Functional Complexity on a Cellular Scale: Why In Situ Analyses Are Indispensable for Our Understanding of Lignified Tissues.

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.

Inducible Pluripotent Suspension Cell Cultures (iPSCs) to Study Plant Cell Differentiation.

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.

Confined-microtubule assembly shapes three-dimensional cell wall structures in xylem vessels.

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.

Different combinations of laccase paralogs nonredundantly control the amount and composition of lignin in specific cell types and cell wall layers in Arabidopsis.

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.

Organochemical Characterization of Peat Reveals Decomposition of Specific Hemicellulose Structures as the Main Cause of Organic Matter Loss in the Acrotelm.

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.

Plant biomechanics and resilience to environmental changes are controlled by specific lignin chemistries in each vascular cell type and morphotype.

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.

Metabolism is a major driver of hydrogen isotope fractionation recorded in tree-ring glucose of Pinus nigra.

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.

CO2 fertilization of Sphagnum peat mosses is modulated by water table level and other environmental factors.

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 (D6S /D6R 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 .

Global CO2 fertilization of Sphagnum peat mosses via suppression of photorespiration during the twentieth century.

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.

Establishment and Utilization of Habituated Cell Suspension Cultures for Hormone-Inducible Xylogenesis.

The development of inducible cell differentiation in suspension cultures led to multiple breakthroughs. It enabled the understanding of the chronology, duration, regulation and interdependency of the multiple events leading to fully functional specialized cells. The most studied cell differentiation in plants using inducible suspension cultures is the formation of tracheary elements (TEs) - the hydro-mineral sap conducting cells. Several in vitro systems established in different plant species have been developed to trigger TE formation on-demand. Here, we describe the establishment, harvesting and analysis of Arabidopsis thaliana stable habituated cell lines inducible by hormones to differentiate into TEs on-demand. Moreover, we explain the means to monitor and modify the chronology, duration and regulation of the progression of TE formation.

Analysis of Lignin Composition and Distribution Using Fluorescence Laser Confocal Microspectroscopy.

Lignin is a polyphenolic polymer specifically accumulating in the cell walls of xylem cells in higher vascular plants. Far from being homogeneous, the lignification of xylem cell walls varies in deposition site, quantity, composition and macromolecular conformation depending on the cell wall compartment, cell type, cell developmental stage and plant species. Here, we describe how confocal microspectroscopy methods using lignin autofluorescence can be used to evaluate the relative lignin amounts, its spatial distribution and composition at the cellular and sub-cellular levels in both isolated cells and histological cross-sections of plant tissues.

The cell biology of lignification in higher plants.

BACKGROUND: Lignin is a polyphenolic polymer that strengthens and waterproofs the cell wall of specialized plant cell types. Lignification is part of the normal differentiation programme and functioning of specific cell types, but can also be triggered as a response to various biotic and abiotic stresses in cells that would not otherwise be lignifying. SCOPE: Cell wall lignification exhibits specific characteristics depending on the cell type being considered. These characteristics include the timing of lignification during cell differentiation, the palette of associated enzymes and substrates, the sub-cellular deposition sites, the monomeric composition and the cellular autonomy for lignin monomer production. This review provides an overview of the current understanding of lignin biosynthesis and polymerization at the cell biology level. CONCLUSIONS: The lignification process ranges from full autonomy to complete co-operation depending on the cell type. The different roles of lignin for the function of each specific plant cell type are clearly illustrated by the multiple phenotypic defects exhibited by knock-out mutants in lignin synthesis, which may explain why no general mechanism for lignification has yet been defined. The range of phenotypic effects observed include altered xylem sap transport, loss of mechanical support, reduced seed protection and dispersion, and/or increased pest and disease susceptibility.

Cooperative lignification of xylem tracheary elements.

The development of xylem tracheary elements (TEs)--the hydro-mineral sap conducting cells--has been an evolutionary breakthrough to enable long distance nutrition and upright growth of vascular land plants. To allow sap conduction, TEs form hollow laterally reinforced cylinders by combining programmed cell death and secondary cell wall formation. To ensure their structural resistance for sap conduction, TE cell walls are reinforced with the phenolic polymer lignin, which is deposited after TE cell death by the cooperative supply of monomers and other substrates from the surrounding living cells.

Non-cell-autonomous postmortem lignification of tracheary elements in Zinnia elegans.

Postmortem lignification of xylem tracheary elements (TEs) has been debated for decades. Here, we provide evidence in Zinnia elegans TE cell cultures, using pharmacological inhibitors and in intact Z. elegans plants using Fourier transform infrared microspectroscopy, that TE lignification occurs postmortem (i.e., after TE programmed cell death). In situ RT-PCR verified expression of the lignin monomer biosynthetic cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase in not only the lignifying TEs but also in the unlignified non-TE cells of Z. elegans TE cell cultures and in living, parenchymatic xylem cells that surround TEs in stems. These cells were also shown to have the capacity to synthesize and transport lignin monomers and reactive oxygen species to the cell walls of dead TEs. Differential gene expression analysis in Z. elegans TE cell cultures and concomitant functional analysis in Arabidopsis thaliana resulted in identification of several genes that were expressed in the non-TE cells and that affected lignin chemistry on the basis of pyrolysis-gas chromatography/mass spectrometry analysis. These data suggest that living, parenchymatic xylem cells contribute to TE lignification in a non-cell-autonomous manner, thus enabling the postmortem lignification of TEs.