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Correlations between lignin content and structural robustness in plants revealed by X-ray ptychography
摘要： Lignin is a heterogeneous aromatic polymer responsible for cell wall stiffness and protection from pathogen attack. However, lignin represents a bottleneck to biomass degradation due to its recalcitrance related to the natural cell wall resistance to release sugars for fermentation or further processing. A biological approach involving genetics and molecular biology was used to disrupt lignin pathway synthesis and decrease lignin deposition. Here, we imaged three-dimensional fragments of the petioles of wild type and C4H lignin mutant Arabidopsis thaliana plants by synchrotron cryo-ptychography. the three-dimensional images revealed the heterogeneity of vessels, parenchyma, and fibre cell wall morphologies, highlighting the relation between disturbed lignin deposition and vessel implosion (cell collapsing and obstruction of water flow). We introduce a new parameter to accurately define cell implosion conditions in plants, and we demonstrate how cryo-ptychographic X-ray computed tomography (cryo-PXCT) provides new insights for plant imaging in three dimensions to understand physiological processes.
关键词： lignin,structural robustness,X-ray ptychography,cryo-PXCT,plants,Arabidopsis thaliana
更新于2025-09-23 15:19:57
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[Methods in Molecular Biology] Phototropism Volume 1924 (Methods and Protocols) || Determination of Phototropism by UV-B Radiation
摘要： UV-B phototropism in etiolated Arabidopsis seedlings has only been shown recently and needs further exploration. Here we elaborate on how to generate a customized setup with a unilateral UV-B light source, the required plant materials, different growth substrates, and a framework for data analysis.
关键词： Medium reflectance,Arabidopsis seedlings,Medium fluorescence,Growth substrates,Phototropism,UV-B,UV-B light sources
更新于2025-09-19 17:15:36
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[IEEE 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) - Munich, Germany (2019.6.23-2019.6.27)] 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) - Terahertz Spectroscopy of Different Phenotypes of Arabidopsis Thaliana
摘要： The recent surge in ‘Precision agriculture’ is fueled by the acute need of food security felt worldwide to feed the global burgeoning population. To this effect, various optical techniques have recently been employed to characterize the biochemical processes in various parts of the plants [1]. Terahertz (THz) region (0.3 – 10 THz) has gathered momentum in this field as it is biologically safe, can penetrate food packaging and most importantly, has fingerprinting ability of various organic and inorganic molecules due to active rotational and vibrational modes [2]. Moreover, unlike other optical spectroscopic techniques, THz time domain spectroscopic (THz-TDS) technique records the information in form of THz electric field; which once converted to frequency domain provides both amplitude and phase information [3]. Additionally, THz waves are highly absorbed by water which in turn can be utilized for quantitative water status monitoring in plants [4]. In this work, we have used THz-TDS with a bandwidth over 5 THz for the detection of biochemicals present in two different T-DNA insertion mutants in two independent genes of Arabidopsis thaliana (Col-0 Columbia ecotype). The plants were grown from mutant seeds in controlled environment. For easier optical access to all parts of the plants, the seedlings were grown parallel to the surface in a petri dish of plant growth medium. Liu et al. has already reported successful discrimination of transgenic Soybean seeds using THz spectroscopy [5]. We have recorded spectroscopic data of several parts of all the mutant plants which were 5, 7, 12 and 13 days old kept under identical conditions. The time domain spectrum obtained from the experiment was converted to frequency domain. Figure 1 shows the frequency domain spectrum of one such set of spectroscopic data collected from the stem part of the two different mutants compared to wild type control Col-0 plants. As it is evident from the spectrum, there are many absorption peaks corresponding to water and other biochemical molecules. For example, the peaks at 1.7 THz and 2.6 THz corresponds to sucrose present in the plants and in the nutri-solution which has been taken as the reference [2]. Our ongoing study involves careful analysis of the variations of the molecular absorption peaks for different mutants which would not only enable a label-free genetic identification, but also would help in our understanding of the underlying shift in biochemical processes contributed by the specific gene.
关键词： Terahertz spectroscopy,Arabidopsis thaliana,THz-TDS,Precision agriculture,biochemical processes
更新于2025-09-16 10:30:52
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Cadmium sulfide quantum dots impact Arabidopsis thaliana physiology and morphology
摘要： The differential mechanisms of CdS QDs (Quantum Dots) and Cd ion toxicity to Arabidopsis thaliana (L.) Heynh were investigated. Plants were exposed to 40 and 60 mg L-1 for CdS QDs and 76.9 and 115.2 mg L-1 CdSO4·7H2O and toxicity was evaluated at 5, 20, 35 (T5, T20, T35) days after exposure. Oxidative stress upon exposure was evaluated by biochemical essays targeting non-enzymatic oxidative stress physiological parameters, including respiration efficiency, total chlorophylls, carotenoids, ABTS and DPPH radicals reduction, total phenolics, GSH redox state, lipid peroxidation. Total Cd in plants was measured with AAS. Root and leaf morphology and element content were assessed in vivo utilizing low-vacuum Environmental Scanning Electron Microscopy (ESEM) with X-ray microanalysis (EDX). This integrated approach allowed identification of unique nanoscale CdS QDs toxicity to the plants that was distinct from CdSO4 exposure. The analyses highlighted that CdS QDs and Cd ions effects are modulated by the developmental stage of the plant, starting from T20 till T35 the plant development was modulated by the treatments, in particular CdS QDs induced early flowering. Both treatments induced Fe accumulation in roots, but at different intensities, while CdS QDs was associated with Mn increase into plant leaf. CdSO4 elicited higher levels of oxidative stress compared with QDs, especially the former treatment caused more intense respiration damages and reduction in chlorophyll and carotenoids than the latter. The two types of treatments impact differently on root and leaf morphology.
关键词： oxidative stress,morphology,Arabidopsis thaliana,Iron,ESEM/EDX,CdS QDs
更新于2025-09-12 10:27:22
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Controlled Growth of CH <sub/>3</sub> NH <sub/>3</sub> PbBr <sub/>3</sub> Perovskite Nanocrystals via a Water-Oil Interfacial Synthesis Method
摘要： Perturbation of the cellular redox state by stress conditions is sensed by redox-sensitive proteins so that the cell can physiologically respond to stressors. However, the mechanisms linking sensing to response remain poorly understood in plants. Here we report that the transcription factor bZIP68 underwent in vivo oxidation in Arabidopsis cells under oxidative stress which is dependent on its redox-sensitive Cys320 residue. bZIP68 is primarily localized to the nucleus under normal growth conditions in Arabidopsis seedlings. Oxidative stress reduces its accumulation in the nucleus and increases its cytosolic localization. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed that bZIP68 primarily binds to promoter regions containing the core G-box (CACGTG) or G-box-like motif of the genes involved in abiotic and biotic stress responses, photosynthesis, biosynthetic processes, and transcriptional regulation. The bzip68 mutant displayed slower growth under normal conditions but enhanced tolerance to oxidative stress. The results from the ChIP-seq and phenotypic and transcriptome comparison between the bzip68 mutant and wildtype indicate that bZIP68 normally suppresses expression of stress tolerance genes and promotes expression of growth-related genes, whereas its inactivation enhances stress tolerance but suppresses growth. bZIP68 might balance stress tolerance with growth through the extent of its oxidative inactivation according to the environment.
关键词： Arabidopsis thaliana,bZIP68,seedling growth,oxidative stress,transcriptional regulation
更新于2025-09-11 14:15:04
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Protocol for rapid clearing and staining of fixed Arabidopsis ovules for improved imaging by confocal laser scanning microscopy
摘要： Background: A salient topic in developmental biology relates to the molecular and genetic mechanisms that underlie tissue morphogenesis. Modern quantitative approaches to this central question frequently involve digital cellular models of the organ or tissue under study. The ovules of the model species Arabidopsis thaliana have long been established as a model system for the study of organogenesis in plants. While ovule development in Arabidopsis can be followed by a variety of different imaging techniques, no experimental strategy presently exists that enables an easy and straightforward investigation of the morphology of internal tissues of the ovule with cellular resolution. Results: We developed a protocol for rapid and robust confocal microscopy of fixed Arabidopsis ovules of all stages. The method combines clearing of fixed ovules in ClearSee solution with marking the cell outline using the cell wall stain SCRI Renaissance 2200 and the nuclei with the stain TO-PRO-3 iodide. We further improved the microscopy by employing a homogenous immersion system aimed at minimizing refractive index differences. The method allows complete inspection of the cellular architecture even deep within the ovule. Using the new protocol we were able to generate digital three-dimensional models of ovules of various stages. Conclusions: The protocol enables the quick and reproducible imaging of fixed Arabidopsis ovules of all developmental stages. From the imaging data three-dimensional digital ovule models with cellular resolution can be rapidly generated using image analysis software, for example MorphographX. Such digital models will provide the foundation for a future quantitative analysis of ovule morphogenesis in a model species.
关键词： Ovule,3D organ models,ClearSee,To-PRO-3 iodide,Imaging,Arabidopsis,SCRI Renaissance 2200,3D reconstruction
更新于2025-09-11 14:15:04
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[American Society of Agricultural and Biological Engineers 2017 Spokane, Washington July 16 - July 19, 2017 - ()] 2017 Spokane, Washington July 16 - July 19, 2017 - <i>Phenotyping of Arabidopsis for drought stress response using kinetic chlorophyll fluorescence imaging</i>
摘要： Drought stress is one of the major concerns in global agricultural production. Developing an efficient phenotyping technology can bridge the knowledge gap between the plant phenotype and genotype, which can promote the progress of breeding for drought tolerant accessions and provide economic benefits for the producers and consumers. This research was aimed to investigate the plant phenotyping for drought stress responses of two different genotypes of Arabidopsis using chlorophyll fluorescence imaging. 59 treatment groups (three plants for each group) of each genotype were withholding being watered for 8 days as the drought stress treatment, and the other 59 groups considered as control were regularly watered with 6 ml 1% nutrient solution every day. The kinetic chlorophyll fluorescence images of the drought treatment groups and the control groups were acquired at day 1, 3, 5, 7 and 8 after the drought stress treatment started. The conventional chlorophyll fluorescence parameters and the leaf area index were then extracted from the images. In addition, associated morphological and physiological parameters were also assayed. To construct combinatorial images, the sequential forward selection (SFS) algorithm was used to select the maximum contrast images between two genotypes and the linear discriminant analysis (LDA) was used to build combinatorial images. Finally, combinatorial images were analyzed, indicating combinatorial images are valuable in drought stress studies. Above all, the study showed that AQ and osca1 presented different drought stress responses during the treatment period based on the conventional chlorophyll parameters and combinatorial images.
关键词： drought stress,Arabidopsis,plant phenotyping,combinatorial imaging,Chlorophyll fluorescence imaging
更新于2025-09-10 09:29:36
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Live cell imaging of <i>Plasmodiophora brassicae</i> -host plant interactions based on a two-step axenic culture system
摘要： Plasmodiophora brassicae, a parasitic protist, induces club‐shaped tumor‐like growth of host Brassicas roots and hypocotyls after infection. Due to its soil‐borne nature and intracellular, biotrophic parasitism the infection biology and early pathogenesis remains in doubt. In this study, we have established a new protocol, based on a two‐step axenic culture of P. brassicae with its host tissues, for easy and in planta observation of cellular interactions between P. brassicae and host plants: first, coculture of P. brassicae with infected canola root tissues, on growth‐medium plates, enables the propagation of P. brassicae that serves as pure inoculum for pathogenicity assays, and second, the pure inoculum is subsequently used for pathogenicity tests on both canola and Arabidopsis seedlings grown on medium plates in Petri dishes. During the first axenic culture, we established a staining protocol by which the pathogen was fluorescently labeled with Nile red and calcofluor white, thus allowing in planta observation of pathogen development. In the pathogenicity assays, our results showed that axenic cultures of P. brassicae, in calli, remains fully virulent and completes its life cycle in both canola and Arabidopsis roots grown in Petri dishes. Combining visualization of fluorescent probe‐labeled P. brassicae structures with fluorescent protein tagging of Arabidopsis cellular components, further revealed dynamic responses of host cells at the early stages of P. brassicae infection. Thus, established protocols for in planta detection of P. brassicae structures and the live cell imaging of P. brassicae—Arabidopsis interactions provide a novel strategy for improving our detailed knowledge of P. brassicae infection in host tissues.
关键词： DAPI,Nile red,resting spores,calcofluor white,canola,primary plasmodia,axenic culture,Plasmodiophora brassicae,Arabidopsis
更新于2025-09-10 09:29:36
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Light-dependent suppression of COP1 multimeric complex formation is determined by the blue-light receptor FKF1 in Arabidopsis
摘要： CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), a multifunctional E3 ligase protein with many target proteins, is involved in diverse developmental processes throughout the plant's lifecycle, including seed germination, the regulation of circadian rhythms, photomorphogenesis, and the control of flowering time. To function, COP1 must form multimeric complexes with SUPPRESSOR OF PHYA1 (SPA1), i.e., [(COP1)2(SPA1)2] tetramers. We recently reported that the blue-light receptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX1) represses COP1 activity by inhibiting its homodimerization, but it is not yet clear whether FKF1 affects the formation of COP1-containing multimeric complexes. To explore this issue, we performed size exclusion chromatography (SEC) of Arabidopsis thaliana proteins and found that the levels and composition of COP1-containing multimeric complexes varied throughout a 24-h period. The levels of 440e669 kDa complexes were dramatically reduced in the late afternoon compared to the morning and at night in wild-type plants. During the daytime, the levels of these complexes were reduced in FKF1-overexpressing plants but not in fkf1-t, a loss-of-function mutant of FKF1, suggesting that FKF1 is closely associated with the destabilization of COP1 multimeric protein complexes in a light-dependent manner. We also analyzed the SEC patterns of COP1 multimeric complexes in transgenic plants overexpressing mutant COP1 variants, including COP1L105A (which forms homodimers) and COP1L170A (which cannot form homodimers), and found that COP1 multimeric complexes were scarce in plants overexpressing COP1L170A. These results indicate that COP1 homodimers serve as basic building blocks that assemble into COP1 multimeric complexes with diverse target proteins. We propose that light-activated FKF1 inhibits COP1 homodimerization, mainly by destabilizing 440e669 kDa COP1 complexes, resulting in the repression of CONSTANS-degrading COP1 activity in the late afternoon in long days, but not in short days, thereby regulating photoperiodic flowering in Arabidopsis.
关键词： Dimerization,Arabidopsis,Multimeric protein complex,COP1,Photoperiodic flowering,FKF1
更新于2025-09-09 09:28:46
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A FRET sensor for live-cell imaging of MAP kinase activity in Arabidopsis
摘要： The catalytic activity of mitogen activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain, and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the F?rster resonance energy transfer (FRET) efficiency of the sensor. FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live-cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22, and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial FRET changes. We also expressed the sensor in a conditional loss-of function double-mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the sensor’s NaCl-induced FRET gain, while other MAPKs are likely contributing to the chitin and flg22-induced FRET increases. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.
关键词： Arabidopsis thaliana,FRET sensor,live-cell imaging,MAP kinase
更新于2025-09-09 09:28:46