We are indebted to Xuefei Yang, Sabine Both, Lin Chen and Kaitian Wang for coordinating the fieldwork and establishing the BEF-China experiment. We are also grateful to the whole BEF-China research group for their general support. BEF-China is mainly funded by the German Research Foundation (DFG FOR 891/1 and 2) and funding for this particular project was provided by the German Research Foundation to H.B. (DFG BR 1698/9-2). We are also thankful for the travel grants and summer schools financed by the Sino-German Centre for Research Promotion in Beijing (GZ 524, 592, 698, 699 and 785). In addition we would like to thank David Eichenberg, Michael Staab, Katja Grotius, Silvana Tornack, Lin Chen, and Shouren Zhang for their support in the field and in the lab.

1. Stomatal Conductance
<ol>
	<li>Measurements of stomatal conductance 
	NOTE: The authors made use of a simple type of steady state porometer (Decagon SC1). The porometer&#8217;s design has the advantage of small size, intuitive manual operation and high reliability. When measuring stomatal conductance in the field, make sure to minimize the distance between measured individuals for a repeated measurement cycle to be the most efficient possible.</li>
	<li>Choose leaves of different species and individuals according to a reproducible pattern (same height, same exposure, same position within the plant, if possible only from the same node, and only from one category; sun or shade leaves, etc.).
	<ol>
		<li>Only measure leaves in healthy, non-damaged and fully developed condition. Mark the leaves on the individuals (e.g., with cable ties or color tape), to ensure that the repeated measurements are done on the same leaf. 
		NOTE: Measurements of stomatal conductance should be made only on leaf surface whilst avoiding the midrib and strong leaf veins.</li>
		<li>Start measurements in the early morning hours before sunrise taking five to ten repeated measurements until stomatal conductance values shows a clear decline at noon. 
		NOTE: A daily course of measurements will deliver good data for analyzing the relationships between vpd and stomatal conductance.</li>
	</ol>
	</li>
	<li>Vpd measurements
	<ol>
		<li>With each gS measurement, record temperature and relative humidity preferably with portable loggers to directly measure the conditions at the position of the same leaf. For calculating the vapor pressure deficit use the August-Roche Magnus formula15.</li>
	</ol>
	</li>
</ol>
<img alt="Equation 1" fo:content-width="3in" src="/files/ftp_upload/52738/52738eq1.jpg" width="300" />
es=saturation water vapor pressure [hPa]
T=temperature [&#176;C]
<ol start="4">
	<li>Model gS - vpd response
	<ol>
		<li>Now plot species-wise all gS data against vpd, combining all daily courses of individual leaves into one analysis per species. Extract the maximum value observed from the stomatal conductance data by searching for the maximum value. To scale the model for species-wise comparability, divide the observed values through the maximum value observed for that species (gS/gSMAX).</li>
		<li>For each species, regress the logits of gS (gS/gSMAX) to vpd and the quadratic term of vpd using a generalized linear model with a binomial error distribution (a, b and c represent regression parameters):</li>
	</ol>
	</li>
</ol>
<img alt="Equation 2" src="/files/ftp_upload/52738/52738eq2.jpg" width="300" />
<img alt="Figure 1" src="/files/ftp_upload/52738/52738fig1highres.jpg" width="700" /> 
Figure 1. Example of the plotted values and the fitted model for gS - vpd. Stomatal conductance plotted as a function of vapor pressure deficit for the species Liquidambar formosana. Empty dots represent the observed values. (A) Maximum stomatal conductance, the vpd at maximum stomatal conductance and mean stomatal conductance was extracted from the absolute thus non-scaled stomatal conductance (gS) data. (B) Scaled stomatal conductance data (gS/gSMAX) were plotted to extract the relative parameters (shown as filled dots) 20. Re-print with permission from20. <a href="https://www.jove.com/files/ftp_upload/52738/52738fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52738/52738fig2highres.jpg" width="400" /> 
Figure 2. All fitted models for all species. Model graphs for the stomatal conductance data to vpd regression for all species. Evergreen species are represented by black lines, deciduous species by red lines 20. Re-print with permission from20.
NOTE: Using the logits instead of regressing gS directly to vpd, leads to the point that modeled maximum values do not exceed gSMAX and that gS approaches 0 at high vpd.
<ol start="5">
	<li>Extract parameter of stomatal regulation for every species
	<ol>
		<li>Calculate absolute modeled gSMAX values (MaxFit in Figure&#160;1B). To do this, calculate the vpd at maximum stomatal conductance from setting the first derivative of 1.4.2 to zero, which gives vpdgsMaxFit = -b/2a. Insert vpdgsMaxFit into the formula of 1.4.2 and raise to the power of e to obtain MaxFit. Calculate the mean of all conductance measurements per species (see Figure 1A). 
		NOTE: Use statistical software R (http://www.r-project.org).</li>
		<li>To calculate relative values, from the scaled model (gS/gSMAX), extract stomatal conductance and vpd values for the following two points: (1) stomatal conductance and vpd at the maximum of the model (MaxFit) and (2) vpd at the second point of inflection of the curve (see Figure 1B). Multiply these values by gSMAX to obtain absolute gS values for these points. See Figure 2 for a complete overlay of the single models of all 39 species analyzed.</li>
	</ol>
	</li>
</ol>
2. Measurements of Stomatal Traits
<ol>
	<li>Take samples preferably from exactly the same leaves that have been used for the measurements of stomatal conductance. If this is not possible, apply the same selection procedure that was applied to choose the leaves for the stomatal conductance measurements, preferably on the same individuals.</li>
	<li>Apply a thin layer of colorless, quickly drying nail polish (test different makes, some are more suitable than others) to a fresh sample. If the samples cannot be processed immediately, store them in 70% alcohol. After the nail polish has dried out, gently peel the impression of from the leaf and proceed for the microscopic analysis, as with a normal leaf sample. &#160; 
	NOTE: In the case of leaves with high trichome density a preceding step of applying sodium hydroxide or a 1:1 solution of acetic acid and oxygen peroxide can lead to better results.</li>
	<li>Optical microscope analytics
	<ol>
		<li>Connect a camera to an optical microscope capable of magnifications between 40X to 400X. After the camera is connected to the microscope, match the pictures taken to the optical magnification and resolution of the picture, e.g., with the help of a scale.</li>
		<li>Employing open source image processing software like ImageJ 16 analyze these pictures.
		<ol>
			<li>Draw a shape with the shape tool from the image analysis tool on the image in an area with no dirt, thumb prints, damaged areas, or large leaf veins. Count stomata in this area and in total of at least 50,000 &#181;m2 per sample.</li>
			<li>Measure stomatal guard cell length and pore length. Calculate the number of stomata per mm2. Calculate stomatal index as the ratio of stomatal density to epidermal cell number per mm2.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Assessment of Leaf Vein Traits
NOTE: For the assessment of leaf vein traits, a modified a protocol from Sack &#38; Scoffoni 17 was employed.
<ol>
	<li>Sample preparation 
	NOTE: To optimize the visibility of the leaf veins, the leaves are first bleached and then stained with safranine and malachite green.
	<ol>
		<li>To bleach the leaves, leave them at least 72 hr in 50% solution of decolorizer (test different makes, again some might work better). Alternatively, use 5% NaOH solution or 10% KOH solution or 25% H2O2 solution.</li>
		<li>Heat the solution up to 30&#176; C or combine the different solutions from step 3.1.1 for better effects. Rinse several times in water afterwards. Adapt the bleaching process for the specific species, depending on their leaf characteristics. 
		NOTE: Thicker leaves may need longer periods of solution bath and or the more aggressive solutions. Thinner and more tender leaves may be bleached to a satisfactorily degree in less than 72 hr.</li>
		<li>To color the leaves place them in 100% ethanol. Color them for 2 &#8211; 30 min in 1% safranine solution. To enhance coloring, add an additional treatment with 1% malachite green solution only for several seconds. Adapt the protocol to every species in terms of timing and intensity to achieve optimal results. Rinse several times in water afterwards. If the leaves are to deeply stained, some time in ethanol or decolorizer may help.</li>
	</ol>
	</li>
	<li>Sample analysis
	<ol>
		<li>Scan the leaves with a backlight scanner at a resolution of ca. 1,200 dpi. Match the scans taken to the resolution of the picture, e.g., with the help of a scale, to ensure that pixel length can be traced back to absolute length measures of the scanned leaf.</li>
		<li>Measure area, circumference, length and width of the leaves. Calculate several indices, e.g., length/width and circumference2/area. Cut a 1 by 1 cm rectangle out of the middle of the picture. Measure diameter of the veins of first and second order (do not include the main middle vein). Measure the length of all veins of first order in this quadrat (vein density).</li>
	</ol>
	</li>
</ol>
4. Assessment of Other Leaf Traits
NOTE: Asses typical leaf traits such as specific leaf area (SLA), leaf dry matter content (LDMC), leaf area, element contents, leaf habit, leaf pinnation, leaf compound type, leaf margin type etc. following established protocols 18,19.
<ol>
	<li>Observational leaf traits
	<ol>
		<li>Assess leaf pinnation, leaf compound type, leaf margin type and the presence of extrafloral nectaries18,19 through observation in the field.</li>
	</ol>
	</li>
	<li>Analytical leaf traits
	<ol>
		<li>Collect fresh leaf samples, preferably on the same individuals as used for the other measurements, for the determination of specific leaf area (SLA), leaf area and leaf dry matter content (LDMC) 18,19. After 48 hr in the drying oven at 80 &#176;C, measure element contents and ratios, preferably on the same leaves.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The parameters of stomatal regulation extracted by the method presented in this paper underline the importance of stomatal traits, such as stomata density and stomata index. These novel relationships demonstrate the potential of linking parameters from physiological models to morphological, anatomical and chemical leaf traits 20. Compared to other methods, the present approach bears the advantage of capturing a unique and unequivocal vpd value at which stomatal conductance is down-regulated to half of the modeled gs maximum.
Of all steps outlined in the protocol the most critical ones are the measurements of stomatal conductance. Due to the multifactorial regulation of stomatal conductance ambient weather conditions have a strong influence on gS. Stomatal conductance measurements at high relative humidity and low light intensity may be unreliable 21-23. With respect to morphological and anatomical traits, the protocol should always be adapted to the target species included in the study. In particular in the vein density analysis, the duration of bleaching and staining of leaves should be varied, depending on leaf structure and toughness. Potential limitations of the method include species, for which measurements of stomatal conductance are impossible or complicated and prone to error owing to extraordinary leaf forms. This may include conifers and grasses with very narrow leaf blades.
Our results partly confirm the first hypothesis of a link between stomatal conductance parameters and leaf traits of the leaf economics spectrum (LES), which corresponds to several other studies. For example, Poorter and Bongers (2006)24 reported a close link between gS and traits represented by the LES, e.g., with gS decreasing with increasing leaf lifespan. Accordingly, Schulze et al. (1994) 1 demonstrated clear links between leaf nitrogen content and gSMAX. Similarly, Juhrbandt et al. (2004) 25 found significant relationships between gSMAX and leaf area and leaf nitrogen content.
Our second hypothesis of clear differences with regard to leaf habit could not be confirmed. The high variation in the measured parameters and traits within evergreen and deciduous leaf habit indicate that leaf habit is not a good descriptor of the LES. Brodribb and Holbrook (2005) 26 discussed that leaf habit and leaf physiological strategies may not be inevitably connected since broad trait variation is common in all types of leaf habit.
The approach may be extended to traits and physiological characteristics of plant organs other than leaves, for example to traits related to xylem hydraulics such as specific xylem hydraulic conductivity and microscopy wood traits 27. Similarly, other types of leaf traits as derived from microscopy such as palisade parenchyma structure and epicuticular wax layer structure could be included 28.
In summary, this study confirmed the close connection between the LES and stomatal regulation. In addition, the method presented here revealed facets of stomatal regulation patterns that are not related to the LES. Especially specific leaf traits such as stomata size, density and index as well as vein length deserve future attention in functional plant studies.

To advance the functional understanding of plant leaves, many recent studies have attempted to relate morphological, anatomical and chemical leaf traits to physiological responses, such as leaf stomatal conductance (gS) 1-4. In addition, to leaf traits, stomatal conductance is strongly affected by environmental conditions, such as photosynthetically active photon flux density, air temperature and vpd 5. Various ways have been proposed to model gs - vpd curves6-8 which are mainly based on linear regression of gs on vpd 6. In contrast, the model presented in this study regresses the logits of relative stomatal conductance (i.e., the ratio of gS to maximum stomatal conductance gSMAX) on vpd and accounts for the non-linearity by adding vpd as a quadratic regressor term.
Compared to other models, the new model allows for deriving parameters that describe the vpd at which gS is down-regulated under water shortage. Similarly, the vpd is obtained at which gS is maximal. As such physiological parameters can be expected to be tightly linked to carbon assimilation 9,10 a close link between these model parameters and the key leaf traits to nutrient and resource allocation as reflected in the LES should be expected 3,11. In consequence, there should be also a tight relationship between strategies of stomatal regulation with LES traits. Such a relationship is expected in particular for leaf habit (evergreen versus deciduous) as leaf habit is both correlated with the LES and with water use efficiency12,13. Evergreen species tend to grow slower, but are more efficient in environments poor in nutrients 14. Thus, leaf habit should translate into differing stomatal regulation patterns, with a more conservative water use strategy than deciduous species.
Comparing a large set of broad-leaved tree species in a common garden situation, the following hypotheses were tested: 1) Model parameters from gS - vpd models are connected to leaf traits related to the leaf economics spectrum. 2) Evergreen species have lower mean gS and gSMAX values than deciduous species.

<table><tbody><tr><td>SC 1 Porometer</td><td>Decagon</td><td></td><td>Any other porometer is suitable</td></tr><tr><td>Cable ties to mark leaves</td><td></td><td></td><td></td></tr><tr><td>Plastic sample bags</td><td></td><td></td><td></td></tr><tr><td>Paper sample bags</td><td></td><td></td><td></td></tr><tr><td>Hygrometer</td><td>Trotec</td><td></td><td>Any other is suitable</td></tr><tr><td>Nail polish</td><td></td><td></td><td></td></tr><tr><td>Axioskop 2 plus</td><td>Zeiss</td><td></td><td>Any other is suitable</td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>Bleach</td><td></td><td></td><td></td></tr><tr><td>5% NaOH</td><td></td><td></td><td></td></tr><tr><td>10% KOH</td><td></td><td></td><td></td></tr><tr><td>25% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>Malachite green</td><td></td><td></td><td></td></tr><tr><td>Safranine</td><td></td><td></td><td></td></tr></tbody></table>

relating stomatal conductance to leaf functional traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Many parameters of stomatal conductance and stomatal regulation were found to be related to morphological, anatomical and chemical leaf traits. In the following, the focus will be on the links to vpd at the point of inflection, which decreased with stomatal density (p=0.04) and stomatal index (p=0.03) and increased with leaf carbon content (p=0.02, See Figure 3). The results show that with decreasing vpd at the point of inflection there was a decrease in stomatal density and stomatal index. In contrast, no parameter of stomatal conductance showed a clear relationship to leaf habit. The high variation within the two groups of leaf habit shows that different regulatory mechanisms exist both within the group of evergreen and deciduous leaf habits.

<img alt="Figure 3" src="/files/ftp_upload/52738/52738fig3highres.jpg" width="700" /> 
Figure 3. Result figure for the links between patterns of stomatal conductance regulation and leaf traits. Vapor pressure deficit (vpd) at the point of inflection of the gS - vpd curve (VpdPoi) as a function of (A) stomata density, (B) stomata index, and (C) leaf carbon content 20. Re-print with permission from20. <a href="https://www.jove.com/files/ftp_upload/52738/52738fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Relating Stomatal Conductance to Leaf Functional Traits at JoVE.com

Relating Stomatal Conductance to Leaf Functional Traits

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

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Research

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Biology

19.0K Views.  Martin-Luther-University Halle-Wittenberg.  Ontrafelen hoe fysiologie en morfologie worden gekoppeld staat een dieper inzicht in de mechanistische werking van de bladeren van de planten. We presenteren zowel een procedure om de parameters van stomataire regelgeving ontlenen stomatale geleidbaarheid metingen en correlaties met traditionele functionele blad kenmerken. 

Video: Relating Stomatal Conductance to Leaf Functional Traits

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Immunology and Infection

The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus area. Biomass capacitance plays an integral role in the avoidance of hydraulic impairment to transpiration. However, high temporal resolution measurements of dynamic changes in the hydraulic capacitance of large trees are rare. Here, we present procedures for the calibration and use of capacitance sensors, typically used to monitor soil water content, to measure the volumetric water content in trees in the field. Frequency domain reflectometry-style observations are sensitive to the density of the media being studied. Therefore, it is necessary to perform species-specific calibrations to convert from the sensor-reported values of dielectric permittivity to volumetric water content. Calibration is performed on a harvested branch or stem cut into segments that are dried or re-hydrated to produce a full range of water contents used to generate a best-fit regression with sensor observations. Sensors are inserted into calibration segments or installed in trees after pre-drilling holes to a tolerance fit using a fabricated template to ensure proper drill alignment. Special care is taken to ensure that sensor tines make good contact with the surrounding media, while allowing them to be inserted without excessive force. Volumetric water content dynamics observed via the presented methodology align with sap flow measurements recorded using thermal dissipation techniques and environmental forcing data. Biomass water content data can be used to observe the onset of water stress, drought response and recovery, and has the potential to be applied to the calibration and evaluation of new plant-level hydrodynamics models, as well as to the partitioning of remotely sensed moisture products into above- and belowground components.

The hydraulic capacitance of biomass is a key component of the vegetation water budget, which serves as a buffer against short and long-term drought stresses. Here, we present a protocol for the calibration and use of soil moisture capacitance sensors to monitor water content in the stems of large trees.

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus ...

Environment

Digital Leaf Physiognomy for Paleoclimate and Paleoecological Reconstruction

Reconstructing Terrestrial Paleoclimate and Paleoecology with Fossil Leaves Using Digital Leaf Physiognomy and Leaf Mass Per Area

Climate and environment strongly influence the size, shape, and toothiness (physiognomy) of plants&#39; leaves. These relationships, particularly in woody non-monocotyledonous angiosperms, have been used to develop leaf-based proxies for paleoclimate and paleoecology that have been applied to reconstruct ancient terrestrial ecosystems for the last ~120 million years of Earth&#39;s history. Additionally, given that these relationships have been documented in living plants, they are important for understanding aspects of plant evolution and how plants respond to climatic and environmental changes. To conduct these types of analyses on modern and fossil plants, leaf physiognomy must be measured accurately using a reproducible methodology. This protocol describes a computer-based method for measuring and analyzing a variety of leaf physiognomic variables in modern and fossil leaves. This method allows for the measurement of leaf physiognomic traits, in particular variables related to leaf serrations, leaf area, leaf dissection, and linearity that are used in the digital leaf physiognomy proxy for reconstructing paleoclimate, as well as petiole width and leaf area, which are used for reconstructing leaf mass per area, a paleoecological proxy. Because this digital leaf trait measurement method can be applied to fossil and living plants, it is not limited to applications related to reconstructing paleoclimate and paleoecology. It can also be used to explore leaf traits that may be informative for understanding the function of leaf morphology, leaf development, phylogenetic relationships of leaf traits, and plant evolution.

Author Spotlight: Leaf Trait Analysis for Climate and Ecology Reconstruction in Modern and Ancient Plant Communities

The protocol presented shows digital measurement and analysis of continuous leaf physiognomic traits on fossil leaves to reconstruct paleoclimate and paleoecology using the digital leaf physiognomy and leaf mass per area reconstruction methods.

Climate and environment strongly influence the size, shape, and toothiness (physiognomy) of plants&#39; leaves. These relationships, particularly in woody ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Automated Stomatal Response Measurement to Bacterial Invasion in Arabidopsis

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.

Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement

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 ...

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of these analyses is of crucial importance, because growth is driven by cell division and cell elongation. Kinematic analysis represents a methodology to quantify these two processes. Moreover, this technique is easy to use in non-specialized laboratories. Here, we present a protocol for performing a kinematic analysis in monocotyledonous maize (Zea mays) leaves. Two aspects are presented: (1) the quantification of cell division and expansion parameters, and (2) the determination of the location of the developmental zones. This could serve as a basis for sampling design and/or could be useful for data interpretation of biochemical and molecular measurements with high spatial resolution in the leaf growth zone. The growth zone of maize leaves is harvested during steady-state growth. Individual leaves are used for meristem length determination using a DAPI stain and cell-length profiles using DIC microscopy. The protocol is suited for emerged monocotyledonous leaves harvested during steady-state growth, with growth zones spanning at least several centimeters. To improve the understanding of plant growth regulation, data on growth and molecular studies must be combined. Therefore, an important advantage of kinematic analysis is the possibility to correlate changes at the molecular level to well-defined stages of cellular development. Furthermore, it allows for a more focused sampling of specified developmental stages, which is useful in case of limited budget or time.

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Quantifying cell division and expansion is of crucial importance to the understanding of whole-plant growth. Here, we present a protocol to calculate cellular parameters determining maize leaf growth rates and highlight the use of these data for investigating molecular growth regulatory mechanisms by directing developmental stage-specific sampling strategies.

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

Spatiotemporal Investigation of ERL1 Dynamics via Confocal Imaging

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Author Spotlight: Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

Potato Resilience Analysis Under Climate-Related Stresses

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions over time. By using multiple imaging sensors, many traits of interest can be assessed, including plant biomass, photosynthetic efficiency, canopy temperature, and leaf reflectance indices. Plants are frequently exposed to multiple stresses under field conditions where severe heat waves, flooding, and drought events seriously threaten crop productivity. When stresses coincide, resulting effects on plants can be distinct due to synergistic or antagonistic interactions. To elucidate how potato plants respond to single and combined stresses that resemble naturally occurring stress scenarios, five different treatments were imposed on a selected potato cultivar (Solanum tuberosum L., cv. Lady Rosetta) at the onset of tuberization, i.e. control, drought, heat, waterlogging, and combinations of heat, drought, and waterlogging stresses. Our analysis shows that waterlogging stress had the most detrimental effect on plant performance, leading to fast and drastic physiological responses related to stomatal closure, including a reduction in the quantum yield and efficiency of photosystem II and an increase in canopy temperature and water index. Under heat and combined stress treatments, the relative growth rate was reduced in the early phase of stress. Under drought and combined stresses, plant volume and photosynthetic performance dropped with an increased temperature and stomata closure in the late phase of stress. The combination of optimized stress treatment under defined environmental conditions together with selected phenotyping protocols allowed to reveal&#160;the dynamics of morphological and physiological responses to single and combined stresses. Here, a useful tool is presented for plant researchers looking to identify plant traits indicative of resilience to several climate change-related stresses.

Author Spotlight: Unraveling Plant Responses to Abiotic Stresses Using the PlantScreen Robotic Platform

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions ...

Transpiration

<h4>Background</h4>

All living organisms must carry out a set of basic functions in order to maintain themselves. One of these processes involves the transportation of materials throughout the organism. Therefore, organisms need to exchange materials with the environment, which can be seen on the small scale of cells transporting protein and other materials among one another or on a larger scale such as the water cycle, where water is continuously moving on, above, and below the surface of Earth. In both examples, the movement of materials from one area to another is a dynamic process regulated by both environmental and biological variables.

<h4>Transpiration: A Driver for Water Transport</h4>

Vascular plants, from the smallest ferns to the giant redwoods of California, transport water and the water-soluble materials throughout the plant by a system of bundles of vascular tissues that run from the roots to the tips of the plant. Specifically, water and nutrients are absorbed by the root hairs and transferred by osmosis into the plant&#39;s xylem, one of the two large vascular systems found in plants. Water is then transported to the tallest point of the plant and outwards into the leaves where photosynthesis takes place. Interestingly, only 1% of the water taken up by the plants is used for photosynthesis. The other 99% of the water is not directly used by the plant and is lost from the plant due to evaporation or guttation, also known as transpiration. Evaporation is the movement of water to the air, where guttation specifically refers to secretion of droplets of water from the pores of plants. Both processes combined make up transpiration in plants.

The leaves of plants play an important role in releasing the by-products of photosynthesis through their stomata, which are openings that allow the exchange of materials between the plant and the atmosphere. The stomata are bordered by guard cells, which regulate when the stomata open and close. This is the active site where the majority of water is lost due to evaporation, as well as the site where gases are exchanged with the atmosphere. Although it might at first seem like a bad strategy for a plant to lose water due to evaporation, it is inevitable in order to maintain the transportation of materials and water in the plant while maximizing gas input. Transpiration creates a lower concentration of water, thus a lower osmotic potential in the leaf. These differences in water concentration are responsible for driving the movement of water into the plant&#39;s leaves and also in releasing water into the atmosphere.

Water potential drives the uptake of water from the root hairs and also in transporting water to the tips of leaves. Water potential is the measure of the free energy of water, where water molecules move from areas of higher water potential to areas of lower water potential. When evaporation is high in leaves, this creates areas with lower water potential, or areas with less water, so water from the roots and stem is driven to the leaves. The cohesion and adhesion properties of water molecules enable this movement of water. Cohesion is the attraction of water molecules to each other and adhesion is the attraction of water molecules to other materials, such as the xylem walls. When water molecules exit through stomata, they pull water molecules underneath, causing water to move towards lower water potential.

<h4>Environmental Factors</h4>

Plant species differ widely in their physical characteristics as well as in their morphology and functions in the ecosystem. These differences among plant species and also similarities among distantly related plant species are shaped over time through evolution and more specifically by selection imposed by herbivores, pollinators, and other climatic and environmental factors. Therefore, differences in transpiration rates is influenced by both the environment and the plant species itself. The main environmental factors that drive the transpiration rate is temperature. Higher temperatures increase the rate of transpiration because water is lost via evaporation more quickly. Plants living in hot environments are prone to losing more water than plants located in cooler climates. Factors such as water availability, wind, sunlight, and others also help influence the transpiration rate in plants.

Plants living in hot and arid environments have specific adaptations that help them control water loss, such as the ability to store or conserve water. A group of plants in these environments, also known as Crassulacean acid metabolism or CAM plants, have evolved strategies, such as only opening their stomata for gas exchange at night in order to reduce water loss1. Some other plant features specific to arid environments include reduced leaf surface area, fewer stomata, or having hairs on their leaves to conserve water. However, there is a tradeoff with limiting water loss and having an optimal rate of transpiration needed for photosynthesis. The rate of photosynthesis relates to a plant&#8217;s rate of growth and energy acquisition, which is related to rate of water intake and loss, so it is extremely important for plants to be able to balance this tradeoff. On the other hand, in environments where water is not a limiting resource, such as tropical rainforests, plants face different selective pressures that drive differences in transpiration rates. In these environments, natural selection may instead favor plant species that can transport water more quickly in order to outgrow their competing neighbors or grow tall enough to avoid being eaten by herbivores.

<h4>Assessing Transpiration Rates</h4>

The transpiration rates can be assessed indirectly by using a potometer, which is a device that measures the rate of water uptake of a leafy plant. The assumption of the potometer measurement is that transpiration will cause the water uptake, the amount of which can be quantified. In addition, scientists can determine relative transpiration rates of plants by observing the leaf structures, such as the sizes and numbers of stomata per unit area.

Examining plant transpiration rates can teach us not only how plants adapt to different environments, but can also provide information about how to best grow crops in different environmental conditions to increase food production and adjust our use of plants to adapt to global climate change and population growth. For example, transpiration rates are altered by global warming and other causes and can affect the global water cycle, which in turn can affect ecosystems as well as human populations2. Thus, understanding these changes would be necessary to develop strategies to remediate their negative effects. Moreover, studying transpiration rates of different crops can help identify crops with high water-use efficiency to increase the food production per unit of water and reduce the need for irrigation3.

<h4>References</h4>

<ol class="references">
	<li>IP, TIng. Crassulacean acid metabolism. Ann REv Plant Physiol. 1985, Vol. 36, 595-622.</li>
	<li>Schlesinger WH, Jasechko S. Transpiration in the global water cycle. Agricultural and Forest Meteorology. 2014, Vols. 189-190, 115-7.</li>
	<li>Coupel-Ledru A, Lebon E, Christophe A, Gallo A, Gago P, Pantin F, Doligez A, Simonneaua T. Reduced nighttime transpiration is a relevant breeding target for high water-use efficiency in grapevine. PNAS. 2016, Vol. 113, 32: 8963-8.</li>
</ol>

Relating Stomatal Conductance to Leaf Functional Traits

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