Fine tuning chloroplast movements through physical interactions between phototropins

Phototropins are plant photoreceptors which regulate numerous responses to blue light, including chloroplast relocation. Weak blue light induces chloroplast accumulation, whereas strong light leads to an avoidance response. Two Arabidopsis phototropins are characterized by different light sensitivities. Under continuous light, both can elicit chloroplast accumulation, but the avoidance response is controlled solely by phot2. As well as continuous light, brief light pulses also induce chloroplast displacements. Pulses of 0.1s and 0.2s of fluence rate saturating the avoidance response lead to transient chloroplast accumulation. Longer pulses (up to 20s) trigger a biphasic response, namely transient avoidance followed by transient accumulation. This work presents a detailed study of transient chloroplast responses in Arabidopsis. Phototropin mutants display altered chloroplast movements as compared with the wild type: phot1 is characterized by weaker responses, while phot2 exhibits enhanced chloroplast accumulation, especially after 0.1s and 0.2s pulses. To determine the cause of these differences, the abundance and phosphorylation levels of both phototropins, as well as the interactions between phototropin molecules are examined. The formation of phototropin homo- and heterocomplexes is the most plausible explanation of the observed phenomena. The physiological consequences of this interplay are discussed, suggesting the universal character of this mechanism that fine-tunes plant reactions to blue light. Additionally, responses in mutants of different protein phosphatase 2A subunits are examined to assess the role of protein phosphorylation in signaling of chloroplast movements

Phototropin2 3’UTR overlaps with the AT5G58150 gene encoding an inactive RLK kinase

ackground This study examines the biological implications of an overlap between two sequences in the Arabidop�sis genome, the 3’UTR of the PHOT2 gene and a putative AT5G58150 gene, encoded on the complementary strand. AT5G58150 is a probably inactive protein kinase that belongs to the transmembrane, leucine-rich repeat receptor-like kinase family. Phot2 is a membrane-bound UV/blue light photoreceptor kinase. Thus, both proteins share their cellular localization, on top of the proximity of their loci. Results The extent of the overlap between 3’UTR regions of AT5G58150 and PHOT2 was found to be 66 bp, using RACE PCR. Both the at5g58150 T-DNA SALK_093781C (with insertion in the promoter region) and 35S::AT5G58150�GFP lines overexpress the AT5G58150 gene. A detailed analysis did not reveal any substantial impact of PHOT2 or AT5G58150 on their mutual expression levels in diferent light and osmotic stress conditions. AT5G58150 is a plasma membrane protein, with no apparent kinase activity, as tested on several potential substrates. It appears not to form homodimers and it does not interact with PHOT2. Lines that overexpress AT5G58150 exhibit a greater reduction in lat‑ eral root density due to salt and osmotic stress than wild-type plants, which suggests that AT5G58150 may partici‑ pate in root elongation and formation of lateral roots. In line with this, mass spectrometry analysis identifed proteins with ATPase activity, which are involved in proton transport and cell elongation, as putative interactors of AT5G58150. Membrane kinases, including other members of the LRR RLK family and BSK kinases (positive regulators of brassinos‑ teroid signalling), can also act as partners for AT5G58150. Conclusions AT5G58150 is a membrane protein that does not exhibit measurable kinase activity, but is involved in signalling through interactions with other proteins. Based on the interactome and root architecture analysis, AT5G58150 may be involved in plant response to salt and osmotic stress and the formation of roots in Arabidopsis

Phototropin interactions with SUMO proteins

The disruption of the sumoylation pathway affects processes controlled by the two phototropins (phots) of Arabidopsis thaliana, phot1 and phot2. Phots, plant UVA/blue light photoreceptors, regulate growth responses and fast movements aimed at optimizing photosynthesis, such as phototropism, chloroplast relocations and stomatal opening. Sumoylation is a posttranslational modification, consisting of the addition of a SUMO (SMALL UBIQUITIN-RELATED MODIFIER) protein to a lysine residue in the target protein. In addition to affecting the stability of proteins, it regulates their activity, interactions and subcellular localization. We examined physiological responses controlled by phots, phototropism and chloroplast movements, in sumoylation pathway mutants. Chloroplast accumulation in response to both continuous and pulse light was enhanced in the E3 ligase siz1 mutant, in a manner dependent on phot2. A significant decrease in phot2 protein abundance was observed in this mutant after blue light treatment both in seedlings and mature leaves. Using plant transient expression and yeast two-hybrid assays, we found that phots interacted with SUMO proteins mainly through their N-terminal parts, which contain the photosensory LOV domains. The covalent modification in phots by SUMO was verified using an Arabidopsis sumoylation system reconstituted in bacteria followed by the mass spectrometry analysis. Lys 297 was identified as the main target of SUMO3 in the phot2 molecule. Finally, sumoylation of phot2 was detected in Arabidopsis mature leaves upon light or heat stress treatment

Phototropin interactions with SUMO proteins

The disruption of the sumoylation pathway affects processes controlled by the two phototropins (phots) of Arabidopsis thaliana, phot1 and phot2. Phots, plant UVA/blue light photoreceptors, regulate growth responses and fast movements aimed at optimizing photosynthesis, such as phototropism, chloroplast relocations and stomatal opening. Sumoylation is a posttranslational modification, consisting of the addition of a SUMO (SMALL UBIQUITIN-RELATED MODIFIER) protein to a lysine residue in the target protein. In addition to affecting the stability of proteins, it regulates their activity, interactions and subcellular localization. We examined physiological responses controlled by phots, phototropism and chloroplast movements, in sumoylation pathway mutants. Chloroplast accumulation in response to both continuous and pulse light was enhanced in the E3 ligase siz1 mutant, in a manner dependent on phot2. A significant decrease in phot2 protein abundance was observed in this mutant after blue light treatment both in seedlings and mature leaves. Using plant transient expression and yeast two-hybrid assays, we found that phots interacted with SUMO proteins mainly through their N-terminal parts, which contain the photosensory LOV domains. The covalent modification in phots by SUMO was verified using an Arabidopsis sumoylation system reconstituted in bacteria followed by the mass spectrometry analysis. Lys 297 was identified as the main target of SUMO3 in the phot2 molecule. Finally, sumoylation of phot2 was detected in Arabidopsis mature leaves upon light or heat stress treatment

Characterizing of PHR2, a putative photolyase/blue-light photoreceptor from Arabidopsis thaliana and investigation of this protein interaction partners

Promieniowanie ultrafioletowe, w szczególności UV-B o długości fali 280 – 320 nm, indukuje powstawanie genotoksycznych fotoproduktów w DNA. Dwa główne typy uszkodzeń wywołanych przez UV to cyklobutanowe dimery pirymidyny (CPDs) i fotoprodukty 6-4 pirymidyno-pirymidynowe (6-4 PPs). U roślin wyższych występują różne strategie umożliwiające minimalizację uszkodzeń wywołanych promieniowaniem UV. Jednym z takich mechanizmów jest fotoreaktywacja, czyli zależna od światła niebieskiego i UVA enzymatyczna naprawa dimerów. Fotoreaktywacja polega na katalitycznej hydrolizie wiązania kowalencyjnego między dimerami pirymidynowymi i prowadzona jest przez enzymy zwane fotoliazami. Niniejsza praca poświęcona była wstępnej charakterystyce białka PHR2 Arabidopsis thaliana. Na podstawie analizy in silico PHR2 jest opisywane jako fotoliaza/fotoreceptor światła niebieskiego. W wysokoprzepustowych badaniach przesiewowych (ang. high throughput screening) z wykorzystaniem drożdżowych testów dwuhybrydowych wykazano, że PHR2 może wchodzić w interakcje z niektórymi białkami biorącymi udział w regulacji szlaków przekazu sygnału takimi jak: ATARCA, AGB1 oraz RGS1, a także z białkiem At5g57170, o niepoznanej dotąd funkcji. Głównym celem pracy było zweryfikowanie powyższych oddziaływań wykorzystując w tym celu dwucząsteczkową komplementację fluorescencji oraz drożdżowy system dwuhybrydowy. Dodatkowo sprawdzano aktywność fotoliazową PHR2, stosując test komplementacji z użyciem bakterii pozbawionych naturalnej fotoliazy, a także badano ekspresję genu kodującego powyższe białko przy pomocy metody PCR w czasie rzeczywistym. Wykazano, że białko PHR2 uzupełnia, w sposób zależny od światła, niedobór naturalnej fotoliazy w naświetlanych UVB bakteriach E. coli, co wskazuje na jego potencjalną aktywność fotoliazową. Względny poziom ekspresji genu PHR2 jest różny w zależności od organu A. thaliana oraz etapu rozwoju rośliny. Najwyższy poziom ekspresji zaobserwowano w siewkach Arabidopsis, najniższy w korzeniach dorosłych roślin. Dodatkowo wykazano, że światło białe pozytywnie reguluje poziom mRNA PHR2. Stosując transfomację przejściową N. benthamiana pokazano, że PHR2 lokalizuje się w jądrze i chloroplastach, sprawdzono także lokalizację wewnątrzkomórkową wszystkich badanych potencjalnych partnerów PHR2. Potwierdzono również zachodzenie oddziaływań pomiędzy PHR2 a At5g57170.Ultraviolet radiation, especially UV-B which have spectrum from 280 nm to 320 nm, induces formation of genotoxic photoproducts in DNA. The two major lesions in DNA induced by UV are cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs). In higher plants several strategies to repair different types of UV-induced damage exist. One of this strategies is a light-dependent process called photoreactivation catalyzed by photolyases. Photoreactivation is an enzymatic repair, which allows catalytic hydrolysis of the covalent bond between the pyrimidine dimers. This work was dedicated to preliminary characteristics of PHR2 protein from Arabidopsis thaliana. Based on in silico analysis this protein is postulated to act as a photolyase and/or blue-light photoreceptor. Some proteins have been shown to interact with PHR2 using high throughput screening assays. These proteins are: ATARCA, RGS1, AGB1 which are involved in signal transduction pathways: and At5g57170, a protein with unknown function. To verify the interactions yeast two-hybrid system and bimolecular fluorescence complementation were used. Moreover, a photolyase complementation assay in E. coli has been performed. Another aim of this work was to check the PHR2 expression in different organs of Arabidopsis as well as influence of light on this gene expression using real-time PCR. It was shown that PHR2 protein complement a photolyase-deficient mutant of Escherichia coli in a light dependent manner. However, a photoreactivation activity of this protein still needs to be confirmed. The relative PHR2 expression varied between Arabidopsis organs and different life stages. The highest mRNA level of this gene was observed in seedlings, the lowest in mature roots. The expression of PHR2 gene was up-regulated by white light. It was shown that PHR2 is located in chloroplasts and nucleus. The subcellular localization of all proteins which potentially interact with PHR2 was also checked. Only an interaction between PHR2 and At5g57170 was confirmed

The Dark Side of UV-Induced DNA Lesion Repair

In their life cycle, plants are exposed to various unfavorable environmental factors including ultraviolet (UV) radiation emitted by the Sun. UV-A and UV-B, which are partially absorbed by the ozone layer, reach the surface of the Earth causing harmful effects among the others on plant genetic material. The energy of UV light is sufficient to induce mutations in DNA. Some examples of DNA damage induced by UV are pyrimidine dimers, oxidized nucleotides as well as single and double-strand breaks. When exposed to light, plants can repair major UV-induced DNA lesions, i.e., pyrimidine dimers using photoreactivation. However, this highly efficient light-dependent DNA repair system is ineffective in dim light or at night. Moreover, it is helpless when it comes to the repair of DNA lesions other than pyrimidine dimers. In this review, we have focused on how plants cope with deleterious DNA damage that cannot be repaired by photoreactivation. The current understanding of light-independent mechanisms, classified as dark DNA repair, indispensable for the maintenance of plant genetic material integrity has been presented

All You Need Is Light. Photorepair of UV-Induced Pyrimidine Dimers

Although solar light is indispensable for the functioning of plants, this environmental factor may also cause damage to living cells. Apart from the visible range, including wavelengths used in photosynthesis, the ultraviolet (UV) light present in solar irradiation reaches the Earth’s surface. The high energy of UV causes damage to many cellular components, with DNA as one of the targets. Putting together the puzzle-like elements responsible for the repair of UV-induced DNA damage is of special importance in understanding how plants ensure the stability of their genomes between generations. In this review, we have presented the information on DNA damage produced under UV with a special focus on the pyrimidine dimers formed between the neighboring pyrimidines in a DNA strand. These dimers are highly mutagenic and cytotoxic, thus their repair is essential for the maintenance of suitable genetic information. In prokaryotic and eukaryotic cells, with the exception of placental mammals, this is achieved by means of highly efficient photorepair, dependent on blue/UVA light, which is performed by specialized enzymes known as photolyases. Photolyase properties, as well as their structure, specificity and action mechanism, have been briefly discussed in this paper. Additionally, the main gaps in our knowledge on the functioning of light repair in plant organelles, its regulation and its interaction between different DNA repair systems in plants have been highlighted

Additional file 1 of Phototropin2 3’UTR overlaps with the AT5G58150 gene encoding an inactive RLK kinase

Additional file 1: Fig. S1. The locus of PHOT2 (AT5G58140) and AT5G58140 genes in the whole-genome pairwise alignments between Arabidopsis and several plant genomes, obtained with the VISTA-Point tool ( https://pipeline.lbl.gov/ ). For each species, the curves show the identity score of the alignment, averaged across a 100 bp moving window. A region is considered to be conserved if the sequence identity is at least 70% over at least 100 bp. Conserved regions are color-coded as dark blue, light blue or orange if they correspond to exons, UTRs, or non-coding sequences, respectively. Fig. S2. Confirmation of at5g58150 mutation (SALK_093781C, insertion in the promoter region) with the three primers in one reaction (Lba1, sequence specific primers Table S1). Fig. S3. Kinase activity assay for AT5G58150 kinase domain. Fig. S4. Laser scanning confocal images of N. benthamiana epidermal cells transiently co-expressing AT5G58150-GFP or Plasma Membrane-mCherry or Tonoplast-mCherry markers. Fig. S5. Western Blot analysis of N. benthamiana epidermal cells transiently co-expressing PHOT2 and AT5G58150 fused with C(N)-terminal YFP fragments in the following configurations: AT5G58150_NtermYFP and AT5G58150_CtermYFP, NtermYFP_PHOT2 and PHOT2_CtermYFP, NtermYFP_PHOT2 and AT5G58150_CtermYFP, CtermYFP_PHOT2 and PHOT2_NtermYFP, PHOT2_CtermYFP and AT5G58150_NtermYFP, AT5G58150_NtermYFP, AT5G58150_CtermYFP, NtermYFP_PHOT2, CtermYFP_PHOT2, PHOT2_NtermYFP, PHOT2_CtermYFP probed with anti-cYFP and anti-nYFP. The white light image was merged with the chemiluminescent signal to show the borders of the membranes and molecular weight marker (PageRuler Prestained Protein, SM #26616, Thermo Scientific). Cropped image in Fig. 4. Fig. S6. AT5G58150 and phototropin2 interactions tested with MYTH assay. Fig. S7. Averaged curves (A) and amplitudes (B) of changes in rosette leaf transmittance T induced by blue light of increasing irradiance of 0.4, 1.6, 4, 20, 40, 80, 120 µmol·m-2·s-1 in wild type, at5g58150 mutant and 35S::AT5G58150-GFP lines. Fig. S8. Co-expression analysis of AT5G58150 based on proteomic data performed with Athena (https://athena.proteomics.wzw.tum.de/master_arabidopsisshiny/). Co-expression of AT5G58150 with BAK1, BSK, BRL1, BRL3 is observed. Fig. S9. Co-expression analysis of AT5G58150 based on transcriptomic data performed with Athena (https://athena.proteomics.wzw.tum.de/master_arabidopsisshiny/). Co-expression of AT5G58150 with ATBS1, BES1, BZR1 is observed. Table S6. List of proteins that co-immunoprecipitated with AT5G59150-GFP, identified with Mass Spectrometry, which fulfill the interaction criteria, together with the values of parameters taken into account in the calculations. Table S7. List of proteins co-immunoprecipitated with AT5G59150-GFP, identified with MS, with their functional descriptions from the String database. Table S8. Primer sequences used in this study. Table S9. Genes co-expressed with AT5G58150 based on published data from transcriptomic analysis performed with the Arabidopsis Co-expression Tool (https://www.michalopoulos.net/act). Genes of the brassinosteroid pathway, involved in root development as well as cell wall and junction formation, are marked in yellow