Monitoring of stomatal function over long term timescales at single stoma level

Photo credit: Lab on a Chip

 

Persistent drought monitoring using a microfluidic-printed electro-mechanical sensor of stomata in planta

by Koman V., Lew T., Wong M. H., Kwak S. Y., Giraldo J. P., Strano M. (2017)

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in Lab Chip 17: 4015-4024 – DOI:10.1039/C7LC00930E

http://pubs.rsc.org/en/content/articlelanding/2017/lc/c7lc00930e#!divAbstract

Abstract

Stomatal function can be used effectively to monitor plant hydraulics, photosensitivity, and gas exchange. Current approaches to measure single stomatal aperture, such as mold casting or fluorometric techniques, do not allow real time or persistent monitoring of the stomatal function over timescales relevant for long term plant physiological processes, including vegetative growth and abiotic stress.

Herein, we utilize a nanoparticle-based conducting ink that preserves stomatal function to print a highly stable, electrical conductometric sensor actuated by the stomata pore itself, repeatedly and reversibly for over 1 week.

This stomatal electro-mechanical pore size sensor (SEMPSS) allows for real-time tracking of the latency of single stomatal opening and closing times in planta, which we show vary from 7.0 ± 0.5 to 25.0 ± 0.5 min for the former and from 53.0 ± 0.5 to 45.0 ± 0.5 min for the latter in Spathiphyllum wallisii. These values are shown to correlate with the soil water potential and the onset of the wilting response, in quantitative agreement with a dynamic mathematical model of stomatal function. A single stoma of Spathiphyllum wallisii is shown to distinguish between incident light intensities (up to 12 mW cm−2) with temporal latency slow as 7.0 ± 0.5 min. Over a seven day period, the latency in opening and closing times are stable throughout the plant diurnal cycle and increase gradually with the onset of drought. The monitoring of stomatal function over long term timescales at single stoma level will improve our understanding of plant physiological responses to environmental factors.

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The monitoring of stomata function over relevant timescales for plant physiology

 

 

A Stomatal Electro-Mechanical Pore Size Sensor (SEMPSS) for Persistent Monitoring of Plant Physiology

by Koman V., Salim Lew T., Wong M. H., Kwak S. Y., Strano M., (2017)

Massachusetts Institute of Technology

at 2017 AIChE Annual Meeting –

https://www.aiche.org/conferences/aiche-annual-meeting/2017/proceeding/paper/615f-stomatal-electro-mechanical-pore-size-sensor-sempss-persistent-monitoring-plant-physiology

Stomatal function can be used effectively to monitor plant hydraulic efficiency, photo-sensitivity and COconductance. Current approaches to measure stomatal aperture size, such as mold casting or fluorometric techniques, do not allow real time or persistent monitoring of the stomata over the timescales relevant for plant physiology including growth and maturation, or gradual changes in soil water potential associated with drought conditions.

Herein, we utilize a nanoparticle-based conducting ink that preserves stomatal function to print a highly stable, electrical conductometric sensor actuated by the stomata pore itself, repeatedly and reversibly for over 1 week.

This Stomatal Electro-Mechanical Pore Size Sensor (SEMPSS) allows for real-time tracking of the latency of stomatal opening and closing times, which we show vary from 7±0.5 to 25±0.5 min for the former and from 53±0.5 to 45±0.5 min for the latter in Spathiphyllum.

These values are shown to correlate with a drop in soil water potential and the onset of the wilting response, in quantitative agreement with a mathematical model of stomata signaling in function.

A single stoma of Spathiphyllum is shown to distinguish between incident light intensities (up to 12 mW/cm2) with temporal latency slow as 7±0.5 min. Over a seven day period, the latency in opening and closing times are stable throughout the plant diurnal cycle and increase gradually with physiological changes associated with drought onset.

The monitoring of stomata function over relevant timescales for plant physiology will improve understanding of plant adaptation to environmental factors.

The physiological significance of the autonomous stomatal behaviour.

Screen Shot 2017-11-24 at 18.36.00
Figure 3. Dissociation of PHOT1 from the plasma membrane in blue-light irradiated cells (A) Stomata blue-light irradiation

 

Use of Confocal Laser as Light Source Reveals Stomata-Autonomous Function

by Cañamero R. C., Boccalandro H., Casal J., Serna L. (2006)

in PLoS ONE1(1): e36.  – https://doi.org/10.1371/journal.pone.0000036

Roberto C. Cañamero, Hernán Boccalandro, Jorge Casal, Laura Serna,

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000036

Abstract

In most terrestrial plants, stomata open during the day to maximize the update of CO2 for photosynthesis, but they close at night to minimize water loss. Blue light, among several environmental factors, controls this process.

Stomata response to diverse stimuli seems to be dictated by the behaviour of neighbour stomata creating leaf areas of coordinated response.

Here individual stomata of Arabidopsis leaves were illuminated with a short blue-light pulse by focusing a confocal argon laser. Beautifully, the illuminated stomata open their pores, whereas their dark-adapted neighbours unexpectedly experience no change.

This induction of individual stomata opening by low fluence rates of blue light was disrupted in the phototropin1 phototropin2 (phot1 phot2) double mutant, which exhibits insensitivity of stomatal movements in blue-illuminated epidermal strips.

The irradiation of all epidermal cells making direct contact with a given stoma in both wild type and phot1 phot2 plants does not trigger its movement.

These results unravel the stoma autonomous function in the blue light response and illuminate the implication of PHOT1 and/or PHOT2 in such response. The micro spatial heterogeneity that solar blue light suffers in partially shaded leaves under natural conditions highlights the physiological significance of the autonomous stomatal behaviour.

Measuring stomatal aperture, morphology and density with silicon polymer and SEM

20170810002548_0261

Figure 1. Silicon polymer impression technique and scanning electron microscopy (SEM) to measure stomatal apertures. A. (a) Mix silicon polymer (reagents A + B) with a toothpick on a slide; (b) Apply on the lower surface of leaf; (c) Negative impression mold made with silicon polymers; (d) The mold seated on a double-sided tape on top of the glass slide. B. (a) Mix Epoxy gel (reagents C + D) with a toothpick; (b) Fill the impression mold with freshly mixed Epoxy gel; (c) Thoroughly harden the Epoxy gel at room temperature (RT) for overnight, or in a 60 °C oven for 1 h; (d) Once hardened, observe using SEM examination. C. The example of image was taken using an SEM. The round shapes on the SEM picture could be air bubbles and foreign particles.

Using Silicon Polymer Impression Technique and Scanning Electron Microscopy to Measure Stomatal Aperture, Morphology, and Density

by Wu H.-C., Huang Y.-C., Liu C.-H., Jinn T.-L. (2017)

Hui-Chen Wu, Ya-Chen Huang, Chia-Hung Liu,

Tsung-Luo Jinn

Tsung-Luo Jinn, 

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in Bio-protocol 7(16): e2449. DOI: 10.21769/BioProtoc.2449.-

http://www.bio-protocol.org/e2449

Abstract

The number of stomata on leaves can be affected by intrinsic development programming and various environmental factors, in addition the control of stomatal apertures is extremely important for the plant stress response.

In response to elevated temperatures, transpiration occurs through the stomatal apertures, allowing the leaf to cool through water evaporation.

As such, monitoring of stomata behavior to elevated temperatures remains as an important area of research. The protocol allows analysis of stomatal aperture, morphology, and density through a non-destructive imprint of Arabidopsis thaliana leaf surface. Stomatal counts were performed and observed under a scanning electron microscope.

20170810003225_5081

Figure 2. The measurement of stomata aperture using the image processing software ImageJ. The basic procedures of image analysis are showing step-by-step for analyzing the objects in the SEM image. Step 1. Download and Run ImageJ program; Step 2. Open stomata image via select File, Open sample and click OK; Step 3. For image processing: (a) Convert image to 8-bit grayscale mode, and then (b) Crop a meaningful part of the image (draw a rectangular shape). (c) It can use a sharpened version of the image before the threshold setting. (d) The threshold new image of stomata was chosen by manually defining the histogram. (e) and (f) Process binary to make black and white images. To refine shapes of samples in this step. (g) The particle size range (minimum size and maximum pixel area size) can be determined for your objects of interest, and therefore to exclude anything that is not interested in the image. Step 4. Results window opens automatically.

Automatic Counting of Stomata

 

Screen Shot 2017-11-21 at 09.22.25

Fig. 1. Detection of stomata by morphological operations. (a) Original image. (b) Leaf epidermis after low-pass filter. (c) Opening-by-reconstruction morphological operation. (d) Opening-closing-by-reconstruction. (e) Regional minima of result image. (f) Final stomata identification after perimeter elimination rule.

Automatic Counting of Stomata in Epidermis Microscopic Images

 

by da Silva Oliveira M. W., da Silva N. R. , Casanova D. , Souza Pinheiro L. F., Kolb R. M., Martinez Bruno O. (2014)

Marcos William da Silva Oliveira∗ , Nubia Rosa da Silva ´ ∗ , Dalcimar Casanova† , Luiz Felipe Souza Pinheiro‡ , Rosana Marta Kolb‡ and Odemir Martinez Bruno†

∗ Institute of Mathematics and Computer Science University of Sao Paulo (USP), Sao Carlos, Sao Paulo, Brazil

†Scientific Computing Group, Sao Carlos Institute of Physics,  University of Sao Paulo (USP), Sao Carlos, Sao Paulo, Brazil

‡Department of Biological Sciences, Faculty of Sciences and Letters, Univ Estadual Paulista Julio de Mesquita Filho, UNESP, Assis, Sao Paulo, Brazil

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in X Workshop de Visao Computacional – WVC 2014 – 253-257 –

http://www.lbd.dcc.ufmg.br/colecoes/wvc/2014/0044.pdf

Screen Shot 2017-11-21 at 09.24.36

Fig. 2. Examples of stomata segmentation. On the left side, one original image of each species. On right side, the result of stomata segmentation using small green squares overlapping each stomata identified. The 5 different plant species tested are Callophyllum brasiliense, Guapira areolata, Maytenus floribunda, Roupala montana, and Xylopia sericea, displayed in this order.

Abstract

Stomata are small pores in epidermis of plants being their diversity an important feature for morphologists and physiologists. Through their diversity of characteristics (size, shape, density, responsiveness to desiccation, pore dimensions, orientation, etc.) the leaf epidermis may be studied.

However, there is a great difficulty in counting stomata, especially when there are a large number of samples to be analysed, since this task is performed manually, depending on the expert experience.

This paper proposes the automatic detection and counting of stomata through morphological operations in epidermis microscopic images. With this approach, an accuracy rate of 94.3% was achieved, detecting and counting the stomata in 24 images from 5 plant species, indicating that the results obtained are promising.

 

TECHNIQUES FOR ANALYSIS OF STOMATAL APERTURE

 

 

Control of Leaf Stomatal Opening

by Johnson R. (2007)

Russell Johnson

in Colby J. Res. Meth. 9: 14-17 –

https://www.colby.edu/academics_cs/courses/BI214/upload/lab5-stomata.pdf

The opening and closing of stomata is a very important mechanism that plants use to control the diffusion of gases in and out of leaves. Ideally stomata must be sufficiently open to allow enough CO2 (needed for photosynthesis) to diffuse in, but sufficiently closed to prevent too much evaporative loss of H2O. This is sometimes a difficult balance to achieve and the amount of stomatal opening is controlled by a large number of factors. The degree to which stomata are open can be observed and measured by making imprints of the leaf epidermis and viewing (and photographing) these imprints using a light microscope. Once this technique is mastered it can then be used to measure the effects on stomatal aperture of a wide variety of environmental and chemical factors. Epidermal imprints can be made by applying a dental resin to the surface of the leaf and allowing it to harden. This negative impression can be archived and used to make a positive image (with clear nail polish) whenever desired.

PART A – TECHNIQUES FOR ANALYSIS OF STOMATAL APERTURE

I. Procedure for making imprints of the leaf epidermis

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II. Procedure for making digital records (movie clips) of epidermal surfaces.

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III. Procedure for measuring stomatal aperture from digital images.

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PART B – ASSESSMENT OF FACTORS THAT INFLUENCE STOMATAL APERTURE