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.

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.

The essential gene EMB1611 maintains shoot apical meristem function during Arabidopsis development.

The Arabidopsis thaliana genome contains hundreds of genes essential for seed development. Because null mutations in these genes cause embryo lethality, their specific molecular and developmental functions are largely unknown. Here, we identify a role for EMB1611/MEE22, an essential gene in Arabidopsis, in shoot apical meristem maintenance. EMB1611 encodes a large, novel protein with N-terminal coiled-coil regions and two putative transmembrane domains. We show that the partial loss-of-function emb1611-2 mutation causes a range of pleiotropic developmental phenotypes, most dramatically a progressive loss of shoot apical meristem function that causes premature meristem termination. emb1611-2 plants display disorganization of the shoot meristem cell layers early in development, and an associated stem cell fate change to an organogenic identity. Genetic and molecular analysis indicates that EMB1611 is required for maintenance of the CLV-WUS stem cell regulatory pathway in the shoot meristem, but also has WUS-independent activity. In addition, emb1611-2 plants have reduced shoot and root growth, and their rosette leaves form trichomes with extra branches, a defect we associate with an increase in endoreduplication. Our data indicate that EMB1611 functions to maintain cells, particularly those in the shoot meristem, roots and developing rosette leaves, in a proliferative or uncommitted state.

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.