Phytochrome B Phosphorylation Expanded: Site‐Specific Kinases are Identified

The phytochrome B (phyB) photoreceptor is a key participant in red and far‐red light sensing, playing a dominant role in many developmental and growth responses throughout the whole life of plants. Accordingly, phyB governs diverse signaling pathways, and although our knowledge about these pathways is constantly expanding, our view about their fine‐tuning is still rudimentary. Phosphorylation of phyB is one of the relevant regulatory mechanisms, and – despite the expansion of the available methodology – it is still not easy to examine. Phosphorylated phytochromes have been detected using various techniques for decades, but the first phosphorylated phyB residues were only identified in 2013. Since then, concentrated attention has been turned toward the functional role of post‐translational modifications in phyB signaling. Very recently in 2023, the first kinases that phosphorylate phyB were identified. These discoveries opened up new research avenues, especially by connecting diverse environmental impacts to light signaling and helping to explain some long‐term unsolved problems such as the co‐action of Ca 2+ and phyB signaling. This review summarizes our recent views about the roles of the identified phosphorylated phyB residues, what we know about the enzymes that modulate the phospho‐state of phyB, and how these recent discoveries impact future investigations

Prooxidáns és antioxidáns növényi gének a nem-gazda betegségrezisztenciában - funkció meghatározás géncsendesítéssel = Prooxidant and antioxidant plant genes in non-host resistance - functional identification by gene silencing

Prooxidáns, antioxidáns és programozott sejthalállal kapcsolatos gének mRNS-szintű kifejeződését vizsgáltuk az ún. nem-gazda rezisztenciában (=rezisztencia a más növényfajokat fertőző kórokozókkal szemben), vírus-, baktérium- és gomba kórokozókkal fertőzött dohányfélékben (Nicotiana spp.) és árpában. Kimutattuk, hogy a prooxidáns/antioxidáns egyensúly ebben a hatékony, gyors lefolyású rezisztenciaformában is döntő szerepet játszik: a prooxidánsok - elsősorban a szuperoxid - korai felhalmozódását antioxidáns (pl. szuperoxid-dizmutáz, glutation-S-transzferáz) és sejthalál-gátló (BAX inhibitor) gének által termelt fehérjék ellensúlyozzák. Kutatásaink szerint tehát a nem-gazda rezisztencia egyik kulcslépése lehet a kórokozókat gátló korai prooxidáns-felhalmozódás, ill. ezzel egyidőben a megtámadott növény antioxidáns kapacitásának gyors indukciója. Igazoltuk, hogy egy vírus kórokozó (TMV) ellen ható rezisztencia gén (N) terméke egy nem-gazda rezisztenciát előidéző másik vírus (TNV) fertőzésekor pontosan ellenkező hatást válthat ki, ugyanis fogékonysági faktorként hathat. Az N gént Nicotiana edwardsonii növényekben csendesítve (expresszióját gátolva) ugyanis a TMV-vel szembeni rezisztencia sérült, a TNV-vel szembeni nem-gazda rezisztencia viszont fokozódott. A vírusokkal szembeni nem-gazda rezisztenciát befolyásoló növényi gének azonosítása/jellemzése a jövő rezisztencia-nemesítését teheti még eredményesebbé. | We have investigated expression of plant genes involved in prooxidant and antioxidant effects and inhibition of programmed cell death during non-host resistance (i.e. resistance to pathogens that infect other plant species) to viral, bacterial and fungal pathogens in Nicotiana spp. and barley. We have shown the pivotal role of prooxidant/antioxidant balance during this quick and effective form of resistance: the early accumulation of prooxidants - primarily superoxide - is counteracted by proteins encoded by antioxidant (e.g. superoxide dismutase and glutathione S-transferase) and cell death inhibitor (BAX inhibitor) genes. According to our research a key component of non-host resistance could be the early prooxidant accumulation that inhibits pathogens and the quick induction of the antioxidant capacity of attacked plants. We have pointed to the fact that the product of a plant resistance gene (N) effective against a virus (TMV) could have a completely opposite effect during infection by a virus (TNV) that elicits non-host resistance: in the latter case the same product may function as a susceptibility factor. Following silencing of the N gene in Nicotiana edwardsonii resistance to TMV was compromised, while non-host resistance to TNV was enhanced. Identification and characterization of plant genes that influence non-host resistance to viruses could provide valuable information for resistance breeding in the future

Optogenetics : past, present and future

The term ‘optogenetics’ was introduced into the scientific literature less than a decade ago by Karl Deisseroth, developer of pioneering optogenetic techniques, who defined optogenetics as “the combination of genetic and optical methods to achieve gain or loss of function of well-defined events in specific cells of living tissue”. Since then this new field of biology has become a very exciting and rapidly developing area producing hundreds of scientific publications. New methods and tools have been developed and long-sought answers found in these new experimental systems. Discussion and full elaboration of every optogenetic approach and application are beyond the scope of this review, instead, it gives a short insight to (i) how light can be used to manipulate the membrane potential of various cells; (ii) how light-sensitive proteins can be used to regulate targeted gene expression, and (iii) how controlled release or spatio-temporal targeting of certain molecules can be modulated by light. Besides, the most widely used light-sensor proteins, including their structure, working mechanism and their involvement in existing optogenetic applications are also discussed

Ultraviolet-B induced phototropism in Arabidopsis seedlings and inflorescence stems

Ultraviolet-B radiation (UV-B - 280-315nm) was only recently described as a phototropism-inducing type of radiation. We have identified that phototropic growth towards UV-B of Arabidopsis hypocotyls and inflorescence stems are both regulated by the same photoreceptor pathways but display a shift in dominance during plant development. The phototropin pathway is dominant in etiolated seedlings (Vanhaelewyn et al., 2016a), while the UVR8 pathway is predominant in inflorescence stems. The role of key-players in the UVR8 pathway, such as HY5 and HYH has been evaluated for their importance in this phototropic response. Unilaterally UV-B irradiated inflorescence stem tissue demonstrates a lateral UVR8-mediated signal gradient. In addition, the function of UVR8 in different cell types was validated by use of cell type specific complementation lines of UVR8 in a uvr8-6 mutant background. This reveals that UVR8 signaling is important in various cell types within the inflorescence stem. As UV-B is known to affect plant hormones (Vanhaelewyn et al., 2016b), we investigated their involvement in this phototropic response. By means of reporter line analysis, mutant analysis, gene expression assays and pharmacological assays, we designate a role for both gibberellins and auxins to UV-B induced phototropism of Arabidopsis inflorescence stems. These combined data provide a mechanistic framework for UV-B induced phototropism

Comparative functional analysis of full-length and N-terminal fragments of phytochrome C, D and E in red light-induced signaling.

Phytochromes (phy) C, D and E are involved in the regulation of red/far-red light-induced photomorphogenesis of Arabidopsis thaliana, but only limited data are available on the mode of action and biological function of these lesser studied phytochrome species. We fused N-terminal fragments or full-length PHYC, D and E to YELLOW FLUORESCENT PROTEIN (YFP), and analyzed the function, stability and intracellular distribution of these fusion proteins in planta. The activity of the constitutively nuclear-localized homodimers of N-terminal fragments was comparable with that of full-length PHYC, D, E-YFP, and resulted in the regulation of various red light-induced photomorphogenic responses in the studied genetic backgrounds. PHYE-YFP was active in the absence of phyB and phyD, and PHYE-YFP controlled responses, as well as accumulation, of the fusion protein in the nuclei, was saturated at low fluence rates of red light and did not require functional FAR-RED ELONGATED HYPOCOTYL1 (FHY-1) and FHY-1-like proteins. Our data suggest that PHYC-YFP, PHYD-YFP and PHYE-YFP fusion proteins, as well as their truncated N-terminal derivatives, are biologically active in the modulation of red light-regulated photomorphogenesis. We propose that PHYE-YFP can function as a homodimer and that low-fluence red light-induced translocation of phyE and phyA into the nuclei is mediated by different molecular mechanisms

Phosphorylation of phytochrome B inhibits light-induced signaling via accelerated dark reversion in Arabidopsis

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (lmax = 730 nm) and inactive Pr (lmax = 660 nm) forms in a red/far-red–dependent fashion and regulates, as molecular switch, many aspects of lightdependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein–protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB–yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyBSer86Asp Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light–induced nuclear import and interaction of phyBSer86Asp-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB–green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling

A deep learning-based approach for high-throughput hypocotyl phenotyping

Hypocotyl length determination is a widely used method to phenotype young seedlings. The measurement itself has advanced from using rulers and millimetre papers to assessing digitized images but remains a labour-intensive, monotonous and time-consuming procedure. To make high-throughput plant phenotyping possible, we developed a deep learning-based approach to simplify and accelerate this method. Our pipeline does not require a specialized imaging system but works well with low-quality images produced with a simple flatbed scanner or a smartphone camera. Moreover, it is easily adaptable for a diverse range of datasets not restricted to Arabidopsis (Arabidopsis thaliana). Furthermore, we show that the accuracy of the method reaches human performance. We not only provide the full code at https://github.com/biomag-lab/hypocotyl-UNet, but also give detailed instructions on how the algorithm can be trained with custom data, tailoring it for the requirements and imaging setup of the user

UVR8-dependent reporters reveal spatial characteristics of signal spreading in plant tissues

The UV Resistance Locus 8 (UVR8) photoreceptor controls UV-B mediated photomorphogenesis in Arabidopsis. The aim of this work is to collect and characterize different molecular reporters of photomorphogenic UV-B responses. Browsing available transcriptome databases, we identified sets of genes responding specifically to this radiation and are controlled by pathways initiated from the UVR8 photoreceptor. We tested the transcriptional changes of several reporters and found that they are regulated differently in different parts of the plant. Our experimental system led us to conclude that the examined genes are not controlled by light piping of UV-B from the shoot to the root or signalling molecules which may travel between different parts of the plant body but by local UVR8 signalling. The initiation of these universal signalling steps can be the induction of Elongated Hypocotyl 5 (HY5) and its homologue, HYH transcription factors. We found that their transcript and protein accumulation strictly depends on UVR8 and happens in a tissue autonomous manner. Whereas HY5 accumulation correlates well with the UVR8 signal across cell layers, the induction of flavonoids depends on both UVR8 signal and a yet to be identified tissue-dependent or developmental determinant

Differential phosphorylation of the N - terminal extension regulates phytochrome B signaling

Viczián, András. Institute of Plant Biology. Biological Research Centre. Szeged, Hungary.Ádám, Éva. Institute of Plant Biology. Biological Research Centre. Szeged, Hungary.Staudt, Anne Marie. University of Freiburg. Institute of Biology II. Freiburg, Germany.Lambert, Dorothee. University of Freiburg. Institute of Biology II. Freiburg, Germany.Klement, Eva. Biological Research Centre. Laboratory of Proteomics Research. Szeged, Hungary.Romero Montepaone, Sofia. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura (IFEVA). Buenos Aires, Argentina.Hiltbrunner, Andreas. University of Freiburg. Institute of Biology II. Freiburg, Germany.Casal, Jorge José. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura (IFEVA). Buenos Aires, Argentina.1635–1650Phytochrome B (phyB) is an excellent light quality and quantity sensor that can detect subtle changes in the light environment. The relative amounts of the biologically active photoreceptor (phyB Pfr) are determined by the light conditions and light independent thermal relaxation of Pfr into the inactive phyB Pr, termed thermal reversion. Little is known about the regulation of thermal reversion and how it affects plants’ light sensitivity. In this study we identified several serine/threonine residues on the N-terminal extension (NTE) of Arabidopsis thaliana phyB that are differentially phosphorylated in response to light and temperature, and examined transgenic plants expressing nonphosphorylatable and phosphomimic phyB mutants. The NTE of phyB is essential for thermal stability of the Pfr form, and phosphorylation of S86 particularly enhances the thermal reversion rate of the phyB Pfr–Pr heterodimer in vivo. We demonstrate that S86 phosphorylation is especially critical for phyB signaling compared with phosphorylation of the more N terminal residues. Interestingly, S86 phosphorylation is reduced in light, paralleled by a progressive Pfr stabilization under prolonged irradiation. By investigating other phytochromes (phyD and phyE) we provide evidence that acceleration of thermal reversion by phosphorylation represents a general mechanism for attenuating phytochrome signaling