Kian Hématy

A Receptor-like Kinase Mediates the Response of Arabidopsis Cells to the Inhibition of Cellulose Synthesis

BACKGROUND A major challenge is to understand how the walls of expanding plant cells are correctly assembled and remodeled, often in the presence of wall-degrading micro-organisms. Plant cells, like yeast, react to cell-wall perturbations as shown by changes in gene expression, accumulation of ectopic lignin, and growth arrest caused by the inhibition of cellulose synthesis. RESULTS We have identified a plasma-membrane-bound receptor-like kinase (THESEUS1), which is present in elongating cells. Mutations in THE1 and overexpression of a functional THE1-GFP fusion protein did not affect wild-type (WT) plants but respectively attenuated and enhanced growth inhibition and ectopic lignification in seedlings mutated in cellulose synthase CESA6 without influencing the cellulose deficiency. A T-DNA insertion mutant for THE1 also attenuated the growth defect and ectopic-lignin production in other but not all cellulose-deficient mutants. The deregulation of a small number of genes in cesA6 mutants depended on the presence of THE1. Some of these genes are involved in pathogen defense, in wall crosslinking, or in protecting the cell against reactive oxygen species. CONCLUSIONS The results show that THE1 mediates the response of growing plant cells to the perturbation of cellulose synthesis and may act as a cell-wall-integrity sensor.

Growth Control and Cell Wall Signaling in Plants

Plant cell walls have the remarkable property of combining extreme tensile strength with extensibility. The maintenance of such an exoskeleton creates nontrivial challenges for the plant cell: How can it control cell wall assembly and remodeling during growth while maintaining mechanical integrity? How can it deal with cell wall damage inflicted by herbivores, pathogens, or abiotic stresses? These processes likely require mechanisms to keep the cell informed about the status of the cell wall. In yeast, a cell wall integrity (CWI) signaling pathway has been described in great detail; in plants, the existence of CWI signaling has been demonstrated, but little is known about the signaling pathways involved. In this review, we first describe cell wall-related processes that may require or can be targets of CWI signaling and then discuss our current understanding of CWI signaling pathways and future prospects in this emerging field of plant biology.

Novel receptor kinases involved in growth regulation.

The growth of plant cells involves a constant adjustment of synthesis and rearrangement of cell wall polymers. Recently, three plasma membrane-bound receptor kinases related to CrRLK1 have been shown to be involved in the negative control of cell growth in different contexts. THESEUS1 is activated in mutants deficient for cellulose and may act as a cell wall integrity sensor inhibiting cell elongation. FERONIA is polarly localized in synergid cells of the female gametophyte and is required for growth cessation of compatible pollen tubes and subsequent delivery of sperm cells. AmRLK is involved in the control of the polar conical outgrowth of epidermal cells of Antirrhinum petals. The conservation of both extracellular and kinase domains suggests that the three receptors bind to related ligands and have similar cellular outputs, which may involve the production of reactive oxygen species.

Sphingolipids Containing Very-Long-Chain Fatty Acids Define a Secretory Pathway for Specific Polar Plasma Membrane Protein Targeting in Arabidopsis

This study shows that Arabidopsis has two classes of ceramide synthases discriminating acyl chain length and also that very-long-acyl-chain sphingolipids are required for polar auxin transport in particular during lateral root emergence. These lipids define a secretory pathway with specific endomembrane compartments and polar auxin transport protein cargoes. Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1 in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2a– and Rab-A1e–labeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes.

Mutations in UDP-Glucose:Sterol Glucosyltransferase in Arabidopsis Cause Transparent Testa Phenotype and Suberization Defect in Seeds

In higher plants, the most abundant sterol derivatives are steryl glycosides (SGs) and acyl SGs. Arabidopsis (Arabidopsis thaliana) contains two genes, UGT80A2 and UGT80B1, that encode UDP-Glc:sterol glycosyltransferases, enzymes that catalyze the synthesis of SGs. Lines having mutations in UGT80A2, UGT80B1, or both UGT80A2 and UGT8B1 were identified and characterized. The ugt80A2 lines were viable and exhibited relatively minor effects on plant growth. Conversely, ugt80B1 mutants displayed an array of phenotypes that were pronounced in the embryo and seed. Most notable was the finding that ugt80B1 was allelic to transparent testa15 and displayed a transparent testa phenotype and a reduction in seed size. In addition to the role of UGT80B1 in the deposition of flavanoids, a loss of suberization of the seed was apparent in ugt80B1 by the lack of autofluorescence at the hilum region. Moreover, in ugt80B1, scanning and transmission electron microscopy reveals that the outer integument of the seed coat lost the electron-dense cuticle layer at its surface and displayed altered cell morphology. Gas chromatography coupled with mass spectrometry of lipid polyester monomers confirmed a drastic decrease in aliphatic suberin and cutin-like polymers that was associated with an inability to limit tetrazolium salt uptake. The findings suggest a membrane function for SGs and acyl SGs in trafficking of lipid polyester precursors. An ancillary observation was that cellulose biosynthesis was unaffected in the double mutant, inconsistent with a predicted role for SGs in priming cellulose synthesis.

CHITINASE-LIKE1/POM-POM1 and Its Homolog CTL2 Are Glucan-Interacting Proteins Important for Cellulose Biosynthesis in Arabidopsis

Cell wall and cellulose structure is imperative for proper cell elongation and, consequently, the architecture of plants, but components regulating cellulose structure are still elusive. This article shows that the secreted CTL1/POM1 and its close homolog CTL2 interact with glucan-based polymers and influence cellulose crystallinity and cell expansion. Plant cells are encased by a cellulose-containing wall that is essential for plant morphogenesis. Cellulose consists of β-1,4-linked glucan chains assembled into paracrystalline microfibrils that are synthesized by plasma membrane–located cellulose synthase (CESA) complexes. Associations with hemicelluloses are important for microfibril spacing and for maintaining cell wall tensile strength. Several components associated with cellulose synthesis have been identified; however, the biological functions for many of them remain elusive. We show that the chitinase-like (CTL) proteins, CTL1/POM1 and CTL2, are functionally equivalent, affect cellulose biosynthesis, and are likely to play a key role in establishing interactions between cellulose microfibrils and hemicelluloses. CTL1/POM1 coincided with CESAs in the endomembrane system and was secreted to the apoplast. The movement of CESAs was compromised in ctl1/pom1 mutant seedlings, and the cellulose content and xyloglucan structures were altered. X-ray analysis revealed reduced crystalline cellulose content in ctl1 ctl2 double mutants, suggesting that the CTLs cooperatively affect assembly of the glucan chains, which may affect interactions between hemicelluloses and cellulose. Consistent with this hypothesis, both CTLs bound glucan-based polymers in vitro. We propose that the apoplastic CTLs regulate cellulose assembly and interaction with hemicelluloses via binding to emerging cellulose microfibrils.

OCTOPUS Negatively Regulates BIN2 to Control Phloem Differentiation in Arabidopsis thaliana

The phloem is a vascular strand that conducts photoassimilates and systemic signals throughout the plant to coordinate growth. To date, few molecular genetic determinants have been identified to control both specification and differentiation of this tissue [1-3]. Among them, OCTOPUS (OPS) protein was previously identified as a polarly localized plasma membrane-associated protein of unknown biochemical function whose broad provascular expression becomes restricted to the phloem upon differentiation [2]. OPS loss-of-function mutants showed an altered vascular network in cotyledons and an intermittent phloem differentiation in the root [2, 4]. Here, we demonstrate a role for OPS as a positive regulator of the brassinosteroid (BR) signaling pathway. Indeed, transgenic lines overexpressing OPS (OPS-OE) display the hallmarks of constitutively overactivated BR mutants. Physiological and genetic analyses place OPS as a positive regulator of the BR signaling pathway upstream of the key transcription factors BES1 and BZR1. Directed protein interactions with known BR signaling proteins identified BIN2, a GSK3 protein involved in multiple signaling pathways, as a partner of OPS. This interaction recruits BIN2 to the plasma membrane, thus preventing its inhibitory activity in the nucleus. Finally, both bikinin (a potent inhibitor of GSK3 [5]) treatment and downstream dominant mutants bes1-D [6] and bzr1-D [7] can rescue phloem defects of ops in the root. Together, our data show that OPS antagonizes BIN2 to promote phloem differentiation.

The Arabidopsis COBRA protein facilitates cellulose crystallization at the plasma membrane.

Background: The COBRA gene is highly coexpressed with cellulose synthase genes, but its function remains unclear. Results: COBRA localizes at the plasma membrane and binds glucan chains. NMR studies indicate structural defects in cellulose in the mutant despite normal polymerization rate. Conclusion: COBRA functions downstream of cellulose biosynthesis. Significance: This work suggests that alignment of glucan chains into cellulose fibrils is facilitated by one or more proteins. Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual β1–4-linked glucan chains with a KD of 3.2 μm. Competition assays suggests that COBRA binds individual β1–4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging β1–4-glucan chains by acting as a “polysaccharide chaperone.”

Inhibition of very long acyl chain sphingolipid synthesis modifies membrane dynamics during plant cytokinesis

Plant cytokinesis requires intense membrane trafficking and remodeling to form a specific membrane structure, the cell plate that will ultimately separate the daughter cells. The nature and the role of lipids involved in the formation of the cell plate remain unclear. Plant membranes are particularly rich in sphingolipids such as glucosyl-ceramides with long (16 carbons) or very long (24 carbons) acyl chains. We reveal here that inhibition of the synthesis of sphingolipids with very long acyl chains induces defective cell plates with persistent vesicular structures and large gaps. Golgi-derived vesicles carrying material toward the cell plate display longer vesicle-vesicle contact time and their cargos accumulate at the cell plate, suggesting membrane fusion and/or recycling defects. In vitro fusion experiments between artificial vesicles show that glycosphingolipids with very long acyl chains stimulate lipid bilayer fusion. Therefore we propose that the very long acyl chains of sphingolipids are essential structural determinants for vesicle dynamics and membrane fusion during cytokinesis.

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

Antagonistic growth effects of two T-DNA alleles in THESEUS1 correlate with the expression of a predicted functional THE1 protein lacking the kinase domain and antisense transcripts, respectively.