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.

WAKs; cell wall associated kinases.

Students of metazoan biology have traditionally viewed the extracellular matrix (ECM) as a substrate with which cells interact to participate in developmental pattern formation and define a specific location. In contrast, the plant cell wall has been viewed as a cage that limits and thus directs plant cell morphology, and perhaps for this reason many have shied away from calling the plant cell wall the ECM. The recent discovery of a variety of receptor molecules and their ligands on the surface of plant cells and the intimate role cell walls play in development should direct our thinking toward a more dynamic view of the plant cell wall. A recent example, is the discovery of wall associated kinases (WAKs), which may well signal between the ECM and the cell and are required for cell expansion.

Disruption of thylakoid-associated kinase 1 leads to alteration of light harvesting in Arabidopsis.

To survive fluctuations in quality and intensity of light, plants and algae are able to preferentially direct the absorption of light energy to either one of the two photosystems, PSI or PSII. This rapid process is referred to as a state transition and has been correlated with the phosphorylation and migration of the light-harvesting complex protein (LHCP) between PSII and PSI. We show here that thylakoid protein kinases (TAKs) are required for state transitions in Arabidopsis. Antisense TAK1 expression leads to a loss of LHCP phosphorylation and a reduction in state transitions. Preferential activation of PSII causes LHCP to accumulate with PSI, and TAK1 mutants disrupt this process. Finally, TAKs also influence the phosphorylation of multiple thylakoid proteins.

Wall-associated kinases are expressed throughout plant development and are required for cell expansion.

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 WAKs 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.

Shuffling the deck: plant signalling plays a club.

The transmission of signals across the plasma membrane of cells plays an integral part in cell communication in unicellular and complex organisms. Protein kinases and their activators serve key roles in this process, and a number of paradigms have been established to describe their mode of action. Signalling in plant cells appears to shuffle these paradigms - as evidenced by two recent reports on the development of the Arabidopsis meristem.

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.

TAKs, thylakoid membrane protein kinases associated with energy transduction.

The phosphorylation of proteins within the eukaryotic photosynthetic membrane is thought to regulate a number of photosynthetic processes in land plants and algae. Both light quality and intensity influence protein kinase activity via the levels of reductants produced by the thylakoid electron transport chain. We have isolated a family of proteins called TAKs, Arabidopsis thylakoid membrane threonine kinases that phosphorylate the light harvesting complex proteins. TAK activity is enhanced by reductant and is associated with the photosynthetic reaction center II and the cytochrome b6f complex. TAKs are encoded by a gene family that has striking similarity to transforming growth factor beta receptors of metazoans. Thus thylakoid protein phosphorylation may be regulated by a cascade of reductant-controlled membrane-bound protein kinases.

Requirement for the induced expression of a cell wall associated receptor kinase for survival during the pathogen response.

Pathogen 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. Wak1 expression is induced when Arabidopsis plants are infected with pathogen, or when the pathogen response is stimulated either by exogenous salicylate (SA) or its analog 2,2-dichloroisonicotinic acid (INA). This Wak1 induction requires the positive regulator NPR1/NIM1. Thus Wak1 is a pathogen-related (PR) protein. Expression of an antisense and a dominant negative allele of Wak1 shows that induced expression of Wak1 is needed for a plant to survive if stimulated by INA. Ectopic expression of the entire Wak1, or the kinase domain alone, can provide resistance to otherwise lethal SA levels. These experiments suggest that Wak1 expression and other PR proteins are protecting plants from detrimental effects incurred during the pathogen response. These results provide a direct link between a protein kinase that could mediate signals from the ECM, to the events that are precipitated by a pathogen infection.

Tip loci: six Chlamydomonas nuclear suppressors that permit the translocation of proteins with mutant thylakoid signal sequences.

Mutations within the signal sequence of cytochrome f (cytf) in Chlamydomonas inhibit thylakoid membrane protein translocation and render cells nonphotosynthetic. Twenty-seven suppressors of the mutant signal sequences were selected for their ability to restore photoautotrophic growth and these describe six nuclear loci named tip1 through 6 for thylakoid insertion protein. The tip mutations restore the translocation of cytf and are not allele specific, as they suppress a number of different cytf signal sequence mutations. Tip5 and 2 may act early in cytf translocation, while Tip1, 3, 4, and 6 are engaged later. The tip mutations have no phenotype in the absence of a signal sequence mutation and there is genetic interaction between tip4, and tip5 suggesting an interaction of their encoded proteins. As there is overlap in the energetic, biochemical and genetic requirements for the translocation of nuclear and chloroplast-encoded thylakoid proteins, the tip mutations likely identify components of a general thylakoid protein translocation apparatus.

Hydrophobic core but not amino-terminal charged residues are required for translocation of an integral thylakoid membrane protein in vivo.

The integral membrane protein cytochrome f contains an amino-terminal signal sequence that is required for translocation into the thylakoid membrane. The signal sequence contains a hydrophobic core neighbored by an amino-terminal charged residue. Mutations that introduce charged amino acids into the hydrophobic core are inhibitory to cytochrome f translocation, and thus render cells non-photosynthetic. We have isolated both nuclear and chloroplast suppressors of these mutations by selecting for restoration of photosynthetic growth of Chlamydomonas. Here we describe the characterization of two chloroplast, second site suppressor mutations. Both suppressors remove the positively charged amino acid that borders the amino terminus of the hydrophobic core, and replace this arginine with either a cysteine or a leucine. The existence of these suppressors suggests that the hydrophobic core can be shifted in position within the signal sequence, and analysis of triple mutants in the signal confirms this hypothesis. Thus this signal that mediates translocation into the thylakoid membrane is characterized by a hydrophobic region whose exact amino acid content is not critical, and that need not be flanked on its amino terminus by a charged residue.

A cell wall-associated, receptor-like protein kinase.

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.

Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors.

The apparatus that permits protein translocation across the internal thylakoid membranes of chloroplasts is completely unknown, even though these membranes have been the subject of extensive biochemical analysis. We have used a genetic approach to characterize the translocation of Chlamydomonas cytochrome f, a chloroplast-encoded protein that spans the thylakoid once. Mutations in the hydrophobic core of the cytochrome f signal sequence inhibit the accumulation of cytochrome f, lead to an accumulation of precursor, and impair the ability of Chlamydomonas cells to grow photosynthetically. One hydrophobic core mutant also reduces the accumulation of other thylakoid membrane proteins, but not those that translocate completely across the membrane. These results suggest that the signal sequence of cytochrome f is required and is involved in one of multiple insertion pathways. Suppressors of two signal peptide mutations describe at least two nuclear genes whose products likely describe the translocation apparatus, and selected second-site chloroplast suppressors further define regions of the cytochrome f signal peptide.

An Arabidopsis serine/threonine kinase homologue with an epidermal growth factor repeat selected in yeast for its specificity for a thylakoid membrane protein.

A number of molecules have recently been described that effect the correct transport and assembly of cytoplasmically synthesized proteins to cellular membranes. To identify proteins that bind or modify other proteins during the process of membrane translocation, we developed a yeast selection scheme that employs the yeast transcriptional activator GAL4. This selection facilitates the isolation of cDNAs that encode proteases and binding proteins for known target peptide sequences. We report the isolation of an Arabidopsis cDNA encoding a polypeptide that can interact with the amino terminus of a ligh-harvesting chlorophyll a/b-binding protein (LHCP), a cytoplasmically synthesized protein that is integral to the chloroplast thylakoid membrane. The cDNA was selected in yeast from an Arabidopsis expression library for its ability to inhibit a transcriptional activator GAL4-LHCP fusion protein, but not inhibit native GAL4 protein. The LHCP amino-terminal sequences included in the fusion protein are known to regulate LHCP biogenesis and function. The Arabidopsis cDNA encodes a 595-amino acid protein with at least two functional domains, one with similarity to the family of protein-serine/threonine kinases and another that contains an epidermal growth factor repeat. The identification of an EGF repeat in Arabidopsis indicates that the motif is conserved between the plant and animal kingdoms. Hybridization studies indicate that this gene is likely to be present in other genera of plants. Its mRNA is detected in green leaves but not in other plant tissues or in etiolated plants. The specificity in yeast and the expression pattern in plants together are suggestive of a role for this protein kinase in the assembly or regulation of LHCP.

Requirement for three membrane-spanning alpha-helices in the post-translational insertion of a thylakoid membrane protein.

The insertion of a protein into a lipid bilayer usually involves a short signal sequence and can occur either during or after translation. A light-harvesting chlorophyll a/b-binding protein (LHCP) is synthesized in the cytoplasm of plant cells as a precursor and is post-translationally imported into chloroplasts where it subsequently inserts into the thylakoid membrane. Only mature LHCP is required for insertion into the thylakoid. To define which sequences of the mature protein are necessary and sufficient for thylakoid integration, fusion and deletion proteins and proteins with internal rearrangements were synthesized and incubated with isolated thylakoids and stroma. No evidence is found for the existence of a short signal sequence within LHCP, and, with the exception of the amino terminus and a short lumenal loop, the entire mature protein with consecutively ordered alpha-helices is required for insertion into thylakoid membranes. The addition of positive charges into stromal but not lumenal segments permits the insertion of mutant LHCPs into isolated thylakoids. Replacement of the LHCP transit peptide with the transit peptide from plastocyanin has no effect on LHCP insertion and does not restore insertion of the lumenal charge addition mutants.

Integration of a chlorophyll-binding protein into Escherichia coli membranes in the absence of chlorophyll.

The mechanism by which a protein integrates posttranslationally into a membrane can involve the composition of the membrane itself, domains within the inserting polypeptide, and a number of associating proteins. Some integral membrane proteins do not accumulate to normal levels when certain pigments are deficient, and this has been interpreted to mean that such proteins may be rapidly degraded when not in a correct complex. Alternatively, pigments could facilitate the movement of some proteins from an aqueous to a lipid environment. To determine whether chlorophyll is absolutely required for the membrane integration of the light-harvesting chlorophyll-binding protein (LHCP) of chloroplast thylakoid membranes, we have expressed LHCP in Escherichia coli that lacks photosynthetic pigments. LHCP is targeted to the bacterial inner membrane by the addition of a bacterial signal peptide and cannot be extracted from these membranes by NaOH, NaBr, or Na2HCO3 but is extracted by 0.2% Triton X-100. Treatment of isolated right-side-out and inside-out bacterial inner membrane vesicles with trypsin reveals that only the amino terminus of LHCP is exposed on the cytoplasmic face, and the remaining portion of the protein is inaccessible. Treatment of the inside-out vesicles with trypsin followed by alkaline extraction shows that LHCP is intrinsic to the membrane and is not anchored solely by the bacterial signal peptide. Chlorophyll, therefore, is not required for LHCP to integrate into a membrane, but in the absence of these pigments this process is observed to be inefficient.

Direct selection for sequences encoding proteases of known specificity.

We have developed a simple genetic selection that could be used to isolate eukaryotic cDNAs encoding proteases that cleave within a defined amino acid sequence. The selection was developed by using the transcription factor GAL4 from Saccharomyces cerevisiae as a selectable marker, a cloned protease from tobacco etch virus (TEV), and an 18-amino acid TEV protease target sequence. In yeast, TEV protease cleaves its target even when the target is fused to internal regions of the GAL4 protein. This cleavage separates the DNA binding domain from the transcription activation domain of GAL4, rendering it transcriptionally inactive. The proteolytic cleavage can be detected phenotypically by the inability of cells to metabolize galactose. Cells expressing the TEV protease can also be selected on the suicide substrate 2-deoxygalactose. DNA binding studies show that the TEV protease decreases the activity of the GAL4/target fusion protein. Because another protease target sequence of 55 amino acids can be inserted into GAL4 without any loss of transcriptional activity, this assay offers the opportunity to use high-efficiency cDNA cloning and expression vectors to select coding sequences of other proteases from various species. The assay could also be used to help define both target specificities and functional domains of proteases.

Chloroplasts of Arabidopsis thaliana homozygous for the ch-1 locus lack chlorophyll b, lack stable LHCPII and have stacked thylakoids.

We are interested in the mechanism of insertion of proteins into the chloroplast thylakoid membrane and the role that accessory pigments may play in this process. For this reason we have begun a molecular analysis of mutant plants deficient in pigments that associate with thylakoid membrane proteins. We have characterized plants that are homozygous for the previously isolated, recessive mutation chlorina-1 (ch-1) of Arabidopsis thaliana. Despite the lack of chlorophyll b and light-harvesting proteins of photosystem II (LHCPII) near normal levels of LHCPII mRNA are found in the mutant, in contrast to LHCPII mRNA levels in carotenoid-deficient mutants. The LHCPII mRNA of chlorina-1 plants can be translated in vitro so it is likely that LHCPII is not stable in ch-1 plants. Moreover, the thylakoid membranes of ch-1 plants remain appressed even though LHCPII levels are drastically reduced.

Replacement of Histidines of Light Harvesting Chlorophyll a/b Binding Protein II Disrupts Chlorophyll-Protein Complex Assembly.

Eukaryotic light harvesting proteins (LHCPs) bind pigments and assemble into complexes (LHCs) that channel light energy into photosynthetic reaction centers. The structures of several prokaryotic LHCPs are known and histidines are important for the binding of the associated pigments. It has been difficult to predict how the eukaryotic LHCPs associate with pigments as the structure of the major LHCP of photosystem II is not yet known. While each LHCPII binds approximately 13 chlorophylls the protein contains only three histidines, one in each putative transmembrane helix. Experiments that use isolated pea (Pisum sativum L.) chloroplasts and mutant LHCPII synthesized in vitro show that the substitution of either an alanine or an arginine for each histidine residue inhibits some aspect of LHCII assembly. The histidine of the first membrane helix, but not the second or third, may be involved in the transport across the chloroplast envelope. No histidine alone is essential for the insertion of LHCP into thylakoid membranes, yet arginine substitutions are more inhibitory than those of alanine. The histidine replacements have their most pronounced effect on the assembly of LHCP into LHCII.

Movement of newly imported light-harvesting chlorophyll-binding protein from unstacked to stacked thylakoid membranes is not affected by light treatment or absence of amino-terminal threonines.

In higher plants and algae, the transduction of captured light energy is highly regulated as excess excitation of photosystem II (PSII) reaction centers can be redirected to photosystem I (PSI) reaction centers. Models that attempt to explain this phenomenon involve light-harvesting chlorophyll-protein complexes (LHCII) that capture light energy and migrate between PSII and PSI. This report shows that in pea chloroplasts, the major protein component of LHCII, light-harvesting chlorophyll-binding protein (LHCP), can indeed migrate within the thylakoid membrane. We show, however, that although newly imported LHCP inserts into both stacked and unstacked thylakoid membranes, it then moves only from the unstacked, PSI-rich membranes to the stacked, PSII-rich membranes. The observed migration is not affected by light treatment that induces a redistribution of captured light energy (state I-state II transition) that previously was thought to induce LHCP to migrate in the opposite direction, from stacked to unstacked membranes. A mutation that removes the site of LHCP phosphorylation, the proposed trigger of state transitions, also has no effect on the integration and movement of LHCP, but does render LHCP more susceptible to proteolytic degradation. These results are not consistent with current models that deal with the short-term change in the distribution of light energy.