Drought-tolerant ecotypes have fewer stomates, illustrating an evolutionary adaptation to drought tolerance

Genetic and environmental influences on stomates of big bluestem (Andropogon gerardii)

Varvel N.A., Hilt C.J., Johnson L., Galliart M., Baer S., Maricle B. (2018)

Nick A. Varvel ^a, Christina J. Hilt ^a, Loretta C. Johnson ^b, Matthew Galliart ^b, Sara G. Baer ^c, Brian R. Maricle ^a

^aDepartment of Biological Sciences, Fort Hays State University, 600 Park St., Hays, KS 67601, USA

^bDivision of Biology, Kansas State University, Manhattan, KS 66502, USA

^cDepartment of Plant Biology and Center for Ecology, Southern Illinois University, Carbondale, IL 62901, USA

===

Environmental and Experimental Botany, 155: 477-487 – https://doi.org/10.1016/j.envexpbot.2018.07.018 –

https://www.sciencedirect.com/science/article/abs/pii/S0098847218300807

Highlights

• Big bluestem (Andropogon gerardii) ecotypes are adapted to water availability.

• Leaves were largely hypostomatous.

• Genetics was more influential than environment for stomatal size and density.

• Drought-tolerant ecotypes had larger and fewer stomates.

• Mesic ecotypes had the greatest density of stomates.

Abstract

Big bluestem (Andropogon gerardii) is a dominant C₄ prairie grass that has wide distribution and several genetically distinct ecotypes. Many of the ecotypic adaptations are related to water availability in the native environment. Stomates facilitate photosynthetic gas exchange and regulate water loss from the plant. As such, stomatal size and density represent possible adaptations to conserve water. We hypothesized drought-tolerant ecotypes of big bluestem would have fewer or smaller stomates compared to more mesic ecotypes. Five ecotypes of big bluestem were planted in four common gardens from western Kansas to southern Illinois, USA to determine genetic and environmental influences on stomates. Leaves of all ecotypes of A. gerardii were largely hypostomatous and genetics was a greater influence than environment for stomatal size and density. The drought-tolerant Sand bluestem had larger stomates on abaxial surfaces of leaves, but a lower density compared to most other ecotypes. The most mesic Illinois ecotype and the Kaw cultivar had the greatest density of stomates on abaxial surfaces of leaves. Sand Bluestem had a greater density of stomates on adaxial surfaces of leaves compared to all other ecotypes. Gas exchange measures followed patterns of stomate distribution, where abaxial CO₂ uptake rates were greater than adaxial CO₂ uptake rates, although differences between leaf surfaces was more pronounced in stomatal density than in CO₂ uptake. There were minor differences in size and density of stomates among sites that corresponded with precipitation, although these differences were minor, illustrating the genetic underpinnings of stomates in big bluestem. There is a genetic predisposition for drought-tolerant ecotypes to have fewer stomates, illustrating an evolutionary adaptation to drought tolerance in an important prairie species.

Stomata in Desert Plants

Trends in Distribution and Size of Stomata in Desert Plants

Sundberg M. (1985) – – in “Desert Plants” –

https://www.semanticscholar.org/paper/Trends-in-Distribution-and-Size-of-Stomata-in-Sundberg/bedb4ec4e1fba4d014c9b3cb7614c93ca310f2d2#paper-header

Stomatal diffusive resistance significantly increased with antitranspirants

The effects of antitranspirants on leaf water status, stomatal resistance and yield in tomato

Rao N. K. S. (1985)

N. K. Srinivasa Rao

Division of Plant Physiology and Biochemistry, Indian Institute of Horticultural Research, Hessaraghatta Lake, P.O. Bangalore 89, India

===

Journal of Horticultural Science 60(1): 89-92 – https://doi.org/10.1080/14620316.1985.11515605

https://www.tandfonline.com/doi/abs/10.1080/14620316.1985.11515605 –

Summary

The effects of three antitranspirants, PMA, 8-HQ and kaolinite, on leaf water potential, sap osmotic potential, stomatal diffusive resistance and yield in different tomato cultivars were examined. Antitranspirants were sprayed at the flower initiation and/or fruit development stages. Stomatal diffusive resistance significantly increased in the treated plants compared with control plants. Although leaf water potential and leaf sap osmotic potential gradually decreased, in both treated and control plants, compared with irrigated plants, the decrease was greater in the control plants. Significant yield differences were observed between control and treated plants. A single spray of anti-transpirant at either the flower initiation or fruit development stage improved yield. The application of antitranspirants at both stages was not beneficial. PMA and kaolinite improved the plant water status and yield.

Errors in the Estimation of Stomatal Conductance from Porometer Data

Sources of Error in the Estimation of Stomatal Conductance and Transpiration from Porometer Data

McDermitt D. K. (1990)

LI-COR, Inc., PO Box 4425, Lincoln, NE 68504

===

HORTSCIENCE 25(12): 1538-1548 –

file:///C:/Users/wille/Downloads/[HortScience]%20Sources%20of%20Error%20in%20the%20Estimation%20of%20Stomatal%20Conductance%20and%20Transpiration%20from%20Porometer%20Data%20(2).pdf

Stomatal control of leaf conductance is an important means by
which plants limit water loss. It is often used as an indicator of
water stress, and it is an important factor in photosynthesis studies.
Automated commercially available equipment for measuring leaf
conductance has become available in recent years from several manufacturers. When properly used, this equipment makes it easy to
obtain reliable field measurements. However, if the instruments are
not used or calibrated properly, they can give conductance estimates
that appear “reasonable,” and yet are in significant error.
The purpose of this paper is to describe a) the effects of sensor
or calibration errors on the accuracy of conductance measurements,
b) the relationship between molar conductance units that are coming
into prominent use and velocity units that are used in the older
literature. and c) some considerations that will help protect the
quality of porometer data. This discussion is limited to porometry
as it relates to water vapor exchange. Up-to-date general reviews
of instrumentation and procedures for gas exchange measurements
can be found in Coombs et al. (1985), Field et al. (1989), Parkinson
(1985b), and Pearcy et al. (1989). Sestak et al. (1971) provides an
exhaustive review of literature and methods up to 1971.
Porometers in general use today fall into two broad categories,
transient or steady state, according to the measurement principle on
which they are based. Transient porometers may be ventilated, in
which a fan provides rapid chamber mixing, or unventilated, in
which cuvette humidity changes occur strictly by diffusion. In ventilated transient porometers (Komer and Cernusca, 1976; Tan and
Black, 1978; Turner and Parlange, 1970). stomatal resistance is
calculated from the rate and magnitude of change in cuvette humidity, and system volume. In unventilated transient porometers
(van Bavel et al., 1965; Kanemasu et al., 1969; Monteith and Bull,
1970; Stiles, 1970; Visscher, et al., 1978). resistance is not computed directly from the magnitude of changes in relative humidity.
Instead, a measured transit time is compared to an empirical calibration curve that relates diffusion path length to measured transit
time. The transit time is determined by leaf resistance, sensor geometry, and temperature.

(Continued)

Stomatal differentiation

“DEVELOPMENTAL MORPHOLOGY AND PHYSIOLOGY OF HETEROPHYLLOUS AQUATIC ANGIOSPERMS (ABSCISIC ACID, HALORAGACEAE, LEAF DEVELOPMENT, WATER STRESS, STOMATA)”

Kane M. E. (1985)

MICHAEL EDWARD KANE, University of Rhode Island

Dissertations and Master’s Theses (Campus Access) – Paper AAI8516677 –

https://digitalcommons.uri.edu/dissertations/AAI8516677

Abstract

Within the watermilfoil family, Haloragaceae, certain species of the amphibious genera Myriophyllum and Proserpinaca develop morphologically distinct juvenile (vegetative) and adult (flowering) growth phases. All species exhibit morphological and anatomical differences between leaves produced on submerged and aerial shoots (heterophylly). Laboratory experiments were designed to: (1) compare the organogenetic potential of whole leaf explants taken from the aerial juvenile and adult growth phases of Myriophyllum heterophyllum using plant tissue culture techniques and (2) study the environmental and hormonal factors regulating aerial leaf development in seven species of Myriophyllum and two of Proserpinaca.

Whole leaf explants from both aerial juvenile and adult growth phases of Myriophyllum heterophyllum regenerated both roots and shoots when cultured on Gamborg B5 medium supplemented with combinations of cytokinins (benzyladenine BA , 6-gamma-gamma-dimethylallyl amino purine 2iP , or zeatin) and auxins (naphthalene acetic acid NAA or indole-3-acetic acid IAA ). Adult leaf explants consistently produced greater numbers of adventitious shoots and roots. The presence of cytokinin in the medium effectively promoted adventitious shoot production on all explants with zeatin > 2iP > BA. Maximum shoot formation occurred when both cytokinin and auxin were present in the medium. Shoots regenerated from both juvenile and adult leaf explants were juvenile as judged by leaf shape and the absence of flowering. Addition of auxin to the medium promoted adventitious root formation from both explant types with NAA being more effective than IAA. Adventitious shoot-buds originated from the leaf epidermal cells and root primordia originated within the perivascular tissue of the explants. Based upon the histological origin of these adventitious shoots and roots, quantitative differences in the capacity for organogenesis between juvenile and adult explants reflected differences in explant size and venation. Although the juvenile and adult growth phases of Myriophyllum heterophyllum are morphologically distinct, whole leaf explants derived from both growth phases exhibited a similar competency for organogenesis. Both the plant hormone abscisic acid (ABA) and water stress caused the development of aerial juvenile leaves on submerged juvenile shoots of Myriophyllum heterophyllum. These aerial-type leaves were characterized by reduced epidermal cell length, induction of stomatal differentiation, and increased thickness of the cuticle. Induction of aerial juvenile leaves by water stress was initiated by reduced cell turgor. (Abstract shortened with permission of author.)

Stomatal responses to humidity

Chapter 9 – Water Transport Dynamics in Trees and Stands

Pallardy S.G., Čermák J., Ewers F. W., Kaufmann M. R., Parker W. C., Sperry J. S. (1995)

in Resource Physiology of Conifers -Acquisition, Allocation, and UtilizationPhysiological Ecology: 301-389 – https://doi.org/10.1016/B978-0-08-092591-2.50014-5 –

https://www.sciencedirect.com/science/article/pii/B978008092591250014

Publisher Summary

Most of the water lost by trees in transpiration is absorbed by roots. The pathway of liquid water movement—often termed the soil-plant-atmosphere continuum—is a complex network of individual pathways that may be divided into segments. This chapter discusses the water transport dynamics in trees and stands. Water transport dynamics in trees and stands of conifers have certain features that are characteristic of this group and are at least rare among angiosperms. Among these features is the xylem transport system that is dependent on tracheids for long-distance water transport. Tracheid-containing xylem is relatively inefficient, a property that can reduce submaximum allowable rates of gas exchange, but tracheids offer substantial capacity for water storage and high resistance to freezing-induced dysfunction. Thus, they are quite compatible with the typical evergreen habit and long transpiration season of conifers. At the stand level, canopy transpiration in conifers is primarily controlled by stomatal conductance. However, in dense canopies of angiosperms, particularly those of tropical forests with limited air mixing, stand transpiration is limited by radiation input rather than by stomatal control. Because of their evergreen habit, a greater proportion of evapotranspiration in conifer forests is associated with evaporation of water intercepted by the tree crowns. Transpiration rates from conifer foliage are often lower than those of deciduous angiosperms, probably because of the lower maximum capacity of tracheid-bearing xylem to transport water.

1 Dehydration Avoidance

Dehydration avoidance adaptations promote high plant water status during climatic drought through regulation of the balance between soil moisture uptake and transpirational water loss and through maintenance of a functional xylem transport system (see Section II,A,3). Numerous adaptations can be identified (Pallardy, 1981), but they can be categorized in functional categories as adaptations for (1) water acquisition [e.g., rooting patterns and growth, mycorrhizal associations, root–shoot allocation changes (Section I)], (2) efficient transport from roots to shoots [i.e., xylem characteristics (Sections II and III)], and (3) conservation of water by shoots.

A well-developed potential for avoidance of foliar water deficits through enhanced stomatal sensitivity to soil and/or atmospheric drought has been invoked to explain the drought tolerance adaptation of Juniperus occidentalis (Miller and Schultz, 1987), Pinus banksiana (Roberts and Dumbroff, 1986), and more xeric ecotypes of Pseudotsuga menziesii (Zavitkovski and Ferrell, 1968, 1970) and P. taeda (Bilan et al., 1977). Increased stomatal control of E also was associated with improved survival in conifer seedlings (Livingston and Black, 1988; Cui and Smith, 1991). An enhanced sensitivity of stomata to leaf ABA and a comparatively gradual decline in ABA (and increase in E) following stress alleviation were associated with dehydration avoidance capacity of P. banksiana (Roberts and Dumbroff, 1986). Prior exposure of some conifer species to water stress results in acclimation whereby stomatal response to subsequent stress is characterized by lower g_s and enhanced stomatal sensitivity to declining shoot Ψ (Seiler and Johnson, 1985; Zwiazek and Blake, 1989).

A direct stomatal response to diurnal changes in atmospheric water saturation vapor pressure deficit (VPD) in the absence of significant changes in bulk leaf water status has been demonstrated for a number of conifers (Sandford and Jarvis, 1986; Grossnickle and Russell, 1991). This response provides a more efficient and sensitive mechanism for conservation of water than stomatal response to bulk leaf water status. Stomatal sensitivity to diurnal changes in VPD differs among conifer species, from very sensitive in Picea glauca (Goldstein et al., 1985) and Chamaecyparis nootkatensis (Grossnickle and Russell, 1991), to little if any response in P. taeda (Teskey et al., 1986). The response of stomatal aperture to VPD also varies with environmental factors such as light (Kaufmann, 1976; Beadle et al., 1985a; Goldstein et al., 1985), air and soil temperature (Beadle et al., 1985a; Miller and Schultz, 1987), and soil Ψ (Tan et al., 1977; Running, 1980c; Graham and Running, 1984). Plant factors such as shoot Ψ (Beadle et al., 1985b; Goldstein et al., 1985) or previous exposure to high VPD or low soil moisture may alter this stomatal response (Teskey et al., 1987; Mansfield and Atkinson, 1990). Generally, the lower the shoot or soil Ψ, the more sensitive the response or the lower the value of g_s at a given VPD. The diurnal sensitivity of stomata to VPD may decrease over time with aging of the foliage (Kaufmann, 1976; Sandford and Jarvis, 1986; Guehl et al., 1991).

Efficient plant water use (high A/E ratios) could be an important adaptation of plants growing in water-limited environments (Cowan, 1977). From the model of Cowan and Farquhar (1977), stomatal “optimization” of gas exchange will occur when the relative sensitivity of A and E to changes in g_s, δA/δE, remains constant despite environmental fluctuations that affect g_s. This stomatal optimization hypothesis has been tested for a number of conifer species, with stomatal response conforming to this hypothesis in some species (Meinzer, 1982; Sandford and Jarvis, 1986; Guehl et al., 1991) but not in others (Sandford and Jarvis, 1986; Guehl and Aussenac, 1987; Fites and Teskey, 1988; Grieu et al., 1988). The variability in results suggests that optimized stomatal behavior may simply be one of many potentially adaptive traits.

Differences among and within conifer species in stomatal response to humidity have been invoked to define comparative drought tolerance and habitat segregation, but conflicting results have been reported. Comparisons among woody species suggest that species with high vigor and growth rate are generally less sensitive to humidity than are more slower growing, evergreen species (Marshall and Waring, 1984; Korner, 1985). Comparatively low sensitivity of stomata to VPD was offered as explanation for the confinement of some conifer species to more humid, mesophytic environments (Grossnickle and Blake, 1987a,b; Higgins et al., 1987). Populations of conifer species that originate in distinct humidity regimes also exhibit different g_s-VPD relationships and stomatal adaptations to humidity. Seedlings of a dry land family of Abies alba were characterized by a stomatal response indicative of greater dehydration tolerance (Guehl and Aussenac, 1987). Conversely, dry site progeny of P. ponderosa had lower g_s values at a given VPD compared with seedlings of more mesic, coastal origin, a stomatal adaptation for enhanced dehydration avoidance (Monson and Grant, 1989).

Stomatal closure at low T_s in the absence of changes in shoot water status also may constitute a drought avoidance mechanism. The involvement of chemical signals transported from the roots to shoots in this stomatal response was investigated in split-root experiments (Day et al., 1991). No evidence for a nonhydraulic root signal was found in P. taeda seedlings when half the root system was exposed to 24°C and half to either 7 or 1°C. Instead, feedback inhibition of A by carbohydrate accumulation due to reduced root metabolic activity and sink strength at low T_s was suggested to be involved in this response (Day et al., 1991). In other species this response may be related to plant growth regulators produced by roots or close coupling between xylem flux, epidermal turgor, and g_s (Teskey et al., 1983; DeLucia et al., 1991). Although the effect of low T_s on gas exchange may not be a direct consequence of plant water deficits, stomatal closure in response to low T_s may effectively avoid foliar water deficits associated with impaired water uptake from cold soil.

Preferential carbon allocation to root systems in drought-prone environments results in a more favorable functional balance between water absorptive and water-expending tissues and improved potential for dehydration avoidance. Seedlings of conifer species or families native to more xeric environments are sometimes characterized by larger root–shoot ratios (Bongarten and Teskey, 1987; Joly et al., 1989), but not always (Strauss and Ledig, 1985; Barton and Teeri, 1993). The abscission of older foliage coupled with fine root mortality during seasonal periods of decreased soil moisture availability results in a readjustment in root–shoot balance. Further, this reduction in leaf area coincident with rapid production of fine roots following replenishment of soil moisture will result in an increased root–shoot ratio and may enhance plant capacity for stress recovery (Bartsch, 1987; Chaves and Pereira, 1992).

CO2 and stomatal conductance

Chapter 4.04 – Effects of Carbon Dioxide Enrichment on Plants

Taub D. R., Wang X. (2013)

Southwestern University, Georgetown, TX, USA / Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA

in Reference Module in Earth Systems and Environmental Sciences – Climate Vulnerability – Understanding and Addressing Threats to Essential Resources 4: 35-50 – https://doi.org/10.1016/B978-0-12-384703-4.00404-4 –

https://www.sciencedirect.com/science/article/pii/B9780123847034004044

Abstract

Increasing concentrations of atmospheric CO₂ over the next decades can be expected to have important implications for plants. Many plant species are likely to exhibit higher rates of photosynthesis and growth, decreased water consumption and changes in tissue chemistry, including decreased concentrations of protein and minerals. The most likely threats to ecosystem services as a result of increased atmospheric CO₂ are (1) decreased crop and forage food values due to decreased concentrations of protein and minerals and (2) undesirable changes in plant community composition, including increases in populations of some major weed species.

4.04.2.1.2 Transpiration and Water Use

Atmospheric concentrations of CO₂ can also affect plant water use. Carbon dioxide concentrations play a central role in controlling the aperture of stomata, small pores on the surfaces of plants, particularly on the underside of leaves. Open stomata provide a pathway for CO₂ to diffuse into leaves for photosynthesis, but also a pathway for water to diffuse out from leaves. Stomatal aperture (closely related to a physiological measure known as stomatal conductance) is therefore regulated to balance the goals of maintaining CO₂ assimilation and minimizing water loss. At higher concentrations of atmospheric CO₂, plants are able to achieve high rates of photosynthesis with relatively low stomatal conductance. In FACE experiments, elevated CO₂ decreases stomatal conductance by an average of 22% (Ainsworth and Rogers 2007).

Effects of elevated CO₂ on water use by whole plants or plant communities will depend not only on effects on stomatal conductance and leaf-level transpiration, but also on other potential effects of elevated CO₂, such as changes in plant size and morphology. For example, increased growth of plants may lead to increased numbers of leaves, offsetting decreased transpiration per leaf (Warren et al. 2011). Nonetheless, the overall effect of elevated CO₂ appears to be a decrease in total plant and ecosystem water usage, by approximately 5–20% in FACE experiments (Kimball 2010; Leakey et al. 2009). This can lead to alterations of ecosystem hydrology, including an increase in soil moisture and perhaps runoff (Leakey et al. 2009; Norby and Zak 2011).

Stomata and stomatal aperture

CHAPTER 8 – SHOOT MORPHOLOGY AND LEAF ANATOMY IN RELATION TO PHOTOSYNTHESIS

Bolhar-Nordenkampf H. R. (1985)

in Techniques in Bioproductivity and Photosynthesis (Second Edition) – https://doi.org/10.1016/B978-0-08-031999-5.50018-2 –

https://www.sciencedirect.com/science/article/pii/B9780080319995500182

Publisher Summary

This chapter discusses shoot morphology and leaf anatomy in relation to photosynthesis. The youngest leaves of a non-shaded plant usually grow in full sunlight. Some weeks later, the same leaves, now totally expanded and mature, may be shaded to a certain extent by the newly developed younger leaves. This would mean that the light intensity available for photosynthesis could be reduced to less than 10% of full sunlight and, therefore, cause very low net leaf photosynthesis. Therefore, plants may show a distinct leaf arrangement along the stem, which guarantees optimal use of the irradiated light and makes full sunlight available for the largest amount of leaf area. In a growing canopy, these differences in available light are even more striking. The other microclimatic factors show comparable gradients because of the height, density, and geometry of the canopy. Some plants have leaves that adapt well to these changes in the microclimate, whereas others lack this ability.

8.3 Experiments

8.3.1 Leaf anatomy

Transverse sections of the leaf blade cut by hand, using a razor blade, are examined in a light microscope. The layers of the palisade parenchyma cells are counted and the relation of the thickness of the palisade parenchyma to the spongy parenchyma can be estimated. Look for sclerenchyma cells and examine the leaf-air system in the different parts of the mesophyll. Do this by drawing a part of the cross-section.

Try to find the stomata and describe their position and arrangement: raised, sunken, in crypts, protected by trichomes or wax; hypostomatic, hyperstomatic or amphistomatic. To count the stomata per unit of leaf area you have to use a good incident light microscope. You can count the stomata on artificial replicas if the leaves have a blade without many trichomes and if the stomata are not deeply sunken. To produce the replicas you can use any transparent paint or varnish. Put a drop of the varnish on the surface of the leaf and pull off the dry transparent film with forceps after a few minutes. Now you can count the stomatal impressions on this replica with the help of a calibrated grid in the eyepiece of the microscope. Take replicas of different parts of the leaf because stomatal frequency varies greatly on one leaf. Less than 60 stomata per mm² and more than 600 stomata per mm² indicate that the plant grows in an extreme habitat (xeromorphic, hygromorphic respectively). Do not measure stomatal widths by the technique of replicas or epidermal strips. It is better to use an incident light microscope with a mirror (reflecting) objective for such measurements. Alternatively stomatal aperture may be determined by indirect methods as detailed in Chapter 5.

8.3.2 Stomatal width

An estimate of the relative stomatal aperture can be obtained by the infiltration method using xylol, alcohol (ethanol) and paraffin oil. Put a drop of each solution on the upper and lower surface of the leaf and judge the degree of infiltration by the size of the area darkened by the liquid entering the internal air spaces. The results may give good response curves of the changes of the climatic factors over one day of one plant only, but you cannot compare the response curves of different plants.

8.3.3 Demonstration of O₂ evolution from whole plants

Fill a large glass container with a 0.01% indigo carmine (indigo disulphonate) solution. While stirring constantly, carefully add single drops of a 10% sodium dithionite solution until the blue indigo carmine is reduced to the yellow form. Place a whole plant or a branch into this solution and seal the container, excluding air. After several minutes of illumination, if there is no excess sodium dithionite, blue areas will appear around all green parts of the plant. The reason for this is that the small amounts of O₂ produced by photosynthesis inside the leaf are enough to reoxidize the yellow indigo carmine back to the blue form.

8.3.4 In situ demonstration of PSII activity

The presence of PS II activity (reducing power) can be demonstrated by using the reduction of tetranitro-blue-tetrazolium chloride (TNBT). Cross-sections of the leaf blade, which need not be very thin, are cut by hand and infiltrated under vacuum with the staining solution (see below). After 5 to 20 minutes of illumination, under a microscope for instance, the areas in the leaf transverse section containing chloroplasts with high PS II activity will be of a dark-blue colour. This occurs first in all wounded cells, then in the mesophyll cells of all plants and finally in the granai bundle-sheath plastids of the C₄ grasses of the NAD-ME and PCK groups. The agranal bundle-sheath plastids of the NADP-ME species turn blue only after several hours of illumination. Misleading results may occur if infiltration is poor because of residual air in the intercellular spaces. Note that the bundle-sheath cells which are longitudinally elongated are more likely to be injured during sectioning than the more rounded mesophyll cells. Thus C₄ plants, especially dicotyledons, may show some coloured bundle-sheath chloroplasts even after a short period of illumination².

Stock solutions:(A)

0.1% TNBT (1.0 mg ml⁻¹). (Do not make up more than 10 ml at one time: store the dye powder and solution in a refrigerator.)(B)

0.1 M phosphate buffer, pH 6.0.(C)

0.3 M sucrose.

Staining solution:

1 part A + 3 parts B + 1 part C.

8.3.5 Differentiation between C₃ and C₄ plants by detection of starch in situ

Cross-sections of the blades of leaves of various types are stained by immersion in iodine solution. Starch will have been formed in the chloroplasts if the leaf has been illuminated for a long time and if net leaf photosynthesis has been proportionally high as compared to translocation of photosynthate. In such leaves chloroplasts will be stained dark blue by iodine. This occurs in chloroplasts of the mesophyll cells of all C₃ plants. In C₄ plants such starch accumulation occurs predominantly in the “Kranz” bundle-sheath cells. The detection of starch in this way is more distinct if chlorophyll is extracted from the leaves, using hot alcohol, before staining.

After 2 days in the dark the chloroplasts in the leaf are generally destarched. Such a leaf can be used to produce high resolution starch prints or pictures. photographic negative with high contrast is mounted on the upper surface of the leaf exposed to light. Starch will be formed only in those areas which are actually reached by light rays. The starch formation will be proportional to incident light intensity. Thus if the leaf is cut off, killed and extracted in hot water, followed by hot alcohol (in a water bath), and stained with iodine, the negative will be reproduced as a positive image. The high resolution image obtained indicates that starch is in general only formed in those cells which are actually illuminated and that translocation of photosynthate from an illuminated chloroplast, to a non-illuminated chloroplast, even when they both lie in the same cell, does not readily occur¹¹.

Solution for iodine stain (Lugol). First dissolve 2.0 g potassium iodide (KI) in 5 ml water, then add 1.0 g iodine and make up to 300 ml with water.

8.3.6 Demonstration of phloem translocation in detached maize leaves

Corn plants, 80 cm high, are transferred to a darkroom for 48 h. After this dark period no starch ought to be detectable with the iodine/KI test. Under dim light 30 cm long segments are cut from the leaf blade. The midrib is removed and the margins are stripped away. The strips are trimmed under water to a final length of 25 cm. Both ends are dipped in Hoagland’s nutrient solution diluted to 1/20th full strength. The central part of the leaf strip is placed in a chamber with several microlitres of 20% KOH. By pressing hot forceps on the tissue, the phloem is interrupted in a 1 cm wide segment of the leaf strip where it enters this CO₂-free chamber on both sides.

The strip is then exposed to sunlight for 4 hours (or to artificial light for 7 hours). It is recommended that eight replicates are set up for this experiment. The iodine/KI test for starch accumulation should be performed 1, 2 and 4 hours (2, 5 and 7 hours) after the start of the experiment.

The result should show starch formation initially in the lateral free parts of the strip. Towards the end of the experiment the starch should also appear in the central, enclosed part, with the exception of the segment where the phloem is disconnected from the parts of the strip outside the chamber. The starch formation in the central part of the maize leaf strip must be caused by active phloem transport from both sides⁷.

Stomata play a pivotal role in controlling the balance between water loss and carbon gain

CHAPTER 5 – WATER RELATIONS

Beadle C. L., Ludlow M. M., Honeysett J. L., (1985)

in Techniques in Bioproductivity and Photosynthesis (Second Edition) – Pergamon International Library of Science, Technology, Engineering and Social Studies: 50-61 – https://doi.org/10.1016/B978-0-08-031999-5.50015-7 –

https://www.sciencedirect.com/science/article/pii/B9780080319995500157

Publisher Summary

This chapter discusses water relations in plants. To absorb CO₂ for photosynthesis, plants expose wet surfaces to a dry atmosphere and, in consequence, suffer evaporative water loss. The resultant cooling often accounts for considerable heat dissipation by leaves and is probably essential for maintaining equable temperatures for photosynthesis. Plants have, therefore, evolved leaves with an epidermis composed of a relatively impermeable cuticle and turgor-operated valves—stomata. The epidermis not only reduces rates of CO₂ and water vapor exchange but also provides a means of controlling assimilation and transpiration through the size of the stomatal pores. Thus, stomata play a pivotal role in controlling the balance between water loss and carbon gain. For a given leaf, plant, or variety, the length and depth of stomata do not vary among stomata in mature tissues. Most of the changes in aperture are associated with changes in width. In practice, it is not possible to make a direct microscopic observation of stomata and, at the same time, preserve natural conditions.

5.1.3 Methods for stomatal aperture

Stomatal aperture is usually measured by direct microscopic observation or by the extent or rate of infiltration of organic solvents¹⁵.

Direct Observation:

For a given leaf, plant or variety, the length and depth of stomata do not vary among stomata in mature tissues. Instead, most of the changes in aperture are associated with changes in width. In practice it is not possible to make a direct microscopic observation of stomata and at the same time preserve natural conditions. The change in conditions could alter the stomatal aperture. However, it is possible to make a stomatal impression before stomata have time to react by applying a quick-drying substance to the leaf surface^34,15,24. The size of the stomatal aperture can be measured under a microscope either from the impression (where the stomatal pore is represented as a raised area in what is equivalent to a photographic negative) or a positive which is made by painting the negative with a substance such as cosmetic nail varnish (stomatal pores appear as holes in the positive). Stomatal aperture can be converted into an equivalent diffusive resistance (or conductance)^19,20.

Infiltration by Liquids:

A series of mixtures of two liquids (0–100%) is made, one with a high, the other low, viscosity. The mixtures are then applied to leaf surfaces in sequence from the most to the least viscous. The first mixture to infiltrate the leaf surfaces is an index of the degree of stomatal opening. This index can be correlated for each species with aperture obtained by direct observation or with diffusive resistance¹⁶. The infiltration method is simple and cheap, but of limited accuracy.

A cell-within-a-cell arrangement unknown among stomata elsewhere in the plant kingdom

Stomata of Alethopteris sullivanti: a new stomatal type among seed ferns and vascular plants

Stidd B. M. (1988)

Benton M. Stidd

Amer. J. Bot. 75(6): 790-796 – https://doi.org/10.2307/2443998 – .

https://www.jstor.org/stable/2443998

Abstract

Observations based on new preparations confirm the presence of four cells in parallel alignment in A. sullivanti stomata but not with two flanking subsidiary cells as in paracytic stomata. Rather, each guard cell contains in its interior a smaller inner cell. The stomatal pore is formed by walls of the larger cells and it is not known what role, if any, the interior cells may have played in opening and closing the pore. This cell-within-a-cell arrangement is unknown among stomata elsewhere in the plant kingdom. The inner cells have the appearance of guard cells, especially when the poral walls of the larger cells are removed, and were so designated by Oestry Stidd and Stidd (1976). Cuticle preparations (Reihman and Schabilion, 1985) leave most of the cell structure of the stomatal apparatus intact among other leaf tissues (are not removed with the cuticle) and therefore do not reveal essential features.