Phenotyping for waterlogging tolerance in crops: current trends and future prospects.

Yield losses to waterlogging are expected to become an increasingly costly and frequent issue in some regions of the world. Despite the extensive work that has been carried out examining the molecular and physiological responses to waterlogging, phenotyping for waterlogging tolerance has proven difficult. This difficulty is largely due to the high variability of waterlogging conditions such as duration, temperature, soil type, and growth stage of the crop. In this review, we highlight use of phenotyping to assess and improve waterlogging tolerance in temperate crop species. We start by outlining the experimental methods that have been utilized to impose waterlogging stress, ranging from highly controlled conditions of hydroponic systems to large-scale screenings in the field. We also describe the phenotyping traits used to assess tolerance ranging from survival rates and visual scoring to precise photosynthetic measurements. Finally, we present an overview of the challenges faced in attempting to improve waterlogging tolerance, the trade-offs associated with phenotyping in controlled conditions, limitations of classic phenotyping methods, and future trends using plant-imaging methods. If effectively utilized to increase crop resilience to changing climates, crop phenotyping has a major role to play in global food security.

Single-Molecule Imaging in Living Plant Cells: A Methodological Review.

Single-molecule imaging is emerging as a revolutionary approach to studying fundamental questions in plants. However, compared with its use in animals, the application of single-molecule imaging in plants is still underexplored. Here, we review the applications, advantages, and challenges of single-molecule fluorescence imaging in plant systems from the perspective of methodology. Firstly, we provide a general overview of single-molecule imaging methods and their principles. Next, we summarize the unprecedented quantitative details that can be obtained using single-molecule techniques compared to bulk assays. Finally, we discuss the main problems encountered at this stage and provide possible solutions.

Performances Evaluation of a Low-Cost Platform for High-Resolution Plant Phenotyping.

This study aims to test the performances of a low-cost and automatic phenotyping platform, consisting of a Red-Green-Blue (RGB) commercial camera scanning objects on rotating plates and the reconstruction of main plant phenotypic traits via the structure for motion approach (SfM). The precision of this platform was tested in relation to three-dimensional (3D) models generated from images of potted maize, tomato and olive tree, acquired at a different frequency (steps of 4°, 8° and 12°) and quality (4.88, 6.52 and 9.77 µm/pixel). Plant and organs heights, angles and areas were extracted from the 3D models generated for each combination of these factors. Coefficient of determination (R2), relative Root Mean Square Error (rRMSE) and Akaike Information Criterion (AIC) were used as goodness-of-fit indexes to compare the simulated to the observed data. The results indicated that while the best performances in reproducing plant traits were obtained using 90 images at 4.88 µm/pixel (R2 = 0.81, rRMSE = 9.49% and AIC = 35.78), this corresponded to an unviable processing time (from 2.46 h to 28.25 h for herbaceous plants and olive trees, respectively). Conversely, 30 images at 4.88 µm/pixel resulted in a good compromise between a reliable reconstruction of considered traits (R2 = 0.72, rRMSE = 11.92% and AIC = 42.59) and processing time (from 0.50 h to 2.05 h for herbaceous plants and olive trees, respectively). In any case, the results pointed out that this input combination may vary based on the trait under analysis, which can be more or less demanding in terms of input images and time according to the complexity of its shape (R2 = 0.83, rRSME = 10.15% and AIC = 38.78). These findings highlight the reliability of the developed low-cost platform for plant phenotyping, further indicating the best combination of factors to speed up the acquisition and elaboration process, at the same time minimizing the bias between observed and simulated data.

Showcasing the application of synchrotron-based X-ray computed tomography in host-pathogen interactions: The role of wheat rachilla and rachis nodes in Type-II resistance to Fusarium graminearum.

Fusarium head blight, caused primarily by Fusarium graminearum (Fg), is one of the most devastating diseases of wheat. Host resistance in wheat is classified into five types (Type-I to Type-V), and a majority of moderately resistant genotypes carry Type-II resistance (resistance to pathogen spread in the rachis) alleles, mainly from the Chinese cultivar Sumai 3. Histopathological studies in the past failed to identify the key tissue in the spike conferring resistance to pathogen spread, and most of the studies used destructive techniques, potentially damaging the tissue(s) under study. In the present study, nondestructive synchrotron-based phase contrast X-ray imaging and computed tomography techniques were used to confirm the part of the wheat spike conferring Type-II resistance to Fg spread, thus showcasing the application of synchrotron-based techniques to image host-pathogen interactions. Seven wheat genotypes of moderate resistance to Fusarium head blight were studied for changes in the void space volume fraction and grayscale/voxel intensity following Fg inoculation. Cell-wall biopolymeric compounds were quantified using Fourier-transform midinfrared spectroscopy for all genotype-treatment combinations. The study revealed that the rachilla and rachis nodes together are structurally important in conferring Type-II resistance. The structural reinforcement was not necessarily observed from lignin deposition but rather from an unknown mechanism.

Structural and functional imaging of large and opaque plant specimens.

Three- and four-dimensional imaging techniques are a prerequisite for spatially resolving the form-structure-function relationships in plants. However, choosing the right imaging method is a difficult and time-consuming process as the imaging principles, advantages and limitations, as well as the appropriate fields of application first need to be compared. The present study aims to provide an overview of three imaging methods that allow for imaging opaque, large and thick (>5 mm, up to several centimeters), hierarchically organized plant samples that can have complex geometries. We compare light microscopy of serial thin sections followed by 3D reconstruction (LMTS3D) as an optical imaging technique, micro-computed tomography (µ-CT) based on ionizing radiation, and magnetic resonance imaging (MRI) which uses the natural magnetic properties of a sample for image acquisition. We discuss the most important imaging principles, advantages, and limitations, and suggest fields of application for each imaging technique (LMTS, µ-CT, and MRI) with regard to static (at a given time; 3D) and dynamic (at different time points; quasi 4D) structural and functional plant imaging.

Synchrotron Radiation Sheds Fresh Light on Plant Research: The Use of Powerful Techniques to Probe Structure and Composition of Plants.

While synchrotron radiation is a powerful tool in material and biomedical sciences, it is still underutilized in plant research. This mini review attempts to introduce the potential of synchrotron-based spectroscopic and imaging methods and their applications to plant sciences. Synchrotron-based Fourier transform infrared spectroscopy, X-ray absorption and fluorescence techniques, and two- and three-dimensional imaging techniques are examined. We also discuss the limitations of synchrotron-based research in plant sciences, specifically the types of plant samples that can be used. Despite limitations, the unique features of synchrotron radiation such as high brightness, polarization and pulse properties offer great advantages over conventional spectroscopic and imaging tools and enable the correlation of the structure and chemical composition of plants with biochemical function. Modern detector technologies and experimental methodologies are thus enabling plant scientists to investigate aspects of plant sciences such as ultrafast kinetics of biochemical reactions, mineral uptake, transport and accumulation, and dynamics of cell wall structure and composition during environmental stress in unprecedented ways using synchrotron beamlines. The potential for the automation of some of these synchrotron technologies and their application to plant phenotyping is also discussed.

Near-infrared fluorescent probe based on the regulatory dye pKa for imaging of H2S in rice roots and living cells.

Hydrogen sulfide (H2S) is a key factor in various biological processes such as plant grow and its response to environmental stress. Here, we develop a novel near-infrared (NIR) fluorescent probe for detecting hydrogen sulfide based on the regulatory NIR dye pKa values. After triggering the H2S substitution response, probe A with introducing the cyano moiety not only exhibits a significant near-infrared emission (Emax: 724 nm) response in physiological environments, but also shows a fast response, high selectivity, and sensitivity (LOD as 0.52 µM). In addition, probe A with low biological cytotoxicity is successfully used for imaging detection of cellular exogenous and endogenous hydrogen sulfide. More importantly, in situ imaging of probe A tracks the H2S fluctuations in the rice root system and its response to environmental stress. Hence, this work offers a new NIR fluorescence imaging monitoring tool for hydrogen sulfide in biological systems.

Evaluating waterlogging stress response and recovery in barley (Hordeum vulgare L.): an image-based phenotyping approach.

Waterlogging is expected to become a more prominent yield restricting stress for barley as rainfall frequency is increasing in many regions due to climate change. The duration of waterlogging events in the field is highly variable throughout the season, and this variation is also observed in experimental waterlogging studies. Such variety of protocols make intricate physiological responses challenging to assess and quantify. To assess barley waterlogging tolerance in controlled conditions, we present an optimal duration and setup of simulated waterlogging stress using image-based phenotyping. Six protocols durations, 5, 10, and 14 days of stress with and without seven days of recovery, were tested. To quantify the physiological effects of waterlogging on growth and greenness, we used top down and side view RGB (Red-Green-Blue) images. These images were taken daily throughout each of the protocols using the PSI PlantScreen™ imaging platform. Two genotypes of two-row spring barley, grown in glasshouse conditions, were subjected to each of the six protocols, with stress being imposed at the three-leaf stage. Shoot biomass and root imaging data were analysed to determine the optimal stress protocol duration, as well as to quantify the growth and morphometric changes of barley in response to waterlogging stress. Our time-series results show a significant growth reduction and alteration of greenness, allowing us to determine an optimal protocol duration of 14 days of stress and seven days of recovery for controlled conditions. Moreover, to confirm the reproducibility of this protocol, we conducted the same experiment in a different facility equipped with RGB and chlorophyll fluorescence imaging sensors. Our results demonstrate that the selected protocol enables the assessment of genotypic differences, which allow us to further determine tolerance responses in a glasshouse environment. Altogether, this work presents a new and reproducible image-based protocol to assess early stage waterlogging tolerance, empowering a precise quantification of waterlogging stress relevant markers such as greenness, Fv/Fm and growth rates.

Targeted and Long-Term Fluorescence Imaging of Plant Cytomembranes Using Main-Chain Charged Polyelectrolytes with Aggregation-Induced Emission.

Fluorescent polyelectrolytes have attracted tremendous attention due to their unique properties and wide applications. However, current research objects of fluorescent polyelectrolytes mainly focus on side-chain charged polyelectrolytes, and the applications of polyelectrolytes in plant cytomembrane imaging with long time and high specificity still remain challenging. Herein, long-time and targeted fluorescence imaging of plant cytomembranes was achieved for the first time using main-chain charged polyelectrolytes (MCCPs) with aggregation-induced emission (AIE). A series of MCCPs were designed and synthesized, among which the red-emissive and AIE-active MCCP with a triphenylamine linker and a cyano group around the cationic ring-fused heterocyclic core showed the best fluorescence imaging performance of plant cells. Unlike other MCCPs and its neutral form of polymer, this cyano-substituted conjugated polyelectrolyte can specifically target the cytomembrane of plant cells within a short staining time with many advantages, including wash-free staining, high photostability and imaging integrity, excellent durability (at least 12 h), and low biotoxicity. In addition to onion epidermal cells, this AIE fluorescence probe also shows good imaging capabilities for other kinds of plant cells such as Glycine max and Vigna radiata. Such an AIE-active MCCP-based imaging system provides an effective design strategy to develop fluorescence probes with high specificity and long-term imaging ability toward plant plasma membranes.

A wireless fluorescent sensing device for on-site closed-loop detection of hydrazine levels in the environment.

Given the extensive need for the detection of hydrazine (N2H4) in the biomedical and chemical-pharmaceutical sectors, there is a necessity to devise a fast, sensitive, specific, and portable technique for precisely quantifying hydrazine at environmental levels. In our work, an "OFF-ON" type fluorescent probe namely 2-(4-(10-(naphthalen-2-yl)anthracen-9-yl)phenyl)isoindole-1,3-dione (NAP), which was inspired by the "Gabriel" reaction, was synthesized. The NAP fluorescent cellulose film successfully achieved the detection of hydrazine vapor with a LOD = 0.658 ppm. Compared to previous qualitative methods for detecting hydrazine, this study successfully achieved quantitative identification of hydrazine at low concentrations. In addition, a portable sensor device based on NAP cellulose film was successfully integrated, enabling ultra-sensitive, wireless, remote, and real-time detection of N2H4 vapor. It was determined that the probe (NAP) exhibited excellent detection performance when applied to various environmental samples including distilled water, tap water, creek water, soil and plants. This study introduces a potentially effective approach for detecting hydrazine in real-world settings.

Naphthalene-based fluorescent probe for on-site detection of hydrazine in the environment.

The widespread occurrence of hydrazine residues in the environment, including in water, soil, and organisms, is a potential health threat to humans. Therefore, the development of an efficient method for the detection of hydrazine in environmental samples is highly desirable although it poses a significant challenge. In this study, we designed and synthesized a series of naphthalene-based fluorescent dyes through structural engineering and developed a novel probe for hydrazine detection. The probe could provide a distinct fluorescence response toward hydrazine in aqueous solution with high sensitivity and selectivity. Moreover, paper-based test strips can be easily fabricated using this probe, enabling the portable on-site detection of hydrazine with the aid of a smartphone. Furthermore, we demonstrated that this probe is capable of recognizing hydrazine in various environmental samples, including water, soil, plants, and zebrafish embryos. This research provides a promising tool for the detection of hydrazine in the environment.

Highly Selective and Rapid "Turn-On" Fluorogenic Chemosensor for Detection of Salicylic Acid in Plants and Food Samples.

Salicylic acid (SA) is one of the chemical molecules, involved in plant growth and immunity, thereby contributing to the control of pests and pathogens, and even applied in fruit and vegetable preservation. However, only a few tools have ever been designed or executed to understand the physiological processes induced by SA or its function in plant immunity and residue detection in food. Hence, three Rh6G-based fluorogenic chemosensors were synthesized to detect phytohormone SA based on the "OFF-ON" mechanism. The probes showed high selectivity, ultrafast response time (<60 s), and nanomolar detection limit for SA. Moreover, the probe possessed outstanding profiling that can be successfully used for SA imaging of callus and plants. Furthermore, the fluorescence pattern indicated that SA could occur in the distal transport in plants. These remarkable results contribute to improving our understanding of the multiple physiological and pathological processes involved in SA for plant disease diagnosis and for the development of immune activators. In addition, SA detection in some agricultural products used probes to extend the practical application because its use is prohibited in some countries and is harmful to SA-sensitized persons. Interestingly, the as-obtained test paper displayed that SA could be imaged by ultraviolet (UV) and was directly visible to the naked eye. Given the above outcomes, these probes could be used to monitor SA in vitro and in vivo, including, but not limited to, plant biology, food residue detection, and sewage detection.

Importance of Dietary Uptake for in Situ Bioaccumulation of Systemic Fungicides Using Gammarus pulex as a Model Organism.

Bioaccumulation of organic contaminants from contaminated food sources might pose an underestimated risk toward shredding invertebrates. This assumption is substantiated by monitoring studies observing discrepancies of predicted tissue concentrations determined from laboratory-based experiments compared with measured concentrations of systemic pesticides in gammarids. To elucidate the role of dietary uptake in bioaccumulation, gammarids were exposed to leaf material from trees treated with a systemic fungicide mixture (azoxystrobin, cyprodinil, fluopyram, and tebuconazole), simulating leaves entering surface waters in autumn. Leaf concentrations, spatial distribution, and leaching behavior of fungicides were characterized using liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) and matrix-assisted laser desorption ionization-mass spectrometric imaging. The contribution of leached fungicides and fungicides taken up from feeding was assessed by assembling caged (no access) and uncaged (access to leaves) gammarids. The fungicide dynamics in the test system were analyzed using LC-HRMS/MS and toxicokinetic modeling. In addition, a summer scenario was simulated where water was the initial source of contamination and leaves contaminated by sorption. The uptake, translocation, and biotransformation of systemic fungicides by trees were compound-dependent. Internal fungicide concentrations of gammarids with access to leaves were much higher than in caged gammarids of the autumn scenario, but the difference was minimal in the summer scenario. In food choice and dissectioning experiments gammarids did not avoid contaminated leaves and efficiently assimilated contaminants from leaves, indicating the relevance of this exposure pathway in the field. The present study demonstrates the potential impact of dietary uptake on in situ bioaccumulation for shredders in autumn, outside the main application period. The toxicokinetic parameters obtained facilitate modeling of environmental exposure scenarios. The uncovered significance of dietary uptake for detritivores warrants further consideration from scientific as well as regulatory perspectives. Environ Toxicol Chem 2023;42:1993-2006. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Highly sensitive fluorescent sensor for hypochlorite in nearly 100% aqueous solution and its application for live-cell, plant and zebrafish imaging.

A new ICT type D-π-A structured chemosensor DTB derived from a bithiophene-benzothiazole derivative has been synthesized. Sensor DTB showed a colorimetric and fluorometric dual-signaling response to hypochlorite (ClO-) in EtOH/HEPES solution (1/99, V/V, pH = 7.4, nearly 100% aqueous solution). Sensor DTB exhibited well specificity, high sensitivity and rapidity (<1 min) for ClO- with a detection limit of 25 nM. Sensor DTB features remarkable color changes and significant fluorescence "turn-on" response (ca. 45 fold) after treating with ClO-. Comprehensive analyses by 1H NMR, TLC, FTIR, HRMS, UV-vis, fluorescence and DFT illustrated that ClO- reacted with the CC bond of DTB, generating fluorophore 2T-CHO, leading to strong blue fluorescence. Interestingly, DTB loaded colorimetric test strips were established for rapid and real-time visual detection of ClO-. Furthermore, the DTB was successfully applied to quantitatively and sensitively detect ClO- in 84 disinfectant, bio-fluids (human serum and urine) and actual water samples. Importantly, the biocompatible DTB has been employed for visualizing and bioimaging ClO- in mung bean sprouts, Arabidopsis, live cells and zebrafish. These investigations demonstrate that DTB has great potentials for detecting ClO- in various biosystems and environments. This work would offer a new idea for developing multifunctional sensors with better performance for chemo/biosensors.

Molecular probes for fluorescent sensing of metal ions in non-mammalian organisms.

While metal ions play an important role in the proper functioning of all life, many questions remain unanswered about exactly how different metals contribute to health and disease. The development of fluorescent probes, which respond to metals, has allowed greater understanding of the cellular location, concentration and speciation of metals in living systems, giving a new appreciation of their function. While the focus of studies using these fluorescent tools has largely been on mammalian organisms, there has been relatively little application of these powerful tools to other organisms. In this review, we highlight recent examples of molecular fluorophores, which have been applied to sensing metals in non-mammalian organisms.

A surfactant-assisted approach enables the fluorescence tracking of benfluralin in plants.

Developing an effective detection method for benfluralin (BFA) is of great significance, since BFA as most widely used herbicides can be bioaccumulated by aquatic organisms in environment, possessing potential risks to human health. Owing to aggregation-caused quenching effect, most fluorescent detection methods based on donor-acceptor organic fluorophores suffered from very low sensitivity towards BFA in water system, hampering the bioimaging application in plants. In this work, we reported a novel surfactant-assisted fluorescent probe enabling detection of BFA in water with a high sensitivity. The involvement of specific surfactant Triton X100 (TX100) could amplify the response signal of probe more than 100-fold. The detection limit for BFA was determined to be 80 nM, satisfying the environmental protection requirements. Moreover, we demonstrated applications of this strategy for the fluorescent imaging of BFA in plant. The absorbance of BFA into roots of Arabidopsis thaliana and castor seedlings was successfully observed based on this method.

Kinetically Consistent Data Assimilation for Plant PET Sparse Time Activity Curve Signals.

Time activity curve (TAC) signal processing in plant positron emission tomography (PET) is a frontier nuclear science technique to bring out the quantitative fluid dynamic (FD) flow parameters of the plant vascular system and generate knowledge on crops and their sustainable management, facing the accelerating global climate change. The sparse space-time sampling of the TAC signal impairs the extraction of the FD variables, which can be determined only as averaged values with existing techniques. A data-driven approach based on a reliable FD model has never been formulated. A novel sparse data assimilation digital signal processing method is proposed, with the unique capability of a direct computation of the dynamic evolution of noise correlations between estimated and measured variables, by taking into explicit account the numerical diffusion due to the sparse sampling. The sequential time-stepping procedure estimates the spatial profile of the velocity, the diffusion coefficient and the compartmental exchange rates along the plant stem from the TAC signals. To illustrate the performance of the method, we report an example of the measurement of transport mechanisms in zucchini sprouts.

Synchrotron tomography applications in agriculture and food sciences research: a review.

Synchrotron imaging is widely used for research in many scientific disciplines. This article introduces the characteristics of synchrotron X-ray imaging and its applications in agriculture and food science research. The agriculture and food sector are a vast area that comprises of plants, seeds, animals, food and their products; soils with thriving microbial communities; and natural resources such as water, fertilizers, and organic matter. These entities have unique internal features, structures and compositions which differentiate them from each other in varieties, species, grades, and types. The use of a bright and tuneable monochromatic source of synchrotron imaging techniques enables researchers to study the internal features and compositions of plants, seeds, soil and food in a quick and non-destructive way to enhance their use, conservation and productivity. Synchrotron's different X-ray imaging techniques offer a wide domain of applications, which make them perfect to enhance the understanding of structures of raw and processed food products to promote food safety and security. Therefore, this paper summarizes the results of major experiments carried out with seeds, plants, soil, food and relevant areas of agricultural sciences with more emphasis on two synchrotron X-ray imaging techniques: absorption and phase-contrast imaging and computed tomography.

Optical sensing method for screening disease in melon seeds by using optical coherence tomography.

We report a noble optical sensing method to diagnose seed abnormalities using optical coherence tomography (OCT). Melon seeds infected with Cucumber green mottle mosaic virus (CGMMV) were scanned by OCT. The cross-sectional sensed area of the abnormal seeds showed an additional subsurface layer under the surface which is not found in normal seeds. The presence of CGMMV in the sample was examined by a blind test (n = 140) and compared by the reverse transcription-polymerase chain reaction. The abnormal layers (n = 40) were quantitatively investigated using A-scan sensing analysis and statistical method. By utilizing 3D OCT image reconstruction, we confirmed the distinctive layers on the whole seeds. These results show that OCT with the proposed data processing method can systemically pick up morphological modification induced by viral infection in seeds, and, furthermore, OCT can play an important role in automatic screening of viral infections in seeds.

Design Study of a Novel Positron Emission Tomography System for Plant Imaging.

Positron Emission Tomography is a non-disruptive and high-sensitive digital imaging technique which allows to measure in-vivo and non invasively the changes of metabolic and transport mechanisms in plants. When it comes to the early assessment of stress-induced alterations of plant functions, plant PET has the potential of a major breakthrough. The development of dedicated plant PET systems faces a series of technological and experimental difficulties, which make conventional clinical and preclinical PET systems not fully suitable to agronomy. First, the functional and metabolic mechanisms of plants depend on environmental conditions, which can be controlled during the experiment if the scanner is transported into the growing chamber. Second, plants need to be imaged vertically, thus requiring a proper Field Of View. Third, the transverse Field of View needs to adapt to the different plant shapes, according to the species and the experimental protocols. In this paper, we perform a simulation study, proposing a novel design of dedicated plant PET scanners specifically conceived to address these agronomic issues. We estimate their expected sensitivity, count rate performance and spatial resolution, and we identify these specific features, which need to be investigated when realizing a plant PET scanner. Finally, we propose a novel approach to the measurement and verification of the performance of plant PET systems, including the design of dedicated plant phantoms, in order to provide a standard evaluation procedure for this emerging digital imaging agronomic technology.