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.     

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.     

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.     

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.     

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.     

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.     

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.     

An Arabidopsis cell cycle -dependent kinase-related gene, CDC2b, plays a role in regulating seedling growth in darkness.

The Arabidopsis CDC2b gene has been defined as a plant-specific cell cycle-dependent kinase-related gene, although it lacks the conserved cyclin binding motif, and its exact function is not known. Here, we report that in etiolated seedlings, the expression of the CDC2b gene is correlated with elongation rate of the hypocotyl. Inhibition of CDC2b gene expression by using an inducible antisense construct resulted in short-hypocotyl and open-cotyledon phenotypes when transgenic seedlings were grown in the dark. The severity of these phenotypes in dark-grown seedlings could be correlated with the level of the antisense gene expression. The short hypocotyl of seedlings underexpressing CDC2b was a result of inhibition of cell elongation rather than a reduction in cell number, whereas in cotyledons, inhibition of CDC2b expression resulted in large, open cotyledons with amyloplasts rather than etioplasts. Although the nuclear DNA was less compact in the antisense hypocotyl cells, DNA content and endoreduplication were not affected. Cell division of the shoot apical meristem also was not affected by antisense expression. The short-hypocotyl phenotype of these transgenic plants was partially rescued by the addition of brassinolide. Brassinolide can only induce CDC2b expression in darkness. These results suggest a role for the CDC2b gene in seedling growth via regulation of hypocotyl cell elongation and cotyledon cell development.     

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 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.     

Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes

Gibberellin (GA) 20-oxidase catalyses consecutive steps late in GA biosynthesis in plants. In Arabidopsis, the enzyme is encoded by a gene family of at least three members (AtGA20ox1, AtGA20ox2 and AtGA20ox3) with differential patterns of expression. The genes are regulated by feedback from bioactive GAs, suggesting that the enzymes may be involved in regulating GA biosynthesis. To investigate this, we produced transgenic Arabidopsis expressing sense or antisense copies of each of the GA 20-oxidase cDNAs. Over-expression of any of the cDNAs gave rise to seedlings with elongated hypocotyls; the plants flowered earlier than controls in both long and short days and were 25% taller at maturity. GA analysis of the vegetative rosettes showed a two- to threefold increase in the level of GA4, indicating that GA 20-oxidase normally limits bioactive GA levels. Plants expressing antisense copies of AtGA20ox1 had short hypocotyls and reduced rates of stem elongation. This was reflected in reduced levels of GA4 in both rosettes and shoot tips. In short days, flowering was delayed and the reduction in the rate of stem elongation was greater. Antisense expression of AtGA20ox2 had no apparent effects in long days, but stem growth in one transgenic line grown in short days was reduced by 20%. Expression of antisense copies of AtGA20ox3 had no visible effect, except for one transgenic line that had short hypocotyls. These results demonstrate that GA levels and, hence, plant growth and development can be modified by manipulation of GA 20-oxidase expression in transgenic plants.     

Involvement of an ABC transporter in a developmental pathway regulating hypocotyl cell elongation in the light

In the dark, plant seedlings follow the skotomorphogenetic developmental program, which results in hypocotyl cell elongation. When the seedlings are exposed to light, a switch to photomorphogenetic development occurs, and hypocotyl cell elongation is inhibited. We have manipulated the expression of the AtPGP1 (for Arabidopsis thaliana P glycoprotein1) gene in transgenic Arabidopsis plants by using sense and antisense constructs. We show that within a certain light fluence rate window, overexpression of the AtPGP1 gene under the control of the cauliflower mosaic virus 35S promoter causes plants to develop longer hypocotyls, whereas expression of the gene in antisense orientation results in hypocotyls shorter than those occurring in the wild type. In the dark, hypocotyls of transgenic and wild-type plants are indistinguishable. Because the AtPGP1 gene encodes a member of the superfamily of ATP binding cassette-containing (ABC) transporters, these results imply that a transport process is involved in a hypocotyl cell elongation pathway active in the light. The AtPGP1 transporter is localized in the plasmalemma, as indicated by immunohistochemical techniques and biochemical membrane separation methods. Analysis of the AtPGP1 expression pattern by using reporter gene constructs and in situ hybridization shows that in wild-type seedlings, AtPGP1 is expressed in both the root and shoot apices.