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The wall-associated kinase (WAK) gene family, one of the receptor-like kinase (RLK) gene families in plants, plays important roles in cell expansion, pathogen resistance, and heavy-metal stress tolerance in Arabidopsis (Arabidopsis thaliana). Through a reiterative database search and manual reannotation, we identified 125 OsWAK gene family members from rice (Oryza sativa) japonica cv Nipponbare; 37 (approximately 30%) OsWAKs were corrected/reannotated from earlier automated annotations. Of the 125 OsWAKs, 67 are receptor-like kinases, 28 receptor-like cytoplasmic kinases, 13 receptor-like proteins, 12 short genes, and five pseudogenes. The two-intron gene structure of the Arabidopsis WAK/WAK-Likes is generally conserved in OsWAKs; however, extra/missed introns were observed in some OsWAKs either in extracellular regions or in protein kinase domains. In addition to the 38 OsWAKs with full-length cDNA sequences and the 11 with rice expressed sequence tag sequences, gene expression analyses, using tiling-microarray analysis of the 20 OsWAKs on chromosome 10 and reverse transcription-PCR analysis for five OsWAKs, indicate that the majority of identified OsWAKs are likely expressed in rice. Phylogenetic analyses of OsWAKs, Arabidopsis WAK/WAK-Likes, and barley (Hordeum vulgare) HvWAKs show that the OsWAK gene family expanded in the rice genome due to lineage-specific expansion of the family in monocots. Localized gene duplications appear to be the primary genetic event in OsWAK gene family expansion and the 125 OsWAKs, present on all 12 chromosomes, are mostly clustered.

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 β-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.

Here, we report the aluminum (Al)-induced organ-specific expression of a WAK1 (cell wall-associated receptor kinase 1) gene and cell type-specific localization of WAK proteins in Arabidopsis. WAK1-specific reverse transcriptase-polymerase chain reaction analysis revealed an Al-induced WAK1 gene expression in roots. Short- and long-term analysis of gene expression in root fractions showed a typical “on” and “off” pattern with a first peak at 3 h of Al exposure followed by a sharp decline at 6 h and a complete disappearance after 9 h of Al exposure, suggesting the WAK1 is a further representative of Al-induced early genes. In shoots, upon root Al exposure, an increased but stable WAK1 expression was observed. Using confocal microscopy, we visualized Al-induced closure of leaf stomata, consistent with previous suggestions that the Al stress primarily experienced in roots associated with the transfer of root-shoot signals. Elevated levels of WAK protein in root cells were observed through western blots after 6 h of Al exposure, indicating a lag time between the Al-induced WAK transcription and translation. WAK proteins are localized abundantly to peripheries of cortex cells within the elongation zone of the root apex. In these root cells, disintegration of cortical microtubules was observed after Al treatment but not after the Al analog lanthanum treatments. Tip-growing control root hairs, stem stomata, and leaf stomatal pores are characterized with high amounts of WAKs, suggesting WAKs are accumulating at plasma membrane domains, which suffer from mechanical stress and lack dense arrays of supporting cortical microtubules. Further, transgenic plants overexpressing WAK1 showed an enhanced Al tolerance in terms of root growth when compared with the wild-type plants, making the WAK1 one of the important candidates for plant defense against Al toxicity.

The cell wall-associated receptor kinase (WAK) and WAK-like kinase (WAKL) gene family members are good candidates for physical linkers that signal between the cell wall and the cytoplasmic compartment. Previous studies have suggested that while some WAK/WAKL members play a role in bacterial pathogen and heavy-metal aluminum responses, others are involved in cell elongation and plant development. Here, we report a functional role for the WAKL4 gene in Arabidopsis (Arabidopsis thaliana) mineral responses. Confocal microscopic studies localized WAKL4-green fluorescent protein fusion proteins on the cell surfaces suggesting that, like other WAK/WAKL proteins, WAKL4 protein is associated with the cell wall. Histochemical analyses of the WAKL4 promoter fused with the β-glucuronidase reporter gene have shown that WAKL4 expression is induced by Na+, K+, Cu2+, Ni2+, and Zn2+. A transgenic line with a T-DNA insertion at 40-bp upstream of the WAKL4 start codon was characterized. While the T-DNA insertion had little effect on the WAKL4 transcript levels under normal growth conditions, it significantly altered the expression patterns of WAKL4 under various conditions of mineral nutrients. Semiquantitative and quantitative reverse transcription-PCR analyses showed that the promoter impairment abolished WAKL4-induced expression by Na+, K+, Cu2+, and Zn2+, but not by Ni2+. Whereas the WAKL4 promoter impairment resulted in hypersensitivity to K+, Na+, Cu2+, and Zn2+, it conferred a better tolerance to toxic levels of the Ni2+ heavy metal. WAKL4 was required for the up-regulation of zinc transporter genes during zinc deficiency, and the WAKL4 T-DNA insertion resulted in a reduction of Zn2+ accumulation in shoots. A WAKL4-green fluorescent protein fusion gene driven by either the WAKL4 native promoter or the 35S constitutive promoter complemented the phenotypes. Our results suggest versatile roles for WAKL4 in Arabidopsis mineral nutrition responses.

In the simplest sense, cell walls are a carbohydrate and protein structures that surround and separate cells. Perhaps the most familiar cell wall structures are cork and wood, where the cell wall remains after the cells within have degraded. Cotton, the fiber that keeps some of us clothed in our