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

BACKGROUND: This study examines the biological implications of an overlap between two sequences in the Arabidopsis 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 different 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 lateral root density due to salt and osmotic stress than wild-type plants, which suggests that AT5G58150 may participate in root elongation and formation of lateral roots. In line with this, mass spectrometry analysis identified 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 brassinosteroid 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.

Molecular insights into the phototropin control of chloroplast movements.

Chloroplast movements are controlled by ultraviolet/blue light through phototropins. In Arabidopsis thaliana, chloroplast accumulation at low light intensities and chloroplast avoidance at high light intensities are observed. These responses are controlled by two homologous photoreceptors, the phototropins phot1 and phot2. Whereas chloroplast accumulation is triggered by both phototropins in a partially redundant manner, sustained chloroplast avoidance is elicited only by phot2. Phot1 is able to trigger only a small, transient chloroplast avoidance, followed by the accumulation phase. The source of this functional difference is not fully understood at either the photoreceptor or the signalling pathway levels. In this article, we review current understanding of phototropin functioning and try to dissect the differences that result in signalling to elicit two distinct chloroplast responses. First, we focus on phototropin structure and photochemical and biochemical activity. Next, we analyse phototropin expression and localization patterns. We also summarize known photoreceptor systems controlling chloroplast movements. Finally, we focus on the role of environmental stimuli in controlling phototropin activity. All these aspects impact the signalling to trigger chloroplast movements and raise outstanding questions about the mechanism involved.

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.

Phosphoproteomic analysis reveals that dehydrins ERD10 and ERD14 are phosphorylated by SNF1-related protein kinase 2.10 in response to osmotic stress.

SNF1-related protein kinases 2 (SnRK2s) regulate the plant responses to abiotic stresses, especially water deficits. They are activated in plants subjected to osmotic stress, and some of them are additionally activated in response to enhanced concentrations of abscisic acid (ABA) in plant cells. The SnRK2s that are activated in response to ABA are key elements of ABA signalling that regulate plant acclimation to environmental stresses and ABA-dependent development. Much less is known about the SnRK2s that are not activated by ABA, albeit several studies have shown that these kinases are also involved in response to osmotic stress. Here, we show that one of the Arabidopsis thaliana ABA-non-activated SnRK2s, SnRK2.10, regulates not only the response to salinity but also the plant sensitivity to dehydration. Several potential SnRK2.10 targets phosphorylated in response to stress were identified by a phosphoproteomic approach, including the dehydrins ERD10 and ERD14. Their phosphorylation by SnRK2.10 was confirmed in vitro. Our data suggest that the phosphorylation of ERD14 within the S-segment is involved in the regulation of dehydrin subcellular localization in response to stress.

Detailed characterization of Synechocystis PCC 6803 ferredoxin:NADP+ oxidoreductase interaction with model membranes.

Direct interaction of ferredoxin:NADP+ oxidoreductase (FNR) with thylakoid membranes was postulated as a part of the cyclic electron flow mechanism. In vitro binding of FNR to digalactosyldiacylglycerol and monogalactosyldiacylglycerol membranes was also shown. In this paper we deal with the latter interaction in more detail describing the effect for two FNR forms of Synechocystis PCC 6803. The so-called short FNR (sFNR) is homologous to FNR from higher plant chloroplasts. The long FNR (lFNR) form contains an additional domain, responsible for the interaction with phycobilisomes. We compare the binding of both sFNR and lFNR forms to native and non-native lipids. We also include factors which could modulate this process: pH change, temperature change, presence of ferredoxin, NADP+ and NADPH and heavy metals. For the lFNR, we also include phycobilisomes as a modulating factor. The membrane binding is generally faster at lower pH. The sFNR was binding faster than lFNR. Ferredoxin isoforms with higher midpoint potential, as well as NADPH and NADP+, weakened the binding. Charged lipids and high phosphate promoted the binding. Heavy metal ions decreased the rate of membrane binding only when FNR was preincubated with them before injection beneath the monolayer. FNR binding was limited to surface lipid groups and did not influence hydrophobic chain packing. Taken together, FNR interaction with lipids appears to be non-specific, with an electrostatic component. This suggests that the direct FNR interaction with lipids is most likely not a factor in directing electron transfer, but should be taken into account during in vitro studies.

6,4-PP Photolyase Encoded by AtUVR3 is Localized in Nuclei, Chloroplasts and Mitochondria and its Expression is Down-Regulated by Light in a Photosynthesis-Dependent Manner.

Pyrimidine dimers are the most important DNA lesions induced by UVB irradiation. They can be repaired directly by photoreactivation or indirectly by the excision repair pathways. Photoreactivation is carried out by photolyases, enzymes which bind to the dimers and use the energy of blue light or UVA to split bonds between adjacent pyrimidines. Arabidopsis thaliana has three known photolyases: AtPHR1, AtCRY3 and AtUVR3. Little is known about the cellular localization and regulation of AtUVR3 expression. We have found that its transcript level is down-regulated by light (red, blue or white) in a photosynthesis-dependent manner. The down-regulatory effect of red light is absent in mature leaves of the phyB mutant, but present in leaves of phyAphyB. UVB irradiation does not increase AtUVR3 expression in leaves. Transiently expressed AtUVR3-green fluorescent protein (GFP) is found in the nuclei, chloroplasts and mitochondria of Nicotiana benthamiana epidermal cells. In the nucleoplasm, AtUVR3-GFP is distributed uniformly, while in the nucleolus it forms speckles. Truncated AtUVR3 and muteins were used to identify the sequences responsible for its subcellular localization. Mitochondrial and chloroplast localization of AtUVR3 is independent of its N-terminal sequence. Amino acids located at the C-terminal loop of the protein are involved in its transport into chloroplasts and its retention inside the nucleolus.

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.

The effect of UV-B on Arabidopsis leaves depends on light conditions after treatment.

BACKGROUND: Ultraviolet B (UV-B) irradiation can influence many cellular processes. Irradiation with high UV-B doses causes chlorophyll degradation, a decrease in the expression of genes associated with photosynthesis and its subsequent inhibition. On the other hand, sublethal doses of UV-B are used in post-harvest technology to prevent yellowing in storage. To address this inconsistency the effect of short, high-dose UV-B irradiation on detached Arabidopsis thaliana leaves was examined. RESULTS: Two different experimental models were used. After short treatment with a high dose of UV-B the Arabidopsis leaves were either put into darkness or exposed to constant light for up to 4 days. UV-B inhibited dark-induced chlorophyll degradation in Arabidopsis leaves in a dose-dependent manner. The expression of photosynthesis-related genes, chlorophyll content and photosynthetic efficiency were higher in UV-B -treated leaves left in darkness. UV-B treatment followed by constant light caused leaf yellowing and induced the expression of senescence-related genes. Irrespective of light treatment a high UV-B dose led to clearly visible cell death 3 days after irradiation. CONCLUSIONS: High doses of UV-B have opposing effects on leaves depending on their light status after UV treatment. In darkened leaves short UV-B treatment delays the appearance of senescence symptoms. When followed by light treatment, the same doses of UV-B result in chlorophyll degradation. This restricts the potential usability of UV treatment in postharvest technology to crops which are stored in darkness.

Expression of enzymes involved in chlorophyll catabolism in Arabidopsis is light controlled.

We found that the levels of mRNA of two enzymes involved in chlorophyll catabolism in Arabidopsis (Arabidopsis thaliana), products of two chlorophyllase genes, AtCLH1 and AtCLH2, dramatically increase (by almost 100- and 10-fold, respectively) upon illumination with white light. The measurements of photosystem II quantum efficiency in 3-(3,4-dichlorophenyl)-1,1-dimethylurea-inhibited leaves show that their expression is not related to photosynthesis but mediated by photoreceptors. To identify the photoreceptors involved, we used various light treatments and Arabidopsis photoreceptor mutants (cry1, cry2, cry1cry2, phot1, phot2, phot1phot2, phyA phyB, phyAphyB). In wild-type Columbia, the amount of transcripts of both genes increase after white-light irradiation but their expression profile and the extent of regulation differ considerably. Blue and red light is active in the case of AtCLH1, whereas only blue light raises the AtCLH2 mRNA level. The fundamental difference is the extent of up-regulation, higher by one order of magnitude in AtCLH1. Both blue and red light is active in the induction of AtCLH1 expression in all mutants, pointing to a complex control network and redundancy between photoreceptors. The blue-specific up-regulation of the AtCLH2 transcript is mediated by cryptochromes and modulated by phototropin1 and phytochromes. Individually darkened leaves were used to test the effects of senescence on the expression of AtCLH1 and AtCLH2. The expression profile of AtCLH1 remains similar to that found in nonsenescing leaves up to 5 d after darkening. In contrast, the light induction of AtCLH2 mRNA declines during dark treatment. These results demonstrate that the expression of enzymes involved in chlorophyll catabolism is light controlled.

The expression of phototropins in Arabidopsis leaves: developmental and light regulation.

Phototropins are blue light receptors, which play different roles during plant development. Two phototropins of Arabidopsis thaliana, phot1 and phot2, have strongly overlapping functions. In seedlings, both photoreceptors are responsible for phototropism. In mature leaves they redundantly regulate leaf shape, stomatal opening, and the accumulation of chloroplasts, whereas phototropin2 alone controls chloroplast avoidance response. Light not only activates phototropins, but also affects the level of their expression. In Arabidopsis seedlings, PHOT1 is downregulated and PHOT2 is upregulated by light. Since data on transcription levels of phototropins in mature Arabidopsis leaves is scarce, a comprehensive real-time PCR study of PHOT1 and PHOT2 expression during development was performed, from seedlings to senescing leaves. So far, neither the phototropin expression nor its modulation by light have been investigated during senescence. The results show that the general regulation pattern remains conserved during Arabidopsis lifecycle, whereas the level of transcripts fluctuates over time, pointing to the significance of the light control for functioning of phototropins. The second part of the study determined the influence of photosynthesis-derived signals and photoreceptor-activated transduction pathways on phototropin mRNA levels. The effects of blue and red light were examined using Arabidopsis mutant lines deficient in photoreceptors. The results reveal a complex network of interactions between these receptors in the regulation of phototropin transcription profiles. Cryptochrome1 and phytochromeB appear to be main photoreceptors involved in the regulation of PHOT1 transcript accumulation. The expression of PHOT2 is dependent on both cryptochromes and phytochromeA.

Blue light signalling in chloroplast movements.

Chloroplast movements are among the mechanisms allowing plants to cope with changes in their environment. Chloroplasts accumulate at illuminated cell areas under weak light while they avoid areas exposed to strong light. These directional responses may be controlled by blue and/or red light, depending on the plant group. In terrestrial angiosperms only the blue light perceived by phototropins is active. The last decade has seen a rapid development of studies on the mechanism of directional chloroplast movements, which started with an identification of the photoreceptors. A forward genetic approach has been used to identify the components which control chloroplast movements. This review summarizes the current state of research into the signalling pathways which lead to chloroplast responses. First, the molecular properties of phototropins are presented, followed by a characterization both of proteins which are active downstream of phototropins and of secondary messengers. Finally, cross-talk between light signalling involved in chloroplast movements and other signalling pathways is discussed.

The Multifaceted Regulation of SnRK2 Kinases.

SNF1-related kinases 2 (SnRK2s) are central regulators of plant responses to environmental cues simultaneously playing a pivotal role in the plant development and growth in favorable conditions. They are activated in response to osmotic stress and some of them also to abscisic acid (ABA), the latter being key in ABA signaling. The SnRK2s can be viewed as molecular switches between growth and stress response; therefore, their activity is tightly regulated; needed only for a short time to trigger the response, it has to be induced transiently and otherwise kept at a very low level. This implies a strict and multifaceted control of SnRK2s in plant cells. Despite emerging new information concerning the regulation of SnRK2s, especially those involved in ABA signaling, a lot remains to be uncovered, the regulation of SnRK2s in an ABA-independent manner being particularly understudied. Here, we present an overview of available data, discuss some controversial issues, and provide our perspective on SnRK2 regulation.

UV-B Induces Chloroplast Movements in a Phototropin-Dependent Manner.

We examined the impact of UV-B irradiation on chloroplast movements in Arabidopsis leaves. Directional chloroplast movements induced by blue light have been described in multiple plant species. In weak light, chloroplasts accumulate at periclinal cell walls to increase light capture. In strong light, chloroplasts exhibit the avoidance response, as they move towards anticlinal walls to protect the photosynthetic apparatus from light-induced damage. In Arabidopsis, chloroplast movements are triggered by phototropins, phot1 and phot2, which are known as blue/UV-A photoreceptors. We found that irradiation with UV-B of 3.3 µmol·m-2·s-1 induced chloroplast accumulation in wild-type plants. UV-B-triggered accumulation was dependent on the presence of phototropins, especially phot1, but not on UVR8 (the canonical UV-B photoreceptor). Irradiation with strong UV-B of 20 µmol·m-2·s-1 did not induce substantial chloroplast relocations in wild-type leaves. However, in the jac1 mutant, which is defective in chloroplast accumulation, strong UV-B elicited chloroplast avoidance. This indicated that UV-B can also activate signaling to the avoidance response. To assess the possibility of indirect effects of UV-B on chloroplast movements, we examined the impact of UV-B on the actin cytoskeleton, which serves as the motile system for chloroplast movements. While irradiation with UV-B of 3.3 µmol·m-2·s-1 did not affect the actin cytoskeleton, strong UV-B disrupted its structure as shown using an Arabidopsis line expressing Lifeact-green fluorescent protein (GFP). In wild-type plants, pretreatment with strong UV-B attenuated chloroplast responses triggered by subsequent blue light irradiation, further indicating that this UV-B intensity also indirectly affects chloroplast movements. Taken together, our results suggest that the effect of UV-B on chloroplast movement is twofold: it directly induces phototropin-mediated movements; however, at higher intensities, it attenuates the movements in a nonspecific manner.

Heterodimerizing helices as tools for nanoscale control of the organization of protein-protein and protein-quantum dots.

In this study, we tested the possibility of creating complexes of two proteins by fusing them with heterodimerizing helices. We used the fluorescent proteins GFP and mCHERRY expressed with a His-tag as our model system. We added heterodimer-forming sequences at the C- or N- termini of the proteins, opposite to the His-tag position. Heterodimerization was tested for both helices at the C-terminus or at the N- terminus and C-terminus. We observed complex formation with a nanomolar dissociation constant in both cases that was higher by one order of magnitude than the Kds measured for helices alone. The binding of two C-terminal helices was accompanied by an increased enthalpy change. The binding between helices could be stabilized by introducing an additional turn of the helix with cysteine, which was capable of forming disulphide bridges. Covalently linked proteins were obtained using this strategy and observed using fluorescence cross-correlation spectroscopy. Finally, we demonstrated the formation of complexes of protein dimers and quantum dots.

Comparison of α-Helix and ß-Sheet Structure Adaptation to a Quantum Dot Geometry: Toward the Identification of an Optimal Motif for a Protein Nanoparticle Cover.

While quantum dots (QDs) are useful as fluorescent labels, their application in biosciences is limited due to the stability and hydrophobicity of their surface. In this study, we tested two types of proteins for use as a cover for spherical QDs, composed of cadmium selenide. Pumilio homology domain (Puf), which is mostly α-helical, and leucine-rich repeat (LRR) domain, which is rich in ß-sheets, were selected to determine if there is a preference for one of these secondary structure types for nanoparticle covers. The protein sequences were optimized to improve their interaction with the surface of QDs. The solubilization of the apoproteins and their assembly with nanoparticles required the application of a detergent, which was removed in subsequent steps. Finally, only the Puf-based cover was successful enough as a QD hydrophilic cover. We showed that a single polypeptide dimer of Puf, PufPuf, can form a cover. We characterized the size and fluorescent properties of the obtained QD:protein assemblies. We showed that the secondary structure of the Puf proteins was not destroyed upon contact with the QDs. We demonstrated that these assemblies do not promote the formation of reactive oxygen species during illumination of the nanoparticles. The data represent advances in the effort to obtain a stable biocompatible cover for QDs.

Photometric Assays for Chloroplast Movement Responses to Blue Light.

Assessment of chloroplast movements by measuring changes in leaf transmittance is discussed, with special reference to the conditions necessary for reliable estimation of blue light-activated chloroplast responses.

Arabidopsis PCNAs form complexes with selected D-type cyclins.

Proliferating Cell Nuclear Antigen (PCNA) is a key nuclear protein of eukaryotic cells. It has been shown to form complexes with cyclin dependent kinases, cyclin dependent kinase inhibitors and the D-type cyclins which are involved in the cell cycle control. In Arabidopsis two genes coding for PCNA1 and PCNA2 proteins have been identified. In this study by analyzing Arabidopsis PCNA/CycD complexes we tested the possible functional differentiation of PCNA1/2 proteins in cell cycle control. Most out of the 10 cyclins investigated showed only nuclear localization except CycD2;1, CycD4;1, and CycD4;2 which were observed both in the nucleus and cytoplasm. Using the Y2H, BiFC and FLIM-FRET techniques we identified D-type cyclins which formed complexes with either PCNA1 or PCNA2. Among the candidates tested only CycD1;1, CycD3;1, and CycD3;3 were not detected in a complex with the PCNA proteins. Moreover, our results indicate that the formation of CycD3;2/PCNA and CycD4;1/PCNA complexes can be regulated by other as yet unidentified factor(s). Additionally, FLIM-FRET analyses suggested that in planta the distance between PCNA1/CycD4;1, PCNA1/CycD6;1, PCNA1/CycD7;1, and PCNA2/CycD4;2 proteins was shorter than that between PCNA2/CycD4;1, PCNA2/CycD6;1, PCNA2/CycD7;1, and PCNA1/CycD4;2 pairs. These data indicate that the nine amino acid differences between PCNA1 and PCNA2 have an impact on the architecture of Arabidopsis CycD/PCNA complexes.

Photoprotective function of chloroplast avoidance movement: in vivo chlorophyll fluorescence study.

Light-induced chloroplast avoidance movement has long been considered to be a photoprotective mechanism. Here, we present an experimental model in which this function can be shown for wild type Arabidopsis thaliana. We used blue light of different fluence rates for chloroplast positioning, and strong red light inactive in chloroplast positioning as a stressing light. The performance of photosystem II was measured by means of chlorophyll fluorescence. After stressing light treatment, a smaller decrease in photosystem II quantum yield was observed for leaves with chloroplasts in profile position as compared with leaves with chloroplasts in face position. Three Arabidopsis mutants, phot2 (no avoidance response), npq1 (impaired zeaxanhtin accumulation) and stn7 (no state transition), were examined for their chloroplast positioning and chlorophyll fluorescence parameters under identical experimental conditions. The results obtained for these mutants revealed additional stressing effects of blue light as compared with red light.