SUMMARY The angiosperm extracellular matrix, or cell wall, is composed of a complex array of cellulose, hemicelluose, pectins and proteins, the modification and regulated synthesis of which are essential for cell growth and division. The wall associated kinases (WAKs) are receptor-like proteins that have an extracellular domain that bind pectins, the more flexible portion of the extracellular matrix, and are required for cell expansion as they have a role in regulating cellular solute concentrations. We show here that both recombinant WAK1 and WAK2 bind pectin in vitro. In protoplasts pectins activate, in a WAK2-dependent fashion, the transcription of vacuolar invertase, and a wak2 mutant alters the normal pectin regulation of mitogen-activated protein kinases. Microarray analysis shows that WAK2 is required for the pectin activation of numerous genes in protoplasts, many of which are involved in cell wall biogenesis. Thus, WAK2 plays a major role in signaling a diverse array of cellular events in response to pectin in the extracellular matrix.

The wall-associated kinases, WAKs, are encoded by five highly similar genes clustered in a 30-kb locus in Arabidopsis. These receptor-like proteins contain a cytoplasmic serine threonine kinase, a transmembrane domain, and a less conserved region that is bound to the cell wall and contains a series of epidermal growth factor repeats. Evidence is emerging that WAKs serve as pectin receptors, for both short oligogalacturonic acid fragments generated during pathogen exposure or wounding, and for longer pectins resident in native cell walls. This ability to bind and respond to several types of pectins correlates with a demonstrated role for WAKs in both the pathogen response and cell expansion during plant development.

Physical connections between higher plant cell walls and the plasma membrane have been identified visually, but the molecules involved in the contact are unknown. We describe here an Arabidopsis thaliana protein kinase, designated Wak1 for wall-associated kinase, whose predicted extracytoplasmic domain contains several epidermal growth factor repeats and identity with a viral movement protein. Wak1 fractionates with insoluble material when plant tissue is ground in a variety of buffers and detergents, suggesting a tight association with the plant extracellular matrix. Immunocytochemistry confirms that Wak1 is associated with the cell wall. Enzymatic digestion of the cell wall allows the release of Wak1 from the insoluble cell wall fraction, and protease experiments indicate that Wak1 likely has a cytoplasmic kinase domain, and the EGF containing domain is extracellular. Wak1 is found in all vegetative tissues of Arabidopsis, and has relatives in other angiosperms, but not Chlamydomonas. We suggest that Wak1 is a good candidate for a physical continuum between the cell wall and the cytoplasm, and since the kinase is cytoplasmic, it also has the potential to mediate signals to the cytoplasm from the cell wall.

The mechanism by which events in the angiosperm cell wall are communicated to the cytoplasm is not well characterized. A family of five Arabidopsis wall-associated kinases (WAKs) have the potential to provide a physical and signaling continuum between the cell wall and the cytoplasm. The WAKs have an active cytoplasmic protein kinase domain, span the plasma membrane, and contain an N terminus that binds the cell wall. We show here that WAK s are expressed at organ junctions, in shoot and root apical meristems, in expanding leaves, and in response to wall disturbances. Leaves expressing an antisense WAK gene have reduced WAK protein levels and exhibit a loss of cell expansion. WAKs are covalently bound to pectin in the cell wall, providing evidence that the binding of a structural carbohydrate by a receptor-like kinase may have significance in the control of cell expansion. INTRODUCTIONIn animal, fungal, and algal systems, the physical connection and the communication between the extracellular matrix (ECM) and the cell plays a fundamental role in cell growth and division (Fowler and Quatrano, 1997;Lukashev and Werb, 1998;Tsai, 1998). Similarly, the plant cell wall forms an ECM of carbohydrate and protein that provides structure for individual cells and whole organs. The cell wall must be dynamic as cells divide and elongate, and modulation of its composition and architecture is required during its synthesis and after it has been deposited (Cosgrove, 1997;Reiter, 1998). The wall must therefore be considered in the context of modulating plant development (Kohorn, 2000). Communication between the cytoplasm and the cell wall is necessary and evident because events like cell expansion (Cosgrove, 1997) and pathogen infection (Hammond-Kosack and Jones, 1996) lead to altered biosynthesis and modification of cell wall components and downstream cytoplasmic events such as systemic acquired resistance. How the dynamics and synthesis of the cell wall are coordinated with cytoplasmic events is largely uncharacterized.Developing cells have walls that are composed of cellulose, hemicellulose, pectin, and proteins. Cellulose is directly secreted by cellulose synthase into the ECM, where it assembles with hemicelluloses and pectins, which are produced in the endomembrane system and secreted by vesicles. The cell wall also includes endoglucanases (Hayashi et al., 1984; Zuo et al., 2000), xyloglucan endotransglycosylases (Fry et al., 1992;Vissenberg et al., 2000), expansins (McQueen-Mason et al., 1992; Cho and Cosgrove, 2000), and a number of other glycosyl transferases that alter carbohydrate linkages and modify secreted cell wall components. Other cell wall proteins, some of which are heavily glycosylated, have been proposed as structural cell wall components or have been implicated in mediating multiple aspects of plant development (reviewed in: Showalter, 1993; Cosgrove, 1997;Kohorn, 2000). These include the families of proline-rich proteins, glycine-rich proteins, hydroxyprolinerich glycoproteins, and arabinogalactan proteins...

SummaryPathogen infection of angiosperms must rely on some interaction between the extracellular matrix (ECM) and the invading agent, and may be accompanied by signaling between the ECM and cytoplasm. An Arabidopsis cell wall associated receptor kinase (Wak1) has an amino-terminal domain that is tightly associated with the ECM, spans the plasma membrane and has a cytoplasmic protein kinase domain.