Different Roles of α- and β-Branch Xanthophylls in Photosystem Assembly and Photoprotection

Xanthophylls (oxygenated carotenoids) are essential components of the plant photosynthetic apparatus, where they act in photosystem assembly, light harvesting, and photoprotection. Nevertheless, the specific function of individual xanthophyll species awaits complete elucidation. In this work, we analyze the photosynthetic phenotypes of two newly isolated Arabidopsis mutants in carotenoid biosynthesis containing exclusively alpha-branch (chy1chy2lut5) or beta-branch (chy1chy2lut2) xanthophylls. Both mutants show complete lack of qE, the rapidly reversible component of nonphotochemical quenching, and high levels of photoinhibition and lipid peroxidation under photooxidative stress. Both mutants are much more photosensitive than npq1lut2, which contains high levels of viola- and neoxanthin and a higher stoichiometry of light-harvesting proteins with respect to photosystem II core complexes, suggesting that the content in light-harvesting complexes plays an important role in photoprotection. In addition, chy1chy2lut5, which has lutein as the only xanthophyll, shows unprecedented photosensitivity even in low light conditions, reduced electron transport rate, enhanced photobleaching of isolated LHCII complexes, and a selective loss of CP26 with respect to chy1chy2lut2, highlighting a specific role of beta-branch xanthophylls in photoprotection and in qE mechanism. The stronger photosystem II photoinhibition of both mutants correlates with the higher rate of singlet oxygen production from thylakoids and isolated light-harvesting complexes, whereas carotenoid composition of photosystem II core complex was not influential. In depth analysis of the mutant phenotypes suggests that alpha-branch (lutein) and beta-branch (zeaxanthin, violaxanthin, and neoxanthin) xanthophylls have distinct and complementary roles in antenna protein assembly and in the mechanisms of photoprotection

Light Sheet Fluorescence Microscopy Quantifies Calcium Oscillations in Root Hairs of Arabidopsis thaliana

Calcium oscillations play a role in the regulation of the development of tip-growing plant cells. Using optical microscopy, calcium oscillations have been observed in a few systems (e.g. pollen tubes, fungal hyphae and algal rhizoids). High-resolution, non-phototoxic and rapid imaging methods are required to study the calcium oscillation in root hairs. We show that light sheet fluorescence microscopy is optimal to image growing root hairs of Arabidopsis thaliana and to follow their oscillatory tip-focused calcium gradient. We describe a protocol for performing live imaging of root hairs in seedlings expressing the cytosol-localized ratiometric calcium indicator Yellow Cameleon 3.6. Using this protocol, we measured the calcium gradient in a large number of root hairs. We characterized their calcium oscillations and correlated them with the rate of hair growth. The method was then used to screen the effect of auxin on the properties of the growing root hairs

Full-aperture extended-depth oblique plane microscopy through dynamic remote focusing

Oblique plane microscopy is a method enabling light-sheet fluorescence imaging through a single microscope objective lens by focusing on a tilted plane within the sample. To focus the fluorescence emitted by the oblique plane on a camera, the light is imaged through a pair of remote objective lenses, facing each other at an angle. The aperture mismatch resulting from this configuration limits the effective numerical aperture of the system, reducing image resolution and signal intensity. This manuscript introduces an alternative method to capture the oblique plane on the camera. Instead of relying on angled objective lenses, an electrically tunable lens is employed. This lens adjusts the focal plane of the microscope synchronously with the rolling shutter of a scientific CMOS camera. In this configuration the entire aperture of the objective is effectively employed, increasing the resolution of the system. Moreover, a variety of objective lenses can be employed, enabling the acquisition of wider axial fields of view compared to conventional oblique plane microscopy

A quadruple mutant of Arabidopsis reveals a β-carotene hydroxylation activity for LUT1/CYP97C1 and a regulatory role of xanthophylls on determination of the PSI/PSII ratio

<p>Abstract</p> <p>Background</p> <p>Xanthophylls are oxygenated carotenoids playing an essential role as structural components of the photosynthetic apparatus. Xanthophylls contribute to the assembly and stability of light-harvesting complex, to light absorbance and to photoprotection. The first step in xanthophyll biosynthesis from α- and β-carotene is the hydroxylation of ε- and β-rings, performed by both non-heme iron oxygenases (CHY1, CHY2) and P450 cytochromes (LUT1/CYP97C1, LUT5/CYP97A3). The Arabidopsis triple <it>chy1chy2lut5 </it>mutant is almost completely depleted in β-xanthophylls.</p> <p>Results</p> <p>Here we report on the quadruple <it>chy1chy2lut2lut5 </it>mutant, additionally carrying the <it>lut2 </it>mutation (affecting lycopene ε-cyclase). This genotype lacks lutein and yet it shows a compensatory increase in β-xanthophylls with respect to <it>chy1chy2lut5 </it>mutant. Mutant plants show an even stronger photosensitivity than <it>chy1chy2lut5</it>, a complete lack of qE, the rapidly reversible component of non-photochemical quenching, and a peculiar organization of the pigment binding complexes into thylakoids. Biochemical analysis reveals that the <it>chy1chy2lut2lut5 </it>mutant is depleted in Lhcb subunits and is specifically affected in Photosystem I function, showing a deficiency in PSI-LHCI supercomplexes. Moreover, by analyzing a series of single, double, triple and quadruple Arabidopsis mutants in xanthophyll biosynthesis, we show a hitherto undescribed correlation between xanthophyll levels and the PSI-PSII ratio. The decrease in the xanthophyll/carotenoid ratio causes a proportional decrease in the LHCII and PSI core levels with respect to PSII.</p> <p>Conclusions</p> <p>The physiological and biochemical phenotype of the <it>chy1chy2lut2lut5 </it>mutant shows that (i) LUT1/CYP97C1 protein reveals a major β-carotene hydroxylase activity <it>in vivo </it>when depleted in its preferred substrate α-carotene; (ii) xanthophylls are needed for normal level of Photosystem I and LHCII accumulation.</p

Seroprevalence of Toxoplasma gondii and Salmonella in Hunted Wild Boars from Two Different Regions in Switzerland

Toxoplasma gondii and Salmonella are zoonotic foodborne pathogens that may be transmitted to humans through the consumption of raw or undercooked meat, including game. The aim of this study was to determine the seroprevalence of T. gondii and Salmonella antibodies in wild boars in two different regions in Switzerland. During the hunting season of 2020, a total of 126 diaphragm muscle samples of hunted wild boars were collected and the meat juice of these samples was analysed for pathogen-specific IgG antibodies using commercial enzyme-linked immunosorbent assay (ELISA) kits. The overall seroprevalences were 35% for T. gondii and 17% for Salmonella, respectively. In general, seropositivity increased with the age of the animals. Seroprevalences of T. gondii were similar for animals from the northern region (29%) to those from the southern region (36.8%), indicating that T. gondii is widespread in the sylvestrian environment. By contrast, Salmonella seropositivity was remarkably higher in wild boars from the north (52%) compared with those from the south (5.3%). The high occurrence of Salmonella may represent a risk of transmission to compatriot domestic animals such free-range farmed pigs as well as to humans. Further, meat of hunted wild boars may present a source of human toxoplasmosis or salmonellosis

Virtual unfolding of light sheet fluorescence microscopy dataset for quantitative analysis of the mouse intestine

Light sheet fluorescence microscopy has proven to be a powerful tool to image fixed and chemically cleared samples, providing in depth and high resolution reconstructions of intact mouse organs. We applied light sheet microscopy to image the mouse intestine. We found that large portions of the sample can be readily visualized, assessing the organ status and highlighting the presence of regions with impaired morphology. Yet, three-dimensional (3-D) sectioning of the intestine leads to a large dataset that produces unnecessary storage and processing overload. We developed a routine that extracts the relevant information from a large image stack and provides quantitative analysis of the intestine morphology. This result was achieved by a three step procedure consisting of: (1) virtually unfold the 3-D reconstruction of the intestine; (2) observe it layer-by-layer; and (3) identify distinct villi and statistically analyze multiple samples belonging to different intestinal regions. Even if the procedure has been developed for the murine intestine, most of the underlying concepts have a general applicability

The study of polyplex formation and stability by time-resolved fluorescence spectroscopy of SYBR Green I-stained DNA

Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency