Epigenetics and plant hormone dynamics - a functional and methodological perspective.

Plant hormones, pivotal regulators of plant growth, development, and response to environmental cues, have recently emerged as central modulators of epigenetic processes governing gene expression and phenotypic plasticity. This review addresses the complex interplay between plant hormones and epigenetic mechanisms, highlighting the diverse methodologies that have been harnessed to decipher these intricate relationships. We present a comprehensive overview to understand how phytohormones orchestrate epigenetic modifications, shaping plant adaptation and survival strategies. Conversely, we explore how epigenetic regulators ensure hormonal balance and regulate the signalling pathways of key plant hormones. Furthermore, our investigation includes a search for novel genes that are regulated by plant hormones under the control of epigenetic processes. Our review offers a contemporary overview of the epigenetic-plant hormone crosstalk, emphasizing its significance in plant growth, development, and potential agronomical applications.

Cytonuclear interactions modulate the plasticity of photosynthetic rhythmicity and growth in wild barley.

In plants, the contribution of the plasmotype (mitochondria and chloroplast) in controlling the circadian clock plasticity and possible consequences on cytonuclear genetic makeup have yet to be fully elucidated. A genome-wide association study in the wild barley (Hordeum vulgare ssp. spontaneum) B1K collection identified overlap with our previously mapped DRIVERS OF CLOCKS (DOCs) loci in wild-cultivated interspecific population. Moreover, we identified non-random segregation and epistatic interactions between nuclear DOCs loci and the chloroplastic RpoC1 gene, indicating an adaptive value for specific cytonuclear gene combinations. Furthermore, we show that DOC1.1, which harbours the candidate SIGMA FACTOR-B (SIG-B) gene, is linked with the differential expression of SIG-B and CCA1 genes and contributes to the circadian gating response to heat. High-resolution temporal growth and photosynthesis measurements of B1K also link the DOCs loci to differential growth, Chl content and quantum yield. To validate the involvement of the Plastid encoded polymerase (PEP) complex, we over-expressed the two barley chloroplastic RpoC1 alleles in Arabidopsis and identified significant differential plasticity under elevated temperatures. Finally, enhanced clock plasticity of de novo ENU (N-Ethyl-N-nitrosourea) -induced barley rpoB1 mutant further implicates the PEP complex as a key player in regulating the circadian clock output. Overall, this study highlights the contribution of specific cytonuclear interaction between rpoC1 (PEP gene) and SIG-B with distinct circadian timing regulation under heat, and their pleiotropic effects on growth implicate an adaptive value.

A kaleidoscope of photosynthetic antenna proteins and their emerging roles.

Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis.

Genetic mapping of the early responses to salt stress in Arabidopsis thaliana.

Salt stress decreases plant growth prior to significant ion accumulation in the shoot. However, the processes underlying this rapid reduction in growth are still unknown. To understand the changes in salt stress responses through time and at multiple physiological levels, examining different plant processes within a single set-up is required. Recent advances in phenotyping has allowed the image-based estimation of plant growth, morphology, colour and photosynthetic activity. In this study, we examined the salt stress-induced responses of 191 Arabidopsis accessions from 1 h to 7 days after treatment using high-throughput phenotyping. Multivariate analyses and machine learning algorithms identified that quantum yield measured in the light-adapted state (Fv' /Fm' ) greatly affected growth maintenance in the early phase of salt stress, whereas the maximum quantum yield (QYmax ) was crucial at a later stage. In addition, our genome-wide association study (GWAS) identified 770 loci that were specific to salt stress, in which two loci associated with QYmax and Fv' /Fm' were selected for validation using T-DNA insertion lines. We characterized an unknown protein kinase found in the QYmax locus that reduced photosynthetic efficiency and growth maintenance under salt stress. Understanding the molecular context of the candidate genes identified will provide valuable insights into the early plant responses to salt stress. Furthermore, our work incorporates high-throughput phenotyping, multivariate analyses and GWAS, uncovering details of temporal stress responses and identifying associations across different traits and time points, which are likely to constitute the genetic components of salinity tolerance.

Glucose uptake to guard cells via STP transporters provides carbon sources for stomatal opening and plant growth.

Guard cells on the leaf epidermis regulate stomatal opening for gas exchange between plants and the atmosphere, allowing a balance between photosynthesis and transpiration. Given that guard cells possess several characteristics of sink tissues, their metabolic activities should largely depend on mesophyll-derived sugars. Early biochemical studies revealed sugar uptake into guard cells. However, the transporters that are involved and their relative contribution to guard cell function are not yet known. Here, we identified the monosaccharide/proton symporters Sugar Transport Protein 1 and 4 (STP1 and STP4) as the major plasma membrane hexose sugar transporters in the guard cells of Arabidopsis thaliana. We show that their combined action is required for glucose import to guard cells, providing carbon sources for starch accumulation and light-induced stomatal opening that are essential for plant growth. These findings highlight mesophyll-derived glucose as an important metabolite connecting stomatal movements with photosynthesis.

Towards Automated Analysis of Grain Spikes in Greenhouse Images Using Neural Network Approaches: A Comparative Investigation of Six Methods.

Automated analysis of small and optically variable plant organs, such as grain spikes, is highly demanded in quantitative plant science and breeding. Previous works primarily focused on the detection of prominently visible spikes emerging on the top of the grain plants growing in field conditions. However, accurate and automated analysis of all fully and partially visible spikes in greenhouse images renders a more challenging task, which was rarely addressed in the past. A particular difficulty for image analysis is represented by leaf-covered, occluded but also matured spikes of bushy crop cultivars that can hardly be differentiated from the remaining plant biomass. To address the challenge of automated analysis of arbitrary spike phenotypes in different grain crops and optical setups, here, we performed a comparative investigation of six neural network methods for pattern detection and segmentation in RGB images, including five deep and one shallow neural network. Our experimental results demonstrate that advanced deep learning methods show superior performance, achieving over 90% accuracy by detection and segmentation of spikes in wheat, barley and rye images. However, spike detection in new crop phenotypes can be performed more accurately than segmentation. Furthermore, the detection and segmentation of matured, partially visible and occluded spikes, for which phenotypes substantially deviate from the training set of regular spikes, still represent a challenge to neural network models trained on a limited set of a few hundreds of manually labeled ground truth images. Limitations and further potential improvements of the presented algorithmic frameworks for spike image analysis are discussed. Besides theoretical and experimental investigations, we provide a GUI-based tool (SpikeApp), which shows the application of pre-trained neural networks to fully automate spike detection, segmentation and phenotyping in images of greenhouse-grown plants.

Estimating heat tolerance of plants by ion leakage: a new method based on gradual heating.

Heat tolerance of plants related to cell membrane thermostability is commonly estimated via the measurement of ion leakage from plant segments after defined heat treatment. To compare heat tolerance of various plants, it is crucial to select suitable heating conditions. This selection is time-consuming and optimizing the conditions for all investigated plants may even be impossible. Another problem of the method is its tendency to overestimate basal heat tolerance. Here we present an improved ion leakage method, which does not suffer from these drawbacks. It is based on gradual heating of plant segments in a water bath or algal suspensions from room temperature up to 70-75°C. The electrical conductivity of the bath/suspension, which is measured continuously during heating, abruptly increases at a certain temperature TCOND (within 55-70°C). The TCOND value can be taken as a measure of cell membrane thermostability, representing the heat tolerance of plants/organisms. Higher TCOND corresponds to higher heat tolerance (basal or acquired) connected to higher thermostability of the cell membrane, as evidenced by the common ion leakage method. The new method also enables determination of the thermostability of photochemical reactions in photosynthetic samples via the simultaneous measurement of Chl fluorescence.

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation.

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

High-Throughput Spike Detection in Greenhouse Cultivated Grain Crops with Attention Mechanisms-Based Deep Learning Models.

Detection of spikes is the first important step toward image-based quantitative assessment of crop yield. However, spikes of grain plants occupy only a tiny fraction of the image area and often emerge in the middle of the mass of plant leaves that exhibit similar colors to spike regions. Consequently, accurate detection of grain spikes renders, in general, a non-trivial task even for advanced, state-of-the-art deep neural networks (DNNs). To improve pattern detection in spikes, we propose architectural changes to Faster-RCNN (FRCNN) by reducing feature extraction layers and introducing a global attention module. The performance of our extended FRCNN-A vs. conventional FRCNN was compared on images of different European wheat cultivars, including "difficult" bushy phenotypes from 2 different phenotyping facilities and optical setups. Our experimental results show that introduced architectural adaptations in FRCNN-A helped to improve spike detection accuracy in inner regions. The mean average precision (mAP) of FRCNN and FRCNN-A on inner spikes is 76.0% and 81.0%, respectively, while on the state-of-the-art detection DNNs, Swin Transformer mAP is 83.0%. As a lightweight network, FRCNN-A is faster than FRCNN and Swin Transformer on both baseline and augmented training datasets. On the FastGAN augmented dataset, FRCNN achieved a mAP of 84.24%, FRCNN-A attained a mAP of 85.0%, and the Swin Transformer achieved a mAP of 89.45%. The increase in mAP of DNNs on the augmented datasets is proportional to the amount of the IPK original and augmented images. Overall, this study indicates a superior performance of attention mechanisms-based deep learning models in detecting small and subtle features of grain spikes.

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato.

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions over time. By using multiple imaging sensors, many traits of interest can be assessed, including plant biomass, photosynthetic efficiency, canopy temperature, and leaf reflectance indices. Plants are frequently exposed to multiple stresses under field conditions where severe heat waves, flooding, and drought events seriously threaten crop productivity. When stresses coincide, resulting effects on plants can be distinct due to synergistic or antagonistic interactions. To elucidate how potato plants respond to single and combined stresses that resemble naturally occurring stress scenarios, five different treatments were imposed on a selected potato cultivar (Solanum tuberosum L., cv. Lady Rosetta) at the onset of tuberization, i.e. control, drought, heat, waterlogging, and combinations of heat, drought, and waterlogging stresses. Our analysis shows that waterlogging stress had the most detrimental effect on plant performance, leading to fast and drastic physiological responses related to stomatal closure, including a reduction in the quantum yield and efficiency of photosystem II and an increase in canopy temperature and water index. Under heat and combined stress treatments, the relative growth rate was reduced in the early phase of stress. Under drought and combined stresses, plant volume and photosynthetic performance dropped with an increased temperature and stomata closure in the late phase of stress. The combination of optimized stress treatment under defined environmental conditions together with selected phenotyping protocols allowed to reveal the dynamics of morphological and physiological responses to single and combined stresses. Here, a useful tool is presented for plant researchers looking to identify plant traits indicative of resilience to several climate change-related stresses.

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus.

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

iReenCAM: automated imaging system for kinetic analysis of photosynthetic pigment biosynthesis at high spatiotemporal resolution during early deetiolation.

Seedling de-etiolation is one of the key stages of the plant life cycle, characterized by a strong rearrangement of the plant development and metabolism. The conversion of dark accumulated protochlorophyllide to chlorophyll in etioplasts of de-etiolating plants is taking place in order of ns to µs after seedlings illumination, leading to detectable increase of chlorophyll levels in order of minutes after de-etiolation initiation. The highly complex chlorophyll biosynthesis integrates number of regulatory events including light and hormonal signaling, thus making de-etiolation an ideal model to study the underlying molecular mechanisms. Here we introduce the iReenCAM, a novel tool designed for non-invasive fluorescence-based quantitation of early stages of chlorophyll biosynthesis during de-etiolation with high spatial and temporal resolution. iReenCAM comprises customized HW configuration and optimized SW packages, allowing synchronized automated measurement and analysis of the acquired fluorescence image data. Using the system and carefully optimized protocol, we show tight correlation between the iReenCAM monitored fluorescence and HPLC measured chlorophyll accumulation during first 4h of seedling de-etiolation in wild type Arabidopsis and mutants with disturbed chlorophyll biosynthesis. Using the approach, we demonstrate negative effect of exogenously applied cytokinins and ethylene on chlorophyll biosynthesis during early de-etiolation. Accordingly, we identify type-B response regulators, the cytokinin-responsive transcriptional activators ARR1 and ARR12 as negative regulators of early chlorophyll biosynthesis, while contrasting response was observed in case of EIN2 and EIN3, the components of canonical ethylene signaling cascade. Knowing that, we propose the use of iReenCAM as a new phenotyping tool, suitable for quantitative and robust characterization of the highly dynamic response of seedling de-etiolation.

Unraveling Epigenetic Changes in A. thaliana Calli: Impact of HDAC Inhibitors.

The ability for plant regeneration from dedifferentiated cells opens up the possibility for molecular bioengineering to produce crops with desirable traits. Developmental and environmental signals that control cell totipotency are regulated by gene expression via dynamic chromatin remodeling. Using a mass spectrometry-based approach, we investigated epigenetic changes to the histone proteins during callus formation from roots and shoots of Arabidopsis thaliana seedlings. Increased levels of the histone H3.3 variant were found to be the major and most prominent feature of 20-day calli, associated with chromatin relaxation. The methylation status in root- and shoot-derived calli reached the same level during long-term propagation, whereas differences in acetylation levels provided a long-lasting imprint of root and shoot origin. On the other hand, epigenetic signs of origin completely disappeared during 20 days of calli propagation in the presence of histone deacetylase inhibitors (HDACi), sodium butyrate, and trichostatin A. Each HDACi affected the state of post-translational histone modifications in a specific manner; NaB-treated calli were epigenetically more similar to root-derived calli, and TSA-treated calli resembled shoot-derived calli.

Seed Priming With Protein Hydrolysates Improves Arabidopsis Growth and Stress Tolerance to Abiotic Stresses.

The use of plant biostimulants contributes to more sustainable and environmentally friendly farming techniques and offers a sustainable alternative to mitigate the adverse effects of stress. Protein hydrolysate-based biostimulants have been described to promote plant growth and reduce the negative effect of abiotic stresses in different crops. However, limited information is available about their mechanism of action, how plants perceive their application, and which metabolic pathways are activating. Here we used a multi-trait high-throughput screening approach based on simple RGB imaging and combined with untargeted metabolomics to screen and unravel the mode of action/mechanism of protein hydrolysates in Arabidopsis plants grown in optimal and in salt-stress conditions (0, 75, or 150 mM NaCl). Eleven protein hydrolysates from different protein sources were used as priming agents in Arabidopsis seeds in three different concentrations (0.001, 0.01, or 0.1 µl ml-1). Growth and development-related traits as early seedling establishment, growth response under stress and photosynthetic performance of the plants were dynamically scored throughout and at the end of the growth period. To effectively classify the functional properties of the 11 products a Plant Biostimulant Characterization (PBC) index was used, which helped to characterize the activity of a protein hydrolysate based on its ability to promote plant growth and mitigate stress, and to categorize the products as plant growth promoters, growth inhibitors and/or stress alleviator. Out of 11 products, two were identified as highly effective growth regulators and stress alleviators because they showed a PBC index always above 0.51. Using the untargeted metabolomics approach, we showed that plants primed with these best performing biostimulants had reduced contents of stress-related molecules (such as flavonoids and terpenoids, and some degradation/conjugation compounds of phytohormones such as cytokinins, auxins, gibberellins, etc.), which alleviated the salt stress response-related growth inhibition.

Integration of Phenomics and Metabolomics Datasets Reveals Different Mode of Action of Biostimulants Based on Protein Hydrolysates in Lactuca sativa L. and Solanum lycopersicum L. Under Salinity.

Plant phenomics is becoming a common tool employed to characterize the mode of action of biostimulants. A combination of this technique with other omics such as metabolomics can offer a deeper understanding of a biostimulant effect in planta. However, the most challenging part then is the data analysis and the interpretation of the omics datasets. In this work, we present an example of how different tools, based on multivariate statistical analysis, can help to simplify the omics data and extract the relevant information. We demonstrate this by studying the effect of protein hydrolysate (PH)-based biostimulants derived from different natural sources in lettuce and tomato plants grown in controlled conditions and under salinity. The biostimulants induced different phenotypic and metabolomic responses in both crops. In general, they improved growth and photosynthesis performance under control and salt stress conditions, with better performance in lettuce. To identify the most significant traits for each treatment, a random forest classifier was used. Using this approach, we found out that, in lettuce, biomass-related parameters were the most relevant traits to evaluate the biostimulant mode of action, with a better response mainly connected to plant hormone regulation. However, in tomatoes, the relevant traits were related to chlorophyll fluorescence parameters in combination with certain antistress metabolites that benefit the electron transport chain, such as 4-hydroxycoumarin and vitamin K1 (phylloquinone). Altogether, we show that to go further in the understanding of the use of biostimulants as plant growth promotors and/or stress alleviators, it is highly beneficial to integrate more advanced statistical tools to deal with the huge datasets obtained from the -omics to extract the relevant information.

Dissecting the interaction of photosynthetic electron transfer with mitochondrial signalling and hypoxic response in the Arabidopsis rcd1 mutant.

The Arabidopsis mutant rcd1 is tolerant to methyl viologen (MV). MV enhances the Mehler reaction, i.e. electron transfer from Photosystem I (PSI) to O2, generating reactive oxygen species (ROS) in the chloroplast. To study the MV tolerance of rcd1, we first addressed chloroplast thiol redox enzymes potentially implicated in ROS scavenging. NADPH-thioredoxin oxidoreductase type C (NTRC) was more reduced in rcd1. NTRC contributed to the photosynthetic and metabolic phenotypes of rcd1, but did not determine its MV tolerance. We next tested rcd1 for alterations in the Mehler reaction. In rcd1, but not in the wild type, the PSI-to-MV electron transfer was abolished by hypoxic atmosphere. A characteristic feature of rcd1 is constitutive expression of mitochondrial dysfunction stimulon (MDS) genes that affect mitochondrial respiration. Similarly to rcd1, in other MDS-overexpressing plants hypoxia also inhibited the PSI-to-MV electron transfer. One possible explanation is that the MDS gene products may affect the Mehler reaction by altering the availability of O2. In green tissues, this putative effect is masked by photosynthetic O2 evolution. However, O2 evolution was rapidly suppressed in MV-treated plants. Transcriptomic meta-analysis indicated that MDS gene expression is linked to hypoxic response not only under MV, but also in standard growth conditions. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Understanding the Biostimulant Action of Vegetal-Derived Protein Hydrolysates by High-Throughput Plant Phenotyping and Metabolomics: A Case Study on Tomato.

Designing and developing new biostimulants is a crucial process which requires an accurate testing of the product effects on the morpho-physiological traits of plants and a deep understanding of the mechanism of action of selected products. Product screening approaches using omics technologies have been found to be more efficient and cost effective in finding new biostimulant substances. A screening protocol based on the use of high-throughput phenotyping platform for screening new vegetal-derived protein hydrolysates (PHs) for biostimulant activity followed by a metabolomic analysis to elucidate the mechanism of the most active PHs has been applied on tomato crop. Eight PHs (A-G, I) derived from enzymatic hydrolysis of seed proteins of Leguminosae and Brassicaceae species were foliarly sprayed twice during the trial. A non-ionic surfactant Triton X-100 at 0.1% was also added to the solutions before spraying. A control treatment foliarly sprayed with distilled water containing 0.1% Triton X-100 was also included. Untreated and PH-treated tomato plants were monitored regularly using high-throughput non-invasive imaging technologies. The phenotyping approach we used is based on automated integrative analysis of photosynthetic performance, growth analysis, and color index analysis. The digital biomass of the plants sprayed with PH was generally increased. In particular, the relative growth rate and the growth performance were significantly improved by PHs A and I, respectively, compared to the untreated control plants. Kinetic chlorophyll fluorescence imaging did not allow to differentiate the photosynthetic performance of treated and untreated plants. Finally, MS-based untargeted metabolomics analysis was performed in order to characterize the functional mechanisms of selected PHs. The treatment modulated the multi-layer regulation process that involved the ethylene precursor and polyamines and affected the ROS-mediated signaling pathways. Although further investigation is needed to strengthen our findings, metabolomic data suggest that treated plants experienced a metabolic reprogramming following the application of the tested biostimulants. Nonetheless, our experimental data highlight the potential for combined use of high-throughput phenotyping and metabolomics to facilitate the screening of new substances with biostimulant properties and to provide a morpho-physiological and metabolomic gateway to the mechanisms underlying PHs action on plants.

A Combined Phenotypic and Metabolomic Approach for Elucidating the Biostimulant Action of a Plant-Derived Protein Hydrolysate on Tomato Grown Under Limited Water Availability.

Plant-derived protein hydrolysates (PHs) are an important category of biostimulants able to increase plant growth and crop yield especially under environmental stress conditions. PHs can be applied as foliar spray or soil drench. Foliar spray is generally applied to achieve a relatively short-term response, whereas soil drench is used when a long-term effect is desired. The aim of the study was to elucidate the biostimulant action of PH application method (foliar spray or substrate drench) on morpho-physiological traits and metabolic profile of tomato grown under limited water availability. An untreated control was also included. A high-throughput image-based phenotyping (HTP) approach was used to non-destructively monitor the crop response under limited water availability (40% of container capacity) in a controlled environment. Moreover, metabolic profile of leaves was determined at the end of the trial. Dry biomass of shoots at the end of the trial was significantly correlated with number of green pixels (R 2 = 0.90) and projected shoot area, respectively. Both drench and foliar treatments had a positive impact on the digital biomass compared to control while the photosynthetic performance of the plants was slightly influenced by treatments. Overall drench application under limited water availability more positively influenced biomass accumulation and metabolic profile than foliar application. Significantly higher transpiration use efficiency was observed with PH-drench applications indicating better stomatal conductance. The mass-spectrometry based metabolomic analysis allowed the identification of distinct biochemical signatures in PH-treated plants. Metabolomic changes involved a wide and organized range of biochemical processes that included, among others, phytohormones (notably a decrease in cytokinins and an accumulation of salicylates) and lipids (including membrane lipids, sterols, and terpenes). From a general perspective, treated tomato plants exhibited an improved tolerance to reactive oxygen species (ROS)-mediated oxidative imbalance. Such capability to cope with oxidative stress might have resulted from a coordinated action of signaling compounds (salicylic acid and hydroxycinnamic amides), radical scavengers such as carotenoids and prenyl quinones, as well as a reduced biosynthesis of tetrapyrrole coproporphyrins.

High-Throughput Plant Phenotyping for Developing Novel Biostimulants: From Lab to Field or From Field to Lab?

Plant biostimulants which include bioactive substances (humic acids, protein hydrolysates and seaweed extracts) and microorganisms (mycorrhizal fungi and plant growth promoting rhizobacteria of strains belonging to the genera Azospirillum, Azotobacter, and Rhizobium spp.) are gaining prominence in agricultural systems because of their potential for improving nutrient use efficiency, tolerance to abiotic stressors, and crop quality. Highly accurate non-destructive phenotyping techniques have attracted the interest of scientists and the biostimulant industry as an efficient means for elucidating the mode of biostimulant activity. High-throughput phenotyping technologies successfully employed in plant breeding and precision agriculture, could prove extremely useful in unraveling biostimulant-mediated modulation of key quantitative traits and would also facilitate the screening process for development of effective biostimulant products in controlled environments and field conditions. This perspective article provides an innovative discussion on how small, medium, and large high-throughput phenotyping platforms can accelerate efforts for screening numerous biostimulants and understanding their mode of action thanks to pioneering sensor and image-based phenotyping techniques. Potentiality and constraints of small-, medium-, and large-scale screening platforms are also discussed. Finally, the perspective addresses two screening approaches, "lab to field" and "field to lab," used, respectively, by small/medium and large companies for developing novel and effective second generation biostimulant products.

High-Throughput Non-destructive Phenotyping of Traits that Contribute to Salinity Tolerance in Arabidopsis thaliana.

Reproducible and efficient high-throughput phenotyping approaches, combined with advances in genome sequencing, are facilitating the discovery of genes affecting plant performance. Salinity tolerance is a desirable trait that can be achieved through breeding, where most have aimed at selecting for plants that perform effective ion exclusion from the shoots. To determine overall plant performance under salt stress, it is helpful to investigate several plant traits collectively in one experimental setup. Hence, we developed a quantitative phenotyping protocol using a high-throughput phenotyping system, with RGB and chlorophyll fluorescence (ChlF) imaging, which captures the growth, morphology, color and photosynthetic performance of Arabidopsis thaliana plants in response to salt stress. We optimized our salt treatment by controlling the soil-water content prior to introducing salt stress. We investigated these traits over time in two accessions in soil at 150, 100, or 50 mM NaCl to find that the plants subjected to 100 mM NaCl showed the most prominent responses in the absence of symptoms of severe stress. In these plants, salt stress induced significant changes in rosette area and morphology, but less prominent changes in rosette coloring and photosystem II efficiency. Clustering of ChlF traits with plant growth of nine accessions maintained at 100 mM NaCl revealed that in the early stage of salt stress, salinity tolerance correlated with non-photochemical quenching processes and during the later stage, plant performance correlated with quantum yield. This integrative approach allows the simultaneous analysis of several phenotypic traits. In combination with various genetic resources, the phenotyping protocol described here is expected to increase our understanding of plant performance and stress responses, ultimately identifying genes that improve plant performance in salt stress conditions.