The ability to enhance photosynthetic capacity remains a recognized bottleneck to improving plant productivity. Phototropin blue light receptors (phot1 and phot2) optimize photosynthetic efficiency ...in Arabidopsis thaliana by coordinating multiple light-capturing processes. In this study, we explore the potential of using protein engineering to improve photoreceptor performance and thereby plant growth. We demonstrate that targeted mutagenesis can decrease or increase the photocycle lifetime of Arabidopsis phototropins in vitro and show that these variants can be used to reduce or extend the duration of photoreceptor activation in planta. Our findings show that slowing the phototropin photocycle enhanced several light-capturing responses, while accelerating it reduced phototropin’s sensitivity for chloroplast accumulation movement. Moreover, plants engineered to have a slow-photocycling variant of phot1 or phot2 displayed increased biomass production under low-light conditions as a consequence of their improved sensitivity. Together, these findings demonstrate the feasibility of engineering photoreceptors to manipulate plant growth and offer additional opportunities to enhance photosynthetic competence, particularly under suboptimal light regimes.
Ascorbate is involved in numerous vital processes, in particular in response to abiotic but also biotic stresses whose frequency and amplitude increase with climate change. Ascorbate levels vary ...greatly depending on species, tissues, or stages of development, but also in response to stress. Since its discovery, the ascorbate biosynthetic pathway has been intensely studied and it appears that GDP-l-galactose phosphorylase (GGP) is the enzyme with the greatest role in the control of ascorbate biosynthesis. Like other enzymes of this pathway, its expression is induced by various environmental and also developmental factors. Although mRNAs encoding it are among the most abundant in the transcriptome, the protein is only present in very small quantities. In fact, GGP translation is repressed by a negative feedback mechanism involving a small open reading frame located upstream of the coding sequence (uORF). Moreover, its activity is inhibited by a PAS/LOV type photoreceptor, the action of which is counteracted by blue light. Consequently, this multi-level regulation of GGP would allow fine control of ascorbate synthesis. Indeed, experiments varying the expression of GGP have shown that it plays a central role in response to stress. This new understanding will be useful for developing varieties adapted to future environmental conditions.
A new miniaturized micro sequential injection coupled with the lab-on-valve (µSIA–LOV) technique was full-blown to perform inhibitory studies on dihydrofolate reductase (DHFR). The system was used to ...evaluate the DHFR inhibition activity of metal-based anticancer compounds. The metal complexes exhibited IC50 values in the range 1.3 ± 0.3–108 ± 7 μM, with half of the complexes lying in the low μM range, i.e., 1.3 ± 0.3–4.4 ± 0.2 μM. For comparison, methotrexate (MTX), a known inhibitor of DHFR, has an IC50 value of 0.38 ± 0.06 μM. The µSIA–LOV is a versatile, robust, rapid, and easy to operate, allowing automated determination of DHFR inhibition. Moreover, the automated system requires very little sample (approximately 40 µL per analysis), uses minimal reagents (5 times less than the batch procedure used), and generates very little waste (around 1.2 mL per analysis) compared with batch methods, considerably reducing costs.
•DHFR is an attractive promising target to differentiate cancer from normal cells.•µSIA-LOV is developed to test DHFR inhibition of synthesized organometallic compounds.•Some of the metal compounds evaluated exhibit noteworthy DHFR inhibitory activity.•The µSIA-LOV system is a useful tool for the evaluation of DHFR enzymatic reaction.•Automation to assess enzymatic assays proved to be advantageous.
ABSTRACT
The problems arising as a result of aging aircraft, rail and civil infrastructure have focused attention on tools for predicting the growth of cracks from small naturally occurring material ...discontinuities. To this end, the present paper discusses on the difference between the analysis tools needed for ab initio design and sustainment, modelling of cracks that grow from small naturally occurring material discontinuities and ways to determine the short crack da/dN versus ΔK data from long crack American Society for Testing and Materials (ASTM) tests. It also discusses how existing equations can be used to predict short crack growth and how to account for the variations seen in crack growth histories. Attention is also focused on the recent Federal Aviation Administration limit of validity ruling and the effect of the environment on widespread fatigue damage in civil transport aircraft.
Optical manipulations are widely used to analyze neuronal functions in vivo. Blue light is frequently used to activate channelrhodopsins or LOV domains, although the degrees of its absorption and ...scattering are higher than those of longer wavelength light. High spatial resolution of optical manipulation is easily achieved in vitro, while the light is unevenly scattered and absorbed in tissues due to many factors. It is difficult to spatially measure a blue light transmission area in vivo. Here, we propose a genetic method to visualize blue light transmission in the brain and other organs using light-induced nuclear translocation of fluorescent proteins with a LOV domain. A light-inducible nuclear localization signal (LINuS) consists of a LOV2 domain fused with a nuclear localization signal (NLS). We confirmed that blue light illumination induced reversible translocation of NES-tdTomato-LINuS from the cytosol to the nucleus within 30 min in HEK293 cells. By employing a PHP.eb capsid that can penetrate the blood-brain barrier, retro-orbital sinus injection of adeno-associated virus (AAV) vectors induced scattered expression of nuclear export signal (NES)-tdTomato-LINuS in the brain. We confirmed that 30-min transcranial blue light illumination induced nuclear translocation of NES-tdTomato-LINuS in the cortex, the hippocampus, and even the paraventricular nucleus of the thalamus. We also found that mice exposed to blue light in a shaved abdominal area exhibited a substantial increase in nuclear translocation in the ventral surface lobe of the liver. These results provide a simple way to obtain useful information on light transmission in tissues without any transgenic animals or skillful procedures.
•We achieved light-dependent nuclear translocation of tdTomato by employing a light-inducible nuclear localization signal.•We achieved scattered expression of NES-tdTomato-LINuS in the brain by simple intravenous injection of AAV vectors.•We achieved visualization of blue light transmission areas by quantifying nuclear translocation of NES-tdTomato-LINuS.
We report natural light–oxygen–voltage (LOV) photoreceptors with a blue light-switched, high-affinity (K
D ∼ 10−7 M), and direct electrostatic interaction with anionic phospholipids. Membrane ...localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure–function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.
Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of ...flavin‐binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time‐resolved and low‐temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein‐flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission, and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low‐temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine the x‐ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color‐tuning mechanisms of flavin‐binding proteins and could be helpful for the engineering of new variants with improved optical properties.
Phototropin (phot) is a blue light (BL) receptor and thermosensor that mediates chloroplast movements in plants. Liverworts, as early‐diverging plant species, have a single copy of PHOT gene, and the ...phot protein in each liverwort activates the signaling pathway adapted to its specific growing environment. In this study, we functionally compared phot from two different liverworts species: Apopellia endiviifolia (Aephot) and Marchantia polymorpha (Mpphot). The BL‐dependent photochemical activity of Aephot was similar to that of Mpphot, whereas the thermochemical activity of Aephot was lower than that of Mpphot. Therefore, the phot‐mediated signaling pathways of the two plant species may differ more in response to temperature than to BL. Furthermore, we analyzed the functional compatibility of Aephot and Mpphot in chloroplast movements by transiently expressing AePHOT or MpPHOT. The transient expression of AePHOT did not mediate chloroplast movement in M. polymorpha, showing the incompatibility of Aephot with the signaling pathway of M. polymorpha. By contrast, the transient expression of MpPHOT mediated chloroplast movement in A. endiviifolia, indicating the compatibility of Mpphot with the signaling pathway of A. endiviifolia. Our findings reveal both functional similarities and differences between Aephot and Mpphot proteins from the closely related liverworts.
The phototropin (phot) of two closed liverworts species: Apopellia endiviifolia (Aephot) and Marchantia polymorpha (Mpphot) have been functionally compared in this study. Both Aephot and Mpphot show similarities in their photocycle, but diversity in the functions of regulating chloroplast movement. The transient expression of AePHOT did not mediate chloroplast movement in M. polymorpha, showing the incompatibility of Aephot with the signaling pathway of M. polymorpha. By contrast, the transient expression of MpPHOT‐mediated chloroplast movement in A. endiviifolia indicates the compatibility of Mpphot with the signaling pathway of A. endiviifolia.
Ferromagnesian chondrules present a remarkable dichotomy between reduced (type I) and oxidized (type II) varieties. How these formed, and how they may be related remains contentious. Many type II ...chondrules, especially in carbonaceous chondrites, contain forsteritic grains in disequilibrium with FeO-rich host olivine grains, which must be relicts of precursor material. In this study, we analyzed the oxygen isotopic composition of magnesian relict and host olivine grains in type II chondrules in CO and CR chondrites. The analyzed Mg-rich relicts are generally more 16O-rich than ferroan olivine (mostly host) grains and plot in the range (in term of chemistry and isotopic composition) of type I chondrules in carbonaceous chondrites. Remarkably, they tend to cluster around the dominant Δ17O peaks of the type I chondrules in their host chondrites, viz. –6 ‰ and –2 ‰ for CO and CR, respectively. With the occurrence of relatively intact type I chondrules within some type II chondrules, this corroborates that local type I chondrules were among the precursors of type II chondrules, and that chondrule formation occurred within the accretion reservoir of the eventual chondrites. This supports the nebular brand of chondrule-forming scenarios. Since not all previous generations of chondrules (or other precursor objects) have been recycled, chondrule formation events must also have been extremely localized.
PDF
