DWD HYPERSENSITIVE TO UV-B 1 is negatively involved in UV-B mediated cellular responses in Arabidopsis

Among T-DNA insertion mutants of various cullin4-RING ubiquitin E3 ligase (CRL4) substrate receptors, one mutant that exhibits enhanced sensitivity in response to ultraviolet-B (UV-B) illumination has been isolated and its corresponding gene has been named DWD HYPERSENSITIVE TO UV-B 1 (DHU1) in Arabidopsis. dhu1 lines showed much shorter hypocotyls than those in wild type under low doses of UV-B. Other light did not alter hypocotyl growth patterns in dhu1, indicating the hypersensitivity of dhu1 is restricted to UV-B. DHU1 was upregulated by more than two times in response to UV-B application of 1.5 μmol m^?2 s^?1, implying its possible involvement in UV-B signaling. DHU1 is able to bind to DDB1, an adaptor of CRL4; accordingly, DHU1 is thought to act as a substrate receptor of CRL4. Microarray data generated from wild-type and dhu1 under low doses of UV-B revealed that 209 or 124 genes were upregulated or downregulated by more than two times in dhu1 relative to wild type, respectively. About 23.4 % of the total upregulated genes in dhu1 were upregulated by more than five times in response to UV-B based on the AtGenExpress Visualization Tool data, while only about 1.4 % were downregulated to the same degree by UV-B, indicating that loss of DHU1 led to the overall enhancement of the upregulation of UV-B inducible genes. dhu1 also showed altered responsiveness under high doses of UV-B. Taken together, these findings indicate that DHU1 is a potent CRL4 substrate receptor that may function as a negative regulator of UV-B response in Arabidopsis.     

The plastid-specific ribosomal proteins of Arabidopsis thaliana can be divided into non-essential proteins and genuine ribosomal proteins

Plastid translation occurs on bacterial-type 70S ribosomes consisting of a large (50S) subunit and a small (30S) subunit. The vast majority of plastid ribosomal proteins have orthologs in bacteria. In addition, plastids also possess a small set of unique ribosomal proteins, so-called plastid-specific ribosomal proteins (PSRPs). The functions of these PSRPs are unknown, but, based on structural studies, it has been proposed that they may represent accessory proteins involved in translational regulation. Here we have investigated the functions of five PSRPs using reverse genetics in the model plant Arabidopsis thaliana. By analyzing T-DNA insertion mutants and RNAi lines, we show that three PSRPs display characteristics of genuine ribosomal proteins, in that down-regulation of their expression led to decreased accumulation of the 30S or 50S subunit of the plastid ribosomes, resulting in plastid translational deficiency. In contrast, two other PSRPs can be knocked out without visible or measurable phenotypic consequences. Our data suggest that PSRPs fall into two types: (i) PSRPs that have a structural role in the ribosome and are bona fide ribosomal proteins, and (ii) non-essential PSRPs that are not required for stable ribosome accumulation and translation under standard greenhouse conditions.     

UV-B signal transduction pathway in Arabidopsis

Plants experience a variety of environmental stresses such as cold, drought, freezing, flooding, wounding, heat and UV-B, all of which result in decreased productivity. Among abiotic stresses, UV-B stress is considered to be a critical factor affecting the rate of plant growth because the amount of UV-B reaching the Earth’s surface is constantly increasing. While high fluence rates of UV-B trigger stress-related processes, low fluence rates of UV-B induce photomorphogenesis, a crucial developmental process at the early seedling stage in plants. Among the signaling components involved in UV-B-mediated cellular response, a clade composed of UVR8-COP1-HY5 has been shown to be a central sequence that effectively transduces the pathway from the primary signal to adaptation response. This review summarizes the most recent progress in studies of UVR8-COP1-HY5 as the key players participating in the UV-B signal transduction pathway. The current understanding of additional UV-B signaling components including substrate receptors of multi-subunit E3 ubiquitin ligase is also discussed.     

Photoreceptor-Mediated Bending towards UV-B in Arabidopsis

Plants reorient their growth towards light to optimize photosynthetic light capture--a process known as phototropism. Phototropins are the photoreceptors essential for phototropic growth towards blue and ultraviolet-A （UV- A） light. Here we detail a phototropic response towards UV-B in etiolated Arabidopsis seedlings. We report that early differential growth is mediated by phototropins but clear phototropic bending to UV-B is maintained in photl phot2 double mutants. We further show that this phototropin-independent phototropic response to UV-B requires the UVoB photoreceptor UVR8. Broad UV-B-mediated repression of auxin-responsive genes suggests that UVR8 regulates directional bending by affecting auxin signaling. Kinetic analysis shows that UVR8-dependent directional bending occurs later than the phototropin response. We conclude that plants may use the full short-wavelength spectrum of sunlight to efficiently reorient photosynthetic tissue with incoming light.     

Crystal structures of mutant ribosomal proteins L1

Nine mutant ribosomal proteins L1 from the bacterium Thermus thermophilus and archaeon Methanococcus jannaschii were obtained and their crystal structures were determined and analyzed. The structure of the S179C TthL1 mutant, determined earlier, was also analyzed. In half of the proteins studied, point mutations of the amino acid residues exposed on the protein surface essentially changed the spatial structure of the protein. This proves that a correct study of biological processes with the help of site-directed mutagenesis requires a preliminary determination or, at least, modeling of the structures of mutant proteins. A detailed comparison of the structures of the L1 mutants and the corresponding wild-type L1 proteins demonstrated that the side chain of a mutated amino acid residue tends to adopt a location similar to that of the side chain of the corresponding residue in the wild-type protein. This observation assists in modeling the structure of mutant proteins.     

Crystal structures of mutant ribosomal proteins L1

Nine mutant forms of ribosomal proteins L1 from the bacterium Thermus thermophilus and the archaeon Methanococcus jannaschii were obtained. Their crystal structures were determined and analyzed. Earlier determined structure of S179C TthL1 was also thoroughly analyzed. Five from ten mutant proteins reveal essential changes of spatial structure caused by surface point mutation. It proves that for correct studies of biological processes by site-directed mutagenesis it is necessary to determine or at least to model spatial structures of mutant proteins. Detailed comparison of mutant L1 structures with that of corresponding wild type proteins reveals that side chain of a mutated amino acid residue tries to locate like the side chain of the original residue in the wild type protein. This observation helps to model the mutant structures.     

The N-terminal sequence of ribosomal protein L10 from the archaebacterium Halobacterium marismortui and its relationship to eubacterial protein L6 and other ribosomal proteins

The amino-terminal sequence of ribosomal protein L10 from Halobacterium marismortui has been determined up to residue 54, using both a liquid- and a gas-phase sequenator. The two sequences are in good agreement. The protein is clearly homologous to protein HcuL10 from the related strain Halobacterium cutirubrum. Furthermore, a weaker but distinct homology to ribosomal protein L6 from Escherichia coli and Bacillus stearothermophilus can be detected. In addition to 7 identical amino acids in the first 36 residues in all four sequences a number of conservative replacements occurs, of mainly hydrophobic amino acids. In this common region the pattern of conserved amino acids suggests the presence of a beta-alpha fold as it occurs in ribosomal proteins L12 and L30. Furthermore, several potential cases of homology to other ribosomal components of the three ur-kingdoms have been found.     

Versatile roles of Arabidopsis plastid ribosomal proteins in plant growth and development

A lack of individual plastid ribosomal proteins (PRPs) can have diverse phenotypic effects in Arabidopsis thaliana, ranging from embryo lethality to compromised vitality, with the latter being associated with photosynthetic lesions and decreases in the expression of plastid proteins. In this study, reverse genetics was employed to study the function of eight PRPs, five of which (PRPS1, ‐S20, ‐L27, ‐L28 and ‐L35) have not been functionally characterised before. In the case of PRPS17, only leaky alleles or RNA interference lines had been analysed previously. PRPL1 and PRPL4 have been described as essential for embryo development, but their mutant phenotypes are analysed in detail here. We found that PRPS20, ‐L1, ‐L4, ‐L27 and ‐L35 are required for basal ribosome activity, which becomes crucial at the globular stage and during the transition from the globular to the heart stage of embryogenesis. Thus, lack of any of these PRPs leads to alterations in cell division patterns, and embryo development ceases prior to the heart stage. PRPL28 is essential at the latest stages of embryo–seedling development, during the greening process. PRPS1, ‐S17 and ‐L24 appear not to be required for basal ribosome activity and the organism can complete its entire life cycle in their absence. Interestingly, despite the prokaryotic origin of plastids, the significance of individual PRPs for plant development cannot be predicted from the relative phenotypic severity of the corresponding mutants in prokaryotic systems.     

Involvement of 50S ribosomal proteins L6 and L10 in the ribosome dependent GTPase activity of elongation factor G

CsCI-prepared 50S cores in the presence of groups of individual split proteins were tested for their capacity to support EF-G dependent GTP hydrolysis. The activity of cores prepared at 40 mM Mg²⁺ could be restored by adding $\text{L7}\text{L12}$ and L10 together, each in an amount of two copies per 50S particle, which abolishes the difference in activity between L7 and L12. In the range of 20-2 mM Mg²⁺, 50S cores lose the protein L6, which is also required for GTP hydrolysis. L10 cannot replace L6, or $\text{vice versa}$.     

Crosslinking of ribosomal proteins to RNA in maize ribosomes by UV-B and its effects on translation

Ultraviolet-B (UV-B) photons can cause substantial cellular damage in biomolecules, as is well established for DNA. Because RNA has the same absorption spectrum for UV as DNA, we have investigated damage to this cellular constituent. In maize (Zea mays) leaves, UV-B radiation damages ribosomes by crosslinking cytosolic ribosomal proteins S14, L23a, and L32, and chloroplast ribosomal protein L29 to RNA. Ribosomal damage accumulated during a day of UV-B exposure correlated with a progressive decrease in new protein production; however, de novo synthesis of some ribosomal proteins is increased after 6 h of UV-B exposure. After 16 h without UV-B, damaged ribosomes were eliminated and translation was restored to normal levels. Ribosomal protein S6 and an S6 kinase are phosphorylated during UV-B exposure; these modifications are associated with selective translation of some ribosomal proteins after ribosome damage in mammalian fibroblast cells and may be an adaptation in maize. Neither photosynthesis nor pigment levels were affected significantly by UV-B, demonstrating that the treatment applied is not lethal and that maize leaf physiology readily recovers.     

Differential phosphorylation of ribosomal proteins in Arabidopsis thaliana plants during day and night

Protein synthesis in plants is characterized by increase in the translation rates for numerous proteins and central metabolic enzymes during the day phase of the photoperiod. The detailed molecular mechanisms of this diurnal regulation are unknown, while eukaryotic protein translation is mainly controlled at the level of ribosomal initiation complexes, which also involves multiple events of protein phosphorylation. We characterized the extent of protein phosphorylation in cytosolic ribosomes isolated from leaves of the model plant Arabidopsis thaliana harvested during day or night. Proteomic analyses of preparations corresponding to both phases of the photoperiod detected phosphorylation at eight serine residues in the C-termini of six ribosomal proteins: S2-3, S6-1, S6-2, P0-2, P1 and L29-1. This included previously unknown phosphorylation of the 40S ribosomal protein S6 at Ser-231. Relative quantification of the phosphorylated peptides using stable isotope labeling and mass spectrometry revealed a 2.2 times increase in the day/night phosphorylation ratio at this site. Phosphorylation of the S6-1 and S6-2 variants of the same protein at Ser-240 increased by the factors of 4.2 and 1.8, respectively. The 1.6 increase in phosphorylation during the day was also found at Ser-58 of the 60S ribosomal protein L29-1. It is suggested that differential phosphorylation of the ribosomal proteins S6-1, S6-2 and L29-1 may contribute to modulation of the diurnal protein synthesis in plants.     

Autogenous regulation of the synthesis of ribosomal proteins, L10 and L7/12, in Escherichia coli

Summary The in vitro synthesis of Escherichia coli ribosomal proteins, L10 and L7/12, is specifically repressed by the addition of the L10-L7/12 complex, while that of other ribosomal proteins encoded by the neighboring operons is not affected. Thus the expression of the rpoBC operon is controlled by two autorepression systems, one for the two ribosomal proteins and the other for RNA polymerase and subunits, both operating probably at the translational level.