Monitoring the Outside: Cell Wall-Sensing Mechanisms

Plant cell walls are established by different polysaccharides and structural proteins. The exact composition of these complex structures is dependent on the type of cell and the developmental stage and therefore is in a constant flow of remodeling. Cell shape is defined by the balance between turgor pressure from the symplast and the strength of the cell wall. Yet, for cell growth to take place, the controlled loosening of the cell wall is a prerequisite (Cosgrove, 2005). Thus, control mechanisms that closely survey the different steps of cell wall remodeling are necessary, implying that signals from the apoplast to the cell wall and vice versa ascertain the exchange of information. The goal of this article is to give an overview on the understanding of the signaling mechanisms that take place between cell walls and the cytoplasm, with a focus on the recent advances in the field. Plant cell walls are also defense structures against different abiological and biological stresses such as pathogens. The mechanisms of recognition of pathogens and the modification of cell walls upon pathogen encounter are reviewed elsewhere (Hematy et al., 2009). A number of phenomena demonstrate that a sensing and signaling system must exist in the extracellular matrix that monitors the structure and integrity of cell walls. For example, cellulose synthesis takes place in rosette-forming protein complexes made of cellulose synthases (CesAs). Mutations in Arabidopsis (Arabidopsis thaliana) CesA genes lead to a reduction in cellulose content and induce compensatory mechanisms, including modifications in lignin deposition, pectins, xyloglucan, and AGPs. Thus, a cell wall-sensing process must recognize the cellulose deficiency and induce appropriate responses. These signaling events are clearly not linear but induce many different reactions, including stress-related processes that depend on intact hormone signaling pathways (Ellis et al., 2002; Cano-Delgado et al., 2003; Bosca et al., 2006; Hernandez-Blanco et al., 2007). Excellent candidates for a function in sensing the structure of cell walls and transducing this information to the cytoplasm are transmembrane proteins. Plant genomes code for a large number of receptor-like kinases (RLKs; Arabidopsis, more than 600; rice (Oryza sativa), more than 1,100; Shiu and Bleecker, 2001), which can relay a signal to the cytoplasm via the cytoplasmic kinase domain (Fig. 1). Some of these proteins should be able to sense changes in the cell wall structure by, for example, a missing interaction partner. Indeed, a role in cell wall-related signaling has been demonstrated for a number of RLKs. Functions of proteins implicated in cell wall-related signaling and development. Receptor-like proteins and extracellular proteins are shown that have a function in cell wall sensing and/or signaling. From left to right: FEI1 and FEI2 are LRR-containing RLKs that influence cellulose content and act in a linear pathway with the GPI-anchored SOS5. A direct interaction, however, has so far not been shown. THE1 senses cellulose deficiencies and, under these conditions, induces changes in cell wall structures. COBRA is a GPI-anchored membrane-associated protein important for the orientation of cellulose microfibrils. WAK1 binds GRP3, a Gly-rich protein, and both WAK1 and WAK2 are able to interact with pectin. WAK2 activates MAPK3 and thus possibly other MAPKs and induces a vacuolar invertase to modify turgor pressure. The membrane-associated PERK4 interacts with pectin and activates Ca2+ channels, leading to increased cytosolic Ca2+ content, which can induce changes in intracellular and extracellular pH and the NADPH oxidase (NOX)-dependent production of ROS. The Arg-Gly-Asp (RGD) tripeptide is a cell-adhesion motif and is bound by at least one LecRK. AGPs are GPI-anchored membrane-associated proteins implicated in cell wall-related signaling. LRX1 is an extracellular receptor binding an unknown interactor and is insolubilized in the cell wall. ANX1 and ANX2 are important for pollen tube rupture involving sudden changes in the cell wall. The TOR kinase is central to the TOR pathway that also influences cell wall structure, by a so far unknown mechanism. Solid, double-arrowed lines indicate experimentally shown interactions or signaling outputs; dashed arrows indicate possible, but not experimentally shown, interactions. Wavy lines represent GPI anchors. [See online article for color version of this figure.] Wall-associated kinases (WAKs) of Arabidopsis are a family of five transmembrane proteins with a cytoplasmic Ser/Thr kinase domain and an extracellular domain with motifs similar to the vertebrate epidermal growth factor repeats. These proteins are involved in cell expansion but are also induced upon pathogen attack or as a stress response (Wagner and Kohorn, 2001; Sivaguru et al., 2003). The reduction of WAK expression has been shown to lead to a reduction in cell growth (Lally et al., 2001; Wagner and Kohorn, 2001; Kohorn et al., 2006b). Most interestingly with respect to cell wall sensing is the observation that WAK proteins interact strongly, in some cases covalently, with pectin (He et al., 1996; Wagner and Kohorn, 2001). For WAK1 and WAK2, pectin binding could be demonstrated in vivo and in vitro (Decreux and Messiaen, 2005; Kohorn et al., 2009). In addition, in a yeast two-hybrid experiment, WAK1 was shown to interact with the Gly-rich protein GRP3, a structural protein in the cell wall (Park et al., 2001). These results strongly indicate a direct interaction of WAK1 and WAK2 with the cell wall and make them likely candidates for a cell wall-sensing function (Fig. 1). A recent study revealed that WAK1-GFP accumulates in a pectin-containing compartment in the cytoplasm. From there, WAK1-GFP migrates to the plasma membrane, but much slower than RLKs that do not associate with cell walls, such as the brassinosteroid receptor BRI1 (Wang et al., 2001). The migration process is dependent on active cellulose biosynthesis and negatively influenced by Fuc, which is a component of pectin (Kohorn et al., 2006a). The regulation of protein localization by cell wall components adds another level of complexity to the control of RLK signaling activity. A further insight into the possible function of WAK proteins was provided by a detailed analysis of Arabidopsis WAK2. A wak2 knockout mutant grew normal under standard conditions. However, on medium with low salt and no Suc, it showed a reduced cell elongation rate. The importance of Suc for the wak2 phenotype could be compensated for by expressing a maize (Zea mays) Suc phosphate synthase (Kohorn et al., 2006b), indicating that the wak2 phenotype is linked to Suc-related physiology. Measurements of Suc-related enzymes revealed that vacuolar invertase activity, which converts Suc to Glc and Fru, is reduced in wak2. Most likely, the low level of these sugars in the vacuole reduces turgor pressure in the cell that affects cell elongation (Martin et al., 2001). Next, the authors aimed at making WAK2 expression inducible to follow its effect on cell turgor. The WAK2 extracellular domain was replaced by the one of the brassinosteroid (BR) receptor BRI1 (Wang et al., 2001), thus making WAK2 signaling BR dependent. In protoplast experiments, BR-mediated WAK2 activity induced vacuolar invertase, which resulted in an increased turgor pressure (Kohorn et al., 2006b). Together, these analyses suggest that WAK2 interacts with pectin at the cell surface, upon which the protein induces a signal transduction cascade (see below) that leads to a modification of the turgor pressure in the cell. Lectin receptor kinases (LecRKs) contain an extracellular lectin domain and thus represent a second class of RLKs with potential carbohydrate-binding properties. Lectins have been identified as proteins that bind carbohydrates, suggesting that, in the context of LecRKs, the lectin domain establishes a direct link to cell wall polysaccharides (André et al., 2005; Bouwmeester and Govers, 2009). However, at least some LecRKs undergo protein-protein interactions. Upon plasmolysis, during which the plasma membrane detaches from the cell wall, connections remain between these two structures. The addition of peptides containing the RGD (Arg-Gly-Asp) tripeptide motif interferes with the integrity of these linkages (Canut et al., 1998). This is of particular interest since the RGD motif is also found in mammalian extracellular matrix proteins involved in cell adhesion. The search for Arabidopsis proteins that are able to interact with the RGD tripeptide via phage display technology revealed several LecRKs as likely candidates (Gouget et al., 2006). Thus, LecRKs potentially represent a group of receptors that have diverse binding specificities and can have roles in sensing of the cell wall structure/plasma membrane-cell wall connection (Fig. 1). For the type of RLKs discussed so far, there is evidence that at least some of their members do interact with polysaccharidic components in cell walls. There are, however, a number of RLKs that rather interact with proteins, at least based on the structure of their extracellular domains. A motif particularly well known for undergoing protein-protein interaction is represented by Leu-rich repeats (LRRs), which have been identified in many different organisms such as plants and metazoans, where they are involved in signal transduction activities. In plants, a number of proteins with an LRR domain have been shown to play a role in pathogen recognition or during developmental processes (Kobe and Deisenhofer, 1994; Jones and Jones, 1997). FEI1 and FEI2 are two out of over 200 RLKs in Arabidopsis with an LRR domain (Morillo and Tax, 2006). They are highly homologous, and fei1 fei2 double mutants develop a swollen-root phenotype due to isotropic (instead of longitudinal) cell growth (Xu et al., 2008). This phenotype and the ectopic deposition of lignin are similar to the cellulose synthase (CesA6) mutant procuste1 (prc1; Fagard et al., 2000). Indeed fei1 fei2 double mutants contain reduced levels of crystalline cellulose. Further analyses revealed that FEI1 and FEI2 function in the regulation of cellulose biosynthesis. Interestingly, the kinase activity of the FEI1 and FEI2 kinase domain is not essential for protein function. Corresponding mutations in the kinase domain still allowed for complementation of the fei1 fei2 mutant phenotype, even though the efficiency of complementation was reduced (Xu et al., 2008). Hence, the mode of action of these RLKs appears to be different from the expected and remains to be determined. Genetic evidence suggests that FEI1 and FEI2 function in the same pathway as SALT OVERLY SENSITIVE5 (SOS5), which was identified in a screen for salt-hypersensitive mutants (Fig. 1). The sos5 mutant showed root growth arrest, root swelling, and an increased width in etiolated hypocotyls under high-salt conditions. These sos5 phenotypes are similar to those observed in fei1 fei2 seedlings, but no additive effects were observed in the fei1 fei2 sos5 triple mutant, suggesting that the proteins function in the same pathway. SOS5 codes for a glycosylphosphatidylinositol (GPI)-anchored, and thus extracellular, protein (Shi et al., 2003). Future experiments will have to reveal whether SOS5 does directly interact with FEI1 and/or FEI2 or is involved in perception of the ligand without a direct interaction with the receptor proteins. THESEUS1 (THE1) is a member of the subfamily of Catharanthus roseus Protein Kinase1-Like receptor kinases, for which a function in cell wall integrity sensing has been demonstrated (Hematy et al., 2007). The the1 mutant was identified as a suppressor of prc1-1, a mutant affected in CesA6 (Fagard et al., 2000). The prc1-1 mutant is characterized by reduced levels of cellulose and develops short hypocotyls when grown in the dark. While the short-hypocotyl phenotype is suppressed by the1, the cellulose deficiency is not, indicating that THE1 is involved in sensing structural defects in the cell wall. Cellulose-deficient mutants tend to accumulate ectopic lignin, and this effect is dependent on THE1, as prc1 the1 mutants failed to accumulate lignin ectopically. Overexpression of THE1 led to an overaccumulation of ectopic lignin, but only in the prc1 mutant background. In addition, the1 was able to partially suppress other cesa mutants, but as a single mutant it did not develop a mutant phenotype. This again indicates that THE1 is involved in sensing cellulose deficiency and adapts cell wall development upon changes or irregularities in the cell wall structure (Fig. 1). THE1 exhibits in vitro phosphorylation activity and was localized to the plasma membrane, further supporting the that THE1 as an active is not at whether THE1 directly interacts with the cell wall or rather as a signaling two RLKs to THE1, and were These genes are influenced by as they were in the BR receptor mutant et al., and in the mutant et al., was shown to interaction during by pollen tube at the cell et al., and plants reduced cell elongation et al., 2009). and THE1 revealed interaction, as a the1 double mutant develops a phenotype. with and showed that THE1, and are likely to function in a pathway to cell elongation that is influenced by, but still signaling. ANX1 and ANX2 represent the two of in These two RLKs are in pollen and to the of the pollen where they are to rupture of the tube to at the The double mutant was characterized by pollen tube rupture et al., et al., 2009). The interaction of ANX1 and ANX2 and the of pollen tube rupture remain to be shown, but it is to that this also sudden changes in the cell wall of the and, that ANX1 and ANX2 are involved in cell wall-related signaling (Fig. 1). receptor proteins are candidates for transducing signals from the extracellular matrix to the cytoplasm. In addition, proteins can be to the of the membrane via the membrane GPI that is to proteins et al., and into the proteins have been identified that play a role in the of cell wall-related information the The Arabidopsis mutants a in the biosynthesis of GPI and develop a phenotype. The mutant a strongly and and can only be as with the cell walls a strongly reduced cellulose content and ectopic of xyloglucan, and et al., 2005). Hence, proteins play an important role during cell wall development. Indeed, several of these proteins have been identified as important for the and of the cell wall. COBRA is a GPI-anchored protein that was identified based on cell growth in and reduced cellulose content in a mutant et al., 2001). A detailed analysis revealed that cellulose is not in the cell wall due to the of this protein (Fig. 1). is a of the of cell as the to the of cell elongation The orientation of cellulose is as they are to be the structure of the cell wall, in with and the cell and Thus, the cellulose deposition in mutants the in root The orientation of the and the of the complexes cellulose in the membrane have been shown to be and it was that the of the and with the by mutations or of also led to cellulose deposition and 2002; et al., Together, these suggest that COBRA the information on the to the cellulose synthase complexes and therefore is involved in a between the and the cell wall et al., 2005). A direct interaction with cell wall polysaccharides appears also to be established by the membrane-associated receptor kinases proteins and are structural cell wall proteins known to in the cell wall et al., and the extracellular of these proteins et al., The PERK4 of Arabidopsis is by indicating a possible interaction with pectin et al., 2009). the the Arabidopsis induces a phenotype by an increased cell indicating that PERK4 negatively influences cell The mutant also a to not other with respect to root Ca2+ and cytosolic Ca2+ levels et al., 2009). Ca2+ is an important signaling and leads to changes in Ca2+ et al., 2007). Ca2+ induces the production of which induce again Ca2+ and pH changes on both of the plasma membrane that cell wall (Fig. et al., 2003; et al., et al., 2009). Together, these for the in which linked to the cell wall, sense stresses in the cell wall and influence Ca2+ the membrane that a number of cell wall-related in the of cell to the family of and of a rather protein that is highly AGPs can be to the composition of the AGPs contain and AGPs from in their AGPs have an GPI (Fig. 2001). different proteins that are to the plasma membrane via a GPI have been shown to be involved in the intracellular and extracellular and thus influence cell wall development et al., 2005; et al., 2005). of function revealed important roles in cell and 2001; et al., 2007). AGPs have been shown to bind components of the cell wall. This interaction can be and suggests that AGPs are able to link the plasma membrane and the cell wall et al., Kohorn, 2000). the analysis of revealed a of the of and the and AGPs with the affects and and with these components changes the of in et al., 2006). These suggest a link between the and Hence, GPI-anchored AGPs are likely not only involved in a connection between the cell wall and the plasma membrane but to this to the cytoplasm, a that as a to relay information between the intracellular and extracellular are extracellular proteins containing an LRR domain and a a structural protein domain et al., et al., the function of LRR in protein-protein interaction, these proteins are candidates for a signaling function in cell wall development 2005). The characterized protein is LRX1 of Arabidopsis, which is in root LRX1 is insolubilized in the cell wall, a function that is provided by the since are known to in the cell wall 1998). For it could be shown that the LRR-containing of the protein an interaction in the cell wall, since expressing this LRX1 protein in the type results in a root phenotype similar to the mutant (Fig. 1). This suggests that this protein out the binding of the LRX1 et al., 2001). Mutations in LRX1 and its in the of cell wall structures et al., a function of proteins in cell wall possibly as a signaling far, the LRX1 interaction remains the of the exact function of LRX1 and possibly other proteins during cell wall development. Genetic evidence at the of LRX1 involved in a This is from the that the mutants were identified as of the root phenotype et al., 2006). The of potential receptor proteins, particularly those localized in the plasma membrane, is based on A number of involved in protein-protein interaction were identified in plants and metazoans, and they can as a in the of potential proteins for a signaling function in cell wall-related In the and have allowed for in this field. However, information on the signals are to other components in the signal transduction cascade is still Yet, recent has on signal transduction pathways that are involved in cell wall WAK2 activates a vacuolar invertase leading to the production of Glc and Fru, which turgor pressure and cell expansion (Kohorn et al., 2006b). is this the of WAK2 activity, were with a of the to the protein This led to a of activity of the upon addition of pectin to the this was not observed in from a wak2 knockout mutant, the expression appears to be WAK2 in vivo evidence for WAK2 a pectin The of the signal is from the WAK2 kinase The protein kinase pathway is an important signaling and some MAPKs are upon pectin and 2008). Indeed, the kinase activity of MAPK3 was induced in upon addition of pectin to the this was not observed in wak2 Further evidence for a connection between MAPK3 and WAK2 was by a A mutant develops no mutant phenotype, a of wak2 a growth phenotype. However, a wak2 double mutant develops a growth phenotype that is by far more that a of both single mutant phenotypes (Kohorn et al., 2009). This observed interaction between the two mutants also a function in a process that WAK2 and MAPK3 are of the same signal transduction 1). for a pathway a role in cell wall sensing has been from yeast et al., where cell wall-related sensing mechanisms have been 2005). Cell walls of the yeast from cell walls but the same In the cell wall is of different Glc or a Glc the is in proteins with this cell wall is a structure, a of wall is to for cell growth to take particular yeast has to undergo changes in cell and cell wall such as during the to growth under conditions. Hence, the cell wall integrity pathway system is a controlled of cell wall development to the and thus developmental The protein kinase pathway has been to as the since it the expression of cell wall-related genes and is induced in response to changes in growth or when cell wall A number of different proteins to the yeast plasma membrane, where they changes in the cell wall or These are by 2005; et al., and not be discussed to out one pathway that is important for cell development in yeast and that out to have a similar function in The of pathway is a of where it adapts growth to the of growth and et al., 2006). The TOR protein, a Ser/Thr kinase central to this is to making a with et al., 2008). was shown for yeast that some proteins involved in the to growth of the cell also have a function in TOR signaling et al., and Thus, it is that TOR is a of cell wall structures. in the yeast identified a of the to be for of under conditions. A in indicating that also has a function in TOR signaling. In addition, the mutant an and a reduced of the pathway upon with cell et al., 2009). In plants, several components of the TOR including the TOR protein, have been though there is evidence that the TOR pathway is different from the TOR pathway in yeast et al., it is likely that it has as well (Fig. 1). In Arabidopsis, the TOR kinase is essential for development. A knockout mutant is but plants with reduced TOR expression in growth et al., 2002; et al., 2007). Mutations in other TOR pathway components also have an effect on development et al., 2006; et al., 2008). There is evidence that the Arabidopsis TOR pathway is involved in cell wall development. A suppressor screen was on the mutant that cell wall development in root et al., A suppressor was identified that induces changes in cell wall structures and leads to the of root in the mutant background. with the of yeast revealed a function of in the TOR pathway. of the TOR pathway in Arabidopsis by with not only leads to of the root phenotype but also induces changes in cell wall structures that are to those of the mutant et al., Future analyses will be to a insight into the and the by which the TOR pathway affects cell wall development in The have led to the of a of information on receptor proteins that signals from the apoplast and them to the cytoplasm. was on WAK2, where the binding one of the signal and a signaling could be have been identified that do not act as receptors but are involved in transducing information from the cytoplasm to the extracellular matrix which in the orientation of the to the of For many the interaction remains to be determined. There is also a of understanding the steps between signal perception and signaling The was in some cases by experiments, the remains to be In some the of plants with other such as yeast will to possible proteins. For it will be to follow the that will be made in the to with a much insight into complex processes of cell wall-sensing for of the