WAKs: cell wall-associated kinases linking the cytoplasm to the extracellular matrix

There are only a few proteins identified at the cell surface that could directly regulate plant cell wall functions. The cell wall-associated kinases (WAKs) of angiosperms physically link the plasma membrane to the carbohydrate matrix and are unique in that they have the potential to directly signal cellular events through their cytoplasmic kinase domain. In Arabidopsis there are five WAKs and each has a cytoplasmic serine/threonine protein kinase domain, spans the plasma membrane, and extends a domain into the cell wall. The WAK extracellular domain is variable among the five isoforms, and collectively the family is expressed in most vegetative tissues. WAK1 and WAK2 are the most ubiquitously and abundantly expressed of the five tandemly arrayed genes, and their messages are present in vegetative meristems, junctions of organ types, and areas of cell expansion. They are also induced by pathogen infection and wounding. Recent experiments demonstrate that antisense WAK expression leads to a reduction in WAK protein levels and the loss of cell expansion. A large amount of WAK is covalently linked to pectin, and most WAK that is bound to pectin is also phosphorylated. In addition, one WAK isoform binds to a secreted glycine-rich protein (GRP). The data support a model where WAK is bound to GRP as a phosphorylated kinase, and also binds to pectin. How WAKs are involved in signaling from the pectin extracellular matrix in coordination with GRPs will be key to our understanding of the cell wall's role in cell growth.     

An Arabidopsis cell wall-associated kinase required for invertase activity and cell growth

The wall-associated kinases (WAK), a family of five proteins that contain extracellular domains that can be linked to pectin molecules of the cell wall, span the plasma membrane and have a cytoplasmic serine/threonine kinase domain. Previous work has shown that a reduction in WAK protein levels leads to a loss of cell expansion, indicating that these receptor-like proteins have a role in cell shape formation. Here it is shown that a single wak2 mutation exhibits a dependence on sugars and salts for seedling growth. This mutation also reduces the expression and activity of vacuolar invertase, often a key factor in turgor and expansion. WAKs may thus provide a molecular mechanism linking cell wall sensing (via pectin attachment) to regulation of solute metabolism, which in turn is known to be involved in turgor maintenance in growing cells.     

Antisense expression of a cell wall-associated protein kinase, WAK4, inhibits cell elongation and alters morphology

The Arabidopsis cell wall-associated receptor-like kinase (WAK) gene family contains five highly related members whose products are suited for exchanging signals between the intracellular and extracellular compartments. WAK members are expressed in specific organs and regulated differentially by various biotic and abiotic factors. To gain further insight into how WAKs function during development, we used a glucocorticoid-inducible system to express ectopically the WAK4 antisense gene. The induced expression of the WAK4 antisense gene resulted in a significant decrease of WAK proteins. Ninety-six hours after the induction of WAK4 antisense expression, WAK proteins became undetectable. Cell elongation was impaired, and lateral root development was blocked. The level of WAK protein could be controlled by the concentration of the applied inducer, dexamethasone, and was correlated with the severity of the cell elongation inhibition phenotype. These results suggest that the WAKs serve a vital role in cell elongation and are required for plant development.     

The state of cell wall pectin monitored by wall associated kinases: A model

The Wall Associated Kinases (WAKs) bind to both cross-linked polymers of pectin in the plant cell wall, but have a higher affinity for smaller fragmented pectins that are generated upon pathogen attack or wounding. WAKs are required for cell expansion during normal seedling development and this involves pectin binding and a signal transduction pathway involving MPK3 and invertase induction. Alternatively WAKs bind pathogen generated pectin fragments to activate a distinct MPK6 dependent stress response. Evidence is provided for a model for how newly generated pectin fragments compete for longer pectins to alter the WAK dependent responses.     

Antisense Expression of a Cell Wall–Associated Protein Kinase, WAK4, Inhibits Cell Elongation and Alters Morphology

The Arabidopsis cell wall–associated receptor-like kinase (WAK) gene family contains five highly related members whose products are suited for exchanging signals between the intracellular and extracellular compartments. WAK members are expressed in specific organs and regulated differentially by various biotic and abiotic factors. To gain further insight into how WAKs function during development, we used a glucocorticoid-inducible system to express ectopically the WAK4 antisense gene. The induced expression of the WAK4 antisense gene resulted in a significant decrease of WAK proteins. Ninety-six hours after the induction of WAK4 antisense expression, WAK proteins became undetectable. Cell elongation was impaired, and lateral root development was blocked. The level of WAK protein could be controlled by the concentration of the applied inducer, dexamethasone, and was correlated with the severity of the cell elongation inhibition phenotype. These results suggest that the WAKs serve a vital role in cell elongation and are required for plant development.     

Plant cell walls: wall-associated kinases and cell expansion

Arabidopsis has a family of five cell wall-associated protein kinases (WAKs) with properties suggestive of transmembrane sensors between the cell wall and the cytoplasm. Recent results show that WAKs are bound to pectin and are necessary for normal leaf cell enlargement and other growth processes.     

Tissue-specific and developmentally regulated expression of a cluster of tandemly arrayed cell wall-associated kinase-like kinase genes in Arabidopsis

The Arabidopsis cell wall-associated kinase (WAK) and WAK-like kinase (WAKL) family of receptor-like kinase genes encodes transmembrane proteins with a cytoplasmic serine/threonine kinase domain and an extracellular region containing epidermal growth factor-like repeats. Previous studies have suggested that some WAK members are involved in plant defense and heavy metal responses, whereas others are required for cell elongation and plant development. The WAK/WAKL gene family consists of 26 members in Arabidopsis and can be divided into four groups. Here, we describe the characterization of group 2 members that are composed of a cluster of seven tandemly arrayed WAKL genes. The predicted WAKL proteins are highly similar in their cytoplasmic region but are more divergent in their predicted extracellular ligand-binding region. WAKL7 encodes a truncated WAKL isoform that is predicted to be secreted from the cytoplasm. Ratios of nonsynonymous to synonymous substitutions suggest that the extracellular region is subject to diversifying selection. Comparison of the WAKL and WAK gene clusters suggests that they arose independently. Protein gel-blot and immunolocalization analyses suggest that WAKL6 is associated with the cell wall. Histochemical analyses of WAKL promoters fused with the beta-glucuronidase reporter gene have shown that the expressions of WAKL members are developmentally regulated and tissue specific. Unlike WAK members whose expressions were found predominately in green tissues, WAKL genes are highly expressed in roots and flowers. The expression of WAKL5 and WAKL7 can be induced by wounding stress and by the salicylic acid analog 2,6-dichloroisonicotinic acid in an nonexpressor of pathogenesis-related gene 1-dependent manner, suggesting that they, like some WAK members, are wound inducible and can be defined as pathogenesis-related genes.     

Arabinogalactan protein and wall-associated kinase in a plasmalemmal reticulum with specialized vertices

Summary Arabinogalactan protein and wall-associated kinase (WAK) are suspected to be regulatory players at the interface between cytoplasm and cell wall. Both WAK(s) and arabinogalactan shown likely to represent arabinogalactan protein(s) have been visualized there with computational optical-sectioning microscopy. The arabinogalactan occurs in a polyhedral array at the external face of the cell membrane. WAK, and other proteins as yet unidentified, appear to fasten the membrane to the wall at vertices of the array. Evidence is presented that the array bears an important part of the mechanical stress experienced by the membrane, and it is speculated that the architectural organization of arabinogalactan protein, WAK, and other components of the array is critical for coordination of endomembrane activities, growth, and differentiation. The array has been named the plasmalemmal reticulum.Abbreviations AGP arabinogalactan protein - (-D-Glc)₃ -D-glucosyl Yariv phenylglycoside - (-D-Man)₃ -D-mannosyl Yariv phenylglycoside - SDS-AGE sodium dodecyl sulfate-agarose gel electrophoresis - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - WAK wall-associated kinase     

A cluster of five cell wall-associated receptor kinase genes,Wak1–5, are expressed in specific organs of Arabidopsis

WAK1 (wall-associated kinase 1) is a cytoplasmic serine/threonine kinase that spans the plasma membrane and extends into the extracellular region to bind tightly to the cell wall. The Wak1 gene was mapped and found to lie in a tight cluster of five highly similar genes (Wak1–5) within a 30 kb region. All of the Wak genes encode a cytoplasmic serine/threonine protein kinase, a transmembrane domain, and an extracytoplasmic region with several epidermal growth factor (EGF) repeats. The extracellular regions also contain limited amino acid identities to the tenascin superfamily, collagen, or the neurexins. RNA blot analysis with gene-specific probes revealed that Wak1, Wak3 and Wak5 are expressed primarily in leaves and stems of Arabidopsis. Wak4 mRNA is only detected in siliques, while Wak2 mRNA is found in high levels in leaves and stems, and in lower levels in flowers and siliques. A trace amount of Wak2 can also be detected in roots. Wak1 is induced by pathogen infection and salicylic acid or its analogue INA and is involved in the plant's response, and Wak2, Wak3 and Wak5 also can be greatly induced by salicylic acid or INA. The WAK proteins have the potential to serve as both linkers of the cell wall to the plasma membrane and as signaling molecules, and since Wak expression is organ-specific and the isoforms vary significantly in the cell wall associated domain this family of proteins may be involved in cell wall-plasma membrane interactions that direct fundamental processes in angiosperms.     

In vitro characterization of the homogalacturonan-binding domain of the wall-associated kinase WAK1 using site-directed mutagenesis

Wall-associated kinase 1--WAK1 is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain in contact with the pectin fraction of the plant cell wall in Arabidopsis thaliana (L.) HEYNH. In a previous paper [Decreux, A., Messiaen, J., 2005. Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation. Plant Cell Physiol. 46, 268-278], we showed that a recombinant peptide expressed in yeast corresponding to amino acids 67-254 of the extracellular domain of WAK1 specifically interacts with commercial non-methylesterified homogalacturonic acid, purified homogalacturonans from Arabidopsis and oligogalacturonides in a calcium-induced conformation. In this report, we used a receptor binding domain sequence-based prediction method to identify four putative binding sites in the extracellular domain of WAK1, in which cationic amino acids were selected for substitution by site-directed mutagenesis. Interaction studies between mutated forms of WAK1 and homogalacturonans allowed us to identify and confirm at least five specific amino acids involved in the interaction with homogalacturonan dimers and multimers. The presence of this homogalacturonan-binding domain within the extracellular domain of WAK1 is discussed in terms of cell wall architecture and signal transduction.     

Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation

Wall-associated kinase 1 (WAK1) is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain in contact with the pectin fraction of the plant cell walls. In order to characterize further the interaction of WAK1 with pectin, a 564 bp DNA sequence corresponding to amino acids 67-254 of the extracellular domain of WAK1 from Arabidopsis thaliana was cloned and expressed as a soluble recombinant peptide in yeast. Using enzyme-linked immunosorbent assays (ELISA), we show that peptide WAK(67-254) binds to polygalacturonic acid (PGA), oligogalacturonides, pectins extracted from A. thaliana cell walls and to structurally related alginates. Our results suggest that both ionic and steric interactions are required to match the relatively linear pectin backbone. Binding of WAK(67-254) to PGA, oligogalacturonides and alginates occurred only in the presence of calcium and in ionic conditions promoting the formation of calcium bridges between oligo-and polymers (also known as 'egg-boxes'). The conditions inhibiting the formation of calcium bridges (EDTA treatment, calcium substitution, high NaCl concentrations, depolymerization and methylesterification of pectins) also inhibited the binding of WAK(67-254) to calcium-induced egg-boxes. The relevance of this non-covalent link between WAK(67-254) and cell wall pectins is discussed in terms of cell elongation, cell differentiation and host-pathogen interactions.     

Comprehensive Analysis of Wall-Associated Kinase Genes and Their Expression Under Abiotic and Biotic Stress in Chinese Cabbage (Brassica rapa ssp. pekinensis)

The wall-associated kinase (WAK) gene family, a subfamily of the receptor-like kinase (RLK) gene family, is associated with the cell wall in plants, and has vital functions in cell expansion, pathogen resistance, and heavy metal stress tolerance because of their roles of the extracellular environment sensors to trigger intracellular signals in Arabidopsis. In the present study, 96 Chinese cabbage (Brassica rapa ssp. pekinensis) BrWAK gene family members were identified from the B. rapa genome using a reiterative database search and manual confirmation. The protein domain characterization, gene structure analysis, and phylogenetic analysis of the BrWAKs classified them into three gene groups. Comparative genomic analysis between WAK genes from Chinese cabbage and Arabidopsis revealed that the BrWAK genes have undergone the gene expansion and deletion events during evolution. Furthermore, the conserved motifs in the kinase domains of the WAK proteins and eukaryotic protein kinase family proteins were compared and some non-RD kinase proteins among the BrWAKs were identified. Ultimately, expression analysis of BrWAK genes in six tissues and under various stress conditions revealed that some tissue-specific WAK genes might function in callus cell growth and reproduction process; Bra012273, Bra016426, Bra016427, and Bra025882 might be involved in downy mildew resistance and high humidity stress; Bra012273, Bra025882, and Bra025883 might be responded to drought and heat stress. Taken together, this research was identified and classified the WAK gene family in Chinese cabbage and provided valuable resources to explore the potential roles of BrWAK genes in plant development and stress responses.

     

A root-specific wall-associated kinase gene, HvWAK1, regulates root growth and is highly divergent in barley and other cereals

Wall-associated receptor-like kinases (WAKs) are important candidates for directly linking the extracellular matrix with intracellular compartments and are involved in developmental processes and stress response. WAK gene family has been identified in plants such as Arabidopsis and rice. Here, we present a detailed analysis of the WAK1 gene from barley cv. Golden Promise, mapped to chromosome 5H. Three BAC clones corresponding to the WAK fragment were sequenced and the full-length WAK1 gene was characterized. The gene has three exons and two short introns with a coding region of 2,178 bp encoding a protein of 725 amino acids. A regulatory region was analyzed in ?1,000 bp sequence upstream to start codon. Using conserved domains database and SMART, various conserved domains such as GUB WAK Bind, epidermal growth factor CA, and protein kinase C as well as other regions like signal peptides, active sites, and transmembrane domains were identified. The gene organization of HvWAK1 was compared with wheat (TaWAK1) and Arabidopsis (AtWAK1), suggesting that the WAK1 gene organization has remained highly conserved. Nonetheless, WAK1 was found to be highly divergent when compared with sequences available from barley cv. Haruna Nijo (50 %), rice (46 %), wheat (21 %), Arabidopsis (25 %), and maize (19 %). This divergence may have facilitated a better adaptation to surrounding environments due to its role in communication between the extracellular matrix, cell, and outer environment. Semiquantitative RT-PCR-based expression analysis indicates HvWAK1 expression is specific to roots. Significant differences in root growth between GP wild type and GP-Ds mutant seedlings were observed under control and salt stress conditions.     

Oxygen-evolving enhancer protein 2 is phosphorylated by glycine-rich protein 3/wall-associated kinase 1 in Arabidopsis

The Arabidopsis wall-associated receptor kinase, WAK1, is a member of WAK family that links the plasma membrane to the extracellular matrix. A glycine-rich secreted protein, AtGRP-3, was previously shown to regulate WAK1 functions through binding to the extracellular domain of WAK1. In this study, we sought to determine the downstream molecules of the AtGRP-3/WAK1 signaling pathway, by using two-dimensional gel electrophoresis combined with Edman sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). We report here that a chloroplast protein, oxygen-evolving enhancer protein 2 (OEE2), specifically interacts with the cytoplasmic kinase domain of WAK1 and becomes phosphorylated in an AtGRP-3-dependent manner. The phosphorylation of OEE2 is also induced in Arabidopsis by treatment with avirulent Pseudomonas syringae. Taken together, these results suggest that OEE2 activity is regulated by AtGRP-3/WAK1.