Name: Forfatter Spotlight: Fremme af stomatal forskning med automatiseret blændemåling
Uploaded: 2024-02-09T13:55:00
Description: Watch this Scientific Journal Video about Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement at JoVE.com

Vi takker alle medlemmer af forskningsprojektet, 'Co-creation of plant adaptive traits via assembly of plant-microbe holobiont', for frugtbare diskussioner. Dette arbejde blev støttet af Grant-in-Aid for Transformative Research Areas (21H05151 og 21H05149 til AM og 21H05152 til Y.T.) og Grant-in-Aid for Challenging Exploratory Research (22K19178 til A.M.).

1. Dyrkning af planter
<ol><li>For at bryde dvale skal du resuspendere A. thaliana (Col-0) frø i deioniseret vand og inkubere dem ved 4 ° C i 4 dage i mørke.</li>
	<li>Så frøene på jorden og vokse i et kammer udstyret med hvidt fluorescerende lys. Oprethold følgende vækstbetingelser: temperatur på 22 °C, lysintensitet på 6.000 lux (ca. 100 μmol/m2/s) i 10 timer og relativ luftfugtighed på 60 %.</li>
	<li>Når det er nødvendigt, vand planterne med en flydende gødning. ...

Automatiseret stomatal responsmåling på bakteriel invasion i Arabidopsis

<ol>
	<li>Shimono, M., Higaki, T., Kaku, H., Shibuya, N., Hasezawa, S., Day, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+stomatal+cytoskeletal+patterns+during+the+activation+of+immune+signaling+in+Arabidopsis+thaliana.">Quantitative evaluation of stomatal cytoskeletal patterns during the activation of immune signaling in Arabidopsis thaliana.</a> PLoS One. 11, e0159291 (2016).</li><li>Bourdais, 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=The+use+of+quantitative+imaging+to+investigate+regulators+of+membrane+trafficking+in+Arabidopsis+stomatal+closure.">The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure.</a> Traffic. 20 (2), 168-180 (2019).</li><li>Higaki, T., Kutsuna, N., 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=CARTA-based+semi-automatic+detection+of+stomatal+regions+on+an+Arabidopsis+cotyledon+surface.">CARTA-based semi-automatic detection of stomatal regions on an Arabidopsis cotyledon surface.</a> Plant Morphology. 26 (1), 9-12 (2014).</li><li>Eisele, J. F., F&#228;&#223;ler, F., B&#252;rgel, F., Chaban, C. A. <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+and+simple+method+for+microscopy-based+stomata+analyses.">A rapid and simple method for microscopy-based stomata analyses.</a> PLoS One. 11, e0164576 (2016).</li><li>Chitraker, R., Melotto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+stomatal+response+to+live+bacterial+cells+using+whole+leaf+imaging.">Assessing stomatal response to live bacterial cells using whole leaf imaging.</a> Journal of Visualized Experiments. 44, 2185 (2010).</li><li>Sai, N., 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=StomaAI:+an+efficient+and+user-friendly+tool+for+measurement+of+stomatal+pores+and+density+using+deep+computer+vision.">StomaAI: an efficient and user-friendly tool for measurement of stomatal pores and density using deep computer vision.</a> New Phytologist. 238 (2), 904-915 (2023).</li><li>Takagi, 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=Image-based+quantification+of+Arabidopsis+thaliana+stomatal+aperture+from+leaf+images.">Image-based quantification of Arabidopsis thaliana stomatal aperture from leaf images.</a> Plant and Cell Physiology. pcad018, (2023).</li><li>Melotto, M., Zhang, L., Oblessuc, P. R., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+defense+a+decade+later.">Stomatal defense a decade later.</a> Plant Physiology. 174 (2), 561-571 (2017).</li><li>Melotto, M., Underwood, W., Koczan, J., Nomura, K., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+stomata+function+in+innate+immunity+against+bacterial+invasion.">Plant stomata function in innate immunity against bacterial invasion.</a> Cell. 126 (5), 969-980 (2006).</li><li>Zeng, W., He, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prominent+role+of+the+flagellin+receptor+FLAGELLIN-SENSING2+in+mediating+stomatal+response+to+Pseudomonas+syringae+pv+tomato+DC3000+in+Arabidopsis.">A prominent role of the flagellin receptor FLAGELLIN-SENSING2 in mediating stomatal response to Pseudomonas syringae pv tomato DC3000 in Arabidopsis.</a> Plant Physiology. 153 (3), 1188-1198 (2010).</li><li>Zheng, 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=Coronatine+promotes+Pseudomonas+syringae+virulence+in+plants+by+activating+a+signaling+cascade+that+inhibits+salicylic+acid+accumulation.">Coronatine promotes Pseudomonas syringae virulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation.</a> Cell Host and Microbe. 11 (6), 587-596 (2012).</li><li>Raffeiner, 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=The+Xanthomonas+type-III+effector+XopS+stabilizes+CaWRKY40a+to+regulate+defense+responses+and+stomatal+immunity+in+pepper+(Capsicum+annuum).">The Xanthomonas type-III effector XopS stabilizes CaWRKY40a to regulate defense responses and stomatal immunity in pepper (Capsicum annuum).</a> The Plant Cell. 34 (5), 1684-1708 (2022).</li><li>Munemasa, S., Hauser, F., Park, J., Waadt, R., Brandt, B., Schroeder, J. I. <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+abscisic+acid-mediated+control+of+stomatal+aperture.">Mechanisms of abscisic acid-mediated control of stomatal aperture.</a> Current Opinion in Plant Biology. 28, 154-162 (2015).</li><li>F&#246;rster, S., 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=Wounding-induced+stomatal+closure+requires+jasmonate-mediated+activation+of+GORK+K++channels+by+a+Ca2++sensor-kinase+CBL1-CIPK5+complex.">Wounding-induced stomatal closure requires jasmonate-mediated activation of GORK K+ channels by a Ca2+ sensor-kinase CBL1-CIPK5 complex.</a> Developmental Cell. 48 (1), 87-99 (2018).</li><li>Cheng, Y. T., Zhang, L., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-microbe+interactions+facing+environmental+challenge.">Plant-microbe interactions facing environmental challenge.</a> Cell Host and Microbe. 26 (2), 183-192 (2019).</li></ol>

Tidligere undersøgelser brugte epidermale skræl, bladskiver eller løsrevne blade til at undersøge stomatale reaktioner på bakterielle invasioner 9,11,12. I modsætning hertil udnytter den metode, der foreslås i denne undersøgelse, den bærbare stomatal billeddannelsesenhed til direkte at observere stomata på et blad, der er fastgjort til planten efter sprayinokulering af Pto, hvilket efterligner naturlige beting...

Stomata er mikroskopiske porer omgivet af et par beskyttelsesceller på overfladen af blade og andre luftdele af planter. Under stadigt skiftende miljøer er regulering af stomatalåbningen central for planter for at kontrollere den kuldioxidoptagelse, der kræves til fotosyntese på bekostning af vandtab via transpiration. Således har kvantificering af stomatalåbningen været medvirkende til at forstå plantemiljøtilpasning. Imidlertid er kvantificering af stomatalåbningen i sagens natur tidskrævende og besværlig, da det kræver menneskelig arbejdskraft at få øje på og måle stomatale porer i et bladbillede taget af et mikroskop. For at omgå disse begrænsninger er der udviklet forskellige metoder til at lette kvantificeringen af stomatal blænde i Arabidopsis thaliana, en modelplante, der i vid udstrækning anvendes til at studere stomatal biologi 1,2,3,4,5,6. For eksempel kan et porometer bruges til at måle transpirationshastighed som en måling af stomatal konduktans. Denne metode giver imidlertid ikke direkte information om stomatalnummeret og blænden, der bestemmer stomatal konduktans. Nogle undersøgelser har brugt konfokale mikroskopiteknikker, der fremhæver stomatale porer ved hjælp af en fluorescerende actinmarkør, et fluorescerende farvestof eller cellevægsautofluorescens 1,2,3,4,5. Mens disse tilgange letter påvisning af stomata, kan omkostningerne ved både drift af en konfokal mikroskopifacilitet og forberedelse af mikroskopiprøver være en hindring for rutinemæssig anvendelse. I et banebrydende arbejde af Sai et al. blev der udviklet en dyb neural netværksmodel til automatisk måling af stomatal blænde fra lysfeltmikroskopiske billeder af A. thaliana epidermale peelinger6. Alligevel fritager denne innovation ikke forskere fra opgaven med at forberede en epidermal skræl til mikroskopisk observation. For nylig blev denne hindring overvundet ved at udvikle en bærbar billeddannelsesenhed, der kan observere stomata ved at klemme et blad af A. thaliana sammen med en dyb læringsbaseret billedanalysepipeline, der automatisk måler stomatal blænde fra bladbilleder taget af enheden7.
Stomata bidrager til plantemedfødt immunitet mod bakterielle patogener. Nøglen til dette immunrespons er stomatal lukning, der begrænser bakteriel indtrængen gennem den mikroskopiske pore ind i bladets indre, hvor bakterielle patogener spredes og forårsager sygdomme8. Stomatal lukning induceres ved genkendelse af mikrobeassocierede molekylære mønstre (MAMP'er), immunogene molekyler, der ofte er fælles for en klasse af mikrober, af plasmamembranlokaliserede mønstergenkendelsesreceptorer (PRR'er)9. En 22 aminosyreepitop af bakteriel flagellin kendt som flg22 er en typisk MAMP, der inducerer stomatal lukning gennem dens genkendelse af PRR FLS210. Som en modforanstaltning, bakterielle patogener såsom Pseudomonas syringae pv. tomat DC3000 (Pto) og Xanthomonas campestris pv. Vesicatoria har udviklet virulensmekanismer til at genåbne stomata 9,11,12. Disse stomatale reaktioner på bakterielle patogener er traditionelt analyseret i assays, hvor enten bladepidermale skræl, bladskiver eller løsrevne blade flyder på bakteriel suspension, og derefter observeres stomata under et mikroskop efterfulgt af manuel måling af stomatal blænde. Disse analyser er imidlertid besværlige og afspejler muligvis ikke stomatale reaktioner på naturlig bakteriel invasion, der forekommer i et blad, der er fastgjort til planten.
Her præsenteres en simpel metode til at undersøge stomatal lukning og genåbning under Pto-invasion under forudsætning af, at den nøje efterligner den naturlige interaktion mellem plante og bakterier. Denne metode udnytter den bærbare billeddannelsesenhed til direkte observation af A. thaliana stomata på et blad, der er fastgjort til planten podet med Pto, sammen med billedanalyserørledningen til automatiseret måling af stomatal blænde.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Nakarai tesque</td>
			<td>01028-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Airbrush kits</td>
			<td>ANEST IWATA</td>
			<td>MX2900</td>
			<td>Accessory kits for SPRINT JET</td>
		</tr>
		<tr>
			<td>Biotron</td>
			<td>Nippon Medical &#38; Chemical Instruments</td>
			<td>LPH-411S</td>
			<td>Plant Growth Chamber with white fluorescent light</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Wako</td>
			<td>072-00626</td>
			<td></td>
		</tr>
		<tr>
			<td>Half tray</td>
			<td>Sakata</td>
			<td>72000113</td>
			<td>A set of tray and lid</td>
		</tr>
		<tr>
			<td>Hyponex</td>
			<td>Hyponex</td>
			<td>No catalogue number available</td>
			<td>Dilute the solution of Hyponex at a ratio of 1:2000 in deionized water for watering plants</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>Natinal Institute of Health</td>
			<td>Download at https://imagej.nih.gov/ij/download.html</td>
			<td>Used for manual measurement of stomatal aperture</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Wako</td>
			<td>164-04295</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Wako</td>
			<td>163-03545</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Wako</td>
			<td>168-21815</td>
			<td>For MES-KOH</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Wako</td>
			<td>343-01621</td>
			<td>For MES-KOH</td>
		</tr>
		<tr>
			<td>Portable stomatal imaging device</td>
			<td>Phytometrics</td>
			<td>Order at https://www.phytometrics.jp/</td>
			<td>Takagi et al.(2023) doi: 10.1093/pcp/pcad018.</td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Wako</td>
			<td>185-01003</td>
			<td>Dissolve in DMSO</td>
		</tr>
		<tr>
			<td>Silwet-L77</td>
			<td>Bio medical science</td>
			<td>BMS-SL7755</td>
			<td>silicone surfactant used in spray inoculation</td>
		</tr>
		<tr>
			<td>SPRINT JET</td>
			<td>ANEST IWATA</td>
			<td>IS-800</td>
			<td>Airbrush used for spray inoculation</td>
		</tr>
		<tr>
			<td>SuperMix A</td>
			<td>Sakata seed</td>
			<td>72000083</td>
			<td>Mix with Vermiculite G20 in equal proportions for preparing soil</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Nakarai tesque</td>
			<td>35640-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Vermiculite G20</td>
			<td>Nittai</td>
			<td>No catalogue number available</td>
			<td>Mix with Super Mix A in equal proportions for preparing soil</td>
		</tr>
		<tr>
			<td>White fluorescent light</td>
			<td>NEC</td>
			<td>FHF32EX-N-HX-S</td>
			<td>Used for Biotron</td>
		</tr>
	</tbody>
</table>

direct observation and automated measurement of stomatal responses to pseudomonas syringae pv. tomato dc3000 in arabidopsis thaliana

Stomata er mikroskopiske porer, der findes i plantens bladepidermis. Regulering af stomatal blænde er afgørende ikke kun for at afbalancere kuldioxidoptagelse til fotosyntese og transpirationelt vandtab, men også for at begrænse bakteriel invasion. Mens planter lukker stomata ved genkendelse af mikrober, patogene bakterier, såsom Pseudomonas syringae pv. tomat DC3000 (Pto), genåbn den lukkede stomata for at få adgang til bladets indre. I konventionelle assays til vurdering af stomatale reaktioner på bakteriel invasion flyder bladepidermale skaller, bladskiver eller løsrevne blade på bakteriel suspension, og derefter observeres stomata under et mikroskop efterfulgt af manuel måling af stomatal blænde. Disse analyser er imidlertid besværlige og afspejler muligvis ikke stomatale reaktioner på naturlig bakteriel invasion i et blad, der er fastgjort til planten. For nylig blev der udviklet en bærbar billeddannelsesenhed, der kan observere stomata ved at klemme et blad uden at løsne det fra planten sammen med en dyb læringsbaseret billedanalysepipeline designet til automatisk at måle stomatal blænde fra bladbilleder taget af enheden. Her, på baggrund af disse tekniske fremskridt, introduceres en ny metode til vurdering af stomatale reaktioner på bakteriel invasion i Arabidopsis thaliana . Denne metode består af tre enkle trin: sprayinokulering af Pto , der efterligner naturlige infektionsprocesser, direkte observation af stomata på et blad af den Pto-inokulerede plante ved hjælp af den bærbare billeddannelsesenhed og automatiseret måling af stomatal blænde ved billedanalyserørledningen. Denne metode blev med succes brugt til at demonstrere stomatal lukning og genåbning under Pto-invasion under forhold, der nøje efterligner den naturlige plante-bakterie-interaktion.

Stomata are microscopic pores surrounded by a pair of guard cells on the surface of leaves and other aerial parts of plants. Under ever-changing environments, regulation of the stomatal aperture is central for plants to control the carbon dioxide uptake required for photosynthesis at the expense of water loss via transpiration. Thus, quantification of the stomatal aperture has been instrumental to understanding plant environmental adaptation. However, quantifying the stomatal aperture is inherently time-consuming and cumbersome as it requires human labor to spot and measure stomatal pores in a leaf image captured by a microscope. To circumvent these limitations, various methods have been developed to facilitate the quantification of stomatal aperture in Arabidopsis thaliana, a model plant extensively used to study stomatal biology1,2,3,4,5,6. For instance, a porometer can be used to measure transpiration rate as a metric of stomatal conductance. However, this method does not provide direct information on the stomatal number and aperture that determine stomatal conductance. Some studies have used confocal microscopy techniques highlighting stomatal pores using a fluorescent actin marker, a fluorescent dye, or cell wall autofluorescence1,2,3,4,5. While these approaches facilitate the detection of stomata, the cost of both operating a confocal microscopy facility and preparing microscopy samples can be an obstacle to routine application. In a ground-breaking work by Sai et al., a deep neural network model was developed to automatically measure stomatal aperture from bright-field microscopic images of A. thaliana epidermal peels6. Yet, this innovation does not exempt researchers from the task of preparing an epidermal peel for microscopic observation. Recently, this obstacle was overcome by developing a portable imaging device that can observe stomata by pinching a leaf of A. thaliana, together with a deep learning-based image analysis pipeline that automatically measures stomatal aperture from leaf images captured by the device7.
Stomata contribute to plant innate immunity against bacterial pathogens. The key to this immune response is stomatal closure that restricts bacterial entry through the microscopic pore into the leaf interior, where bacterial pathogens proliferate and cause diseases8. Stomatal closure is induced upon recognition of microbe-associated molecular patterns (MAMPs), immunogenic molecules that are often common to a class of microbes, by plasma membrane-localized pattern recognition receptors (PRRs)9. A 22 amino acid epitope of bacterial flagellin known as flg22 is a typical MAMP that induces stomatal closure through its recognition by the PRR FLS210. As a countermeasure, bacterial pathogens such as Pseudomonas syringae pv. tomato DC3000 (Pto) and Xanthomonas campestris pv. vesicatoria have evolved virulence mechanisms to reopen stomata9,11,12. These stomatal responses to bacterial pathogens have been conventionally analyzed in assays in which either 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 that occur in a leaf attached to the plant.
Here, a simple method is presented to investigate stomatal closure and reopening during Pto invasion under the condition that closely mimics the natural plant-bacteria interaction. This method leverages the portable imaging device for direct observation of A. thaliana stomata on a leaf attached to the plant inoculated with Pto, together with the image analysis pipeline for automated measurement of stomatal aperture.

Forfatter Spotlight: Fremme af stomatal forskning med automatiseret blændemåling

Direkte observation og automatiseret måling af stomatale responser på Pseudomonas syringae pv. tomat DC3000 i Arabidopsis thaliana

Plantal stomata play central roles in adaptation to stress conditions, so we need to measure stomata aperture to understand how plants respond to biotic and abiotic stresses. However, stomata aperture measurement is time-consuming and cumbersome. To measure stomata aperture, we peel the epidermis and observe stomata under a microscope.Then we manually measure stomata aperture, which takes a long time even for very experienced researchers. We challenged these barriers and developed a tool and a technique that can automatically measure stomata aperture in intact Arabidopsis leaves. So we strongly believe that stomata measuring device and deep-learning algorithm that we developed will facilitate functional analysis of stomata responses to various biotic and abiotic stresses because these technical advances significantly reduce time and human labor required for stomata aperture measurement.

Efter spraypodning af Pto blev stomata på blade, der var fastgjort til de podede planter, observeret direkte af den bærbare stomatal billeddannelsesenhed. Ved hjælp af manuelle og automatiserede målinger blev de samme bladbilleder brugt til at beregne stomatal blænde ved at tage forhold mellem bredde og længde på ca. 60 stomata. Manuelle og automatiserede målinger indikerede konsekvent et fald i stomatalåbningen i Pto-podede planter sammenlignet med mock-podede planter 1 time efter podning (hpi...

Watch this Scientific Journal Video about Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement at JoVE.com

Her præsenterer vi en simpel metode til direkte observation og automatiseret måling af stomatale reaktioner på bakteriel invasion i Arabidopsis thaliana. Denne metode udnytter en bærbar stomatal billeddannelsesenhed sammen med en billedanalysepipeline designet til bladbilleder, der er taget af enheden.

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

Plantal stomata spiller centrale roller i tilpasningen til stressforhold, så vi er nødt til at måle stomata blænde for at forstå, hvordan planter reagerer på biotiske og abiotiske belastninger. Måling af stomata-blænde er dog tidskrævende og besværlig. For at måle stomata blænde skræller vi epidermis og observerer stomata under et mikroskop.Derefter måler vi manuelt stomata blænde, hvilket tager lang tid selv for meget erfarne forskere. Vi udfordrede disse barrierer og udviklede et værktøj og en teknik, der automatisk kan måle stomata-blænde i intakte Arabidopsis-blade. Så vi er overbeviste om, at stomatamåleenhed og dyb læringsalgoritme, som vi udviklede, vil lette funktionel analyse af stomata-responser på forskellige biotiske og abiotiske belastninger, fordi disse tekniske fremskridt reducerer tid og menneskelig arbejdskraft, der kræves til måling af stomata-blænde, betydeligt.

direct-observation-automated-measurement-stomatal-responses-to

Research

JoVE Journal

Immunology and Infection

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.

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.

Induction of Pseudomonas syringae pv. tomato DC3000 Infection in Arabidopsis thaliana for Automated Stomatal Response Measurement

Induktion af Pseudomonas syringae pv. tomat DC3000-infektion i Arabidopsis thaliana til automatiseret måling af stomatal respons

Automated Measurement of Stomatal Response in Arabidopsis thaliana in Response to Pseudomonas syringae pv. tomato DC3000 Infection