Lignin is an abundant and essential polymer in land plants. It is a prime factor in the recalcitrance of lignocellulosic biomass to agricultural and industrial end-uses such as forage, pulp and papermaking, and biofuels. To better understand lignification at the molecular level, we are developing a lignin spectroscopic and imaging toolbox on one "negligible" auxiliary. Toward that end, we describe the design, synthesis, and characterization of a new designer monolignol, 3-O-propargylcaffeyl alcohol, which contains a bioorthogonal alkynyl functional group at the 3-O-position. Importantly, our data indicate that this monolignol does not alter the fidelity of lignification. We demonstrate that the designer monolignol provides a platform for multiple spectroscopic and imaging approaches to reveal temporal and spatial details of lignification, the knowledge of which is critical to reap the potential of energy-rich renewable plant biomass for sustainable liquid fuels and other diverse economic applications.
Lignin is a key structural component of plant cell walls that provides rigidity, strength, and resistance against microbial attacks. This hydrophobic polymer also serves a crucial role in water transport. Despite its abundance and essential functions, several aspects of lignin biosynthesis and deposition remain cryptic. Lignin precursors are known to be synthesized in the cytoplasm by complex biosynthetic pathways, after which they are transported to the apoplastic space, where they are polymerized via free radical coupling reactions into polymeric lignin. However, the lignin deposition process and the factors controlling it are unclear. In this study, the biochemical and developmental dependencies of lignification were investigated using a click-compatible monolignol analog, 3-O-propargylcaffeyl alcohol (3-OPC), which can incorporate into both in vitro polymerized lignin and Arabidopsis thaliana tissues. Fluorescence labeling of 3-OPC using click chemistry followed by confocal fluorescence microscopy enabled the detection and imaging of 3-OPC incorporation patterns. These patterns were consistent with endogenous lignification observed in different developmental stages of Arabidopsis stems. However, the concentration of supplied monolignols influenced where lignification occurred at the subcellular level, with low concentrations being deposited in cell corners and middle lamellae and high concentrations also being deposited in secondary walls. Experimental inhibition of multiple lignification factors confirmed that 3-OPC incorporation proceeds via a free radical coupling mechanism involving peroxidases/laccases and reactive oxygen species (ROS). Finally, the presence of peroxide-producing enzymes determined which cell walls lignified: adding exogenous peroxide and peroxidase caused cells that do not naturally lignify in Arabidopsis stems to lignify. In summary, 3-OPC accurately mimics natural lignification patterns in different developmental stages of Arabidopsis stems and allows for the dissection of key biochemical and enzymatic factors controlling lignification.
Lignin plays important structural and functional roles in plants by forming a hydrophobic matrix in secondary cell walls that enhances mechanical strength and resists microbial decay. While the importance of the lignin matrix is well documented and the biosynthetic pathways for monolignols are known, the process by which lignin precursors or monolignols are transported and polymerized to form this matrix remains a subject of considerable debate. In this study, we have synthesized and tested an analog of coniferyl alcohol that has been modified to contain an ethynyl group at the C-3 position. This modification enables fluorescent tagging and imaging of this molecule after its incorporation into plant tissue by click chemistry-assisted covalent labeling with a fluorescent azide dye, and confers a distinct Raman signature that could be used for Raman imaging. We found that this monolignol analog is incorporated into in vitro-polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old Arabidopsis seedlings. Incorporation of the analog in stem sections of 6-week-old Arabidopsis thaliana plants and labeling with an Alexa-594 azide dye revealed the precise locations of new lignin polymerization. Results from this study indicate that this molecule can provide high-resolution localization of lignification during plant cell wall maturation and lignin matrix assembly.
The calcium (Ca2+) signaling is a crucial event during plant-herbivore interaction, which involves a transient change in cytosolic Ca2+ concentration, which is sensed by Ca2+-sensors, and the received message is transduced to downstream target proteins leading to appropriate defense response. Calmodulin-like proteins (CMLs) are calcium-sensing plant-specific proteins. Although CMLs have been identified in a few plants, they remained uncharacterized in leguminous crop plants. Therefore, a wide-range analysis of CMLs of soybean was performed, which identified 41 true CMLs with greater than 50% similarity with Arabidopsis CMLs. The phylogenetic study revealed their evolutionary relatedness with known CMLs. Further, the identification of conserved motifs, gene structure analysis, and identification of cis-acting elements strongly supported their identity as members of this family and their involvement in stress responses. Only a few Glycine max CMLs (GmCMLs) exhibited differential expression in different tissue types, and rest of them had minimal expression. Additionally, differential expression patterns of GmCMLs were observed during Spodoptera litura-feeding, wounding, and signaling compound treatments, indicating their role in plant defense. The three-dimensional structure prediction, identification of interactive domains, and docking with Ca2+ ions of S. litura-inducible GmCMLs, indicated their identity as calcium sensors. This study on the characterization of GmCMLs provided insights into their roles in calcium signaling and plant defense during herbivory.
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