We are grateful to Prof. Seiichiro Hasezawa for his kind support of our work. This work was supported by grants from the Japan Society for the Promotion of Science (JSPS) KAKENHgrant numbers 17K19380 and 18H05492, from The Sumitomo Foundation for a Grant for Basic Science Research Projects grant number 160146, and The Canon Foundation to T.H. This experimental system was developed under a financial support from the JSPS KAKENHgrant number 26891006 to K. A. We thank Robbie Lewis, MSc, from Edanz Group (www.edanzediting.com/ac) for editing a draft of the manuscript.

1. Preparation of 3% Sucrose-containing 1/2 Murashige-Skoog Medium Solution
<ol>
	<li>Add 1.1 g of Murashige-Skoog medium salts and 15 g of sucrose to a beaker.</li>
	<li>Add 490 mL of distilled water and mix well using a stir bar.</li>
	<li>Adjust the pH to 5.8 using KOH.</li>
	<li>Dilute to 500 mL with distilled water and transfer the solution into a medium bottle.</li>
	<li>Sterilize the solution by autoclaving (121 &#176;C, 20 min). If not used immediately, this solution may be kept at 4 &#176;C after sterilization.</li>
</ol>
2. Induction of Clustered Stomata by Sucrose-Containing Medium Solution Immersion Treatment
<ol>
	<li>Sterilize the seeds.
	<ol>
		<li>Prepare the sterilization solution by adding 500 &#181;L of 5% active chlorine NaClO solution and 1 &#181;L of 10% Triton X-100 to 500 &#181;L sterile water.</li>
		<li>Place ca. 50 transgenic A. thaliana seeds carrying a fluorescent marker such as CT-GFP11 or GFP-TUB612 into a 1.5-mL tube.</li>
		<li>Add 1 mL of 70% ethanol solution and mix well by inverting five times. Leave for 1 min.</li>
		<li>The seeds will sink to the bottom of the tube. On a clean bench, gently remove the 70% ethanol using a micropipette, and add 1 mL of sterilization solution. Mix well by inverting five times and leave for 5 min.</li>
		<li>Wash the seeds. Still working under aseptic conditions on a clean bench, gently remove the solution using a micropipette, and add 1 mL of sterile water. Repeat this step five times.</li>
	</ol>
	</li>
	<li>Add 1.5 mL of sterilized 3% sucrose-containing 1/2 Murashige-Skoog medium solution to each well of a 24-well plate on a clean bench.</li>
	<li>Add two sterilized seeds into each well. Tape the lid onto the 24-well plate using two layers of parafilm.</li>
	<li>Transfer the 24-well plate to a growth chamber set at 23.5 &#176;C with a 12-h/12-h light-dark cycle using 100 &#181;mol m&#8722;2 s&#8722;1 white light and incubate for 14 days.</li>
</ol>
3. Microscopic Observation of Clustered Stomata
<ol>
	<li>Place 30 &#181;L of 3% sucrose-containing 1/2 Murashige-Skoog medium solution from a well of the 24-well plate onto the center of a glass slide (size: 76 &#215; 26 mm, thickness: 1.0-1.2 mm).</li>
	<li>Remove a cotyledon from a 14-day-old seedling using dissecting scissors. Float the cotyledon with the observation side facing up on the solution drop.</li>
	<li>Prepare the cotyledon specimen according to our previous method13. Essentially, place 30 &#181;L of the solution on the center of a cover glass (size: 18 &#215; 18 mm, thickness: 0.12-0.17 mm). Turn the cover glass upside down and place it on the cotyledon gently. Wipe off excess buffer using a lint-free tissue.</li>
	<li>Set a specimen on the stage of a confocal laser microscope and select clustered guard cells for observation using bright field illumination.</li>
	<li>Acquire confocal images of fluorescently labelled intracellular structures according to the microscope manufacturer&#39;s instructions.</li>
</ol>

<ol><li>Raschke, K., Fellows, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stomatal+movement+in+Zea+mays%3A+shuttle+of+potassium+and+chloride+between+guard+cells+and+subsidiary+cells%5BTitle%5D)+AND+%22Planta%22%5BJournal%5D)">Stomatal movement in Zea mays: shuttle of potassium and chloride between guard cells and subsidiary cells.</a> Planta. 101 (4), 296-316 (1971).</li>
<li>Higaki, T., Hashimoto-Sugimoto, M., Akita, K., Iba, K., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamics+and+environmental+responses+of+PATROL1+in+Arabidopsis+subsidiary+cells%5BTitle%5D)+AND+%22Plant+and+Cell+Physiology%22%5BJournal%5D)">Dynamics and environmental responses of PATROL1 in Arabidopsis subsidiary cells.</a> Plant and Cell Physiology. 55 (4), 773-780 (2013).</li>
<li>Bergmann, D. C., Sack, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stomatal+development%5BTitle%5D)+AND+%22Annual+Review+of+Plant+Biology%22%5BJournal%5D)">Stomatal development.</a> Annual Review of Plant Biology. 58, 163-181 (2007).</li>
<li>Pillitteri, L. J., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+stomatal+development%5BTitle%5D)+AND+%22Annual+Review+of+Plant+Biology%22%5BJournal%5D)">Mechanisms of stomatal development.</a> Annual Review of Plant Biology. 63, 591-614 (2012).</li>
<li>Sakai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+chemical+compound+bubblin+induces+stomatal+mispatterning+in+Arabidopsis+by+disrupting+the+intrinsic+polarity+of+stomatal+lineage+cells%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The chemical compound bubblin induces stomatal mispatterning in Arabidopsis by disrupting the intrinsic polarity of stomatal lineage cells.</a> Development. 144 (3), 499-506 (2017).</li>
<li>Akita, K., Hasezawa, S., Higaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Breaking+of+plant+stomatal+one-cell-spacing+rule+by+sugar+solution+immersion%5BTitle%5D)+AND+%22PLOS+One%22%5BJournal%5D)">Breaking of plant stomatal one-cell-spacing rule by sugar solution immersion.</a> PLOS One. 8 (9), 72456(2013).</li>
<li>Chen, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Arabidopsis+callose+synthase+gene+GSL8+is+required+for+cytokinesis+and+cell+patterning%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">The Arabidopsis callose synthase gene GSL8 is required for cytokinesis and cell patterning.</a> Plant Physiology. 150 (1), 105-113 (2009).</li>
<li>Guseman, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dysregulation+of+cell-to-cell+connectivity+and+stomatal+patterning+by+loss-of-function+mutation+in+Arabidopsis+chorus+%28glucan+synthase-like+8%29%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Dysregulation of cell-to-cell connectivity and stomatal patterning by loss-of-function mutation in Arabidopsis chorus (glucan synthase-like 8).</a> Development. 137 (10), 1731-1741 (2010).</li>
<li>Akita, K., Hasezawa, S., Higaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+microtubules+and+fusicoccin+response+in+clustered+stomatal+guard+cells+induced+by+sucrose+solution+immersion%5BTitle%5D)+AND+%22Plant+Signaling+and+Behavior%22%5BJournal%5D)">Cortical microtubules and fusicoccin response in clustered stomatal guard cells induced by sucrose solution immersion.</a> Plant Signaling and Behavior. 13 (4), 1454815(2018).</li>
<li>Akita, K., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sugar+solution+induces+clustered+lips%5BTitle%5D)+AND+%22Cytologia%22%5BJournal%5D)">Sugar solution induces clustered lips.</a> Cytologia. 79 (2), 125-126 (2014).</li>
<li>Holzinger, A., Buchner, O., Lütz, C., Hanson, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temperature-sensitive+formation+of+chloroplast+protrusions+and+stromules+in+mesophyll+cells+of+Arabidopsis+thaliana%5BTitle%5D)+AND+%22Protoplasma%22%5BJournal%5D)">Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana.</a> Protoplasma. 230 (1-2), 23-30 (2007).</li>
<li>Abe, T., Hashimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+microtubule+dynamics+by+expression+of+modified+%CE%B1-tubulin+protein+causes+right-handed+helical+growth+in+transgenic+Arabidopsis+plants%5BTitle%5D)+AND+%22The+Plant+Journal%22%5BJournal%5D)">Altered microtubule dynamics by expression of modified α-tubulin protein causes right-handed helical growth in transgenic Arabidopsis plants.</a> The Plant Journal. 43 (2), 191-204 (2005).</li>
<li>Higaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+imaging+of+plant+cell+surface+dynamics+with+variable-angle+epifluorescence+microscopy%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Real-time imaging of plant cell surface dynamics with variable-angle epifluorescence microscopy.</a> Journal of Visualized Experiments. (106), 53437(2015).</li>
</ol>

The authors have nothing to disclose.

We have presented protocols for induction of clustered stomata in A. thaliana seedlings by immersion treatment with a sucrose-containing medium solution. As shown here, this method is very simple and requires no specialized skill but can efficiently induce clustered stomata. More than 45% of guard cells are clustered with 3% sucrose-containing medium solution (mean values of more than 20 independent observations)6. Moreover, this experimental system can be directly applied to transgenic o...

The plant stoma is an essential organ for gas exchange for photosynthesis and transpiration, and stomatal movement is accomplished by significant changes in guard cells through ion-driven uptake and release of water. Under a microscope, we can observe a spaced distribution pattern of stomata on the surfaces of leaves and stems. This spaced distribution of stomata is considered to help stomatal movement, which is regulated by ion and water exchange between guard cells and neighboring epidermal cells1,2. Experimental induction systems for clustered stomata are useful for investigating the importance of the spaced distribution of stomata.
It has been reported that spatial clustering of stomata can be induced by genetic modification of key genes for guard cell differentiation3,4 or treatment with a chemical compound5. We also reported that immersion treatment with a medium solution supplemented with sugars including sucrose, glucose, and fructose caused stomatal clustering in cotyledons of Arabidopsis thaliana seedlings6. Reduced callose in new cell walls separating meristemoids and epidermal cells was observed in the sucrose-treated cotyledon epidermis, suggesting that sucrose solution immersion treatment negatively affects the cell wall, which prevents the leakage and ectopic action of key gene products for guard cell differentiation (e.g. transcription factors) towards adjacent epidermal cells6. A similar mechanism was suggested from studies on gsl8/chor mutants7,8. Our experimental system for reproducible induction of clustered stomata using sucrose-containing medium solution is quite easy and cheap. It can also be used to investigate intracellular structures such as organelles and the cytoskeleton in the clustered guard cells when applied to transgenic lines expressing fluorescent markers that label intracellular structures9,10.

<table><tbody><tr><td>24-well plate</td><td>Sumitomo Bakelite</td><td>MS-0824R</td><td></td></tr><tr><td>488 nm laser</td><td>Furukawa Denko</td><td>HPU-50101-PFS2</td><td></td></tr><tr><td>488 nm laser</td><td>Olympus</td><td>Sapphire488-20/O</td><td></td></tr><tr><td>510 nm long-pass filter</td><td>Olympus</td><td>BA510IF</td><td></td></tr><tr><td>524 - 546 nm band-pass filter</td><td>Semrock</td><td>FF01-535/22-25</td><td></td></tr><tr><td>530 nm short-pass filter</td><td>Olympus</td><td>BA530RIF</td><td></td></tr><tr><td>561 nm laser</td><td>CVI Melles Griot</td><td>85-YCA-025-040</td><td></td></tr><tr><td>604 - 644 nm band-pass filter</td><td>Semrock</td><td>FF01-624/40-25</td><td></td></tr><tr><td>Confocal laser scanning head</td><td>Yokogawa</td><td>CSU10</td><td></td></tr><tr><td>Confocal laser scanning head</td><td>Olympus</td><td>FV300</td><td></td></tr><tr><td>Cooled CCD camera</td><td>Photometrics</td><td>CoolSNAP HQ2</td><td></td></tr><tr><td>Image acquisition software</td><td>Molecular Devices</td><td>MetaMorph version 7.8.2.0</td><td></td></tr><tr><td>Image acquisition software</td><td>Olympus</td><td>FLUOVIEW v5.0</td><td></td></tr><tr><td>Immersion oil</td><td>Olympus</td><td>Immersion Oil Type-F</td><td>ne = 1.518 (23 degrees)</td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX-70</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX-71</td><td></td></tr><tr><td>Murashige and Skoog Plant Salt Mixture</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>392-00591</td><td>Murashige T and Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3), 473-497.</td></tr><tr><td>Objective lens&amp;nbsp;</td><td>Olympus</td><td>UPlanApo 100x / 1.35 NA Oil Iris 1.35</td><td>NA = 1.35</td></tr><tr><td>Objective lens&amp;nbsp;</td><td>Olympus</td><td>UPlanAPO 40x / 0.85 NA</td><td>NA = 0.85</td></tr><tr><td>Sucrose</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>196-00015</td><td></td></tr></tbody></table>

an induction system for clustered stomata by sugar solution immersion treatment in arabidopsis thaliana seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

Here, the protocol for a simple method of inducing stomatal clustering with sucrose-containing medium solution in A. thaliana seedlings has been presented. The clustered guard cells grown in sucrose-containing medium solution (Figure 1B) have larger chloroplasts than guard cells grown in sucrose-free control conditions (Figure 1A). The enlargement of chloroplasts was confirmed with CT-GF...

Watch this Scientific Journal Video about An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings at JoVE.com

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...

an-induction-system-for-clustered-stomata-sugar-solution-immersion

Research

JoVE Journal

Environment

5.8K Views.  The University of Tokyo. The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

Video: An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Genetics

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

Developmental Biology

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through nanoindentations with an atomic force microscope (AFM) coupled with an optical inverted fluorescence microscope. The method consists of applying controlled forces to the sample while measuring its deformation, allowing quantifying parameters such as the apparent Young's modulus of cell walls at subcellular resolutions. It requires a careful mechanical immobilization of the sample and correct selection of indenters and indentation depths. Although it can be used only in external tissues, this method allows characterizing mechanical changes in plant cell walls during development and enables the correlation of these microscopic changes with the growth of an entire organ.

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

The atomic force microscopy indentation protocol offers the possibility to dissect the role of the physical properties of the cell wall of a particular cell of a tissue or organ during normal or constrained growth (i.e., under water deficit).

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Biology

Automated Stomatal Response Measurement to Bacterial Invasion in Arabidopsis

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

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

Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement

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

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

Immunology and Infection

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called thigmomorphogenesis. In past decades, several signaling components have been identified and reported for being involved in the mechanotransduction (e.g., calcium ion binding proteins and jasmonic acid biosynthesis enzymes). However, the relatively slow pace of research in the study of force signaling or thigmomorphogenesis is largely attributed to two reasons: the requirement for laborious human hand-manipulated touch induction of thigmomorphogenesis and the force strength errors associated with people&#8217;s hand-touch. To enhance the efficiency of external force loading on a plant organism, an automatic touch-force loading machine was built. This robotic arm-driven hair brush touches provide a labor-saving and easily repeatable touch-force simulation, unlimited rounds of touch repetition and adjustable touch strength. This hair touch-force loading machine can be used for both large scale screening of touch-force signaling mutants and the phenomics study of plant thigmomorphogenesis. In addition, touch materials such as human hair, can be replaced with other natural materials like animal hair, silk threads and cotton fibers. The automated moving arms on the machine may be equipped with water sprinkling nozzles and air blowers to mimic the natural forces of rain drops and wind, respectively. By using this automatic hair touch-force loading machine in combination with the hand-performed cotton swab touching, we have investigated the touch response of two force signaling mutants, MAP KINASE KINASE 1 (MKK1) and MKK2 plants. The phenomes of the touch-force loaded wild type plants and two mutants were evaluated statistically. They have exhibited significant differences in touch response.

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Summary

Explore More Videos

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

A Method for Characterizing Embryogenesis in Arabidopsis

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

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

Measuring Spatial and Temporal Ca²⁺ Signals in Arabidopsis Plants

Lateral Root Inducible System in Arabidopsis and Maize

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus