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...

<ol>
	<li>Raschke, K., Fellows, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+movement+in+Zea+mays:+shuttle+of+potassium+and+chloride+between+guard+cells+and+subsidiary+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+environmental+responses+of+PATROL1+in+Arabidopsis+subsidiary+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+stomatal+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+compound+bubblin+induces+stomatal+mispatterning+in+Arabidopsis+by+disrupting+the+intrinsic+polarity+of+stomatal+lineage+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+of+plant+stomatal+one-cell-spacing+rule+by+sugar+solution+immersion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+callose+synthase+gene+GSL8+is+required+for+cytokinesis+and+cell+patterning.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+cell-to-cell+connectivity+and+stomatal+patterning+by+loss-of-function+mutation+in+Arabidopsis+chorus+(glucan+synthase-like+8).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+microtubules+and+fusicoccin+response+in+clustered+stomatal+guard+cells+induced+by+sucrose+solution+immersion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar+solution+induces+clustered+lips.">Sugar solution induces clustered lips.</a> Cytologia. 79 (2), 125-126 (2014).</li><li>Holzinger, A., Buchner, O., L&#252;tz, C., Hanson, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+formation+of+chloroplast+protrusions+and+stromules+in+mesophyll+cells+of+Arabidopsis+thaliana.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+microtubule+dynamics+by+expression+of+modified+&#945;-tubulin+protein+causes+right-handed+helical+growth+in+transgenic+Arabidopsis+plants.">Altered microtubule dynamics by expression of modified &#945;-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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+plant+cell+surface+dynamics+with+variable-angle+epifluorescence+microscopy.">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 border="0" cellpadding="0" cellspacing="0" style="width:685px;" width="686">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">24-well plate</td>
			<td style="width:135px;">Sumitomo Bakelite</td>
			<td style="width:168px;">MS-0824R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">488 nm laser</td>
			<td style="width:135px;">Furukawa Denko</td>
			<td style="width:168px;">HPU-50101-PFS2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">488 nm laser</td>
			<td style="width:135px;">Olympus</td>
			<td>Sapphire488-20/O</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">510 nm long-pass filter</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">BA510IF</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">524 - 546 nm band-pass filter</td>
			<td style="width:135px;">Semrock</td>
			<td style="width:168px;">FF01-535/22-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">530 nm short-pass filter</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">BA530RIF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">561 nm laser</td>
			<td style="width:135px;">CVI Melles Griot</td>
			<td style="width:168px;">85-YCA-025-040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">604 - 644 nm band-pass filter</td>
			<td style="width:135px;">Semrock</td>
			<td style="width:168px;">FF01-624/40-25</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Confocal laser scanning head</td>
			<td style="width:135px;">Yokogawa</td>
			<td style="width:168px;">CSU10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Confocal laser scanning head</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">FV300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cooled CCD camera</td>
			<td style="width:135px;">Photometrics</td>
			<td style="width:168px;">CoolSNAP HQ2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Image acquisition software</td>
			<td style="width:135px;">Molecular Devices</td>
			<td style="width:168px;">MetaMorph version 7.8.2.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Image acquisition software</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">FLUOVIEW v5.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Immersion oil</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">Immersion Oil Type-F</td>
			<td>ne = 1.518 (23 degrees)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Inverted microscope</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">IX-70</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Inverted microscope</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">IX-71</td>
			<td></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;width:216px;">Murashige and Skoog Plant Salt Mixture</td>
			<td style="width:135px;">FUJIFILM Wako Pure Chemical Corporation</td>
			<td style="width:168px;">392-00591</td>
			<td style="width:167px;">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 height="40">
			<td height="40" style="height:40px;width:216px;">Objective lens&#160;</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">UPlanApo 100x / 1.35 NA Oil Iris 1.35</td>
			<td>NA = 1.35</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Objective lens&#160;</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">UPlanAPO 40x / 0.85 NA</td>
			<td>NA = 0.85</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Sucrose</td>
			<td style="width:135px;">FUJIFILM Wako Pure Chemical Corporation</td>
			<td style="width:168px;">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 ...

This method have answered a key question of plant developmental biology. That is, what is the importance of spatial distribution of stomata? Our induction system for clustered stomata using sucrose solution is fairly easy and directly applicable to transgenic or mutant lines of Arabidopsis thaliana.To begin, add 1.1 grams of Murashige-Skoog medium salts and 15 grams of sucrose to a beaker. Add 490 milliliters of distilled water to the beaker and mix with the stir bar. Use potassium hydroxide to adjust the pH to 5.8.Then dilute the solution with 500 milliliters of distilled water and transfer the diluted solution to a medium bottle. After this, autoclave the solution. Prepare the sterilization solution according the the text protocol.Then, working under aseptic conditions, place about 50 transgenic A.thaliana seeds carrying a fluorescent marker into a 1.5 milliliter tube. Next add one milliliter of 70%ethanol to the tube and mix by inverting the tube five times. After allowing the seeds to sink to the bottom of the tube, gently remove the ethanol with a micropipette.Add one milliliter of sterilization solution to the seeds and invert the tube five times to mix. Gently remove the sterilization solution from the seeds with a micropipette. Then add one milliliter of sterile water.Add 1.5 milliliters of the Murashige-Skoog solution to each well of a 24 well plate. Then add two sterilized seeds to each well. Using two layers of parafilm, tape the lid to the plate.Next, transfer the plate to a growth chamber to incubate for 14 days. First, transfer 30 microliters of Murashige-Skoog solution from the 24 well plate to the center of a glass slide. Using dissecting scissors, remove the cotyledon from a 14-day old seedling.Float the cotyledon with the observation side facing up on the drop of Murashige-Skoog solution. Prepare the cotyledon according to the methods previously described in the Journal of Visualized Experiments. Set the specimen on the stage of a confocal laser microscope.Finally, use bright field illumination to select clustered guard cells for observation. In this protocol, stomatal clustering was induced in A.thaliana seedlings. The clustered guard cells grown in the sucrose containing medium having larger chloroplasts than guard cells grown in sucrose-free conditions.Enlargement of the chloroplasts was confirmed with a chloroplast stroma marker and chlorophyll autofluorescents, suggesting that sucrose treatment resulted in starch grain accumulation in the chloroplasts. Additionally, confocal observation of GFP tub-6 revealed that the cortical microtubules were radially oriented in the sucrose-treated cells, just like in the sucrose-free guard cells. Actually we discovered this method by accident.After the finding, we examined the environmental response of clustered stomata. We believe our system could help to examine the significance of the stomata distribution.

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

Research

JoVE Journal

Environment

5.7K visualizações.  The University of Tokyo. Este método respondeu a uma questão-chave da biologia do desenvolvimento vegetal. Ou seja, qual é a importância da distribuição espacial de stomata? Nosso sistema de indução para stomata agrupada usando solução de sacarose é bastante fácil e diretamente aplicável a linhas transgênicas ou mutantes de Arabidopsis thaliana.Para começar, adicione 1,1 gramas de sais médios Murashige-Skoog e 15 gramas de sacarose a um béquer. Adicione 490 mililitros de água destilada ao bé...