RUS6, a DUF647-containing protein, is essential for early embryonic development in Arabidopsis thaliana.

BACKGROUND: The Arabidopsis RUS (ROOT UV-B SENSITIVE) gene family contains six members, each of which encodes a protein containing a DUF647 (domain of unknown function 647) that is commonly found in eukaryotes. Previous studies have demonstrated that RUS1 and RUS2 play critical roles in early seedling development. All six RUS genes are expressed throughout the plant, but little is known about the functional roles of RUS3, RUS4, RUS5 and RUS6. RESULTS: We used a reverse-genetic approach to identify knockout mutants for RUS3, RUS4, RUS5 and RUS6. Each mutant was confirmed by direct DNA sequencing and genetic segregation analysis. No visible phenotypic differences were observed in rus3, rus4, or rus5 knockout mutants under standard growth conditions, but rus6 knockout mutants displayed a strong embryo-lethal phenotype. Two independent knockout lines for RUS6 were characterized. The rus6 mutations could only be maintained through a heterozygote, because rus6 homozygous mutants did not survive. Closer examinations of homozygous rus6 embryos from rus6/ + parent plants revealed that RUS6 is required for early embryo development. Loss of RUS6 resulted in embryo lethality, specifically at the mid-globular stage. The embryo-lethality phenotype was complemented by a RUS6::RUS6-GFP transgene, and GFP signal was detected throughout the embryo. Histological analyses with the ß-glucuronidase reporter gene driven by the RUS6 promoter showed tissue- and development-specific expression of RUS6, which was highest in floral tissues. CONCLUSION: Our data revealed that RUS6 is essential for early embryo development in Arabidopsis, and that the RUS gene family functions in multiple stages of plant development.

Alternative Seamless Cloning Strategies in Fusing Gene Fragments Based on Overlap-PCR.

Gene fragment swapping and site-directed mutagenesis are commonly required in dissecting functions of gene domains. While there are many approaches for seamless fusion of different gene fragments, new methods are yet to be developed to offer higher efficiency, better simplicity, and more affordability. In this study, we showed that in most cases overlap-PCR was highly effective in creating site-directed mutagenesis, gene fragment deletion, and substitutions using RUS1 and RUS2 as example. While for cases where the overlap-PCR approach is not feasible due to complex secondary structure of gene fragments, a unique restriction site can be generated at the overlapped region of the primers through synonymous mutations. Then different gene fragments can be seamlessly fused through traditional restriction digestion and subsequent ligation. In conclusion, while the classical overlap-PCR is not feasible, the modified overlap-PCR approaches can provide effective and alternative ways to seamlessly fuse different gene fragments.

root uv-b sensitive mutants are suppressed by specific mutations in ASPARTATE AMINOTRANSFERASE2 and by exogenous vitamin B6.

Vitamin B6 (vitB6) serves as an essential cofactor for more than 140 enzymes. Pyridoxal 5'-phosphate (PLP), active cofactor form of vitB6, can be photolytically destroyed by trace amounts of ultraviolet-B (UV-B). How sun-exposed organisms cope with PLP photosensitivity and modulate vitB6 homeostasis is currently unknown. We previously reported on two Arabidopsis mutants, rus1 and rus2, that are hypersensitive to trace amounts of UV-B light. We performed mutagenesis screens for second-site suppressors of the rus mutant phenotype and identified mutations in the ASPARTATE AMINOTRANSFERASE2 (ASP2) gene. ASP2 encodes for cytosolic aspartate aminotransferase (AAT), a PLP-dependent enzyme that plays a key role in carbon and nitrogen metabolism. Genetic analyses have shown that specific amino acid substitutions in ASP2 override the phenotypes of rus1 and rus2 single mutants as well as rus1 rus2 double mutant. These substitutions, all shown to reside at specific positions in the PLP-binding pocket, resulted in no PLP binding. Additional asp2 mutants that abolish AAT enzymatic activity, but which alter amino acids outside of the PLP-binding pocket, fail to suppress the rus phenotype. Furthermore, exogenously adding vitB6 in growth media can rescue both rus1 and rus2. Our data suggest that AAT plays a role in vitB6 homeostasis in Arabidopsis.

ROOT UV-B SENSITIVE2 acts with ROOT UV-B SENSITIVE1 in a root ultraviolet B-sensing pathway.

Ultraviolet B light (UV-B; 280-320 nm) perception and signaling are well-known phenomena in plants, although no specific UV-B photoreceptors have yet been identified. We previously reported on the root UV-B sensitive1 (rus1) mutants in Arabidopsis (Arabidopsis thaliana), which display a block to development under very-low-fluence-rate UV-B (<0.1 mumol m(-2) s(-1)) after the seedling emerges from the seed. Here, we report the analysis and cloning of the rus2-1 mutation in Arabidopsis. The phenotype of rus2-1 mutant seedlings is virtually indistinguishable from the phenotype of rus1 seedlings. A map-based approach was used to clone RUS2. RUS2 encodes a domain of unknown function (DUF647)-containing protein that is homologous to the RUS1 protein. rus1-2 rus2-1 double mutant seedlings have the same phenotype as both rus1 and rus2 single mutants, suggesting that the two genes work in the same pathway. RUS2-Green Fluorescent Protein shows a similar expression pattern as that of RUS1-Green Fluorescent Protein, and RUS1 and RUS2 proteins interact physically in yeast. This protein-protein interaction depends on the DUF647 domain, and site-directed mutagenesis identified specific residues in DUF647 that are required for both protein-protein interaction and physiological function. Six RUS genes are found in Arabidopsis, rice (Oryza sativa), and moss (Physcomitrella patens), and one RUS member, RUS3, is conserved in plants and animals. Our results demonstrate that RUS2 works with RUS1 in a root UV-B-sensing pathway that plays a vital role in Arabidopsis early seedling morphogenesis and development.

Role of root UV-B sensing in Arabidopsis early seedling development.

All sun-exposed organisms are affected by UV-B [(UVB) 280-320 nm], an integral part of sunlight. UVB can cause stresses or act as a developmental signal depending on its fluence levels. In plants, the mechanism by which high-fluence-rate UVB causes damages and activates DNA-repair systems has been extensively studied. However, little is known about how nondamaging low-fluence-rate UVB is perceived to regulate plant morphogenesis and development. Here, we report the identification of an Arabidopsis mutant, root UVB sensitive 1 (rus1), whose primary root is hypersensitive to very low-fluence-rate (VLF) UVB. Under standard growth-chamber fluorescent white light, rus1 displays stunted root growth and fails to form postembryonic leaves. Experiments with different monochromatic light sources showed that rus1 phenotypes can be rescued if the seedlings are allowed to grow in light conditions with minimum UVB. We determined that roots, not other organs, perceive the UVB signal. Genetic and molecular analyses confirmed that the root light-sensitive phenotypes are independent of all other known plant photoreceptors. Three rus1 alleles have been identified and characterized. A map-based approach was used to identify the RUS1 locus. RUS1 encodes a protein that contains DUF647 (domain of unknown function 647), a domain highly conserved in eukaryotes. Our results demonstrate a root VLF UVB-sensing mechanism that is involved in Arabidopsis early seedling morphogenesis and development.

Involvement of a cell wall-associated kinase, WAKL4, in Arabidopsis mineral responses.

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 -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.

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.

Aluminum-induced gene expression and protein localization of a cell wall-associated receptor kinase in Arabidopsis.

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.