolabeled monolignols were not detected in the cytoplasm or vacuoles of tracheary elements or neighbors. To experimentally define which cells in lignifying tissues contribute to lignification in intact plants, a microRNA against CINNAMOYL CoA-REDUCTASE1 driven by the promoter from CELLULOSE SYNTHASE7 (ProCESA7:miRNA CCR1) was used to silence monolignol biosynthesis specifically in cells developing lignified secondary cell walls. When monolignol biosynthesis in ProCESA7:miRNA CCR1 lines was silenced in the lignifying cells themselves, but not in the neighboring cells, lignin was still deposited in the xylem secondary cell walls. Surprisingly, a dramatic reduction in cell wall lignification of extraxylary fiber cells demonstrates that extraxylary fibers undergo cell autonomous lignification.     

Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction mediated by peroxidases. The most important of these is the cationic peroxidase from Zinnia elegans (ZePrx), an enzyme considered to be responsible for the last step of lignification in this plant. Bibliographical evidence indicates that the arabidopsis peroxidase 72 (AtPrx72), which is homolog to ZePrx, could have an important role in lignification. For this reason, we performed a bioinformatic, histochemical, photosynthetic, and phenotypical and lignin composition analysis of an arabidopsis knock-out mutant of AtPrx72 with the aim of characterizing the effects that occurred due to the absence of expression of this peroxidase from the aspects of plant physiology such as vascular development, lignification, and photosynthesis. In silico analyses indicated a high homology between AtPrx72 and ZePrx, cell wall localization and probably optimal levels of translation of AtPrx72. The histochemical study revealed a low content in syringyl units and a decrease in the amount of lignin in the atprx72 mutant plants compared to WT. The atprx72 mutant plants grew more slowly than WT plants, with both smaller rosette and principal stem, and with fewer branches and siliques than the WT plants. Lastly, chlorophyll a fluorescence revealed a significant decrease in Φ_PSII and q _L in atprx72 mutant plants that could be related to changes in carbon partitioning and/or utilization of redox equivalents in arabidopsis metabolism. The results suggest an important role of AtPrx72 in lignin biosynthesis. In addition, knock-out plants were able to respond and adapt to an insufficiency of lignification.     

LtuCAD1 Is a Cinnamyl Alcohol Dehydrogenase Ortholog Involved in Lignin Biosynthesis in Liriodendron tulipifera L., a Basal Angiosperm Timber Species

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis and catalyzes the final step in the synthesis of monolignols. Seven CAD homologs (LtuCAD1 to LtuCAD7) have been previously identified from a basal angiosperm species Liriodendron tulipifera L., which is an important timber tree species with significant ecological and economic values. The phylogenetic analysis indicates that LtuCAD1 is the only Liriodendron CAD grouped with the bona fide CADs, the primary CAD genes involved in lignification. In this study, the predicted protein sequence of LtuCAD1 was found to have conserved domains and the same key determinant site with the bona fide CADs in other plant species. Additionally, LtuCAD1 had the highest expression level in xylem as revealed by quantitative RT-PCR analysis. The expression of beta-glucuronidase (GUS) driven by the LtuCAD1 promoter was largely localized in vascular tissues in Arabidopsis. In stem cross sections, GUS staining was found exclusively in xylem and phloem. When expressed in the Arabidopsis cad4 cad5 double mutant, LtuCAD1 was able to restore the total lignin content and decrease the S/G lignin ratio. Our data indicate that LtuCAD1 is a CAD ortholog involved in lignin biosynthesis in Liriodendron.     

Ectopic deposition of lignin in the pith of stems of two Arabidopsis mutants

The biosynthesis of lignin in vascular plants is regulated both developmentally and environmentally. In the inflorescence stems of Arabidopsis, lignin is mainly deposited in the walls of xylem cells and interfascicular fiber cells during normal plant growth and development. The mechanisms controlling the spatial deposition of lignin remain unknown. By screening ethyl methanesulfonate-mutagenized populations of Arabidopsis, we have isolated two allelic elp1 (ectopic deposition of lignin in pith) mutants with altered lignin deposition patterns. In elp1 stems, lignin was ectopically deposited in the walls of pith parenchyma cells in addition to its normal deposition in the walls of xylem and fiber cells. Lignin appeared to be deposited in patches of parenchyma cells in the pith of both young and mature elp1 stems. The ectopic deposition of lignin in the pith of elp1 stems was accompanied by an increase in the activities of enzymes in the lignin biosynthetic pathway and with the ectopic expression of caffeoyl coenzyme A O-methyltransferase in pith cells. These results indicate that the ELP1 locus is involved in the repression of the lignin biosynthetic pathway in the pith. Isolation of the elp1 mutants provides a novel means with which to study the molecular mechanisms underlying the spatial control of lignification.     

Free galactose and galactosidase activity in the course of flax fiber development

Composition and content of free monosaccharides and β-galactosidase activity were determined in the course of development of the flax (Linum usitatissimum L.) tissues containing bast fibers. In the stem regions where the fibers were at the stage of formation of the secondary cell wall of gelatinous type, the content of free galactose was high (14 mM) and 13–20 times greater than in the upper part of the stem where the fibers were at the stage of intrusive growth. Pulse-chase experiments demonstrated the differences in the metabolism of individual low-molecular sugars. In respect to glucose and sucrose, all the examined characteristics (content, absolute and specific radioactivity, and the temporal changes of these indices) were identical in the stem regions wherein the fibers were at different stages of development. Labeled galactose was detected only in the stem regions where the fibers were at the stage of secondary cell wall formation. The specific radioactivity of glucose and sucrose reached the maximum immediately after photosynthesis in the presence of ¹⁴CO₂ and changed in the same way as in the primary products of photosynthesis. The time-course of label incorporation into galactose indicated that this monosaccharide arose as a result of hydrolytic processes. At the stage of secondary cell wall formation, high activity of β-galactosidase was observed, with tissue- and stage-specific fiber β-1,4-galactan as its substrate.     

Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers.     

The Role of Auxin and Gibberellin in Controlling Lignin Formation in Primary Phloem Fibers and in Xylem of Coleus blumei Stems

The hypothesis that auxin (IAA) and gibberellic acid (GA₃) control the formation of lignin is confirmed for the primary phloem fibers and for the secondary xylem in the stem of Coleus blumel Benth. Indoleacetic acid alone, or a combination of high IAA/low GA₃ (w/w), induced short phloem fibers with thick secondary walls, that contained lignin rich in syringyl units (high ratio of syringyl/guaiacyl). On the other hand, a combination of high GA₃/low IAA (w/w), which promoted the differentiation of long phloem fibers with thin walls, decreased the relative content of the syringyl units (low syringyl/guaiacyl ratio). In the secondary xylem, these hormonal treatments yielded only slight changes in the noncondensed monomeric guaiacyl units, confirming the relative stability of the guaiacyl lignification pattern in this tissue. In the xylem, indoleacetic acid alone, or a combination of high IAA/low GA₃ induced lignin poor in syringyl units (low syringyl/guaiacyl ratio). A combination of high GA₃/low IAA promoted a relatively slight increase in syringyl yield, indicating greater responsiveness of the syringyl lignification pattern to growth regulators. The possible functional and technological significance of our results is discussed.