This work was supported by the ISF-NSFC joint research program (grant No. 2436/18) and was also partially supported by the Israel Ministry of Agriculture and Rural Development (Eugene Kandel Knowledge Centers) as part of the Root of the Matter &#8211; The Root Zone Knowledge Center for Leveraging Modern Agriculture.

<ol>
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The authors have nothing to disclose.

The genotype&#8211;phenotype knowledge gap reflects the complexity of genotype x environment interactions (reviewed by18,24). It might be possible to bridge this gap through the use of high-resolution, HTP-telemetric diagnostic and phenotypic screening platforms that can be used to study whole-plant physiological performance and water-relation kinetics8,9. The complexity of genotype x environment interactions makes phenotyping a challenge, particularly in light of how rapidly plants respond to their changing environments. Although various phenotyping systems are currently available, most of those systems are based on remote sensing and advanced imaging techniques. Although those systems provide simultaneous measurements, to a certain extent, their measurements are limited to morphological and indirect physiological traits25. Physiological traits are very important in the context of responsiveness or sensitivity to environmental conditions26. Therefore, direct measurements taken continuously and simultaneously at a very high resolution (e.g., 3 min intervals) can provide a very accurate description of a plant&#8217;s physiological behavior. Despite those substantial advantages of the gravimetric system, the fact that this system has some potential disadvantages must also be taken into account. The main disadvantages result from the need to work with pots and in greenhouse conditions, which can present major challenges for treatment-regulation (particularly the regulation of drought treatments) and experimental-repeatability.
In order to address these issues, one should standardize the applied stresses, create a truly randomized experimental structure, minimize pot effects and compare multiple dynamic behaviors of plants under changing environmental conditions within a short period of time. The HTP-telemetric functional phenotyping approach described in this paper addresses those issues as noted below.
In order to correlate the plant&#8217;s dynamic response with its dynamic environment and capture a complete, big picture of complex plant&#8211;environment interactions, both environmental conditions (Figure 4) and plant responses (Supplementary Figure 9B) must be measured continuously. This method enables the measurement of physical changes in the potting medium and atmosphere continuously and simultaneously, alongside plant traits (soil&#8211;plant&#8211;atmosphere continuum, SPAC).
To best predict how plants will behave in the field, it is important to perform the phenotyping process under conditions that are as similar as possible to those found in the field18. We conduct the experiments in a greenhouse under semi-controlled conditions to mimic field conditions as much as possible. One of the most important conditions is the growing or potting medium. Selecting the most suitable potting medium for the gravimetric-system experiment is crucial. It is advisable to choose a soil medium that drains quickly, allows for the rapid achievement of pot capacity and has a highly stable pot capacity, as those features allow for more accurate measurements by the gravimetric system. In addition, the different treatments to be applied in the experiment must also be considered. For example, treatments involving salts, fertilizers or chemicals call for the use of an inert potting medium, preferably one with a low cation-exchange capacity. Drought treatments applied to low-transpiring plant species would work best with potting media with relatively low VWC levels. In contrast, slow drought treatments applied to high-transpiring plants would work best with potting media with relatively high VWC levels. If the roots are required for post-experiment analysis (e.g., root morphology, dry weight, etc.), the use of a medium with relatively low organic matter content (i.e., sand, porous ceramic or perlite) will make it easier to wash the roots without damaging them. For experiments that will continue for longer periods, it is advisable to avoid media that are rich in organic matter, as that organic matter may decompose with time. Please see Table 1 and Table 2 for more detailed information on this topic.
Field phenotyping and greenhouse phenotyping (pre-field) have their own objectives and require different experimental set-ups. Pre-field phenotyping assists the selection of promising candidate genotypes that have a high probability of doing well in the field, to help make field trials more focused and cost-effective. However, pre-field phenotyping involves a number of limitations (e.g., pot effects) that can cause plants to perform differently than they would under field conditions18,27. Small pot size, water loss by evaporation and heating of the lysimeter scales are examples of factors in greenhouse experiments that may lead to pot effects18. The method described here is designed to minimize those potential effects in the following manner:
(a) The pot size is chosen based on the genotype to be examined. The system is capable of supporting various pot sizes (up to 25 L) and irrigation treatments, which enables the examination of any type of crop plant. 
(b) The pots and the lysimeter scales are insulated to prevent heat from being transferred and any warming of the pots. 
(c) This system involves a carefully designed irrigation and drainage system. 
(d) There is a separate controller for each pot, to enable true randomization with self-irrigating and self-monitored treatments. 
(e) The software takes into account the plants&#8217; local VPD in calculating the canopy stomatal conductance. Please see the multiple VPD stations localization in Figure 1J.
This system involves direct physiological measurements at field-like plant densities, which eliminates the need for either large spaces between the plants or moving the plants for image-based phenotyping. This system includes real-time data analysis, as well as the ability to accurately detect the physiological stress point (&#952;) of each plant. This enables the researcher to monitor the plants and make decisions regarding how the experiment is to be conducted and how any samples are to be collected over the course of the experiment. The system&#8217;s easy and simple weight calibration facilitates efficient calibration. High-throughput systems generate massive amounts of data, which present additional data-handling and analytical challenges11,12. The real-time analysis of the big data that is directly fed to the software from the controller is an important step in the translation of data into knowledge14 that has great value for practical decision-making.
This HTP-telemetric physiological phenotyping method might be helpful for conducting greenhouse experiments under close-to-field conditions. The system is able to measure and directly calculate water-related physiological responses of plants to their dynamic environment, while efficiently overcoming most of the problems associated with the pot effect. This system&#8217;s abilities are extremely important in the pre-field phenotyping stage, as they offer the possibility to predict yield penalties during early stages of plant growth.

Ensuring food security for a growing global population under deteriorating environmental conditions is currently one of the major goals of agriculture research1,2,3. The availability of new molecular tools has greatly enhanced crop-improvement programs. However, while genomic tools provide a massive amount of data, the limited understanding of actual phenotypic traits creates a significant knowledge gap. Bridging this gap is one of the greatest challenges facing modern plant science4,5,6. To meet the challenges that arise in the process of crop improvement and minimize the genotype&#8211;phenotype knowledge gap, we must balance the genotypic approach with a phenocentric one7,8.
Recently, various high-throughput phenotyping (HTP) platforms have made possible the nondestructive phenotyping of large plant populations over time and these platforms may help us to reduce the genotype&#8211;phenotype knowledge gap6,8,9,10. HTP screening techniques allow the measurement of traits in massive numbers of plants within a relatively short period of time, thanks to robotics and conveyor belts or gantries used to move the plants or sensors (respectively), as opposed to hand-operated techniques based on gas exchange or photography. Nevertheless, the massive amounts of data produced by HTP systems present additional data-handling and analytical challenges11,12.
Most of these HTP platforms involve the assessment of phenotypic traits through electronic sensors or automated image acquisition13,14. Advanced field phenomics involve the deployment of proximal sensors and imaging technologies in the field, as well as a high-resolution, precise and large-population scale of measurement15. Sensor and image data need to be integrated with other multi-omics data to create a holistic, second-generation phenomic approach16. However, methodological advances in data acquisition, handling and processing are becoming increasingly important, as the challenges of translating sensor information into knowledge have been grossly underestimated during the first years of plant phenomics research13. However, the reliability and accuracy of currently available imaging techniques for in depth phenotyping of dynamic genotype&#8211;environment interactions and plant stress responses are questionable17,18. Moreover, the results from controlled environments are often very different than those observed in the field, especially when it comes to drought-stress phenotyping. This is due to differences in the situation the plants experience in terms of soil volume, soil environment and mechanical impedance due to declining soil moisture during drought stress. Therefore, results from controlled environments are difficult to extrapolate to the field19. Finally, the entry price of image-based HTP systems is very high, not only due to the price of sensors, but also due to the robotics, conveyor belts and gantries, which also require higher standards of growth-facility infrastructure and significant maintenance (many moving parts working in a greenhouse environment).
In this paper, we present an HTP-telemetric phenotyping platform designed to solve many of the problems mentioned above. Telemetry technology enables the automatic measurement and transmission of data from remote source(s) to a receiving station for recording and analysis. Here, we demonstrate a nondestructive HTP-telemetric platform that includes multiple weighing lysimeters (a gravimetric system) and environmental sensors. This system can be used for the collection and immediate calculation (image-analysis is not needed) of a wide range of data, such as whole-plant biomass gain, transpiration rates, stomatal conductance, root fluxes and water-use efficiency (WUE). The real-time analysis of the big data that is directly fed to the software from the controller in the system represents an important step in the translation of data into knowledge14 that has great value for practical decision-making, substantially extending the knowledge that can be acquired from controlled environment phenotyping experiments, in general, and greenhouse studies of drought stress, in particular.
Other advantages of the telemetry platform are its scalability and ease of installation and its minimal growth-facility infrastructure requirements (i.e., it can be easily installed in most growth facilities). Moreover, as this sensor-based system has no moving parts, maintenance costs are relatively low, including both the entry price and long-term maintenance costs. For example, the price of a 20-unit gravimetric system, including the feedback fertigation system for each plant, meteorological station and software, will be similar to the price of one portable gas-exchange system of a leading brand.
Rice (Oryza sativa L.) was used as a model crop and drought was the examined treatment. Rice was chosen as it is a major cereal crop with wide genetic diversity and it is the staple food for over half of the world&#39;s population20. Drought is a major environmental abiotic stress factor that can impair plant growth and development, leading to reduced crop yields21. This crop&#8211;treatment combination was used to demonstrate the platform&#8217;s capabilities and the amount and quality of data that it can produce. For more information regarding the theoretical background for this method, please see 22.

<table><tbody><tr><td>Atmospheric Probes </td><td>SpectrumTech/Meter group</td><td>3686WD</td><td>Watchdog 2475</td></tr><tr><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>40027</td><td>VP4</td></tr><tr><td>Array Randomizer</td><td>&amp;nbsp;</td><td>None</td><td>The software &quot;Array Randomizer&quot; can be used for creating an experimental design of a randomized block design, or fully random design. It was developed to have better control over the random distribution of the experimental samples (plants) in order to normalize the atmospheric microvariation inside the greenhouse.</td></tr><tr><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>Free download and more information, please click on the following link: https://drive.google.com/open?id=1y4QbTpxRK5Lx430xzu1RFdrlcL8pz_1q</td></tr><tr><td>Cavity trays</td><td>Danish size with curved rim for nursery</td><td>30162</td><td>4X4X7 Cell, 84 cell per tray&lt;br/&gt;https://desch.nl/en/products/seed_propagation_trays/danish-size-with-curved-rim-for-nursery~p92</td></tr><tr><td>Coarse sand</td><td>Negev Industrial Minerals Ltd., Israel </td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Compost</td><td>Tuff Marom Golan, Israel</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Data Analysis software </td><td>Plant-Ditech Ltd., Israel</td><td>&amp;nbsp;</td><td>SPAC Analytics</td></tr><tr><td>Drippers</td><td>Netafim</td><td>21500-001520</td><td>PCJ 8L/h</td></tr><tr><td>Fine sand</td><td>Negev Industrial Minerals Ltd., Israel </td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Loamy soil (natural soil)</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Nylon mesh</td><td>Not relevant (generic products)</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Operating software </td><td>Plant-Ditech Ltd., Israel</td><td>&amp;nbsp;</td><td>Plantarray Feedback Control (PFC)</td></tr><tr><td>Peat-based soil</td><td>Klasmann-Deilmann GmbH, Germany</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Perlite</td><td>Agrekal , Israel</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Plantarray 3.0 system</td><td>Plant-Ditech Ltd., Israel</td><td>SCA400s</td><td>Weighing lysimeters</td></tr><tr><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>PLA300S</td><td>Planter unit container</td></tr><tr><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>CON100</td><td>Control unit </td></tr><tr><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>part of the planter set</td><td>Fiberglass stick</td></tr><tr><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>part of the planter set</td><td>Gasket ring</td></tr><tr><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>Operating software</td></tr><tr><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>SPAC Analytics software</td></tr><tr><td>Porous, ceramic, mixed-sized medium</td><td>Greens Grade, PROFILE Products LLC., USA</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Porous, ceramic, small-sized medium</td><td>Greens Grade, PROFILE Products LLC., USA</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Pots</td><td>Not relevant (generic products)</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Soil</td><td>Bental 11 by Tuff Marom Golan </td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Soil Probes </td><td>Meter group</td><td>40567</td><td>5TE</td></tr><tr><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>40636</td><td>5TM</td></tr><tr><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>40478</td><td>GS3</td></tr><tr><td>Vermiculite</td><td>Agrekal , Israel</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr></tbody></table>

a telemetric, gravimetric platform for real-time physiological phenotyping of plant&ndash;environment interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

Watch this Scientific Journal Video about A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plantâ€“Environment Interactions at JoVE.com

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...

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JoVE Journal

Environment

11.4K Views.  The Hebrew University of Jerusalem. Planten reageren op zeer complexe manieren op de omgeving. Fysiologische eigenschappen behoren tot de snelst te reageren. Meestal kunnen veel eerder dan morfologische verschillen worden gedetecteerd.Deze functionele fenotyperingsmethode met hoge resolutie maakt het mogelijk om de omgevingsinteracties van de plant continu en destructief te kwantificeren onder vele behandelingen. Dit systeem kan worden gebruikt voor real-time metingen van een breed scala aan fysiologische eigenschappen, ...

Video: A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant–Environment Interactions

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop cultivars against environmental stresses. An experimental protocol is presented for RGB and hyperspectral imaging of root systems. The approach uses rhizoboxes where plants grow in natural soil over a longer time to observe fully developed root systems. Experimental settings are exemplified for assessing rhizobox plants under water stress and studying the role of roots. An RGB imaging setup is described for cheap and quick quantification of root development over time. Hyperspectral imaging improves root segmentation from the soil background compared to RGB color based thresholding. The particular strength of hyperspectral imaging is the acquisition of chemometric information on the root-soil system for functional understanding. This is demonstrated with high resolution water content mapping. Spectral imaging however is more complex in image acquisition, processing and analysis compared to the RGB approach. A combination of both methods can optimize a comprehensive assessment of the root system. Application examples integrating root and aboveground traits are given for the context of plant phenotyping and plant physiological research. Further improvement of root imaging can be obtained by optimizing RGB image quality with better illumination using different light sources and by extension of image analysis methods to infer on root zone properties from spectral data.

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop ...

Manufacturing Simple and Inexpensive Soil Surface Temperature and Gravimetric Water Content Sensors

Quantifying temperature and moisture at the soil surface is essential for understanding how soil surface biota respond to changes in the environment. However, at the soil surface these variables are highly dynamic and standard sensors do not explicitly measure temperature or moisture in the upper few millimeters of the soil profile. This paper describes methods for manufacturing simple, inexpensive sensors that simultaneously measure the temperature and moisture of the upper 5 mm of the soil surface. In addition to sensor construction, steps for quality control, as well as for calibration for various substrates, are explained. The sensors incorporate a Type E thermocouple to measure temperature and assess soil moisture by measuring the resistance between two gold-plated metal probes at the end of the sensor at a depth of 5 mm. The methods presented here can be altered to customize probes for different depths or substrates. These sensors have been effective in a variety of environments and have endured months of heavy rains in tropical forests as well as intense solar radiation in deserts of the southwestern U.S. Results demonstrate the effectiveness of these sensors for evaluating warming, drying, and freezing of the soil surface in a global change experiment.

Accurately measuring temperature and water content of the upper 5 mm of the soil surface can improve our understanding of environmental controls on biological, chemical, and physical processes. Here we describe a protocol for manufacturing, calibrating, and conducting measurements with soil surface temperature and moisture sensors.

Quantifying temperature and moisture at the soil surface is essential for understanding how soil surface biota respond to changes in the environment. ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

Biology

Automated Stomatal Response Measurement to Bacterial Invasion in Arabidopsis

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake for photosynthesis and transpirational water loss but also for restricting bacterial invasion. While plants close stomata upon recognition of microbes, pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000 (Pto), reopen the closed stomata to gain access into the leaf interior. In conventional assays for assessing stomatal responses to bacterial invasion, leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion in a leaf attached to the plant. Recently, a portable imaging device was developed that can observe stomata by pinching a leaf without detaching it from the plant, together with a deep learning-based image analysis pipeline designed to automatically measure stomatal aperture from leaf images captured by the device. Here, building on these technical advances, a new method to assess stomatal responses to bacterial invasion in Arabidopsis thaliana is introduced. This method consists of three simple steps: spray inoculation of Pto mimicking natural infection processes, direct observation of stomata on a leaf of the Pto-inoculated plant using the portable imaging device, and automated measurement of stomatal aperture by the image analysis pipeline. This method was successfully used to demonstrate stomatal closure and reopening during Pto invasion under conditions that closely mimic the natural plant-bacteria interaction.

Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement

Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device.

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake ...

Immunology and Infection

Potato Resilience Analysis Under Climate-Related Stresses

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&#160;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.

Author Spotlight: Unraveling Plant Responses to Abiotic Stresses Using the PlantScreen Robotic Platform

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions ...

Imaging and Analysis for Quantifying Maize (Zea mays) Abiotic Stress Phenotypes

Measuring quantitative plant phenotypes, or traits, is essential for understanding plant responses to environmental factors, including biotic and abiotic stresses. However, traditional methods of measuring plants can be time-consuming, inaccurate, and destructive, particularly when assessing traits such as biomass and height on an individual basis. Image-based analysis offers an opportunity to reduce labor, improve accuracy and precision, and enable repeated measurements of the same plant over multiple time points. While high-throughput phenotyping facilities provide a solution, they are often prohibitively expensive and limited in availability worldwide. A method is described for capturing plant images using low-cost single-board computers and digital cameras within a photo studio setup, accompanied by an image analysis pipeline based on the free and open-source package PlantCV. This method offers a fast and accurate protocol for image capture, labeling, and transfer, along with best practices for ensuring high-quality image acquisition. Through image analysis, more than 10 phenotypes were quantified for all images in the experiment simultaneously, including traits such as plant size, height, and color. These methods were used to characterize the response of the Zea mays (maize) inbred variety B73 to heat, drought, and combined heat and drought abiotic stress conditions.

A method is presented for low-cost imaging of plants to measure phenotypes, along with best practices for image capture and an image analysis pipeline for quantifying plant traits. These methods were applied to measure maize (Zea mays) phenotypes under heat, drought, and combined abiotic stress conditions.

Measuring quantitative plant phenotypes, or traits, is essential for understanding plant responses to environmental factors, including biotic and abiotic ...

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant–Environment Interactions

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