UVR8-TCP4-LOX2 module regulates UV-B tolerance in Arabidopsis.

The phytohormone jasmonate (JA) coordinates stress and growth responses to increase plant survival in unfavorable environments. Although JA can enhance plant UV-B stress tolerance, the mechanisms underlying the interaction of UV-B and JA in this response remain unknown. In this study, we demonstrate that the UV RESISTANCE LOCUS 8 - TEOSINTE BRANCHED1, Cycloidea and PCF 4 - LIPOXYGENASE2 (UVR8-TCP4-LOX2) module regulates UV-B tolerance dependent on JA signaling pathway in Arabidopsis thaliana. We show that the nucleus-localized UVR8 physically interacts with TCP4 to increase the DNA-binding activity of TCP4 and upregulate the JA biosynthesis gene LOX2. Furthermore, UVR8 activates the expression of LOX2 in a TCP4-dependent manner. Our genetic analysis also provides evidence that TCP4 acts downstream of UVR8 and upstream of LOX2 to mediate plant responses to UV-B stress. Our results illustrate that the UV-B-dependent interaction of UVR8 and TCP4 serves as an important UVR8-TCP4-LOX2 module, which integrates UV-B radiation and JA signaling and represents a new UVR8 signaling mechanism in plants.

Ultrafast structural changes direct the first molecular events of vision.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

CeLINC, a fluorescence-based protein-protein interaction assay in Caenorhabditis elegans.

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein's function. We present Caenorhabditis elegans light-induced coclustering (CeLINC), an optical binary protein-protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein-protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Colocalization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.

Regulation of plant phototropic growth by NPH3/RPT2-like substrate phosphorylation and 14-3-3 binding.

Polarity underlies all directional growth responses in plants including growth towards the light (phototropism). The plasma-membrane associated protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by phototropin (phot) AGC kinases. Here we demonstrate that NPH3 is directly phosphorylated by phot1 within a conserved C-terminal consensus sequence (RxS) that is necessary to promote phototropism and petiole positioning in Arabidopsis. RxS phosphorylation also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Mutants of NPH3 that are unable to bind or constitutively bind 14-3-3 s show compromised functionality consistent with a model where phototropic curvature is established by signalling outputs arising from a gradient of NPH3 RxS phosphorylation across the stem. Our findings therefore establish that NPH3/RPT2-Like (NRL) proteins are phosphorylation targets for plant AGC kinases. Moreover, RxS phosphorylation is conserved in other members of the NRL family, suggesting a common mechanism of regulating plant growth to the prevailing light environment.

Direct photoresponsive inhibition of a p53-like transcription activation domain in PIF3 by Arabidopsis phytochrome B.

Photoactivated phytochrome B (PHYB) binds to antagonistically acting PHYTOCHROME-INTERACTING transcription FACTORs (PIFs) to regulate hundreds of light responsive genes in Arabidopsis by promoting PIF degradation. However, whether PHYB directly controls the transactivation activity of PIFs remains ambiguous. Here we show that the prototypic PIF, PIF3, possesses a p53-like transcription activation domain (AD) consisting of a hydrophobic activator motif flanked by acidic residues. A PIF3mAD mutant, in which the activator motif is replaced with alanines, fails to activate PIF3 target genes in Arabidopsis, validating the functions of the PIF3 AD in vivo. Intriguingly, the N-terminal photosensory module of PHYB binds immediately adjacent to the PIF3 AD to repress PIF3's transactivation activity, demonstrating a novel PHYB signaling mechanism through direct interference of the transactivation activity of PIF3. Our findings indicate that PHYB, likely also PHYA, controls the stability and activity of PIFs via structurally separable dual signaling mechanisms.

HY5 and ABI5 transcription factors physically interact to fine tune light and ABA signaling in Arabidopsis.

KEY MESSAGE: Cross-talk between light and ABA signaling is mediated by physical interaction between HY5 and ABI5 Arabidopsis. Plants undergo numerous transitions during their life-cycle and have developed a very complex network of signaling to integrate information from their surroundings to effectively survive in the ever-changing environment. Light signaling is one of the crucial factors that govern the plant growth and development from the very first step of that is from seedling germination to the flowering. Similarly, Abscisic acid (ABA) signaling transduces the signals from external unfavorable condition to the internal developmental pathways and is crucial for regulation of seed maturation, dormancy germination and early seedling development. These two fundamental factors coordinately regulate plant wellbeing, but the underlying molecular mechanisms that drive this regulation are poorly understood. Here, we identified that two bZIP transcription factors, ELONGATED HYPOCOTYLE 5 (HY5), a positive regulator of light signaling and ABA-INSENSITIVE 5 (ABI5), a positive regulator of ABA signaling interacts and integrates the two pathways together. Our phenotypic data suggest that ABI5 may act as a negative regulator during photomorphogenesis in contrast, HY5 acts as a positive regulator of ABA signaling in an ABA dependent manner. We further showed that over-expression of HY5 leads to ABA-hypersensitive phenotype and late flowering phenotype. Taken together, our data provides key insights regarding the mechanism of interaction between ABI5-HY5 that fine tunes the stress and developmental response in Arabidopsis.

Sequence of Events during Peptide Unbinding from RNase S: A Complete Experimental Description.

The phototriggered unbinding of the intrinsically disordered S-peptide from the RNase S complex is studied with the help of transient IR spectroscopy, covering a wide range of time scales from 100 ps to 10 ms. To that end, an azobenzene moiety has been linked to the S-peptide in a way that its helicity is disrupted by light, thereby initiating its complete unbinding. The full sequence of events is observed, starting from unfolding of the helical structure of the S-peptide on a 20 ns time scale while still being in the binding pocket of the S-protein, S-peptide unbinding after 300 µs, and the structural response of the S-protein after 3 ms. With regard to the S-peptide dynamics, the binding mechanism can be classified as an induced fit, while the structural response of the S-protein is better described as conformational selection.

HY5 regulates light-responsive transcription of microRNA163 to promote primary root elongation in Arabidopsis seedlings.

MicroRNAs (miRNAs) play key roles in the post-transcriptional regulation of gene expression in plants. Many miRNAs are responsive to environmental signals. Light is the first environmental signal perceived by plants after emergence from the soil. However, less is known about the roles and regulatory mechanism of miRNAs in response to light signal. Here, using small RNA sequencing, we determined that miR163 is significantly rapidly induced by light signaling in Arabidopsis thaliana seedlings. The light-inducible response of miR163 functions genetically downstream of LONG HYPOCOTYL 5 (HY5), a central positive regulator of photomorphogenesis. HY5 directly binds to the two G/C-hybrid elements in the miR163 promoter with unequal affinity; one of these elements, which is located next to the transcription start site, plays a major role in light-induced expression of miR163. Overexpression of miR163 rescued the defective primary root elongation of hy5 seedlings without affecting lateral root growth, whereas overexpressing of miR163 target PXMT1 inhibited primary root elongation. These findings provide insight into understanding the post-transcriptional regulation of root photomorphogenesis mediated by the HY5-miR163-PXMT1 network.

UV-induced activation of ATR is mediated by UHRF2.

UHRF1 (Ubiquitin-like with PHD and ring finger domains 1) regulates DNA methylation and histone modifications and plays a key role in cell proliferation and the DNA damage response. However, the function of UHRF2, a paralog of UHRF1, in the DNA damage response remains largely unknown. Here, we show that UHRF2 is essential for maintaining cell viability after UV irradiation, as well as for the proliferation of cancer cells. UHRF2 was found to physically interact with ATR in a DNA damage-dependent manner through UHRF2's TTD domain. In addition, phosphorylation of threonine at position 1989, which is required for UV-induced activation of ATR, was impaired in cells depleted of UHRF2, suggesting that UHRF2 is essential in ATR activation. In conclusion, these results suggest a new regulatory mechanism of ATR activation mediated by UHRF2.

Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases.

Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4COP1/SPAs, has been reported for plant CRYs so far. Here we show that Cul3LRBs is the second E3 ligase of CRY2 in Arabidopsis. We demonstrate the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2, at the CCE domain of CRY2, to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4COP1/SPAs and Cul3LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature.

Structural Determinants for Light-Dependent Membrane Binding of a Photoswitchable Polybasic Domain.

OptoPB is an optogenetic tool engineered by fusion of the phosphoinositide (PI)-binding polybasic domain of Rit1 (Rit-PB) to a photoreactive light-oxygen-voltage (LOV) domain. OptoPB selectively and reversibly binds the plasma membrane (PM) under blue light excitation, and in the dark, it releases back to the cytoplasm. However, the molecular mechanism of optical regulation and lipid recognition is still unclear. Here using nuclear magnetic resonance (NMR) spectroscopy, liposome pulldown assay, and surface plasmon resonance (SPR), we find that OptoPB binds to membrane mimetics containing di- or triphosphorylated phosphatidylinositols, particularly phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), an acidic phospholipid predominantly located in the eukaryotic PM. In the dark, steric hindrance prevented this protein-membrane interaction, while 470 nm blue light illumination activated it. NMR titration and site-directed mutagenesis revealed that both cationic and hydrophobic Rit-PB residues are essential to the membrane interaction, indicating that OptoPB binds the membrane via a specific PI(4,5)P2-dependent mechanism.

The PIF1-miR408-PLANTACYANIN repression cascade regulates light-dependent seed germination.

Light-dependent seed germination is a vital process for many seed plants. A decisive event in light-induced germination is degradation of the central repressor PHYTOCHROME INTERACTING FACTOR 1 (PIF1). The balance between gibberellic acid (GA) and abscisic acid (ABA) helps to control germination. However, the cellular mechanisms linking PIF1 turnover to hormonal balancing remain elusive. Here, employing far-red light-induced Arabidopsis thaliana seed germination as the experimental system, we identified PLANTACYANIN (PCY) as an inhibitor of germination. It is a blue copper protein associated with the vacuole that is both highly expressed in mature seeds and rapidly silenced during germination. Molecular analyses showed that PIF1 binds to the miR408 promoter and represses miR408 accumulation. This in turn posttranscriptionally modulates PCY abundance, forming the PIF1-miR408-PCY repression cascade for translating PIF1 turnover to PCY turnover during early germination. Genetic analysis, RNA-sequencing, and hormone quantification revealed that PCY is necessary and sufficient to maintain the PIF1-mediated seed transcriptome and the low-GA-high-ABA state. Furthermore, we found that PCY domain organization and regulation by miR408 are conserved features in seed plants. These results revealed a cellular mechanism whereby PIF1-relayed external light signals are converted through PCY turnover to internal hormonal profiles for controlling seed germination.

Light-Dependent Translation Change of Arabidopsis psbA Correlates with RNA Structure Alterations at the Translation Initiation Region.

mRNA secondary structure influences translation. Proteins that modulate the mRNA secondary structure around the translation initiation region may regulate translation in plastids. To test this hypothesis, we exposed Arabidopsis thaliana to high light, which induces translation of psbA mRNA encoding the D1 subunit of photosystem II. We assayed translation by ribosome profiling and applied two complementary methods to analyze in vivo RNA secondary structure: DMS-MaPseq and SHAPE-seq. We detected increased accessibility of the translation initiation region of psbA after high light treatment, likely contributing to the observed increase in translation by facilitating translation initiation. Furthermore, we identified the footprint of a putative regulatory protein in the 5' UTR of psbA at a position where occlusion of the nucleotide sequence would cause the structure of the translation initiation region to open up, thereby facilitating ribosome access. Moreover, we show that other plastid genes with weak Shine-Dalgarno sequences (SD) are likely to exhibit psbA-like regulation, while those with strong SDs do not. This supports the idea that changes in mRNA secondary structure might represent a general mechanism for translational regulation of psbA and other plastid genes.

A short ORF-encoded transcriptional regulator.

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.

GUN4 Affects the Circadian Clock and Seedlings Adaptation to Changing Light Conditions.

The chloroplast is a key organelle for photosynthesis and perceiving environmental information. GENOME UNCOUPLED 4 (GUN4) has been shown to be required for the regulation of both chlorophyll synthesis, reactive oxygen species (ROS) homeostasis and plastid retrograde signaling. In this study, we found that growth of the gun4 mutant was significantly improved under medium strong light (200 µmol photons m-2s-1) compared to normal light (100 µmol photons m-2s-1), in marked contrast to wild-type (WT). Further analysis revealed that GUN4 interacts with SIGNAL RECOGNITION PARTICLE 54 KDA SUBUNIT (SRP43) and SRP54. RNA-seq analysis indicated that the expression of genes for light signaling and the circadian clock is altered in gun4 compared with (WT). qPCR analysis confirmed that the expression of the clock genes CLOCK-RELATED 1 (CCA1), LATE ELONGATION HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1) and PSEUDO RESPONSE REGULATOR 7 (PRR7) is significantly changed in the gun4 and srp54 mutants under normal and medium strong light conditions. These results suggest that GUN4 may coordinate the adaptation of plants to changing light conditions by regulating the biological clock, although it is not clear whether the effect is direct or indirect.

OsBIC1 Directly Interacts with OsCRYs to Regulate Leaf Sheath Length through Mediating GA-Responsive Pathway.

Cryptochrome 1 and 2 (CRY1 and CRY2) are blue light receptors involved in the regulation of hypocotyl elongation, cotyledon expansion, and flowering time in Arabidopsisthaliana. Two cryptochrome-interacting proteins, Blue-light Inhibitor of Cryptochrome 1 and 2 (BIC1 and BIC2), have been found in Arabidopsis. BIC1 plays critical roles in suppressing the physiological activities of CRY2, which include the blue light-dependent dimerization, phosphorylation, photobody formation, and degradation process, but the functional characterization of BIC protein in other crops has not yet been performed. To investigate the function of BIC protein in rice (Oryza sativa), two homologous genes of Arabidopsis BIC1 and BIC2, namely OsBIC1 and OsBIC2 (OsBICs), were identified. The overexpression of OsBIC1 and OsBIC2 led to increased leaf sheath length, whereas mutations in OsBIC1 displayed shorter leaf sheath in a blue light intensity-dependent manner. OsBIC1 regulated blue light-induced leaf sheath elongation through direct interaction with OsCRY1a, OsCRY1b, and OsCRY2 (OsCRYs). Longitudinal sections of the second leaf sheath demonstrated that OsBIC1 and OsCRYs controlled leaf sheath length by influencing the ratio of epidermal cells with different lengths. RNA-sequencing (RNA-seq) and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) analysis further proved that OsBIC1 and OsCRYs regulated similar transcriptome changes in regulating Gibberellic Acids (GA)-responsive pathway. Taken together, these results suggested that OsBIC1 and OsCRYs worked together to regulate epidermal cell elongation and control blue light-induced leaf sheath elongation through the GA-responsive pathway.

Binding of the synaptic vesicle radiotracer [11C]UCB-J is unchanged during functional brain activation using a visual stimulation task.

The positron emission tomography radioligand [11C]UCB-J binds to synaptic vesicle glycoprotein 2 A (SV2A), a regulator of vesicle release. Increased neuronal firing could potentially affect tracer concentrations if binding site availability is altered during vesicle exocytosis. This study assessed whether physiological brain activation induces changes in [11C]UCB-J tissue influx (K1), volume of distribution (VT), or binding potential (BPND). Healthy volunteers (n = 7) underwent 60-min [11C]UCB-J PET scans at baseline and during intermittent presentation of 8-Hz checkerboard visual stimulation. Sensitivity to intermittent changes in kinetic parameters was assessed in simulations, and visual stimulation was repeated using functional magnetic resonance imaging to characterize neural responses. VT and K1 were determined using the one-tissue compartment model and BPND using the simplified reference tissue model. In primary visual cortex, K1 increased 34.3 ± 15.5% (p = 0.001) during stimulation, with no change in other regions (ps > 0.12). K1 change was correlated with fMRI BOLD response (r = 0.77, p = 0.043). There was no change in VT (-3.9 ± 8.8%, p = 0.33) or BPND (-0.2 ± 9.6%, p = 0.94) in visual cortex nor other regions (ps > 0.19). Therefore, despite robust increases in regional tracer influx due to blood flow increases, binding measures were unchanged during stimulation. [11C]UCB-J VT and BPND are likely to be stable in vivo measures of synaptic density.

The expression of Rpb10, a small subunit common to RNA polymerases, is modulated by the R3H domain-containing Rbs1 protein and the Upf1 helicase.

The biogenesis of eukaryotic RNA polymerases is poorly understood. The present study used a combination of genetic and molecular approaches to explore the assembly of RNA polymerase III (Pol III) in yeast. We identified a regulatory link between Rbs1, a Pol III assembly factor, and Rpb10, a small subunit that is common to three RNA polymerases. Overexpression of Rbs1 increased the abundance of both RPB10 mRNA and the Rpb10 protein, which correlated with suppression of Pol III assembly defects. Rbs1 is a poly(A)mRNA-binding protein and mutational analysis identified R3H domain to be required for mRNA interactions and genetic enhancement of Pol III biogenesis. Rbs1 also binds to Upf1 protein, a key component in nonsense-mediated mRNA decay (NMD) and levels of RPB10 mRNA were increased in a upf1Δ strain. Genome-wide RNA binding by Rbs1 was characterized by UV cross-linking based approach. We demonstrated that Rbs1 directly binds to the 3' untranslated regions (3'UTRs) of many mRNAs including transcripts encoding Pol III subunits, Rpb10 and Rpc19. We propose that Rbs1 functions by opposing mRNA degradation, at least in part mediated by NMD pathway. Orthologues of Rbs1 protein are present in other eukaryotes, including humans, suggesting that this is a conserved regulatory mechanism.

Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry.

Protein-DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein-DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein-RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future.

Adsorption of bovine serum albumin on gold nanoprisms: interaction and effect of NIR irradiation on protein corona.

Because of their photothermal properties, gold nanoparticles (AuNPs) have gained attention regarding their use in drug delivery and therapeutic applications. In this sense, it is interesting to consider their interactions with biologically available proteins, such as serum albumin, as well as the effects of irradiation and photothermal conversion on the protein structure that can lead to a loss of function or generate an immune response. Gold nanoprisms (AuNPrs) have gained interest due to their low toxicity, ease of synthesis, and excellent stability, promoting their use in bioapplications such as surface-enhanced Raman spectroscopy (SERS), drug delivery, and photothermal therapy. The interaction between AuNPrs, with plasmon bands centred in the near-infrared region (NIR), and bovine serum albumin (BSA) has not been explored yet. UV-Vis spectroscopy, dynamic light scattering (DLS) and fluorescence spectroscopy were used to study the interaction between AuNPrs and BSA in addition to estimation of the adsorption rate and kinetic and thermodynamic parameters (K, ΔH°, ΔG°, ΔS°, and Ea) using adsorption isotherms and Langmuir and Freundlich models. The results suggest spontaneous cooperative binding in multilayer adsorption, achieved by the chemisorption of BSA on the AuNPr surface through the S-Au interaction, as confirmed by Raman spectroscopy. On the other hand, the photothermal conversion efficiency (PE) of the coated nanoparticles after NIR irradiation was assessed, resulting in a slight decrease in the PE of BSA coated on AuNPrs in comparison with that of noncapped nanoparticles. The effect of the irradiation on the protein conformation of capped nanoparticles was also assessed; circular dichroism showed BSA unfolding upon interaction with AuNPrs, with a decrease in the α-helix and ß-sheet contents, as well as an increase in random coil conformations. Changes in the Raman spectrum suggest a modification of the disposition of the protein residues exposed to the gold surface after NIR irradiation; but at the secondary structure level, no relevant changes were observed. This provides possibilities for the use of NPs-BSA for bioapplications based on the photothermal effect promoted by laser irradiation, since the biological identity of the protein is preserved after NIR irradiation.