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.

The photoprotective dilemma of a chloroplast: to avoid high light or to quench the fire?

The safe and smooth functioning of photosynthesis in plants is ensured by the operation of numerous regulatory mechanisms that adjust the density of excitation resulting from photon absorption to the capabilities of the photosynthetic apparatus. Such mechanisms include the movement of chloroplasts inside cells and the quenching of electronic excitations in the pigment-protein complexes. Here, we address the problem of a possible cause-and-effect relationship between these two mechanisms. Both the light-induced chloroplast movements and quenching of chlorophyll excitations were analyzed simultaneously with the application of fluorescence lifetime imaging microscopy of Arabidopsis thaliana leaves, wild-type and impaired in chloroplast movements or photoprotective excitation quenching. The results show that both regulatory mechanisms operate over a relatively wide range of light intensities. By contrast, impaired chloroplast translocations have no effect on photoprotection at the molecular level, indicating the direction of information flow in the coupling of these two regulatory mechanisms: from the photosynthetic apparatus to the cellular level. The results show also that the presence of the xanthophyll zeaxanthin is necessary and sufficient for the full development of photoprotective quenching of excessive chlorophyll excitations in plants.

Transformation of European Ash (Fraxinus excelsior L.) Callus as a Starting Point for Understanding the Molecular Basis of Ash Dieback.

The population of European ash (Fraxinus excelsior L.) is currently facing the risk of collapse, mainly due to ash dieback, a disease caused by a pathogenic fungus, Hymenoscyphus fraxineus. To facilitate studies into the molecular basis of ash dieback and design breeding strategies for a generation of resistant trees, it is necessary to develop tools enabling the study of gene function in F. excelsior. Despite this, a method for the genetic engineering of F. excelsior is still missing. Here, we report the first successful genetic transformation of F. excelsior callus and a selection process enabling the formation of stable transgenic callus lines. The protocol relies on the use of Agrobacterium tumefaciens to transform callus tissue derived from embryos of F. excelsior. In our experiments, we used the ß-glucuronidase (GUS) reporter system to demonstrate the transformation of callus cells and performed RT-PCR experiments to confirm the stable expression of the transgene. Since ash dieback threatens the long-term stability of many native F. excelsior populations, we hope that the transformation techniques described in this manuscript will facilitate rapid progress in uncovering the molecular basis of the disease and the validation of gene targets previously proposed to be linked to the resistance of trees to H. fraxineus pathogenicity.

Arabidopsis Phototropins Participate in the Regulation of Dark-Induced Leaf Senescence.

Senescence is the final stage of plant development, affecting individual organs or the whole organism, and it can be induced by several environmental factors, including shading or darkness. Although inevitable, senescence is a complex and tightly regulated process, ensuring optimal remobilization of nutrients and cellular components from senescing organs. Photoreceptors such as phytochromes and cryptochromes are known to participate in the process of senescence, but the involvement of phototropins has not been studied to date. We investigated the role of these blue light photoreceptors in the senescence of individually darkened Arabidopsis thaliana leaves. We compared several physiological and molecular senescence markers in darkened leaves of wild-type plants and phototropin mutants (phot1, phot2, and phot1phot2). In general, all the symptoms of senescence (lower photochemical activity of photosystem II, photosynthetic pigment degradation, down-regulation of photosynthetic genes, and up-regulation of senescence-associated genes) were less pronounced in phot1phot2, as compared to the wild type, and some also in one of the single mutants, indicating delayed senescence. This points to different mechanisms of phototropin operation in the regulation of senescence-associated processes, either with both photoreceptors acting redundantly, or only one of them, phot1, playing a dominant role.

Dynamics of Etiolation Monitored by Seedling Morphology, Carotenoid Composition, Antioxidant Level, and Photoactivity of Protochlorophyllide in Arabidopsis thaliana.

Although etiolated Arabidopsis thaliana seedlings are widely used as a model to study the de-etiolation process, the etiolation itself at the molecular level still needs elucidation. Here, we monitored the etiolation dynamics for wild type A. thaliana seedlings and lutein-deficient (lut2) mutant between 2 and 12 days of their growth in the absence of light. We analyzed the shape of the apex, the growth rate, the carotenoids and protochlorophyllide (Pchlide) accumulation, and the light-dependent protochlorophyllide oxidoreductase (LPOR) transcripts. Differences concerning the apical hook curvature and cotyledon opening among seedlings of the same age were observed, mostly after day 6 of the culture. We categorized the observed apex shapes and presented quantitatively how distribution among the categories changed during 12 days of seedling growth. The Pchlide654/Pchlide633 ratio, corresponding to the amount of the photoactive Pchlide, was the highest in the youngest seedlings, and decreased with their age. LPORA, LPORB, and LPORC transcripts were detected in etiolated seedlings, and their content decreased during seedling growth. Expression of SAG12 or SAG13 senescence markers, depletion in antioxidants, and excess ion leakage were not observed during the etiolation. Lack of lutein in the lut2 mutant resulted in slow Pchlide accumulation and affected other xanthophyll composition.

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.

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.

ASG2 is a farnesylated DWD protein that acts as ABA negative regulator in Arabidopsis.

The tagging-via-substrate approach designed for the capture of mammal prenylated proteins was adapted to Arabidopsis cell culture. In this way, proteins are in vivo tagged with an azide-modified farnesyl moiety and captured thanks to biotin alkyne Click-iT® chemistry with further streptavidin-affinity chromatography. Mass spectrometry analyses identified four small GTPases and ASG2 (ALTERED SEED GERMINATION 2), a protein previously associated to the seed germination gene network. ASG2 is a conserved protein in plants and displays a unique feature that associates WD40 domains and tetratricopeptide repeats. Additionally, we show that ASG2 has a C-terminal CaaX-box that is farnesylated in vitro. Protoplast transfections using CaaX prenyltransferase mutants show that farnesylation provokes ASG2 nucleus exclusion. Moreover, ASG2 interacts with DDB1 (DAMAGE DNA BINDING protein 1), and the subcellular localization of this complex depends on ASG2 farnesylation status. Finally, germination and root elongation experiments reveal that asg2 and the farnesyltransferase mutant era1 (ENHANCED RESPONSE TO ABSCISIC ACID (ABA) 1) behave in similar manners when exposed to ABA or salt stress. To our knowledge, ASG2 is the first farnesylated DWD (DDB1 binding WD40) protein related to ABA response in Arabidopsis that may be linked to era1 phenotypes.