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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R., Stavness, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+Plant+Phenomics:+A+Deep+Learning+Platform+for+Complex+Plant+Phenotyping+Tasks.">Deep Plant Phenomics: A Deep Learning Platform for Complex Plant Phenotyping Tasks.</a> Frontiers in Plant Science. , (2017).</li><li>Danzi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+High+Throughput+Phenotyping+Help+Food+Security+in+the+Mediterranean+Area.">Can High Throughput Phenotyping Help Food Security in the Mediterranean Area.</a> Frontiers in Plant Science. , (2019).</li><li>Miflin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crop+improvement+in+the+21st+century.">Crop improvement in the 21st century.</a> Journal of Experimental Botany. 51, 1-8 (2000).</li><li>Dalal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Physiological+Phenotyping+of+Drought-Stressed+Pepper+Plants+Treated+With+"Productivity-Enhancing"+and+"Survivability-Enhancing"+Biostimulants.">Dynamic Physiological Phenotyping of Drought-Stressed Pepper Plants Treated With "Productivity-Enhancing" and "Survivability-Enhancing" Biostimulants.</a> Frontiers in Plant Science. , (2019).</li><li>Moshelion, M., Altman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+challenges+and+future+perspectives+of+plant+and+agricultural+biotechnology.">Current challenges and future perspectives of plant and agricultural biotechnology.</a> Trends in Biotechnology. 33, 337-342 (2015).</li><li>Singh, A., Ganapathysubramanian, B., Singh, A. K., Sarkar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine+Learning+for+High-Throughput+Stress+Phenotyping+in+Plants.">Machine Learning for High-Throughput Stress Phenotyping in Plants.</a> Trends in Plant Science. 21, 110-124 (2016).</li><li>Houle, D., Govindaraju, D. 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E., Wang, J., Spangenberg, G. C., Smith, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospects+for+measurement+of+dry+matter+yield+in+forage+breeding+programs+using+sensor+technologies.">Prospects for measurement of dry matter yield in forage breeding programs using sensor technologies.</a> Agronomy. 9, 65 (2019).</li><li>Roitsch, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+sensors+and+data-driven+approaches-A+path+to+next+generation+phenomics.">New sensors and data-driven approaches-A path to next generation phenomics.</a> Plant Science. 282, 2-10 (2019).</li><li>Li, L., Zhang, Q., Huang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+imaging+techniques+for+plant+phenotyping.">A review of imaging techniques for plant phenotyping.</a> Sensors (Switzerland). 14, 20078-20111 (2014).</li><li>Gosa, S. C., Lupo, Y., Moshelion, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+comparative+analysis+of+whole-plant+performance+for+functional+physiological+traits+phenotyping:+New+tools+to+support+pre-breeding+and+plant+stress+physiology+studies.">Quantitative and comparative analysis of whole-plant performance for functional physiological traits phenotyping: New tools to support pre-breeding and plant stress physiology studies.</a> Plant Science. 282, 49-59 (2019).</li><li>Araus, J. L., Cairns, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+high-throughput+phenotyping:+the+new+crop+breeding+frontier.">Field high-throughput phenotyping: the new crop breeding frontier.</a> Trends in Plant Science. 19, 52-61 (2014).</li><li>Ito, V. C., Lacerda, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Black+rice+(Oryza+sativa+L.):+A+review+of+its+historical+aspects,+chemical+composition,+nutritional+and+functional+properties,+and+applications+and+processing+technologies.">Black rice (Oryza sativa L.): A review of its historical aspects, chemical composition, nutritional and functional properties, and applications and processing technologies.</a> Food Chemistry. 301, 125304 (2019).</li><li>Anjum, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=physiological+and+biochemical+responses+of+plants+to+drought+stress.">physiological and biochemical responses of plants to drought stress.</a> African Journal of Agricultural Research. , (2011).</li><li>Halperin, O., Gebremedhin, A., Wallach, R., Moshelion, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+physiological+phenotyping+and+screening+system+for+the+characterization+of+plant-environment+interactions.">High-throughput physiological phenotyping and screening system for the characterization of plant-environment interactions.</a> The Plant Journal. 89, 839-850 (2017).</li><li>Yaaran, A., Negin, B., Moshelion, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+guard-cell+ABA+in+determining+steady-state+stomatal+aperture+and+prompt+vapor-pressure-deficit+response.">Role of guard-cell ABA in determining steady-state stomatal aperture and prompt vapor-pressure-deficit response.</a> Plant Science. 281, 31-40 (2019).</li><li>Dalal, A., Attia, Z., Moshelion, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+produce+or+to+survive:+how+plastic+is+your+crop+stress+physiology.">To produce or to survive: how plastic is your crop stress physiology.</a> Frontiers in Plant Science. 8, 2067 (2017).</li><li>Araus, J. L., Kefauver, S. C., Zaman-Allah, M., Olsen, M. S., Cairns, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+High-Throughput+Phenotyping+into+Genetic+Gain.">Translating High-Throughput Phenotyping into Genetic Gain.</a> Trends in Plant Science. 23, 451-466 (2018).</li><li>Ghanem, M. E., Marrou, H., Sinclair, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+phenotyping+of+plants+for+crop+improvement.">Physiological phenotyping of plants for crop improvement.</a> Trends in Plant Science. 20, 139-144 (2015).</li><li>Sinclair, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pot+binding+as+a+variable+confounding+plant+phenotype:+theoretical+derivation+and+experimental+observations.">Pot binding as a variable confounding plant phenotype: theoretical derivation and experimental observations.</a> Planta. 245, 729-735 (2017).</li></ol>

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>&nbsp;</td>
<td>&nbsp;</td>
<td>40027</td>
<td>VP4</td>
</tr>
<tr>
<td>Array Randomizer</td>
<td>&nbsp;</td>
<td>None</td>
<td>The software "Array Randomizer" 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>&nbsp;</td>
<td>&nbsp;</td>
<td>&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 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>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Compost</td>
<td>Tuff Marom Golan, Israel</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Data Analysis software </td>
<td>Plant-Ditech Ltd., Israel</td>
<td>&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>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Loamy soil (natural soil)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Nylon mesh</td>
<td>Not relevant (generic products)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Operating software </td>
<td>Plant-Ditech Ltd., Israel</td>
<td>&nbsp;</td>
<td>Plantarray Feedback Control (PFC)</td>
</tr>
<tr>
<td>Peat-based soil</td>
<td>Klasmann-Deilmann GmbH, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Perlite</td>
<td>Agrekal , Israel</td>
<td>&nbsp;</td>
<td>&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>&nbsp;</td>
<td>&nbsp;</td>
<td>PLA300S</td>
<td>Planter unit container</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>CON100</td>
<td>Control unit </td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>part of the planter set</td>
<td>Fiberglass stick</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>part of the planter set</td>
<td>Gasket ring</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Operating software</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&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>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Porous, ceramic, small-sized medium</td>
<td>Greens Grade, PROFILE Products LLC., USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Pots</td>
<td>Not relevant (generic products)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Soil</td>
<td>Bental 11 by Tuff Marom Golan </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Soil Probes </td>
<td>Meter group</td>
<td>40567</td>
<td>5TE</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>40636</td>
<td>5TM</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>40478</td>
<td>GS3</td>
</tr>
<tr>
<td>Vermiculite</td>
<td>Agrekal , Israel</td>
<td>&nbsp;</td>
<td>&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 PlantÃ¢â‚¬â€œEnvironment 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

11.3K Views.  The Hebrew University of Jerusalem. 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–environment interactions.

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 ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

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 ...

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

Combined Size and Density Fractionation of Soils for Investigations of Organo-Mineral Interactions

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF relies on sequential density separation and sedimentation steps to isolate (1) the free light fraction (uncomplexed organic matter), (2) the occluded light fraction (uncomplexed organic matter trapped in soil aggregates) and (3) a variable number of heavy fractions (soil minerals and their associated organic matter) differing in composition. Provided that the parameters of the CSDF (dispersion energy, density cut-offs, sedimentation time) are properly selected, the method yields heavy fractions of relatively homogeneous mineral composition. Each of these fractions is expected to have a different complexing ability towards organic matter, rendering this a useful method to isolate and study the nature of organo-mineral interactions. Combining density and particle size separation brings an improved resolution compared to simple size or density fractionation methods, allowing the separation of heavy components according to both mineralogy and size (related to surface area) criteria. As is the case for all physical fractionation methods, it may be considered as less disruptive or aggressive than chemically-based extraction methods. However, CSDF is a time-consuming method and furthermore, the quantity of material obtained in some fractions can be limiting for subsequent analysis. Following CSDF, the fractions may be analyzed for mineralogical composition, soil organic carbon concentration and organic matter chemistry. The method provides quantitative information about organic carbon distribution within a soil sample and brings light to the sorptive capacity of the different, naturally-occurring mineral phases, thus providing mechanistic information about the preferential nature of organo-mineral interactions in soils (i.e., which minerals, what type of organic matter).

Combined size and density fractionation (CSDF) is a method to physically separate soil into fractions differing in texture (particle size) and mineralogy (density). The purpose is to isolate fractions with different reactivities towards soil organic matter (SOM), in order to better understand organo-mineral interactions and SOM dynamics.

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF ...