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Transpiration is the process of water movement through a plant and its evaporation from aerial parts, such as leaves , stems and flowers. Water is necessary for plants but only a small amount of water taken up by the roots is used for growth and metabolism. The remaining 97— The stomata are bordered by guard cells and their stomatal accessory cells together known as stomatal complex that open and close the pore.

Function of ABA in Stomatal Defense against Biotic and Drought Stresses

There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures.

Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required. Our aim was to develop a protocol based on the minimization of leaf manipulation and the reduction of analysis completion time, while still producing consistent results.

We applied rhodamine 6G staining of Arabidopsis thaliana leaves for stomata visualization, which greatly simplifies the measurement of stomatal apertures. By using this staining protocol, we successfully conducted analyses of stomatal responses in Arabidopsis leaves to both closure and opening stimuli. We performed long-term monitoring of living stomata and were able to document the same leaf before and after treatment.

Moreover, we developed a protocol for rapid-fixation of epidermal peels, which enables high throughput data analysis. The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements, and will facilitate the analysis of several lines in parallel. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. The opening and closing of stomata is a fine-controlled masterpiece of plant evolution driven by the transition of a chemical signal into a mechanical movement. By changing the osmotic pressure in the guard cells, these tiny pores regulate leaf temperature, water evaporation and gas exchange, processes essential for plant survival and growth [ 1 — 3 ].

Due to uptake of CO 2 , stomata participate in providing a carbon source for photosynthetic reactions, whereas the coinciding transpiration of water is essential for nutrient uptake from soil to the plant body. On the other hand, excess water loss from plants under drought stress is disadvantageous and might exert damaging effects resulting in plant death. Because of the great importance of proper stomatal movement, numerous signaling systems inside the plant co-participate in the regulation of stomatal opening and stomatal closure.

As a consequence, a mutation of individual components of any of these systems often modulates stomatal movement, which can be either beneficial or unfavorable for the plant. Therefore, the monitoring of stomatal behavior is very important for plant scientists. Stomatal aperture is tightly regulated by divergent exogenous stimuli, such as light, drought stress, pathogens, temperature and others.

Abscisic acid ABA is among the major players in terms of stress related stomatal closure [ 4 ]. These signaling cascades lead to modifications in the activity of ion channels, decrease of the osmotic pressure in guard cells and, thereby, closure of stomata [ 1 , 2 , 8 ]. Other phytohormones, such as ethylene, jasmonates and salicylic acid, also function in the regulation of stomatal aperture.

Signaling pathways triggered by hormones, as well as by pathogen attack, often involve the generation of second messengers like NO and H 2 O 2. Treatment of plants with exogenous H 2 O 2 alone can trigger stomatal closure [ 6 , 7 ]. One pathway by which H 2 O 2 , derived from endogenous and environmental stimuli, is sensed and transduced to effect stomatal closure involves histidine kinase AHK5 [ 9 ].

Arabidopsis mutants lacking AHK5 have been shown to exhibit impaired stomatal closure in response to H 2 O 2. Abiotic or hormone signals able to generate endogenous H 2 O 2 , such as darkness or ethylene, also caused reduced stomatal closure in the ahk5 mutants, whereas stomatal movement was rescued by over-expression of AHK5. Auxin is known to be a positive regulator of stomatal opening although it can also inhibit stomatal opening when applied exogenously at high concentrations [ 1 , 2 ].

In spite of the critical role played by stomata in the regulation of plant gas exchange and water use efficiency, the measurement of stomatal aperture is difficult and depends on various environmental factors.

Therefore, it is important to have a fast and reliable method to properly monitor stomatal aperture. Here we present a new staining method for stomata, which enables imaging of stomata within minutes after its application. It allows the in situ capture of stomata without removing them from their natural surroundings. Additionally, it provides a possibility to analyze stomatal density and stomatal index owing to a staining of pavement cells. As the staining of the stomata takes only 1—2 minutes, one may create snapshots of stomata at short time intervals.

Especially, almost instant cell fixation, when desirable, makes it possible to analyze a great number of stomata without the risk of artefact creation due to the prolonged time necessary for the microscopic analyses.

Arabidopsis thaliana L. Four to six week old plants were used for the analyses. The lines are described in more detail in Desikan et al. For cell staining, rhodamine 6G Sigma at final concentration of 0. It was freshly prepared from 1mM stock solution in water , which was kept in darkness. Either whole leaves or epidermal peels were dipped into the staining solution for 1—2 minutes. When desired, staining was followed by short rinsing in corresponding incubation solution.

Three fully expanded leaves at a comparable developmental stage one leaf per plant were used per line for each treatment. Afterwards the leaves were cut along the midrib. After treatment, both leaf parts were stained as described above and examined under microscope. The data are presented in comparison to the corresponding controls mock treatments. Then the leaves were cut along the midrib and treated as follows: one leaf half was stained in the staining solution and immediately examined under microscope corresponds to 0 h treatment ; then it was incubated either in ABA or IAA for 2 h followed by examination under microscope corresponds to 2 h treatment.

Another leaf half was incubated either in ABA or IAA for 2 h without pre-staining, then stained in the staining solution and examined under microscope corresponds to 2 h post-stained. The stomatal aperture for 0 h time point was measured before ABA treatment.

Epidermis peels were prepared as described in Wu et al. The adaxial epidermal leaf surface was affixed to a strip of laboratory tape TimeMed Labeling Systems, Inc. A strip of transparent universal adhesive tesa film solvent free Tesa SE was gently applied to the abaxial surface of the affixed leaf.

The tesa adhesive tape was then carefully pulled away from the laboratory tape, peeling away the abaxial epidermal cell layer. At the end of the incubation period, the epidermal cell peels were stained and analyzed under microscope. To enable a direct comparison, the intact leaves were treated in parallel with the peels. For the stomatal aperture closure kinetics in response to ABA, the images of guard cells were taken under microscope at 0 h, 0. The Petri dishes were maintained either under light or in darkness for 2 h.

The peels were imaged employing an epi-fluorescence microscope Nikon Eclipse 90i and returned to the corresponding petri dishes. The petri dishes previously maintained under light were transferred into darkness, whereas those from darkness were transferred into light conditions for 30 min.

At the end of the treatment the peels were again analyzed under microscope. Peels from three leaves per treatment were used for analysis. The width and the length of the stomatal aperture were measured as shown on Fig 1A , and the stomatal aperture index SAI was calculated by division of the aperture width through the length.

The confocal images were captured under Leica SP8 microscope at following settings: excitation at nm, emission — nm green fluorescence ; excitation at nm, emission at — nm red fluorescence , 20x objective, using Leica Application Suite LAS software. Rhodamine 6G belongs to a group of fluorescent dyes widely used in different fields including molecular and cell biology. Different rhodamine derivatives are most often applied as fluorescent reporters fused to antibodies or other molecules in order to visualize their intracellular localization.

Rhodamine 6G is characterized by a high quantum yield 0. From preliminary experiments we found that the optimal staining of guard cells was achieved at 0. At this concentration the guard cells are often more strongly stained than the pavement cells Fig 1B. Although very low dye concentrations were used for staining, there was still the chance of rhodamine 6G affecting stomatal aperture. In consistence with previously published results, ahk5 mutant lines failed to close stomata in response to H 2 O 2, irrespective of the ecotype background.

Moreover, ectopic expression of AHK5 in ahk background led to the reversion of the mutant phenotype, i. The obtained data indicate that applied rapid staining of leaves immediately before analysis allows visualization of stomatal pores on intact leaves and proper evaluation of stomatal aperture without detectable side effects on stomatal behavior. Three fully expanded leaves at a similar developmental stage one leaf per plant per treatment were analyzed for each line. A total of six leaves c and nine leaves d at a similar developmental stage one leaf per plant were used for the analyses.

Our next question was whether rhodamine influences stomatal movement in pre-stained leaves. Therefore, after detachment, the leaves of Col-0 plants were either stained with rhodamine and then incubated in ABA- or IAA-containing buffer, or incubated with phytohormones without rhodamine pre-staining.

In the latter case, the leaves were stained immediately before image acquisition further referred as to post-stained. Auxin treatment caused further opening of pores compared to untreated control 0 h IAA.

The increase of aperture was small yet significant. As in the case of ABA, we did not detect any difference between pre-stained and post-stained stomatal apertures Fig 2C. In order to see if the response kinetics of stomata is not impeded by the staining, the monitoring of stomatal closure at several time points in response to ABA was conducted.

The decrease of stomatal aperture was already detectable after 30 min of the ABA treatment irrespective of rhodamine pre-staining. The kinetics of stomatal closure was similar in the pre-stained and post-stained leaves Fig 2D.

In conclusion, pre-staining of leaves with rhodamine 6G influenced neither stomatal closure nor opening. As mentioned above, low-concentration rhodamine 6G staining of intact leaves often results in the preferential staining of guard cells as compared to pavement cells. Such differential staining is especially observed in leaves with open stomata. Although this can be advantageous for stomatal aperture measurement, it causes difficulties in analyzing stomatal density or stomatal index.

Thus, rhodamine-stained epidermis peels can be used for calculating stomatal index and stomatal density. In the case of stomatal aperture measurement, however, the complications with focusing on numerous cells still persist, since the mounting of leaves in glycerol solution would affect stomata aperture.

This results in a considerable prolongation of the microscopic analysis. In order to overcome this problem, leaf epidermis peels are often used [ 14 — 16 ]. To prepare the epidermis peels we applied a very easy method previously used for protoplast isolation [ 11 ]. By this method, a peel of broad leaf surface can be readily obtained Fig 3C. Due to the leveled arrangement of cells in such peels, staining them with rhodamine 6G results in a simultaneous visualization of guard cells throughout the entire section of a sample Fig 3D.

Our next question was whether such peels could be used for the analysis of stomatal movement. Therefore, we compared ABA-induced stomatal closure in intact leaves and epidermal peels. As shown on the Fig 4A , upon 2 h of ABA treatment stomatal aperture significantly decreased in both intact leaves and epidermis peels. Measurement of the kinetics of ABA-induced stomatal closure showed that it was comparable in both material samples Fig 4B.

Transpiration

Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Stomatal movements are regulated by many environmental signals, such as light, CO 2 , temperature, humidity, and drought.

As plants evolved to function on land, they developed stomata for effective gas exchange, for photosynthesis and for controlling water loss. We have recently shown that sugars, as the end product of photosynthesis, close the stomata of various angiosperm species, to coordinate sugar production with water loss. In the current study, we examined the sugar responses of the stomata of phylogenetically different plant species and species that employ different photosynthetic mechanisms i. To examine the effect of sucrose on stomata, we treated leaves with sucrose and then measured their stomatal apertures. Sucrose reduced stomatal aperture, as compared to an osmotic control, suggesting that regulation of stomata by sugars is a trait that evolved early in evolutionary history and has been conserved across different groups of plants. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information file.

A Rapid and Simple Method for Microscopy-Based Stomata Analyses

There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures. Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required.

PubMed Central. Stomata are pores on the leaf surface, which are formed by a pair of curved, tubular guard cells; an increase in turgor pressure deforms the guard cells, resulting in the opening of the stomata. Recent studies employed numerical simulations, based on experimental data, to analyze the effects of various structural, chemical, and mechanical features of the guard cells on the stomatal opening characteristics; these studies all support the well-known qualitative observation that the mechanical anisotropy of the guard cells plays a critical role in stomatal opening. Here, we propose a computationally based analytical model that quantitatively establishes the relations between the degree of anisotropy of the guard cell, the bio-composite constituents of the cell wall, and the aperture and area of stomatal opening.

Shigeo Toh and Shinpei Inoue authors contributed equally to this work. Regulation of stomatal aperture is essential for plant growth and survival in response to environmental stimuli. Opening of stomata induces uptake of CO 2 for photosynthesis and transpiration, which enhances uptake of nutrients from roots. Light is the most important stimulus for stomatal opening. Under drought stress, the plant hormone ABA induces stomatal closure to prevent water loss. However, the molecular mechanisms of stomatal movements are not fully understood. In this study, we screened chemical libraries to identify compounds that affect stomatal movements in Commelina benghalensis and characterize the underlying molecular mechanisms.

The glucosinolate—myrosinase system is a well-known defense system that has been shown to induce stomatal closure in Brassicales. It remains unknown whether AITC inhibits light-induced stomatal opening. AITC induced stomatal closure and inhibited light-induced stomatal opening in a dose-dependent manner. Stomata, surrounded by pairs of guard cells, function as the main window for gas exchange and are in the frontline for defense against microbe invasion in the phyllosphere. To deal with the changing growth and environmental cues, guard cells have evolved to be specialist responders to various stimuli, such as light, drought stress, CO 2 , phytohormones, and microbe-derived signals, resulting in stomatal movement Murata et al. Myrosinases are highly abundant proteins in guard cells and required for abscisic acid ABA -induced stomatal movement Zhao et al.


The tested heavy metal ions, such as Hg2+, Pb2+, Zn2+, and La3+, partly inhibited stomatal opening in light or closing in darkness at.


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