A pilot study on non-invasive in situ detection of phytochemicals and plant endogenous status using fiber optic infrared spectroscopy.

Traditional methods for assessing plant health often lack the necessary attributes for continuous and non-destructive monitoring. In this pilot study, we present a novel technique utilizing a customized fiber optic probe based on attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) with a contact force control unit for non-invasive and continuous plant health monitoring. We also developed a normalized difference mid-infrared reflectance index through statistical analysis of spectral features, enabling differentiation of drought and age conditions in plants. Our research aims to characterize phytochemicals and plant endogenous status optically, addressing the need for improved analytical measurement methods for in situ plant health assessment. The probe configuration was optimized with a triple-loop tip and a 3 N contact force, allowing sensitive measurements while minimizing leaf damage. By combining polycrystalline and chalcogenide fiber probes, a comprehensive wavenumber range analysis (4000-900 cm-1) was achieved. Results revealed significant variations in phytochemical composition among plant species, for example, red spinach with the highest polyphenolic content and green kale with the highest lignin content. Petioles displayed higher lignin and cellulose absorbance values compared to veins. The technique effectively monitored drought stress on potted green bok choy plants in situ, facilitating the quantification of changes in water content, antioxidant activity, lignin, and cellulose levels. This research represents the first demonstration of the potential of fiber optic ATR-FTIR probes for non-invasive and rapid plant health measurements, providing insights into plant health and advancements in quantitative monitoring for indoor farming practices, bioanalytical chemistry, and environmental sciences.

Miniaturized Vis-NIR handheld spectrometer for non-invasive pigment quantification in agritech applications.

Advanced precision agriculture requires the objective measurement of the structural and functional properties of plants. Biochemical profiles in leaves can differ depending on plant growing conditions. By quantitatively detecting these changes, farm production processes can be optimized to achieve high-yield, high-quality, and nutrient dense agricultural products. To enable the rapid and non-destructive detection on site, this study demonstrates the development of a new custom-designed portable handheld Vis-NIR spectrometer that collects leaf reflectance spectra, wirelessly transfers the spectral data through Bluetooth, and provides both raw spectral data and processed information. The spectrometer has two preprogramed methods: anthocyanin and chlorophyll quantification. Anthocyanin content of red and green lettuce estimated with the new spectrometer showed an excellent correlation coefficient of 0.84 with those determined by a destructive gold standard biochemical method. The differences in chlorophyll content were measured using leaf senescence as a case study. Chlorophyll Index calculated with the handheld spectrometer gradually decreased with leaf age as chlorophyll degrades during the process of senescence. The estimated chlorophyll values were highly correlated with those obtained from a commercial fluorescence-based chlorophyll meter with a correlation coefficient of 0.77. The developed portable handheld Vis-NIR spectrometer could be a simple, cost-effective, and easy to operate tool that can be used to non-invasively monitor plant pigment and nutrient content efficiently.

Classification of Plant Endogenous States Using Machine Learning-Derived Agricultural Indices.

Leaf color patterns vary depending on leaf age, pathogen infection, and environmental and nutritional stresses; thus, they are widely used to diagnose plant health statuses in agricultural fields. The visible-near infrared-shortwave infrared (VIS-NIR-SWIR) sensor measures the leaf color pattern from a wide spectral range with high spectral resolution. However, spectral information has only been employed to understand general plant health statuses (e.g., vegetation index) or phytopigment contents, rather than pinpointing defects of specific metabolic or signaling pathways in plants. Here, we report feature engineering and machine learning methods that utilize VIS-NIR-SWIR leaf reflectance for robust plant health diagnostics, pinpointing physiological alterations associated with the stress hormone, abscisic acid (ABA). Leaf reflectance spectra of wild-type, ABA2-overexpression, and deficient plants were collected under watered and drought conditions. Drought- and ABA-associated normalized reflectance indices (NRIs) were screened from all possible pairs of wavelength bands. Drought associated NRIs showed only a partial overlap with those related to ABA deficiency, but more NRIs were associated with drought due to additional spectral changes within the NIR wavelength range. Interpretable support vector machine classifiers built with 20 NRIs predicted treatment or genotype groups with an accuracy greater than those with conventional vegetation indices. Major selected NRIs were independent from leaf water content and chlorophyll content, 2 well-characterized physiological changes under drought. The screening of NRIs, streamlined with the development of simple classifiers, serves as the most efficient means of detecting reflectance bands that are highly relevant to characteristics of interest.

Drug Delivery in Plants Using Silk Microneedles.

New systems for agrochemical delivery in plants will foster precise agricultural practices and provide new tools to study plants and design crop traits, as standard spray methods suffer from elevated loss and limited access to remote plant tissues. Silk-based microneedles can circumvent these limitations by deploying a known amount of payloads directly in plants' deep tissues. However, plant response to microneedles' application and microneedles' efficacy in deploying physiologically relevant biomolecules are unknown. Here, it is shown that gene expression associated with Arabidopsis thaliana wounding response decreases within 24 h post microneedles' application. Additionally, microinjection of gibberellic acid (GA3 ) in A. thaliana mutant ft-10 provides a more effective and efficient mean than spray to activate GA3 pathways, accelerating bolting and inhibiting flower formation. Microneedle efficacy in delivering GA3 is also observed in several monocot and dicot crop species, i.e., tomato (Solanum lycopersicum), lettuce (Lactuca sativa), spinach (Spinacia oleracea), rice (Oryza Sativa), maize (Zea mays), barley (Hordeum vulgare), and soybean (Glycine max). The wide range of plants that can be successfully targeted with microinjectors opens the doors to their use in plant science and agriculture.

G protein signaling and metabolic pathways as evolutionarily conserved mechanisms to combat calcium deficiency.

Calcium (Ca) deficiency causes necrotic symptoms of foliar edges known as tipburn; however, the underlying cellular mechanisms have been poorly understood due to the lack of an ideal plant model and research platform. Using a phenotyping system that quantitates growth and tipburn traits in the model bryophyte Marchantia polymorpha, we evaluated metabolic compounds and the Gß-null mutant (gpb1) that modulate the occurrence and expansion of the tipburn. Transcriptomic comparisons between wild-type and gpb1 plants revealed the phenylalanine/phenylpropanoid biosynthesis pathway and reactive oxygen species (ROS) important for Ca deficiency responses. gpb1 plants reduced ROS production possibly through transcriptomic regulations of class III peroxidases and induced the expression of phenylpropanoid pathway enzymes without changing downstream lignin contents. Supplementation of intermediate metabolites and chemical inhibitors further confirmed the cell-protective mechanisms of the phenylpropanoid and ROS pathways. Marchantia polymorpha, Arabidopsis thaliana, and Lactuca sativa showed comparable transcriptomic changes where genes related to phenylpropanoid and ROS pathways were enriched in response to Ca deficiency. In conclusion, our study demonstrated unresolved signaling and metabolic pathways of Ca deficiency response. The phenotyping platform can speed up the discovery of chemical and genetic pathways, which could be widely conserved between M. polymorpha and angiosperms.

Handheld Multifunctional Fluorescence Imager for Non-invasive Plant Phenotyping.

Fluorescence imaging has shown great potential in non-invasive plant monitoring and analysis. However, current systems have several limitations, such as bulky size, high cost, contact measurement, and lack of multifunctionality, which may hinder its applications in a wide range of settings including indoor vertical farming. Herein, we developed a compact handheld fluorescence imager enabling multipurpose plant phenotyping, such as continuous photosynthetic activity monitoring and non-destructive anthocyanin quantification. The compact imager comprises of pulse-amplitude-modulated multi-color light emitting diodes (LEDs), optimized light illumination and collection, dedicated driver circuit board, miniaturized charge-coupled device camera, and associated image analytics. Experiments conducted in drought stressed lettuce proved that the novel imager could quantitatively evaluate the plant stress by the non-invasive measurement of photosynthetic activity efficiency. Moreover, a non-invasive and fast quantification of anthocyanins in green and red Batavia lettuce leaves had excellent correlation (>84%) with conventional destructive biochemical analysis. Preliminary experimental results emphasize the high throughput monitoring capability and multifunctionality of our novel handheld fluorescence imager, indicating its tremendous potential in modern agriculture.

Effect of Ergothioneine on 7-Ketocholesterol-Induced Endothelial Injury.

Ergothioneine (ET) is a naturally occurring antioxidant that is synthesized by non-yeast fungi and certain bacteria. ET is not synthesized by animals, including humans, but is avidly taken up from the diet, especially from mushrooms. In the current study, we elucidated the effect of ET on the hCMEC/D3 human brain endothelial cell line. Endothelial cells are exposed to high levels of the cholesterol oxidation product, 7-ketocholesterol (7KC), in patients with cardiovascular disease and diabetes, and this process is thought to mediate pathological inflammation. 7KC induces a dose-dependent loss of cell viability and an increase in apoptosis and necrosis in the endothelial cells. A relocalization of the tight junction proteins, zonula occludens-1 (ZO-1) and claudin-5, towards the nucleus of the cells was also observed. These effects were significantly attenuated by ET. In addition, 7KC induces marked increases in the mRNA expression of pro-inflammatory cytokines, IL-1ß IL-6, IL-8, TNF-α and cyclooxygenase-2 (COX2), as well as COX2 enzymatic activity, and these were significantly reduced by ET. Moreover, the cytoprotective and anti-inflammatory effects of ET were significantly reduced by co-incubation with an inhibitor of the ET transporter, OCTN1 (VHCL). This shows that ET needs to enter the endothelial cells to have a protective effect and is unlikely to act via extracellular neutralizing of 7KC. The protective effect on inflammation in brain endothelial cells suggests that ET might be useful as a nutraceutical for the prevention or management of neurovascular diseases, such as stroke and vascular dementia. Moreover, the ability of ET to cross the blood-brain barrier could point to its usefulness in combatting 7KC that is produced in the CNS during neuroinflammation, e.g. after excitotoxicity, in chronic neurodegenerative diseases, and possibly COVID-19-related neurologic complications.

Ultrastructural Characteristics of DHA-Induced Pyroptosis.

Microglial cells are resident macrophages of the central nervous system (CNS) that respond to bioactive lipids such as docosahexaenoic acid (DHA). Low micromolar concentrations of DHA typically promote anti-inflammatory functions of microglia, but higher concentrations result in a form of pro-inflammatory programmed cell death known as pyroptosis. This study used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to investigate the morphological characteristics of pyroptosis in BV-2 microglial cells following exposure to 200 µM DHA. Vehicle-treated cells are characterized by extended processes, spine-like projections or 0.4 to 5.2 µm in length, and numerous extracellular vesicles (EVs) tethered to the surface of the plasma membrane. In contrast to vehicle-treated cells, gross abnormalities are observed after treating cells with 200 µM DHA for 4 h. These include the appearance of numerous pits or pores of varying sizes across the cell surface, structural collapse and flattening of the cell shape. Moreover, EVs and spines were lost following DHA treatment, possibly due to release from the cell surface. The membrane pores appear after DHA treatment initially measured ~ 30 nm, consistent with the previously reported gasdermin D (GSDMD) pore complexes. Complete collapse of cytoplasmic organization and loss of nuclear envelope integrity were also observed in DHA-treated cells. These processes are morphologically distinct from the changes that occur during cisplatin-induced apoptosis, such as the appearance of apoptotic bodies and tightly packed organelles, and the maintenance of EVs and nuclear envelope integrity. Cumulatively, this study provides a systematic description of the ultrastructural characteristics of DHA-induced pyroptosis, including distinguishing features that differentiate this process from apoptosis.