JoVE Logo
Faculty Resource Center

Sign In





Representative Results





Immunology and Infection

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

Published: February 9th, 2024



1Graduate School of Agriculture, Kyoto University, 2Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, 3Phytometrics Co., Ltd.
* These authors contributed equally

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 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.

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, vario....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Growing plants

  1. To break dormancy, resuspend A. thaliana (Col-0) seeds in deionized water and incubate them at 4 °C for 4 days in the dark.
  2. Sow the seeds on the soil and grow in a chamber equipped with white fluorescent light. Maintain the following growth conditions: temperature of 22 °C, light intensity of 6,000 lux (ca. 100 µmol/m2/s) for 10 h, and relative humidity of 60%.
  3. When needed, water the plants with a liquid fertilizer. Refr.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Following spray inoculation of Pto, stomata on leaves attached to the inoculated plants were directly observed by the portable stomatal imaging device. Using manual and automated measurements, the same leaf images were used to calculate stomatal aperture by taking ratios of width to length of approximately 60 stomata. Manual and automated measurements consistently indicated a decrease in the stomatal aperture in Pto-inoculated plants compared with mock-inoculated plants at 1 hour post inoculation (hpi) .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Previous studies used epidermal peels, leaf discs, or detached leaves to investigate stomatal responses to bacterial invasions9,11,12. In contrast, the method proposed in this study leverages the portable stomatal imaging device to directly observe stomata on a leaf attached to the plant after spray inoculation of Pto, mimicking natural conditions of bacterial invasion. In addition, because this method does not involve .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

We thank all the members of the research project, 'Co-creation of plant adaptive traits via assembly of plant-microbe holobiont', for fruitful discussions. This work was supported by Grant-in-Aid for Transformative Research Areas (21H05151 and 21H05149 to A.M. and 21H05152 to Y.T.) and Grant-in-Aid for Challenging Exploratory Research (22K19178 to A. M.).


Log in or to access full content. Learn more about your institution’s access to JoVE content here

NameCompanyCatalog NumberComments
AgarNakarai tesque01028-85
Airbrush kitsANEST IWATAMX2900Accessory kits for SPRINT JET
BiotronNippon Medical & Chemical InstrumentsLPH-411SPlant Growth Chamber with white fluorescent light
Half traySakata72000113A set of tray and lid
HyponexHyponexNo catalogue number availableDilute the solution of Hyponex at a ratio of 1:2000 in deionized water for watering plants
Image JNatinal Institute of HealthDownload at for manual measurement of stomatal aperture
KOHWako168-21815For MES-KOH
MESWako343-01621For MES-KOH
Portable stomatal imaging devicePhytometricsOrder at et al.(2023) doi: 10.1093/pcp/pcad018.
RifampicinWako185-01003Dissolve in DMSO
Silwet-L77Bio medical scienceBMS-SL7755silicone surfactant used in spray inoculation
SPRINT JETANEST IWATAIS-800Airbrush used for spray inoculation
SuperMix ASakata seed72000083Mix with Vermiculite G20 in equal proportions for preparing soil
TryptoneNakarai tesque35640-95
Vermiculite G20NittaiNo catalogue number availableMix with Super Mix A in equal proportions for preparing soil
White fluorescent lightNECFHF32EX-N-HX-SUsed for Biotron

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

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved