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Lignins are a key adaptation that enables vascular plants to thrive in terrestrial habitats. Lignin is heterogeneous, containing upward of 30 different monomers, and its function is multifarious: It provides structural support, predetermined breaking points, ultraviolet protection, diffusion barriers, pathogen resistance, and drought resilience. Recent studies, carefully characterizing lignin in situ, have started to identify specific lignin compositions and ultrastructures with distinct cellular functions, but our understanding remains fractional. We summarize recent works and highlight where further in situ lignin analysis could provide valuable insights into plant growth and adaptation. We also summarize strengths and weaknesses of lignin in situ analysis methods.

Inducing the differentiation of specific cell type(s) synchronously and on-demand is a great experimental system to understand the sequential progression of the cellular processes, their timing and their resulting properties for distinct isolated plant cells independently of their tissue context. The inducible differentiation in cell suspension cultures, moreover, enables to obtain large quantities of distinct cell types at specific development stage, which is not possible when using whole plants. The differentiation of tracheary elements (TEs) - the cell type responsible for the hydro-mineral sap conduction and skeletal support of plants in xylem tissues - has been the most studied using inducible cell suspension cultures. We herein describe how to establish and use inducible pluripotent suspension cell cultures (iPSCs) in Arabidopsis thaliana to trigger on-demand different cell types, such as TEs or mesophyll cells. We, moreover, describe the methods to establish, monitor, and modify the sequence, duration, and properties of differentiated cells using iPSCs.

Properly patterned deposition of cell wall polymers is prerequisite for the morphogenesis of plant cells. A cortical microtubule array guides the two-dimensional pattern of cell wall deposition. Yet, the mechanism underlying the three-dimensional patterning of cell wall deposition is poorly understood. In metaxylem vessels, cell wall arches are formed over numerous pit membranes, forming highly organized three-dimensional cell wall structures. Here, we show that the microtubule-associated proteins, MAP70-5 and MAP70-1, regulate arch development. The map70-1 map70-5 plants formed oblique arches in an abnormal orientation in pits. Microtubules fit the aperture of developing arches in wild-type cells, whereas microtubules in map70-1 map70-5 cells extended over the boundaries of pit arches. MAP70 caused the bending and bundling of microtubules. These results suggest that MAP70 confines microtubules within the pit apertures by altering the physical properties of microtubules, thereby directing the growth of pit arches in the proper orientation. This study provides clues to understanding how plants develop three-dimensional structure of cell walls.

Vascular plants reinforce the cell walls of the different xylem cell types with lignin phenolic polymers. Distinct lignin chemistries differ between each cell wall layer and each cell type to support their specific functions. Yet the mechanisms controlling the tight spatial localization of specific lignin chemistries remain unclear. Current hypotheses focus on control by monomer biosynthesis and/or export, while cell wall polymerization is viewed as random and nonlimiting. Here, we show that combinations of multiple individual laccases (LACs) are nonredundantly and specifically required to set the lignin chemistry in different cell types and their distinct cell wall layers. We dissected the roles of Arabidopsis thaliana LAC4, 5, 10, 12, and 17 by generating quadruple and quintuple loss-of-function mutants. Loss of these LACs in different combinations led to specific changes in lignin chemistry affecting both residue ring structures and/or aliphatic tails in specific cell types and cell wall layers. Moreover, we showed that LAC-mediated lignification has distinct functions in specific cell types, waterproofing fibers, and strengthening vessels. Altogether, we propose that the spatial control of lignin chemistry depends on different combinations of LACs with nonredundant activities immobilized in specific cell types and cell wall layers.

• Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and biogeosciences.
• To address these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree-ring series (1961–1995).
• We found fractionation signals (i.e. temporal variability in deuterium abundance) at glucose H¹ and H² introduced by closely related metabolic processes. Regression analysis indicates that these signals (and thus metabolism) respond to drought and atmospheric CO₂ concentration beyond a response change point. They explain ≈ 60% of the whole-molecule deuterium variability. Altered metabolism is associated with below-average yet not exceptionally low growth.
• We propose the signals are introduced at the leaf level by changes in sucrose-to-starch carbon partitioning and anaplerotic carbon flux into the Calvin–Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose.

Peatlands store carbon in the form of dead organic residues. Climate change and human impact impose risks on the sustainability of the peatlands carbon balance due to increased peat decomposition. Here, we investigated molecular changes in the upper peat layers (0-40 cm), inferred from high-resolution vertical depth profiles, from a boreal peatland using two-dimensional 1H-13C nuclear magnetic resonance (NMR) spectroscopy, and comparison to δ13C, δ15N, and carbon and nitrogen content. Effects of hydrological conditions were investigated at respective sites: natural moist, drainage ditch, and natural dry. The molecular characterization revealed preferential degradation of specific side-chain linkages of xylan-type hemicelluloses within 0-14 cm at all sites, indicating organic matter losses up to 25%. In contrast, the xylan backbone, galactomannan-type hemicelluloses, and cellulose were more resistant to degradation and accumulated at the natural moist and drainage site. δ13C, δ15N, and carbon and nitrogen content did not correlate with specific hemicellulose structures but reflected changes in total carbohydrates. Our analysis provides novel insights into peat carbohydrate decomposition and indicates substantial organic matter losses in the acrotelm due to the degradation of specific hemicellulose structures. This suggests that variations in hemicellulose content and structure influence peat stability, which may have important implications with respect to climate change.

The biopolymer lignin is deposited in the cell walls of vascular cells and is essential for long-distance water conduction and structural support in plants. Different vascular cell types contain distinct and conserved lignin chemistries, each with specific aromatic and aliphatic substitutions. Yet, the biological role of this conserved and specific lignin chemistry in each cell type remains unclear. Here, we investigated the roles of this lignin biochemical specificity for cellular functions by producing single cell analyses for three cell morphotypes of tracheary elements, which all allow sap conduction but differ in their morphology. We determined that specific lignin chemistries accumulate in each cell type. Moreover, lignin accumulated dynamically, increasing in quantity and changing in composition, to alter the cell wall biomechanics during cell maturation. For similar aromatic substitutions, residues with alcohol aliphatic functions increased stiffness whereas aldehydes increased flexibility of the cell wall. Modifying this lignin biochemical specificity and the sequence of its formation impaired the cell wall biomechanics of each morphotype and consequently hindered sap conduction and drought recovery. Together, our results demonstrate that each sap-conducting vascular cell type distinctly controls their lignin biochemistry to adjust their biomechanics and hydraulic properties to face developmental and environmental constraints.

Sphagnum mosses account for most accumulated dead organic matter in peatlands. Therefore, understanding their responses to increasing atmospheric CO₂ is needed for estimating peatland C balances under climate change. A key process is photorespiration: a major determinant of net photosynthetic C assimilation that depends on the CO₂ to O₂ ratio. We used climate chambers to investigate photorespiratory responses of Sphagnum fuscum hummocks to recent increases in atmospheric CO₂ (from 280 to 400 ppm) under different water table, temperature, and light intensity levels. We tested the photorespiratory variability using a novel method based on deuterium isotopomers (D6^S/D6^R ratio) of photosynthetic glucose. The effect of elevated CO₂ on photorespiration was highly dependent on water table. At low water table (−20 cm), elevated CO₂ suppressed photorespiration relative to C assimilation, thus substantially increasing the net primary production potential. In contrast, a high water table (~0 cm) favored photorespiration and abolished this CO₂ effect. The response was further tested for Sphagnum majus lawns at typical water table levels (~0 and −7 cm), revealing no effect of CO₂ under those conditions. Our results indicate that hummocks, which typically experience low water table levels, benefit from the 20th century's increase in atmospheric CO₂.

Natural peatlands contribute significantly to global carbon sequestration and storage of biomass, most of which derives from Sphagnum peat mosses. Atmospheric CO₂ levels have increased dramatically during the twentieth century, from 280 to > 400 ppm, which has affected plant carbon dynamics. Net carbon assimilation is strongly reduced by photorespiration, a process that depends on the CO₂ to O₂ ratio. Here we investigate the response of the photorespiration to photosynthesis ratio in Sphagnum mosses to recent CO₂ increases by comparing deuterium isotopomers of historical and contemporary Sphagnum tissues collected from 36 peat cores from five continents. Rising CO₂ levels generally suppressed photorespiration relative to photosynthesis but the magnitude of suppression depended on the current water table depth. By estimating the changes in water table depth, temperature, and precipitation during the twentieth century, we excluded potential effects of these climate parameters on the observed isotopomer responses. Further, we showed that the photorespiration to photosynthesis ratio varied between Sphagnum subgenera, indicating differences in their photosynthetic capacity. The global suppression of photorespiration in Sphagnum suggests an increased net primary production potential in response to the ongoing rise in atmospheric CO₂, in particular for mire structures with intermediate water table depths.

- Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and Earth sciences.

- To close these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree-ring series (1961 to 1995).

- We found fractionation signals at glucose H¹ and H² introduced by closely related metabolic processes. These signals (and thus metabolism) respond to drought and atmospheric CO₂ concentration beyond a response change point. They explain ≈60% of the whole-molecule deuterium variability. Altered metabolism is associated with below-average yet not exceptionally low growth.

- We propose the signals are introduced at the leaf-level by changes in sucrose-to-starch carbon partitioning and anaplerotic carbon flux into the Calvin-Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose.