Name: high-throughput analysis of non-photochemical quenching in crops using pulse amplitude modulated chlorophyll fluorometry
Uploaded: 2022-07-06T12:50:00
Description: Watch this Scientific Journal Video about High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry at JoVE.com

This work is supported by the research project Realizing Increased Photosynthetic Efficiency (RIPE) that is funded by the Bill &#38; Melinda Gates Foundation, Foundation for Food and Agriculture Research, and the U.K. Foreign, Commonwealth &#38; Development Office under grant number OPP1172157.

1. Seed planting
<ol>
	<li>Choose a field site with fertile, well-drained, but not sandy soil, and with a pH of nearly 6.5. Mark out 1.2 m row plots with 0.75 m spacing by scoring the ground with a hoe.</li>
	<li>Plant 50 seeds/m of G. max cv. IA3023 at 3 cm depth along each plot at the beginning of the growing season when soil temperatures are between 25 to 30 &#176;C. 
	NOTE: For the purpose of screening genetic diversity, it is expected that multiple different genotypes are grown and c...

<ol>
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L., 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=An+atypical+short-chain+dehydrogenase-reductase+functions+in+the+relaxation+of+photoprotective+qH+in+Arabidopsis.">An atypical short-chain dehydrogenase-reductase functions in the relaxation of photoprotective qH in Arabidopsis.</a> Nature Plants. 6 (2), 154-166 (2020).</li><li>Dall'Osto, L., Cazzaniga, S., Wada, M., Bassi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+a+slowly+reversible+fluorescence+decay+component+in+the+Arabidopsis+npq4+mutant.">On the origin of a slowly reversible fluorescence decay component in the Arabidopsis npq4 mutant.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 369 (1640), 20130221 (2014).</li><li>Chekanov, K., 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=Non-photochemical+quenching+in+the+cells+of+the+carotenogenic+chlorophyte+Haematococcus+lacustris+under+favorable+conditions+and+under+stress.">Non-photochemical quenching in the cells of the carotenogenic chlorophyte Haematococcus lacustris under favorable conditions and under stress.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1863 (10), 1429-1442 (2019).</li><li>Allorent, G., 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=A+dual+strategy+to+cope+with+high+light+in+Chlamydomonas+reinhardtii.">A dual strategy to cope with high light in Chlamydomonas reinhardtii.</a> The Plant Cell. 25 (2), 545-557 (2013).</li><li>Holzwarth, A. 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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+environmental+photosynthetic+imaging+reveals+emergent+phenotypes.">Dynamic environmental photosynthetic imaging reveals emergent phenotypes.</a> Cell Systems. 2 (6), 365-377 (2016).</li><li>McAusland, L., Atkinson, J. A., Lawson, T., Murchie, E. H. <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+procedure+utilising+chlorophyll+fluorescence+imaging+to+phenotype+dynamic+photosynthesis+and+photoprotection+in+leaves+under+controlled+gaseous+conditions.">High throughput procedure utilising chlorophyll fluorescence imaging to phenotype dynamic photosynthesis and photoprotection in leaves under controlled gaseous conditions.</a> Plant Methods. 15 (1), 109 (2019).</li><li>Woo, N. S., Badger, M. R., Pogson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+non-invasive+procedure+for+quantitative+assessment+of+drought+survival+using+chlorophyll+fluorescence.">A rapid, non-invasive procedure for quantitative assessment of drought survival using chlorophyll fluorescence.</a> Plant Methods. 4 (1), 27 (2008).</li><li>Bielczynski, L. W., &#321;&#261;cki, M. K., Hoefnagels, I., Gambin, A., Croce, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+and+plant+age+affects+photosynthetic+performance+and+photoprotective+capacity.">Leaf and plant age affects photosynthetic performance and photoprotective capacity.</a> Plant Physiology. 175 (4), 1634-1648 (2017).</li><li>Niyogi, K. K., Truong, T. 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Careful choice and handling of leaf disks are critical to obtain reliable measurements of NPQ. First, damage to the tissue, such as rough handling with tweezers, will introduce stress, resulting in low values for the maximum quantum efficiency of photosynthesis. Non-stressed plants typically have Fv/Fm values of around 0.8318, with significant declines indicating a reduction in photosynthetic performance9. However, plants grown under ...

Photosynthesis consists of light absorption, primary electron transfer, energy stabilization, and the synthesis and transport of photosynthetic products1. Understanding each step is vital to guide efforts to increase crop photosynthetic efficiency. Light affects the rate of photosynthesis, requiring balancing energy supply, in the form of photons, with demand for reducing equivalents. When supply exceeds demand, for example under high-light or during reduced CO2 fixation caused by stomatal closure, build-up of reducing power increases the probability of reactive oxygen species formation with the potential to damage the photosynthetic apparatus and impair electron transport. Therefore, to prevent damage, plants have developed several photo-protective mechanisms, including detoxification of reactive oxygen species and non-photochemical quenching of the excited chlorophyll states (NPQ)2.
Maintaining high rates of photosynthesis is challenging under a field environment. Seasonal and diurnal changes, along with environmental fluctuations such as wind-induced leaf movements and transient cloud cover, cause shifts in the amount and intensity of light received by plants for photosynthesis3. NPQ dissipates excess light energy and can help prevent photo-damage while allowing for sustained rates of photosynthesis at high-light4. However, prolonged NPQ during high- to low-light transitions continues to dissipate energy that could be used for carbon reduction5. As a result, speeding up the relaxation of NPQ can increase the efficiency of photosynthesis6, making NPQ relaxation an attractive target for crop improvement.
Pulse amplitude modulated chlorophyll fluorescence (PAM) analysis can be used to calculate NPQ from measurable parameters (Supplementary Table 1 and Supplementary Table 2)7,8,9. This article focuses on determining NPQ relaxation rates in field-grown plants for the purpose of screening natural variation in germplasm. However, PAM chlorophyll fluorometry analysis can also be used for a wide variety of purposes, applied to species ranging from algae to higher plants, and is reviewed elsewhere7,8,9.
In a dark-adapted leaf or cell, photosystem II (PSII) reaction centers are open to receive electrons and there is no NPQ. Switching on a low-intensity measuring light elicits chlorophyll fluorescence while avoiding electron transport through PSII. The recorded minimum fluorescence in this dark-adapted state is described by the parameter Fo. Applying a high-intensity light pulse to a dark-adapted leaf can rapidly reduce the first stable electron acceptor pool of quinones bound to the quinone A site. This temporarily blocks electron transfer capacity in PSII reaction centers, which are then said to be closed and unable to receive electrons from water-splitting. By using a short pulse duration, there is insufficient time to stimulate NPQ. The resulting chlorophyll fluorescence is equivalent to the maximum value obtainable in the absence of NPQ, or maximum fluorescence, Fm. The difference between minimal and maximal fluorescence is referred to as variable fluorescence, Fv. The maximum photochemical quantum yield of photosystem II (Fv/Fm) is calculated from these two parameters using the following equation:
Fv/Fm = (Fm-Fo)/Fm
This can provide an important indicator of photosystem function and stress. Turning on an actinic (photosynthetic) light stimulates non-photochemical quenching, and subsequent application of a saturating flash allows for the measurement of light-adapted maximal fluorescence, Fm&#39;. By comparing the difference between dark and light-adapted maximum fluorescence, NPQ can be calculated according to the Stern-Volmer equation10:
NPQ = Fm/Fm&#39; - 1
In higher plants, NPQ has been described as consisting of at least five distinct components, including qE, qT, qZ, qI and qH. The precise mechanisms involved in NPQ are not fully understood; however, qE is considered to be the major component of NPQ in most plants. Crucial factors for full engagement of qE have been found to include the build-up of a proton gradient across the thylakoid membrane, the activity of photosystem II subunit S11,12, and de-epoxidated xanthophylls, antheraxanthin, lutein, and in particular zeaxanthin13. qE relaxes the fastest of any NPQ component (&#60; 2 min)14, and reversible activation of qE is therefore particularly important for adaptation to shifting light intensities. A second slower phase of NPQ relaxation (~2-30 min) encompasses both qT, related to state transitions, and qZ, involving interconversion of zeaxanthin to violaxanthin15. Slow relaxing (&#62; 30 min) of NPQ may include both photoinhibitory quenching (qI)16 and processes independent of photodamage17,18, such as qH, which is sustained quenching in the peripheral antennae of PSII mediated by a plastid lipocalin protein19,20.
NPQ increases during exposure to high light. Subsequent transfer to low light can result in downregulation of NPQ. The decay of fast, intermediate, and slow relaxing phases can be captured in the parameters of a bi-exponential function15,21,22,23
NPQ = Aq1(-t/&#964;1) + Aq2(-t/&#964;2) + Aq3
The theoretical basis for the bi-exponential function is based on the assumption of first-order utilization of hypothetical quenchers, including qE (Aq1), the combined relaxation of qZ and qT (Aq2), with the corresponding time-constants &#964;q1 and &#964;q2, and long-term NPQ, which includes qI and photodamage independent processes (Aq3). As such, the bi-exponential function provides a more realistic representation of the multiple connected biological processes involved in quenching chlorophyll fluorescence compared to a simpler Hill equation which lacks a theoretical basis24.
NPQ can be measured using a variety of commercially available PAM fluorometers25,26, from simple hand-held devices27 to more advanced closed systems28. However, a limitation of several of these approaches is a relatively low throughput, which makes screening large collections of plants challenging without multiple devices and a team of researchers. To address this issue, McAusland et al. developed a procedure based on excised leaf tissue and used it to identify differences in chlorophyll fluorescence between two wheat cultivars29. The attraction of this approach is that imaging leaf disks, taken from multiple plants with a single device, can facilitate screening hundreds of genotypes within a day. This makes it possible to assess variation in NPQ relaxation as part of genome wide association studies, or for screening breeding populations with the potential to increase crop photosynthetic efficiency and ultimately yield.
Building on the findings of McAusland et al.29, we use PAM chlorophyll fluorescence analysis of leaf disks for high-throughput screening of NPQ relaxation rates in Glycine max (G. max; soybean). This protocol uses the CF Imager25, which is comparable to other commercially available closed-PAM systems, such as the popular FluorCam26. With a dark room for adaptation of samples, users can image 96-well plates, Petri dishes, and small plants. The key advantage of this approach is the increase in throughput afforded by using leaf disks compared to sequential analysis of individual plants. Herein we present representative results, and a method for sampling, measuring, and analysis of NPQ in field-grown plants.

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			<td>Fisher Scientific</td>
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			<td>96 well tissue culture plate</td>
			<td>Fisher Scientific</td>
			<td>FB012931</td>
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			<td>Technologica Ltd.</td>
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			<td>imaging software 
			Country of Origin: United Kingdom</td>
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			<td>iHank-Nose 100-Pack of Premium Nasal Aspirator Hygiene Filters</td>
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			<td>John Henry Company</td>
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			<td>Bemis Company Inc.</td>
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			<td>Fisher Scientific</td>
			<td>14-130M</td>
			<td>Country of Origin: United States of America</td>
		</tr>
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</table>

high-throughput analysis of non-photochemical quenching in crops using pulse amplitude modulated chlorophyll fluorometry

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of non-photochemical quenching (NPQ) has proven to be an effective strategy to increase photosynthetic performance. However, the potential to breed for improved NPQ and a complete understanding of the genetic basis of NPQ relaxation is lacking due to limitations of oversampling and data collection from field-grown crop plants. Building on previous reports, we present a high-throughput assay for analysis of NPQ relaxation rates in Glycine max (soybean) using pulse amplitude modulated (PAM) chlorophyll fluorometry. Leaf disks are sampled from field-grown soybeans before transportation to a laboratory where NPQ relaxation is measured in a closed PAM-fluorometer. NPQ relaxation parameters are calculated by fitting a bi-exponential function to the measured NPQ values following a transition from high to low light. Using this method, it is possible to test hundreds of genotypes within a day. The procedure has the potential to screen mutant and diversity panels for variation in NPQ relaxation, and can therefore be applied to both fundamental and applied research questions.

Figure 1A depicts a typical measurement of NPQ in field-grown soybean. Plants were grown in Urbana, IL (latitude 40.084604&#176;, longitude -88.227952&#176;) during summer 2021, with seeds planted on June 5th. 2021. The leaf discs were sampled after 30 days of planting seeds, and measurements were made with the protocol provided (Table 1). Fv/Fm and NPQ values were calculated for each leaf disk (Supplementary Table 4) and NPQ relaxation par...

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High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry

The protocol introduces a high-throughput method for measuring the relaxation of non-photochemical quenching by pulse amplitude modulated chlorophyll fluorometry. The method is applied to field-grown Glycine max and can be adapted to other species to screen for genetic diversity or breeding populations.

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of ...

This method can provide an insight into the genetic variation in non-photochemical quenching available within genetic diversity panels or breeding populations. The main advantage of this technique is the potential to screen a large number of genotypes for variation in non-photochemical quenching within a single day. The protocol is presented for glycine max or soybean, but can be adapted to analyze other plant spaces from which lift discs can be collected.For somebody performing the protocol for the first time, the key is handling the lift disc to prevent damage or over-saturating sponges with water. To begin, sample the plants in the field site 30 days after germination. Fill 1/3-level of distilled water in all the wells of a 24-well plate.Label the lid and the side of the plate with the replicates to be sampled. Hold the youngest full-expanded leaf present at the top of the plant against a rubber stopper. Press a number two Humboldt cork borer through the leaf and twist to cut a disc, avoiding the mid-rib.Consecutively collect five discs from the same leaf for technical replicates. Push the leaf discs out of the cork borer into a single well of a 24-well plate using a cotton swab and repeat this for a different plant of the same genotype. Check that all leaf discs are floating in the water.If not, gently move leaf discs sticking to the side of a well into a floating position with a cotton swab. Move on to sampling discs from the following plot. Place the lid and seal with a semi-transparent flexible film.after completing the sampling in the entire 24-well plate. Store the plate out of direct sunlight in a bag, box, or empty cooler. In a clean laboratory space, tap the lid of the sealed plate to dislodge leaf discs stuck to the lid during transport.Unwrap the film and remove the lid. Transfer a leaf disc from the first position of the 24-well plate into a fresh 96-well plate with the leaf disc facing flat down at the bottom of the well. Cut a nasal aspirator filter in half.Dip the resulting filter halfway into water and dab on a paper towel to remove excess liquid. Insert the filter into the well with the leaf disc to maintain humidity. Take a second leaf disc from the first position of the 24-well plate and place it face down in the next available position of the the 96-well plate.Dip the remaining half of the nasal filter produced in water and dab it on a paper towel before inserting it in the well with the second leaf disc. Tape the top-right corner to help orient the plate in the dark for imaging. Seal plates with a semi-transparent flexible film and wrap the plate in aluminum foil.Write the plot IDs and plate ID on the aluminum foil. Place plates in a dark box or cabinet for a minimum of 30 minutes for the relaxation of the first two phases of non-photochemical quenching. Use a prolonged dark incubation period of one hour before imaging if long-term phases of non-photochemical quenching are of interest.Prepare an additional dummy plate for focusing during analysis by placing a leaf disc in each corner of a fresh 96-well plate and one in the center. Secure leaf discs with nasal filters as done with previous plates, seal the plate, and incubate in the dark at room temperature and 50%relative humidity. Turn on the imager and open the imaging software.Click on Settings followed by Protocol to open a window for the entry of steps in the PAM experimental protocol. Set the technical specifications of the machine provided in the manuscript. Set the program to start with a saturating pulse to measure the maximum quantum efficiency of dark-adapted photosynthesis by entering 20 seconds into the box named After a delay of.Click the box Apply pulse and enter 1 into the box titled This number of times. Set the pulse PPFD to 6, 152, the pulse length to 800 milliseconds, and check the box Take F prime and FM prime images with all pulses. Check Insert after to add a second step to the protocol.Enter 30 seconds into the box titled After a delay of. Then, select the option Change actinic and enter 50 into the box titled Actinic PPFD to set light intensity to the chamber to 50 PPFD. Click Insert after to add a new step to the protocol and enter 150 seconds into the box titled After a delay of.Select Apply pulse and enter 4 into the box titled This number of times to apply measuring pulses every 150 seconds four times in a row while the actinic light is held at 50 PPFD. Adjust the delay and light intensity for each step according to the values provided in the manuscript before saving the protocol as a pcl file in the desired location. Turn off the light and place the dummy plate face down on the sample stage so that the adaxial surface of the leaf points up towards the detector and that the sponge is facing down towards the platform.Set the sample stage height so that the leaf discs are 140 millimeters above the base of the instrument. Click the connect/disconnect to imager camera and hardware camera icon to start the camera. Click the focus fluorescence symbol, represented by a red-colored, two-sided arrow icon with two green lines at the base, and adjust the lens and exposure to bring the plate into focus.Click the focus fluorescence icon again to turn off the flashing light. Working in the dark, replace the dummy plate with the plate to be analyzed. Place it face down with the tape on the outside used to orient the top right corner and click the map image camera icon.Adjust the image exposure by opening or closing the aperture until the bar in the popup window is positioned in the green zone. Click the Try Again button after each adjustment of the aperture until the exposure is correctly adjusted and the instrument takes an image. Right click on the image and select Apply image isolation to block out background signals.The focused leaf area will be displayed in gray and the background in blue. Select the area or pixels of interest to include only the leaf discs by adjusting the histogram and gamma level from the Modify image pull down menu by right clicking on the image. Right click on the image and select Delete high and low cuts to delete the light blue highlighted areas.Right click on the image and select Delete strays to remove any pixels not touching the last three other pixels. Any areas on the image that appear as isolated islands will be analyzed separately and included in the final data output. Click the run protocol icon to start the program.A timer will appear at the bottom of the screen informing you how long the protocol has left to run. Wait until the protocol is finished. Click File and Save as to save the data as a igr file.Close the window by clicking on the red cross in the top right of the window before starting another sample plate. To export the chlorophyll fluorescence data, open the igr file in the imaging software and click on File followed by Export to Folder to create a new folder with all the necessary files. A typical measurement of non-photochemical quenching in field-grown soybeans showed that after an initial saturating flash to determine Fv/Fm when the leaf discs were exposed to low light of 50 micromoles per meter per second, the non-photochemical quenching was less than one.Furthermore, upon transfer to high light of 2, 000 micromoles per meter per second, non-photochemical quenching increased up to the maximum value of four after 15 minutes. A failed measurement showed a minimal increase in non-photochemical quenching upon transfer to high light. Orienting plates within the imager and a clearly defined plan for sampling and plating leaf discs is essential to ensure that the data is linked to the correct genotype.This protocol can quantify the impact of environmental variables such as drought and shedding on non-photochemical quenching or evaluate non-photochemical quenching at different canopy levels to refine crop models.

high-throughput-analysis-non-photochemical-quenching-crops-using

Research

JoVE Journal

Biology

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Evaluation of Photosynthetic Efficiency in Photorespiratory Mutants by Chlorophyll Fluorescence Analysis

Photosynthesis and photorespiration represent the largest carbon fluxes in plant primary metabolism and are necessary for plant survival. Many of the enzymes and genes important for photosynthesis and photorespiration have been well studied for decades, but some aspects of these biochemical pathways and their crosstalk with several subcellular processes are not yet fully understood. Much of the work that has identified the genes and proteins important in plant metabolism has been conducted under highly controlled environments that may not best represent how photosynthesis and photorespiration function under natural and farming environments. Considering that abiotic stress results in impaired photosynthetic efficiency, the development of a high-throughput screen that can monitor both abiotic stress and its impact on photosynthesis is necessary.
Therefore, we have developed a relatively fast method to screen for abiotic stress-induced changes to photosynthetic efficiency that can identify uncharacterized genes with roles in photorespiration using chlorophyll fluorescence analysis and low CO2 screening. This paper describes a method to study changes in photosynthetic efficiency in transferred DNA (T-DNA) knockout mutants in Arabidopsis thaliana. The same method can be used for screening ethyl methanesulfonate (EMS)-induced mutants or suppressor screening. Utilizing this method can identify gene candidates for further study in plant primary metabolism and abiotic stress responses. Data from this method can provide insight into gene function that may not be recognized until exposure to increased stress environments.

We describe an approach to measure changes in photosynthetic efficiency in plants after treatment with low CO2 using chlorophyll fluorescence.

Photosynthesis and photorespiration represent the largest carbon fluxes in plant primary metabolism and are necessary for plant survival. Many of the ...

Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and fertilization. However, identifying suitable varieties to serve as pollinizers is a time-consuming process. To solve this problem, we have developed a rapid method for identifying pollination-compatible citrus cultivars that utilizes agarose gel electrophoresis and aniline blue staining. Pollen compatibility is determined based on the identification of S genotypes by extracting total DNA and performing PCR-based genotyping assays with specific primers. Additionally, styles are collected 3-4 days after manual pollination, and aniline blue staining is performed. Finally, the growth status of the pollen tubes is observed with a fluorescence microscope. Pollen compatibility and incompatibility can be established by observing whether the pollen tube growth is normal or suppressed, respectively. Due to its simplicity and cost-effectiveness, this method is an effective tool for determining the pollen compatibility and incompatibility of different citrus varieties to establish incompatibility groups and incompatibility relationships between different cultivars. This method provides information essential for the successful selection of suitable pollinizer trees and, thus, facilitates the establishment of new orchards and the selection of appropriate parents for breeding programs.

Determination of Self-Incompatibility and Inter-Incompatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

This protocol provides a rapid method for determining pollen compatibility and incompatibility in citrus cultivars.

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and ...

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Developing Gene Transformation and Editing Methods in Bamboo

In Planta Gene Expression and Gene Editing in Moso Bamboo Leaves

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and regeneration processes. This method involves Agrobacterium-mediated gene expression via wounding and vacuum for bamboo seedlings. It successfully demonstrated the expression of exogenous genes, such as the RUBY reporter and Cas9 gene, in bamboo leaves. The highest transformation efficiency for the accumulation of betalain in RUBY seedlings was achieved using the GV3101 strain, with a percentage of 85.2% after infection. Although the foreign DNA did not integrate into the bamboo genome, the method was efficient in expressing the exogenous genes. Furthermore, a gene editing system has also been developed with a native reporter using this method, from which an in situ mutant generated by the edited bamboo violaxanthin de-epoxidase gene (PeVDE) in bamboo leaves, with a mutation rate of 17.33%. The mutation of PeVDE resulted in decreased non-photochemical quenching (NPQ) values under high light, which can be accurately detected by a fluorometer. This makes the edited PeVDE a potential native reporter for both exogenous and endogenous genes in bamboo. With the reporter of PeVDE, a cinnamoyl-CoA reductase gene was successfully edited with a mutation rate of 8.3%. This operation avoids the process of tissue culture or callus induction, which is quick and efficient for expressing exogenous genes and endogenous gene editing in bamboo. This method can improve the efficiency of gene function verification and will help reveal the molecular mechanisms of key metabolic pathways in bamboo.

Author Spotlight: Advancing Gene Editing in Bamboo Leaves for Sustainable Plastic Alternatives 

In this study, a novel in planta gene expression and gene editing method mediated by Agrobacterium was developed in bamboo. This method greatly improved the efficiency of gene function validation in bamboo, which has significant implications for accelerating the process of bamboo breeding.

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and ...