Microscale thermophoresis (MST) is a simple but powerful tool to study the in vitro interaction among biomolecules, and to quantify binding affinities. MST curves describe the change in the fluorescence level of a fluorescent target as a result of an IR-laser-induced temperature change. The degree and nature of the change in fluorescence signal depends on the size, charge, and solvation shell of the molecules, properties that change in function of the binding of a ligand to the fluorescent target. We used MST to describe the interaction between components of a regulatory module involved in plant cell wall integrity control. This module comprises the secreted peptide Rapid Alkalinization Factor 23 (RALF23) and its receptor complex consisting of the GPI-anchored receptor Lorelei-Like Glycoprotein 1 (LLG1) and a receptor kinase of the CrRLK1L family, FERONIA. Here we show how MST can also be used to study three-partner interactions.

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. The authors show that RALF22 has a dual role in cell wall assembly in root hairs: as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners.

P4B (2-phenyl-1-[4-(6-(piperidin-1-yl) pyridazin-3-yl) piperazin-1-yl] butan-1-one) is a novel cellulose biosynthesis inhibitor (CBI) discovered in a screen for molecules to identify inhibitors of Arabidopsis (Arabidopsis thaliana) seedling growth. Growth and cellulose synthesis inhibition by P4B were greatly reduced in a novel mutant for the cellulose synthase catalytic subunit gene CESA3 (cesa3pbr1). Cross-tolerance to P4B was also observed for isoxaben-resistant (ixr) cesa3 mutants ixr1-1 and ixr1-2. P4B has an original mode of action as compared with most other CBIs. Indeed, short-term treatments with P4B did not affect the velocity of cellulose synthase complexes (CSCs) but led to a decrease in CSC density in the plasma membrane without affecting their accumulation in microtubule-associated compartments. This was observed in the wild type but not in a cesa3pbr1 background. This reduced density correlated with a reduced delivery rate of CSCs to the plasma membrane but also with changes in cortical microtubule dynamics and orientation. At longer timescales, however, the responses to P4B treatments resembled those to other CBIs, including the inhibition of CSC motility, reduced growth anisotropy, interference with the assembly of an extensible wall, pectin demethylesterification, and ectopic lignin and callose accumulation. Together, the data suggest that P4B either directly targets CESA3 or affects another cellular function related to CSC plasma membrane delivery and/or microtubule dynamics that is bypassed specifically by mutations in CESA3.

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.

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.

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.

Summary 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, curiously, bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSINs (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signaling 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 (LLG1) and FERONIA (FER), triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a signaling molecule that regulates this process. This mechanism may also underlie wall assembly and expansion in other plant cell types.

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.

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)-β-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)-β-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.

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.

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.

The plant steroid hormones brassinosteroids (BRs) regulate growth in part through altering the properties of the cell wall, the extracellular matrix of plant cells. Conversely, cell wall signalling 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. BR-induced defects in the orientation of newly placed walls are associated with aberrant localization of the cortical division zone but with normal specification of its positioning. 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.

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.

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.

Micronutrients provide a potentially interesting alternative to fungicides for the protection of crops against fungal pathogens. Here we studied the effect of foliar-applied manganese (Mn) in the form of MnSO4 on severity of anthracnose disease, caused by Colletotrichum lagenarium in cucumber (Cucumis sativus L.) plant. The study was done aimed to characterize the optimum dose and application time of Mn fertilizer on disease suppression as well as to identify the defense mechanisms by which Mn-treated plants resist to fungal disease. In preliminary tests, Mn was applied at different concentrations (1.8, 4.5 and 7.2 mM) and various time points (three days before or two hours before inoculation, or three days after inoculation). Results showed that application of Mn either before or after inoculation suppressed the fungal infection in leaves and cotyledons, with a higher efficiency when applied three days prior to inoculation. However, all applied concentrations of Mn equally reduced the disease severity. Mn treatment in the absence of the pathogen promoted lignification and reactive oxygen species (ROS) accumulation. Also, pre-inoculation Mn treatment enhanced pathogen-induced lignification, callose or ROS production and reduced pathogen-induced cell death. The increase of lignin, callose and ROS induction by Mn application were 34, 30 and 31 % compared to control, respectively. Together, the results suggested the effectiveness of Mn treatments on anthracnose alleviation in cucumber plants. The findings here have a practical importance in plant physiology studies to identify the resistance-relevant mechanisms to pathogens and in sustainable agriculture to control the fungal diseases by a safe method.

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

The cells of all organisms are sugarcoated with polysaccharides, glycoproteins, and glycolipids. In multicellular organisms these polymers not only have a structural role in the organization of tissues and organs, but also have signaling activities. In the animal extracellular matrix for instance, the negatively charged hyaluronic acid (HA) polymers contribute to morphogenesis through mechanosensing, whereas HA fragments that are released from the polymer under certain conditions trigger inflammatory responses and angiogenesis (1). In plants, pectins are the major charged polysaccharides in the cell wall and thus can be considered the equivalent of HA. In addition, pectin-derived oligosaccharides released from the host cell wall during pathogen infection have been shown to trigger immune responses (2⇓–4). Gallego-Giraldo et al. (5) in PNAS now provide evidence for a role of pectin oligosaccharide signaling also in the response of plant cells to cell wall perturbation. The study is part of a larger effort of Gallego-Giraldo et al. (5) to tailor plant biomass for improved digestibility (for animal feed) or conversion into fermentable sugars (saccharification, to produce second-generation biofuels and chemicals) (6). Plants, in contrast to animals, contain lignins in their extracellular matrix, besides polysaccharides and glycoproteins. Lignins consist of oxidatively cross-linked phenolic molecules, which form a hydrophobic network attached to the polysaccharides. Previous studies by Gallego-Giraldo et al. and others have shown that lignins are a major obstacle for biomass digestibility/saccharification, mainly because they limit the access of … [↵][1]1To whom correspondence may be addressed. Email: hermanus.hofte{at}inra.fr. [1]: #xref-corresp-1-1