Rapid alkalinization factor 22 has a structural and signalling role in root hair cell wall assembly.

Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types.

Top five unanswered questions in plant cell surface research.

Plant cell wall researchers were asked their view on what the major unanswered questions are in their field. This article summarises the feedback that was received from them in five questions. In this issue you can find equivalent syntheses for researchers working on bacterial, unicellular parasite and fungal systems.

The European Polysaccharide Network of Excellence (EPNOE) research roadmap 2040: Advanced strategies for exploiting the vast potential of polysaccharides as renewable bioresources.

Polysaccharides are among the most abundant bioresources on earth and consequently need to play a pivotal role when addressing existential scientific challenges like climate change and the shift from fossil-based to sustainable biobased materials. The Research Roadmap 2040 of the European Polysaccharide Network of Excellence (EPNOE) provides an expert's view on how future research and development strategies need to evolve to fully exploit the vast potential of polysaccharides as renewable bioresources. It is addressed to academic researchers, companies, as well as policymakers and covers five strategic areas that are of great importance in the context of polysaccharide related research: (I) Materials & Engineering, (II) Food & Nutrition, (III) Biomedical Applications, (IV) Chemistry, Biology & Physics, and (V) Skills & Education. Each section summarizes the state of research, identifies challenges that are currently faced, project achievements and developments that are expected in the upcoming 20 years, and finally provides outlines on how future research activities need to evolve.

Plant cell wall patterning and expansion mediated by protein-peptide-polysaccharide interaction.

Assembly of cell wall polysaccharides into specific patterns is required for plant growth. A complex of RAPID ALKALINIZATION FACTOR 4 (RALF4) and its cell wall-anchored LEUCINE-RICH REPEAT EXTENSIN 8 (LRX8)-interacting protein is crucial for cell wall integrity during pollen tube growth, but its molecular connection with the cell wall is unknown. Here, we show that LRX8-RALF4 complexes adopt a heterotetrametric configuration in vivo, displaying a dendritic distribution. The LRX8-RALF4 complex specifically interacts with demethylesterified pectins in a charge-dependent manner through RALF4's polycationic surface. The LRX8-RALF4-pectin interaction exerts a condensing effect, patterning the cell wall's polymers into a reticulated network essential for wall integrity and expansion. Our work uncovers a dual structural and signaling role for RALF4 in pollen tube growth and in the assembly of complex extracellular polymers.

Contrasting Cd accumulation of Arabidopsis halleri populations: a role for (1→4)-ß-galactan in pectin.

Cadmium (Cd) accumulation is highly variable among Arabidopsis halleri populations. To identify cell wall (CW) components that contribute to the contrasting Cd accumulation between PL22-H (Cd-hyperaccumulator) and I16-E (Cd-excluder), Cd absorption capacity of CW polysaccharides, CW mono- and poly- saccharides contents and CW glycan profiles were compared between these two populations. PL22-H pectin contained 3-fold higher Cd concentration than I16-E pectin in roots, and (1→4)-ß-galactan pectic epitope showed the biggest difference between PL22-H and I16-E. CW-related differentially expressed genes (DEGs) between PL22-H and I16-E were identified and corresponding A. thaliana mutants were phenotyped for Cd tolerance and accumulation. A higher Cd translocation was observed in GALACTAN SYNTHASE1 A. thaliana knockout and overexpressor mutants, which both showed a lengthening of the RG-I sidechains after Cd treatment, contrary to the wild-type. Overall, our results support an indirect role for (1→4)-ß-galactan in Cd translocation, possibly by a joint effect of regulating the length of RG-I sidechains, the pectin structure and interactions between polysaccharides in the CW. The characterization of other CW-related DEGs between I16-E and PL22-H selected allowed to identify a possible role in Zn translocation for BIIDXI and LEUNIG-HOMOLOG genes, which are both involved in pectin modification.

The receptor kinase FERONIA regulates phosphatidylserine localization at the cell surface to modulate ROP signaling.

Cells maintain a constant dialog between the extracellular matrix and their plasma membrane to fine tune signal transduction processes. We found that the receptor kinase FERONIA (FER), which is a proposed cell wall sensor, modulates phosphatidylserine plasma membrane accumulation and nano-organization, a key regulator of Rho GTPase signaling in Arabidopsis. We demonstrate that FER is required for both Rho-of-Plant 6 (ROP6) nano-partitioning at the membrane and downstream production of reactive oxygen species upon hyperosmotic stimulus. Genetic and pharmacological rescue experiments indicate that phosphatidylserine is required for a subset of, but not all, FER functions. Furthermore, application of FER ligand shows that its signaling controls both phosphatidylserine membrane localization and nanodomains formation, which, in turn, tunes ROP6 signaling. Together, we propose that a cell wall-sensing pathway controls via the regulation of membrane phospholipid content, the nano-organization of the plasma membrane, which is an essential cell acclimation to environmental perturbations.

A dive into the cell wall with Arabidopsis.

One of the many legacies of the work of Michel Caboche is our understanding of plant cell wall synthesis and metabolism thanks to the use of Arabidopsis mutants. Here I describe how he was instrumental in initiating the genetic study of plant cell walls. I also show, with a few examples for cellulose and pectins, how this approach has led to important new insights in cell wall synthesis and how the metabolism of pectins contributes to plant growth and morphogenesis. I also illustrate the limitations of the use of mutants to explain processes at the scale of cells, organs or whole plants in terms of the physico-chemical properties of cell wall polymers. Finally, I sketch how new approaches can cope with these limitations.

L'un des nombreux héritages des travaux de Michel Caboche est notre compréhension de la synthèse et du métabolisme des parois cellulaires végétales grâce à l'utilisation de mutants d'Arabidopsis. Je décris ici comment il a joué un rôle déterminant dans le lancement de l'étude génétique des parois cellulaires végétales. Je montre également, avec quelques exemples pour la cellulose et les pectines, comment cette approche a conduit à de nouvelles connaissances importantes sur la synthèse de la paroi cellulaire et comment le métabolisme des pectines contribue à la croissance et à la morphogenèse des plantes. J'illustre également les limites de l'utilisation de mutants pour expliquer des processus à l'échelle de cellules, d'organes ou de plantes entières en termes de propriétés physico-chimiques de polymères de parois cellulaires. Enfin, j'esquisse comment de nouvelles approches peuvent faire face à ces limitations.

Biochemical characterization of Pectin Methylesterase Inhibitor 3 from Arabidopsis thaliana.

The de-methylesterification of the pectic polysaccharide homogalacturonan (HG) by pectin methylesterases (PMEs) is a critical step in the control of plant cell expansion and morphogenesis. Plants have large gene families encoding PMEs but also PME inhibitors (PMEIs) with differ in their biochemical properties. The Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR 3 (PMEI3) gene is frequently used as a tool to manipulate pectin methylesterase activity in studies assessing its role in the control of morphogenesis. One limitation of these studies is that the exact biochemical activity of this protein has not yet been determined. In this manuscript we produced the protein in Pichia pastoris and characterized its activity in vitro. Like other PMEIs, PMEI3 inhibits PME activity at acidic pH in a variety of cell wall extracts and in purified PME preparations, but does not affect the much stronger PME activity at neutral pH. The protein is remarkable heat stable and shows higher activity against PME3 than against PME2, illustrating how different members of the large PMEI family can differ in their specificities towards PME targets. Finally, growing Arabidopsis thaliana seedlings in the presence of purified PMEI3 caused a dose-dependent inhibition of root growth associated with the overall inhibition of HG de-methylesterification of the root surface. This suggests an essential in vivo role for PME activity at acidic pH in HG de-methylesterification and growth control. These results show that purified recombinant PMEI3 is a powerful tool to study the connection between pectin de-methylesterification and cell expansion.

Optimal BR signalling is required for adequate cell wall orientation in the Arabidopsis root meristem.

Plant brassinosteroid hormones (BRs) regulate growth in part through altering the properties of the cell wall, the extracellular matrix of plant cells. Conversely, feedback signalling from the wall connects the state of cell wall homeostasis to the BR receptor complex and modulates BR activity. Here, we report that both pectin-triggered cell wall signalling and impaired BR signalling result in altered cell wall orientation in the Arabidopsis root meristem. Furthermore, both depletion of endogenous BRs and exogenous supply of BRs triggered these defects. Cell wall signalling-induced alterations in the orientation of newly placed walls appear to occur late during cytokinesis, after initial positioning of the cortical division zone. Tissue-specific perturbations of BR signalling revealed that the cellular malfunction is unrelated to previously described whole organ growth defects. Thus, tissue type separates the pleiotropic effects of cell wall/BR signals and highlights their importance during cell wall placement.

The role of pectin phase separation in plant cell wall assembly and growth.

A rapidly increasing body of literature suggests that many biological processes are driven by phase separation within polymer mixtures. Liquid-liquid phase separation can lead to the formation of membrane-less organelles, which are thought to play a wide variety of roles in cell metabolism, gene regulation or signaling. One of the characteristics of these systems is that they are poised at phase transition boundaries, which makes them perfectly suited to elicit robust cellular responses to often very small changes in the cell's "environment". Recent observations suggest that, also in the semi-solid environment of plant cell walls, phase separation not only plays a role in wall patterning, hydration and stress relaxation during growth, but also may provide a driving force for cell wall expansion. In this context, pectins, the major polyanionic polysaccharides in the walls of growing cells, appear to play a critical role. Here, we will discuss (i) our current understanding of the structure-function relationship of pectins, (ii) in vivo evidence that pectin modification can drive critical phase transitions in the cell wall, (iii) how such phase transitions may drive cell wall expansion in addition to turgor pressure and (iv) the periodic cellular processes that may control phase transitions underlying cell wall assembly and expansion.

Erratum to "The cell surface - A new journal for transkingdom cell wall research" [Cell Surface 1 (2018) 1].

[This corrects the article DOI: 10.1016/j.tcsw.2017.05.001.].

The Proline-Rich Family Protein EXTENSIN33 Is Required for Etiolated Arabidopsis thaliana Hypocotyl Growth.

Growth of etiolated Arabidopsis hypocotyls is biphasic. During the first phase, cells elongate slowly and synchronously. At 48 h after imbibition, cells at the hypocotyl base accelerate their growth. Subsequently, this rapid elongation propagates through the hypocotyl from base to top. It is largely unclear what regulates the switch from slow to fast elongation. Reverse genetics-based screening for hypocotyl phenotypes identified three independent mutant lines of At1g70990, a short extensin (EXT) family protein that we named EXT33, with shorter etiolated hypocotyls during the slow elongation phase. However, at 72 h after imbibition, these dark-grown mutant hypocotyls start to elongate faster than the wild type (WT). As a result, fully mature 8-day-old dark-grown hypocotyls were significantly longer than WTs. Mutant roots showed no growth phenotype. In line with these results, analysis of native promoter-driven transcriptional fusion lines revealed that, in dark-grown hypocotyls, expression occurred in the epidermis and cortex and that it was strongest in the growing part. Confocal and spinning disk microscopy on C-terminal protein-GFP fusion lines localized the EXT33-protein to the ER and cell wall. Fourier-transform infrared microspectroscopy identified subtle changes in cell wall composition between WT and the mutant, reflecting altered cell wall biomechanics measured by constant load extensometry. Our results indicate that the EXT33 short EXT family protein is required during the first phase of dark-grown hypocotyl elongation and that it regulates the moment and extent of the growth acceleration by modulating cell wall extensibility.

A Mutant Allele Uncouples the Brassinosteroid-Dependent and Independent Functions of BRASSINOSTEROID INSENSITIVE 1.

Plants depend on various cell surface receptors to integrate extracellular signals with developmental programs. One of the best-studied receptors is BRASSINOSTEROID INSENSITIVE 1 (BRI1) in Arabidopsis (Arabidopsis thaliana). Upon binding of its hormone ligands, BRI1 forms a complex with a shape-complementary coreceptor and initiates a signal transduction cascade, which leads to a variety of responses. At the macroscopic level, brassinosteroid (BR) biosynthetic and receptor mutants have similar growth defects, which initially led to the assumption that the signaling pathways were largely linear. However, recent evidence suggests that BR signaling is interconnected with several other pathways through various mechanisms. We recently described that feedback from the cell wall is integrated at the level of the receptor complex through interaction with RECEPTOR-LIKE PROTEIN 44 (RLP44). Moreover, BRI1 is required for another function of RLP44: the control of procambial cell fate. Here, we report a BRI1 mutant, bri1 cnu4 , which differentially affects canonical BR signaling and RLP44 function in the vasculature. Although BR signaling is only mildly impaired, bri1 cnu4 mutants show ectopic xylem in place of procambium. Mechanistically, this is explained by an increased association between RLP44 and the mutated BRI1 protein, which prevents the former from acting in vascular cell fate maintenance. Consistent with this, the mild BR response phenotype of bri1 cnu4 is a recessive trait, whereas the RLP44-mediated xylem phenotype is semidominant. Our results highlight the complexity of plant plasma membrane receptor function and provide a tool to dissect BR signaling-related roles of BRI1 from its noncanonical functions.

Oligogalacturonide production upon Arabidopsis thaliana-Botrytis cinerea interaction.

Despite an ever-increasing interest for the use of pectin-derived oligogalacturonides (OGs) as biological control agents in agriculture, very little information exists-mainly for technical reasons-on the nature and activity of the OGs that accumulate during pathogen infection. Here we developed a sensitive OG profiling method, which revealed unsuspected features of the OGs generated during infection of Arabidopsis thaliana with the fungus Botrytis cinerea Indeed, in contrast to previous reports, most OGs were acetyl- and methylesterified, and 80% of them were produced by fungal pectin lyases, not by polygalacturonases. Polygalacturonase products did not accumulate as larger size OGs but were converted into oxidized GalA dimers. Finally, the comparison of the OGs and transcriptomes of leaves infected with B. cinerea mutants with reduced pectinolytic activity but with decreased or increased virulence, respectively, identified candidate OG elicitors. In conclusion, OG analysis provides insights into the enzymatic arms race between plant and pathogen and facilitates the identification of defense elicitors.

A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation.

Somatic polyploidy caused by endoreplication is observed in arthropods, molluscs, and vertebrates but is especially prominent in higher plants, where it has been postulated to be essential for cell growth and fate maintenance. However, a comprehensive understanding of the physiological significance of plant endopolyploidy has remained elusive. Here, we modeled and experimentally verified a high-resolution DNA endoploidy map of the developing Arabidopsis thaliana root, revealing a remarkable spatiotemporal control of DNA endoploidy levels across tissues. Fitting of a simplified model to publicly available data sets profiling root gene expression under various environmental stress conditions suggested that this root endoploidy patterning may be stress-responsive. Furthermore, cellular and transcriptomic analyses revealed that inhibition of endoreplication onset alters the nuclear-to-cellular volume ratio and the expression of cell wall-modifying genes, in correlation with the appearance of cell structural changes. Our data indicate that endopolyploidy might serve to coordinate cell expansion with structural stability and that spatiotemporal endoreplication pattern changes may buffer for stress conditions, which may explain the widespread occurrence of the endocycle in plant species growing in extreme or variable environments.

Receptor Kinase THESEUS1 Is a Rapid Alkalinization Factor 34 Receptor in Arabidopsis.

The growth of plants, like that of other walled organisms, depends on the ability of the cell wall to yield without losing its integrity. In this context, plant cells can sense the perturbation of their walls and trigger adaptive modifications in cell wall polymer interactions. Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) THESEUS1 (THE1) was previously shown in Arabidopsis to trigger growth inhibition and defense responses upon perturbation of the cell wall, but so far, neither the ligand nor the role of the receptor in normal development was known. Here, we report that THE1 is a receptor for the peptide rapid alkalinization factor (RALF) 34 and that this signaling module has a role in the fine-tuning of lateral root initiation. We also show that RALF34-THE1 signaling depends, at least for some responses, on FERONIA (FER), another RALF receptor involved in a variety of processes, including immune signaling, mechanosensing, and reproduction [1]. Together, the results show that RALF34 and THE1 are part of a signaling network that integrates information on the integrity of the cell wall with the coordination of normal morphogenesis.

T-DNA alleles of the receptor kinase THESEUS1 with opposing effects on cell wall integrity signaling.

Perturbation of cellulose synthesis in plants triggers stress responses, including growth retardation, mediated by the cell wall integrity-sensing receptor-like kinase (RLK) THESEUS1 (THE1). The analysis of two alleles carrying T-DNA insertions at comparable positions has led to conflicting conclusions concerning the impact of THE1 signaling on growth. Here we confirm that, unlike the1-3 and other the1 alleles in which cellular responses to genetic or pharmacological inhibition of cellulose synthesis are attenuated, the1-4 showed enhanced responses, including growth inhibition, ectopic lignification, and stress gene expression. Both the1-3 and the1-4 express a transcript encoding a predicted membrane-associated truncated protein lacking the kinase domain. However, the1-3, in contrast to the1-4, strongly expresses antisense transcripts, which are expected to prevent the expression of the truncated protein as suggested by the genetic interactions between the two alleles. Seedlings overexpressing such a truncated protein react to isoxaben treatment similarly to the1-4 and the full-length THE overexpressor. We conclude that the1-4 is a hypermorphic allele; that THE1 signaling upon cell wall damage has a negative impact on cell expansion; and that caution is required when interpreting the phenotypic effects of T-DNA insertions in RLK genes.

Plant cell walls.

Plants are able to generate large leaf surfaces that act as two-dimensional solar panels with a minimum investment in building material, thanks to a hydrostatic skeleton. This requires high intracellular pressures (up to 1 MPa), which depend on the presence of strong cell walls. The walls of growing cells (also called primary walls), are remarkably able to reconcile extreme tensile strength (up to 100 MPa) with the extensibility necessary for growth. All walled organisms are confronted with this dilemma - the need to balance strength and extensibility - and bacteria, fungi and plants have evolved independent solutions to cope. In this Primer, we discuss how plant cells have solved this problem, allowing them to support often very large increases in volume and to develop a broad variety of shapes (Figure 1A,B,D). This shape variation reflects the targeted deposition of wall material combined with local variations in cell-wall extensibility, processes that remain incompletely understood. Once the cell has reached its final size, it can lay down secondary wall layers, the composition and architecture of which are optimized to exert specific functions in different cell types (Figure 1E-G). Such functions include: providing mechanical support, for instance, for fibre cells in tree trunks or grass internodes; impermeabilising and strengthening vascular tissue to resist the negative pressure of the transpiration stream; increasing the surface area of the plasma membrane to facilitate solute exchange between cells (Figure 1C); or allowing important elastic deformation, for instance, to support the opening and closing of stomates. Specialized secondary walls, such as those constituting seed mucilage, are stored in a dehydrated form in seedcoat epidermis cells and show rapid swelling upon hydration of the seed. Other walls, in particular in reserve tissues, can accommodate large amounts of storage polysaccharides, which can be easily mobilized as a carbon source. Here we will discuss some general principles underlying wall architecture and wall growth that have emerged from recent studies, as well as future questions for investigation (Box 1).