Photoreceptor-induced sinapate synthesis contributes to photoprotection in Arabidopsis.

Plants must balance light capture for photosynthesis with protection from potentially harmful ultraviolet radiation (UV). Photoprotection is mediated by concerted action of photoreceptors, but the underlying molecular mechanisms are not fully understood. In this study, we provide evidence that UV RESISTANCE LOCUS 8 (UVR8) UV-B-, phytochrome red-, and cryptochrome blue-light photoreceptors converge on the induction of FERULIC ACID 5-HYDROXYLASE 1 (FAH1) that encodes a key enzyme in the phenylpropanoid biosynthesis pathway, leading to the accumulation of UV-absorbing sinapate esters in Arabidopsis (Arabidopsis thaliana). FAH1 induction depends on the bZIP transcription factors ELONGATED HYPOCOTYL 5 (HY5) and HY5-HOMOLOG (HYH) that function downstream of all three photoreceptors. Noticeably, mutants with hyperactive UVR8 signaling rescue fah1 UV sensitivity. Targeted metabolite profiling suggests that this phenotypic rescue is due to the accumulation of UV-absorbing metabolites derived from precursors of sinapate synthesis, namely coumaroyl-glucose and feruloyl-glucose. Our genetic dissection of the phenylpropanoid pathway combined with metabolomic and physiological analyses show that both sinapate esters and flavonoids contribute to photoprotection with sinapates playing a major role for UV screening. Our findings indicate that photoreceptor-mediated regulation of FAH1 and subsequent accumulation of sinapate "sunscreen" compounds is a key protective mechanism to mitigate damage, preserving photosynthetic performance, and ensuring plant survival under UV.

UV RESISTANCE LOCUS 8-Mediated UV-B Response Is Required Alongside CRYPTOCHROME 1 for Plant Survival in Sunlight under Field Conditions.

As sessile, photoautotrophic organisms, plants are subjected to fluctuating sunlight that includes potentially detrimental ultraviolet-B (UV-B) radiation. Experiments under controlled conditions have shown that the UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) controls acclimation and tolerance to UV-B in Arabidopsis thaliana; however, its long-term impact on plant fitness under naturally fluctuating environments remain poorly understood. Here, we quantified the survival and reproduction of different Arabidopsis mutant genotypes under diverse field and laboratory conditions. We found that uvr8 mutants produced more fruits than wild type when grown in growth chambers under artificial low-UV-B conditions but not under natural field conditions, indicating a fitness cost in the absence of UV-B stress. Importantly, independent double mutants of UVR8 and the blue light photoreceptor gene CRYPTOCHROME 1 (CRY1) in two genetic backgrounds showed a drastic reduction in fitness in the field. Experiments with UV-B attenuation in the field and with supplemental UV-B in growth chambers demonstrated that UV-B caused the cry1 uvr8 conditional lethal phenotype. Using RNA-seq data of field-grown single and double mutants, we explicitly identified genes showing significant statistical interaction of UVR8 and CRY1 mutations in the presence of UV-B in the field. They were enriched in Gene Ontology categories related to oxidative stress, photoprotection and DNA damage repair in addition to UV-B response. Our study demonstrates the functional importance of the UVR8-mediated response across life stages in natura, which is partially redundant with that of cry1. Moreover, these data provide an integral picture of gene expression associated with plant responses under field conditions.

NRF2 Shortage in Human Skin Fibroblasts Dysregulates Matrisome Gene Expression and Affects Collagen Fibrillogenesis.

NRF2 is a master regulator of the antioxidative response that was recently proposed as a potential regulator of extracellular matrix (ECM) gene expression. Fibroblasts are major ECM producers in all connective tissues, including the dermis. A better understanding of NRF2-mediated ECM regulation in skin fibroblasts is thus of great interest for skin homeostasis maintenance and aging protection. In this study, we investigate the impact of NRF2 downregulation on matrisome gene expression and ECM deposits in human primary dermal fibroblasts. RNA-sequencing‒based transcriptome analysis of NRF2 silenced dermal fibroblasts shows that ECM genes are the most regulated gene sets, highlighting the relevance of the NRF2-mediated matrisome program in these cells. Using complementary light and electron microscopy methods, we show that NRF2 deprivation in dermal fibroblasts results in reduced collagen I biosynthesis and impacts collagen fibril deposition. Moreover, we identify ZNF469, a putative transcriptional regulator of collagen biosynthesis, as a target of NRF2. Both ZNF469 silenced fibroblasts and fibroblasts derived from Brittle Corneal Syndrome patients carrying variants in ZNF469 gene show reduced collagen I gene expression. Our study shows that NRF2 orchestrates matrisome expression in human skin fibroblasts through direct or indirect transcriptional mechanisms that could be prioritized to target dermal ECM homeostasis in health and disease.

A perfusable vascularized full-thickness skin model for potential topical and systemic applications.

Vascularization of reconstructed tissues is one of the remaining hurdles to be considered to improve both the functionality and viability of skin grafts and the relevance ofin vitroapplications. Our study, therefore, sought to develop a perfusable vascularized full-thickness skin equivalent that comprises a more complex blood vasculature compared to existing models. We combined molding, auto-assembly and microfluidics techniques in order to create a vascularized skin equivalent representing (a) a differentiated epidermis with a physiological organization and correctly expressing K14, K10, Involucrin, TGM1 and Filaggrin, (b) three perfusable vascular channels with angiogenic sprouts stained with VE-Caderin and Collagen IV, (c) an adjacent microvascular network created via vasculogenesis and connected to the sprouting macrovessels. Histological analysis and immunostaining of CD31, Collagen IV, Perlecan and Laminin proved the integrity of vascular constructs. In order to validate the vascularized skin potential of topical and systemic applications, caffeine and minoxidil, two compounds with different chemical properties, were topically applied to measure skin permeability and benzo[a]pyrene pollutant was systemically applied to evaluate systemic delivery. Our results demonstrated that perfusion of skin reconstructs and the presence of a complex vascular plexus resulted in a more predictive and reliable model to assess respectively topical and systemic applications. This model is therefore aimed at furthering drug discovery and improving clinical translation in dermatology.

Sulphur availability modulates Arabidopsis thaliana responses to iron deficiency.

Among the mineral nutrients that are required for plant metabolism, iron (Fe) and sulphur (S) play a central role as both elements are needed for the activity of several proteins involved in essential cellular processes. A combination of physiological, biochemical and molecular approaches was employed to investigate how S availability influences plant response to Fe deficiency, using the model plant Arabidopsis thaliana. We first observed that chlorosis symptom induced by Fe deficiency was less pronounced when S availability was scarce. We thus found that S deficiency inhibited the Fe deficiency induced expression of several genes associated with the maintenance of Fe homeostasis. This includes structural genes involved in Fe uptake (i.e. IRT1, FRO2, PDR9, NRAMP1) and transport (i.e. FRD3, NAS4) as well as a subset of their upstream regulators, namely BTS, PYE and the four clade Ib bHLH. Last, we found that the over accumulation of manganese (Mn) in response to Fe shortage was reduced under combined Fe and S deficiencies. These data suggest that S deficiency inhibits the Fe deficiency dependent induction of the Fe uptake machinery. This in turn limits the transport into the root and the plant body of potentially toxic divalent cations such as Mn and Zn, thus limiting the deleterious effect of Fe deprivation.

Cryptochrome-mediated blue-light signalling modulates UVR8 photoreceptor activity and contributes to UV-B tolerance in Arabidopsis.

UV-B constitutes a critical part of the sunlight reaching the earth surface. The homodimeric plant UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) monomerizes in response to UV-B and induces photomorphogenic responses, including UV-B acclimation and tolerance. REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 are negative feedback regulators that operate by facilitating UVR8 ground state reversion through re-dimerization. Here we show that RUP1 and RUP2 are transcriptionally induced by cryptochrome photoreceptors in response to blue light, which is dependent on the bZIP transcriptional regulator ELONGATED HYPOCOTYL 5 (HY5). Elevated RUP1 and RUP2 levels under blue light enhance UVR8 re-dimerization, thereby negatively regulating UVR8 signalling and providing photoreceptor pathway cross-regulation in a polychromatic light environment, as is the case in nature. We further show that cryptochrome 1, as well as the red-light photoreceptor phytochrome B, contribute to UV-B tolerance redundantly with UVR8. Thus, photoreceptors for both visible light and UV-B regulate UV-B tolerance through an intricate interplay allowing the integration of diverse sunlight signals.

Transcriptional integration of the responses to iron availability in Arabidopsis by the bHLH factor ILR3.

Iron (Fe) homeostasis is crucial for all living organisms. In mammals, an integrated posttranscriptional mechanism couples the regulation of both Fe deficiency and Fe excess responses. Whether in plants an integrated control mechanism involving common players regulates responses both to deficiency and to excess is still to be determined. In this study, molecular, genetic and biochemical approaches were used to investigate transcriptional responses to both Fe deficiency and excess. A transcriptional activator of responses to Fe shortage in Arabidopsis, called bHLH105/ILR3, was found to also negatively regulate the expression of ferritin genes, which are markers of the plant's response to Fe excess. Further investigations revealed that ILR3 repressed the expression of several structural genes that function in the control of Fe homeostasis. ILR3 interacts directly with the promoter of its target genes, and repressive activity was conferred by its dimerisation with bHLH47/PYE. Last, this study highlighted that important facets of plant growth in response to Fe deficiency or excess rely on ILR3 activity. Altogether, the data presented herein support that ILR3 is at the centre of the transcriptional regulatory network that controls Fe homeostasis in Arabidopsis, in which it acts as both transcriptional activator and repressor.

TCTP and CSN4 control cell cycle progression and development by regulating CULLIN1 neddylation in plants and animals.

Translationally Controlled Tumor Protein (TCTP) controls growth by regulating the G1/S transition during cell cycle progression. Our genetic interaction studies show that TCTP fulfills this role by interacting with CSN4, a subunit of the COP9 Signalosome complex, known to influence CULLIN-RING ubiquitin ligases activity by controlling CULLIN (CUL) neddylation status. In agreement with these data, downregulation of CSN4 in Arabidopsis and in tobacco cells leads to delayed G1/S transition comparable to that observed when TCTP is downregulated. Loss-of-function of AtTCTP leads to increased fraction of deneddylated CUL1, suggesting that AtTCTP interferes negatively with COP9 function. Similar defects in cell proliferation and CUL1 neddylation status were observed in Drosophila knockdown for dCSN4 or dTCTP, respectively, demonstrating a conserved mechanism between plants and animals. Together, our data show that CSN4 is the missing factor linking TCTP to the control of cell cycle progression and cell proliferation during organ development and open perspectives towards understanding TCTP's role in organ development and disorders associated with TCTP miss-expression.

Distinct molecular cues ensure a robust microtubule-dependent nuclear positioning in the Drosophila oocyte.

Controlling nucleus localization is crucial for a variety of cellular functions. In the Drosophila oocyte, nuclear asymmetric positioning is essential for the reorganization of the microtubule (MT) network that controls the polarized transport of axis determinants. A combination of quantitative three-dimensional live imaging and laser ablation-mediated force analysis reveal that nuclear positioning is ensured with an unexpected level of robustness. We show that the nucleus is pushed to the oocyte antero-dorsal cortex by MTs and that its migration can proceed through distinct tracks. Centrosome-associated MTs favour one migratory route. In addition, the MT-associated protein Mud/NuMA that is asymmetrically localized in an Asp-dependent manner at the nuclear envelope hemisphere where MT nucleation is higher promotes a separate route. Our results demonstrate that centrosomes do not provide an obligatory driving force for nuclear movement, but together with Mud, contribute to the mechanisms that ensure the robustness of asymmetric nuclear positioning.

Coordination between Intra- and Extracellular Forces Regulates Focal Adhesion Dynamics.

Focal adhesions (FAs) are important mediators of cell-substrate interactions. One of their key functions is the transmission of forces between the intracellular acto-myosin network and the substrate. However, the relationships between cell traction forces, FA architecture, and molecular forces within FAs are poorly understood. Here, by combining Förster resonance energy transfer (FRET)-based molecular force biosensors with micropillar-based traction force sensors and high-resolution fluorescence microscopy, we simultaneously map molecular tension across vinculin, a key protein in FAs, and traction forces at FAs. Our results reveal strong spatiotemporal correlations between vinculin tension and cell traction forces at FAs throughout a wide range of substrate stiffnesses. Furthermore, we find that molecular tension within individual FAs follows a biphasic distribution from the proximal (toward the cell nucleus) to distal end (toward the cell edge). Using super-resolution imaging, we show that such a distribution relates to that of FA proteins. On the basis of our experimental data, we propose a model in which FA dynamics results from tension changes along the FAs.

Fast and Efficient Cloning of Cis-Regulatory Sequences for High-Throughput Yeast One-Hybrid Analyses of Transcription Factors.

Yeast one-hybrid (Y1H) assay has been proven to be a powerful technique to characterize in vivo the interaction between a given transcription factor (TF), or its DNA-binding domain (DBD), and target DNA sequences. Comprehensive characterization of TF/DBD and DNA interactions should allow designing synthetic promoters that would undoubtedly be valuable for biotechnological approaches. Here, we use the ligation-independent cloning system (LIC) in order to enhance the cloning efficiency of DNA motifs into the pHISi Y1H vector. LIC overcomes important limitations of traditional cloning technologies, since any DNA fragment can be cloned into LIC compatible vectors without using restriction endonucleases, ligation, or in vitro recombination.

Diversity in cell motility reveals the dynamic nature of the formation of zebrafish taste sensory organs.

Taste buds are sensory organs in jawed vertebrates, composed of distinct cell types that detect and transduce specific taste qualities. Taste bud cells differentiate from oropharyngeal epithelial progenitors, which are localized mainly in proximity to the forming organs. Despite recent progress in elucidating the molecular interactions required for taste bud cell development and function, the cell behavior underlying the organ assembly is poorly defined. Here, we used time-lapse imaging to observe the formation of taste buds in live zebrafish larvae. We found that tg(fgf8a.dr17)-expressing cells form taste buds and get rearranged within the forming organs. In addition, differentiating cells move from the epithelium to the forming organs and can be displaced between developing organs. During organ formation, tg(fgf8a.dr17) and type II taste bud cells are displaced in random, directed or confined mode relative to the taste bud they join or by which they are maintained. Finally, ascl1a activity in the 5-HT/type III cell is required to direct and maintain tg(fgf8a.dr17)-expressing cells into the taste bud. We propose that diversity in displacement modes of differentiating cells acts as a key mechanism for the highly dynamic process of taste bud assembly.

Visualizing Microtubule Networks During Drosophila Oogenesis Using Fixed and Live Imaging.

The microtubule cytoskeleton is a plastic network of polarized cables. These polymers of tubulin provide orientated routes for the dynamic transport of cytoplasmic molecules and organelles, through which cell polarity is established and maintained. The role of microtubule-mediated transport in the asymmetric localization of axis polarity determinants, in the Drosophila oocyte, has been the subject of extensive studies in the past years. However, imaging the distribution of microtubule fibers in a large cell, where vitellogenesis ensures the uptake of a thick and hazy yolk, presents a series of technical challenges. This chapter briefly reviews some of these aspects and describes two methods designed to circumvent these difficulties. We provide a detailed protocol for the visualization by immunohistochemistry of the three-dimensional organization of tubulin cables in the oocyte. Additionally, we detail the stepwise procedure for the live imaging of microtubule dynamics and network remodeling, using fluorescently labeled microtubule-associated proteins.

Integration of P, S, Fe, and Zn nutrition signals in Arabidopsis thaliana: potential involvement of PHOSPHATE STARVATION RESPONSE 1 (PHR1).

Phosphate and sulfate are essential macro-elements for plant growth and development, and deficiencies in these mineral elements alter many metabolic functions. Nutritional constraints are not restricted to macro-elements. Essential metals such as zinc and iron have their homeostasis strictly genetically controlled, and deficiency or excess of these micro-elements can generate major physiological disorders, also impacting plant growth and development. Phosphate and sulfate on one hand, and zinc and iron on the other hand, are known to interact. These interactions have been partly described at the molecular and physiological levels, and are reviewed here. Furthermore the two macro-elements phosphate and sulfate not only interact between themselves but also influence zinc and iron nutrition. These intricated nutritional cross-talks are presented. The responses of plants to phosphorus, sulfur, zinc, or iron deficiencies have been widely studied considering each element separately, and some molecular actors of these regulations have been characterized in detail. Although some scarce reports have started to examine the interaction of these mineral elements two by two, a more complex analysis of the interactions and cross-talks between the signaling pathways integrating the homeostasis of these various elements is still lacking. However, a MYB-like transcription factor, PHOSPHATE STARVATION RESPONSE 1, emerges as a common regulator of phosphate, sulfate, zinc, and iron homeostasis, and its role as a potential general integrator for the control of mineral nutrition is discussed.

Iron around the clock.

Carbon assimilation, a key determinant of plant biomass production, is under circadian regulation. Light and temperature are major inputs of the plant clock that control various daily rhythms. Such rhythms confer adaptive advantages to the organisms by adjusting their metabolism in anticipation of environmental fluctuations. The relationship between the circadian clock and nutrition extends far beyond the regulation of carbon assimilation as mineral nutrition, and specially iron homeostasis, is regulated through this mechanism. Conversely, iron status was identified as a new and important input regulating the central oscillator, raising the question of the nature of the Fe-dependent signal that modulates the period of the circadian clock. Several lines of evidence strongly suggest that fully developed and functional chloroplasts as well as early light signalling events, involving phytochromes, are essential to couple the clock to Fe responses. Nevertheless, the exact nature of the signal, which most probably involves unknown or not yet fully characterized elements of the chloroplast-to-nucleus retrograde signalling pathway, remains to be identified. Finally, this regulation may also involves epigenetic components.

Arabidopsis ferritin 1 (AtFer1) gene regulation by the phosphate starvation response 1 (AtPHR1) transcription factor reveals a direct molecular link between iron and phosphate homeostasis.

A yeast one-hybrid screening allowed the selection of PHR1 as a factor that interacted with the AtFer1 ferritin gene promoter. In mobility shift assays, PHR1 and its close homologue PHL1 (PHR1-like 1) interact with Element 2 of the AtFer1 promoter, containing a P1BS (PHR1 binding site). In a loss of function mutant for genes encoding PHR1 and PHL1 (phr1 phl1 mutant), the response of AtFer1 to phosphate starvation was completely lost, showing that the two transcription factors regulate AtFer1 expression upon phosphate starvation. This regulation does not involve the IDRS (iron-dependent regulatory sequence) present in the AtFer1 promoter and involved in the iron-dependent regulation. The phosphate starvation response of AtFer1 is not linked to the iron status of plants and is specifically initiated by phosphate deficiency. Histochemical localization of iron, visualized by Perls DAB staining, was strongly altered in a phr1 phl1 mutant, revealing that both PHR1 and PHL1 are major factors involved in the regulation of iron homeostasis.

Fluorescence excitation analysis by two-photon confocal laser scanning microscopy: a new method to identify fluorescent nanoparticles on histological tissue sections.

In the present study, we make use of the ability of two-photon confocal laser scanning microscopes (CLSMs) equipped with tunable lasers to produce spectral excitation image sequences. Furthermore, unmixing, which is usually performed on emission image sequences, is performed on these excitation image sequences. We use factor analysis of medical image sequences (FAMIS), which produces factor images, to unmix spectral image sequences of stained structures in tissue sections to provide images of characterized stained cellular structures. This new approach is applied to histological tissue sections of mouse aorta containing labeled iron nanoparticles stained with Texas Red and counterstained with SYTO13, to obtain visual information about the accumulation of these nanoparticles in the arterial wall. The possible presence of Texas Red is determined using a two-photon CLSM associated with FAMIS via the excitation spectra. Texas Red and SYTO13 are thus differentiated, and corresponding factor images specify their possible presence and cellular localization. In conclusion, the designed protocol shows that sequences of images obtained by excitation in a two-photon CLSM enables characterization of Texas Red-stained nanoparticles and other markers. This methodology offers an alternative and complementary solution to the conventional use of emission spectra unmixing to localize fluorescent nanoparticles in tissue samples.

Impact of 7-ketocholesterol and very long chain fatty acids on oligodendrocyte lipid membrane organization: evaluation via LAURDAN and FAMIS spectral image analysis.

In the context of multiple sclerosis and X-linked adrenoleukodystrophy, 7-ketocholesterol (7KC) and very long chain fatty acids (C24:0, C26:0) are supposed to induce side effects respectively on oligodendrocytes which are myelin (which is a lipoproteic complex) synthesizing cells. The effects of 7KC (25, 50 µM), C24:0 and C26:0 (10, 20 µM) on cell viability and lipid membrane organization were investigated on 158N murine oligodendrocytes. Concerning 7KC and fatty acids (at 20 µM only): 1) cell growth was strongly inhibited; 2) marked induction of cell death was revealed with propidium iodide (PI); 3) no apoptotic cells were found with C24:0 and C26:0 (absence of cells with condensed and/or fragmented nuclei, of FLICA positive cells and of PI negative/SYTO16 negative cells); 4) some apoptotic cells were detected with 7KC. Fatty acids (at 20 µM only) and 7KC also induced a disorganization of lipid membranes revealed with Merocyanine 540. So, to point out the effects of 7KC (25 µM), C24:0 and C26:0 (20 µM) on the lateral organization of lipid membranes, we used LAURDAN, which gives simultaneous information about morphology and phase state of lipid domains: its emission is blue in the ordered lipid phase, green in the disordered lipid phase. To overcome the qualitative filtering settings of blue and green emission colors, data obtained by mono- and bi-photon confocal microscopy were analyzed by spectral analysis. Sequences of emission images were obtained on both mono- and bi-photon confocal microscopes and processed by means of Factor Analysis of Medical Image Sequences (FAMIS), which is a relevant tool to unmix emission spectra and provide pure color images. Only 7KC was capable to induce a green emission with LAURDAN. Thus, at concentrations inducing oligodendrocyte cell death, 7KC (25 µM) is more efficient than C24:0 and C26:0 (20 µM), to trigger lateral lipid membrane disorganization.