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AGRICULTURAL RESEARCH AND EDUCATION CENTER
IFAS, University of Florida
Bradenton, Florida .....
Bradenton AREC Research Report GC1980-4 M' a i .. 'ch 1980
STOMATAL ACTION AND GAS EXCHANGE CHARACTERISTICS.OFIPLANT980G
RELATIVE TO THE RESPONSE TO AIRBORNE SULFUR DIOXIDE*
T. K. Howe and S. S. Woltz -
SULFUR DIOXIDE POLLUTION AND GAS EXCHANGE IN PLANTS
Sulfur dioxide is the single most investigated air pollutant with respect to
vegetation. The responses of plants to atmospheric SO include 1) beneficial effects
due to a supply of an indispensable nutrient, sulfur; 2) hidden or invisible injury
reflected by reduced growth rate and impaired metabolism; 3) chronic injury visible
as foliar chlorosis, stunting, and poor produce quality; and 4) acute injury causing
the death of plant tissues, especially foliage, resulting in a characteristic inter-
veinal "scorch" of leaf laminae (10). Annually, airborne sulfur dioxide causes sig-
nificant economic loss to agronomic and horticultural crops. Vegetation in the area
of a pollutant source will be much more likely to sustain SO injury under certain
predisposing environmental conditions to be discussed later ?n this report. Poten-
tial pollutant source types include: utilities, residences and industries which
utilize fossil fuels for heat or power; incineration sites; petroleum fueled trans-
portation; and specific industrial sources such as metal smelting, chemical, and
Different plant species have varying degrees of susceptibility to sulfur dioxide
under optimal conditions for SO2 absorption. Sensitive plant types may be used as
bioindicators of SO occurrence in the vicinity of a source. The injury is most
severe nearest the source and least severe some distance away. Some of the suscep-
tibility-resistance characteristics of different plants may be related to the plant's
ability to regulate the exchange of gases with the atmosphere and its interior.
Sulfur dioxide is a gas and enters the plant through open stomates, and to a lesser
degree through cuticle, for damage to occur.
Stomates are small pores bounded by two specialized guard cells, in the epidermis of
all aerial plant parts. Leaves in particular have a high number of stomates. Plants
may have them on both the upper and lower leaf surface or simply on the lower sur-
face. Generally, open stomates range from 3-12 u (micron) in width to 10-30 u in
length. They comprise about 0.1% to 0.3% of the total leaf surface. Stomates vary
from 2,000-50,000 per square centimeter. During leaf growth and expansion, stomates
develop and increase in pore size and frequency. The ultimate numbers and charac-
teristics of stomates depend on environment and culture during growth.
Strictly speaking, a stomate is a pore. The two guard cells which flank the
pore are part of the stomatal apparatus. Stomates can be opened to varying degrees
or closed by the swelling or shrinking, respectively, of the guard cells. The exact
mechanism of stomatal response has not been elucidated entirely. An environmental
stimulus or diurnal rhythm may act as a trigger, producing salt balances and pH
changes in the guard cells which cause the influx or efflux of water and induces a
change in guard cell turgor and thus, aperture size. Most gas exchange between plants
and the surrounding atmosphere takes place via the stomates. Gases such as CO, H 0,
and O are relatively impermeable to the cuticle and rely almost exclusively oh pas-
sage through stomates. Sulfur dioxide (S02) is also largely dependent upon open
stomates for plant entry.
*Supported in part by a grant from the University of Florida Gatorade Fund to the
Interdisciplinary Center for Aeronomy and (other) Atmospheric Sciences and by
funding from the IFAS Center for Environmental and Natural Resources Program.
The character of the teaf anatomy may influence gas diffusion. Hairy or scaly
surfaces or sunken stomates will cause a lengthening of the path of gas diffusion
through still air. Exchange is enhanced by a minimal layer of still air adjacent
to leaf surfaces, and impeded by a larger layer. Stomates are very sensitive to
environmental conditions and changes in stomatal aperture will determine physically
how much gas can diffuse through them.
STOMATAL RESPONSE TO ENVIRONMENT AND SO2
There are numerous factors which contribute to the susceptibility of plants to
sulfur dioxide injury. Probably the single most important factor is the rate at
which this major air pollutant is absorbed by leaves. As previously mentioned,
stomata are the major pathway for gaseous exchange, and anything which influences
the physiology of stomatal action will affect foliar absorption. Environmental
parameters such as temperature, nutritional history, relative humidity, light, water
availability, and the concentration of CO in the air can modify stomatal function.
Less gas exchange is possible when stomata are closed due to darkness, drought, low
temperatures, or low relative humidity, and consequently this closure will afford
protection to the leaves from SO damage. The degree of stomatal opening is of such
importance in SO injury to leaves during episodes of high level fumigation, that in
short exposures 9f 1-4 hours, the pattern of injury coincides approximately with the
pattern of leaf areas with open stomata. The youngest and oldest leaves frequently
escape acute damage because stomates are immature and non-functional in young leaves
or closed because of senescence in old leaves. Environmental factors which favor
increased stomatal opening will enhance the susceptibility to fumigation damage from
SO2. An adequately watered, healthy plant on a sunny day with high relative humidity
and mild temperatures is more susceptible to SO injury because conditions are optimal
for stomates to be open, thereby providing access for the fumigant.
Atmospheric SO itself elicits stomatal responses; it can cause an increased
degree of stomatal opening as in field bean (Vicia faba), corn, tobacco, soybean,
radish, cucumber, sunflower, spinach, perilla and barley, or it can cause closure
as in pine, tobacco, geranium, petunia, bean (Phaseolus vulgaris), tomato, and poin-
settia. Superimposing an SO2 exposure on other environmental conditions can modify
stomatal actions to SO. In Vicia faba SO2 causes stomatal closure at relative humid-
ities below 40% while causing opening at higher relative humidities (5). Even the
effects of SO2 on the stomatal action of leaves on the same plant may be different
in magnitude. Recently matured Vicia faba leaves have more open stomates, while
older leaves have more closed stomates before fumigation. Once exposed to SO the
stomates of the older leaves open, while there is only a slight opening in the young
leaves (1). This indicates that the younger leaves were almost fully open before SO2
exposure and the gas could cause little additional increase in aperture size. So
the physiological age of the leaves must be considered when monitoring stomatal
response to SO2.
It is also pertinent to note that response of the stomates may not be consistent
when the concentration of SO is varied. Black and Black (2) found that Vicia faba
stomata open at concentrations below 200 ug m- and closed at concentrations above
500 ug m" This disagrees with work done by Malrnik (4) who reported open stomata
at concentrations as high as 10 ppm (26,000 ug nr ). Death of epidermal cells may be
a factor in this case. Not only is concentration a key variable, but new evidence
suggests that the peak and short term average concentration may play a role in sto-
matal response, if theories of stomatal response are valid (6). Since numerous
variables control stomatal function, it is expected that contradictory findings will
be found in the literature on SO2-stomatal response relations. The type of plant
being studied must be considered also.
As mentioned before, stomates in-some plants open, some close, and some possibly
are not affected by atmospheric SO2. For instance, Vicia faba is a plant in which
the stomata open in response to SO fumigation at relative humidities above 40% (5).
Under similar conditions, Phaseolu& vulgaris Pinto bean, Will react to SO exposure
with stomatal closure (8). Any review of suggested mechanisms must be considered
carefully because different Circumstances may be involved in different cases. Two
theories predominate, namely that SO elicits its effect on stomata via interference
with a) photosynthesis, or b) cell viability. In the first case it has been sub-
stantiated that SO inhibits photosynthesis. It disrupts chloroplast membranes,
inhibits photosynthetic enzymes, and it also has been suggested that SO competes
directly with bicarbonate in the photosynthetic process. Since a decrease in photo-
synthetic activity means that less CO is needed, it has been proposed that CO
builds up in the leaf and causes stomatal closure (3). This is plausible since
high CO2 concentrations do cause stomatal closure. While this theory explains SO -
induced stomatal closure, it cannot explain SO2-induced stomatal opening. The second
theory (2) intends to explain stomatal opening and closing. T;e response is viewed
as a concentration dependent reaction. At levels of 200 ug m or below, SO2 causes
destruction of epidermal tissue surrounding the stomatal apparatus. The loss of cell
viability and accompanying loss of turgor causes passive stomatal opening. At con-
centrations of 500 ug m or higher the guard cells also become damaged and the loss
of turgor results in stomatal closure. As noted earlier, Black and Black (2) dis-
pute the claim by Mansfield and Majernik (5) that excessive concentrations still
maintain more open stomata. Also, they cite work to show a variety of plants respond
differently to SO2 and the responses fall into their general concentration-response
categories. While individual plant thresholds for an "open versus close" response
may differ, the assumption cannot be made that damage to leaf tissue is the only
basis for reaction of stomata to SO. From our experience at AREC-Bradenton, sto-
matal closure in particular can occur with no visible damage to the leaf blade in
bean, zinnia, tomato, and poinsettia. This lends support to the first theory, and
not to the second; admittedly, other mechanisms could be postulated.
STOMATAL MEASUREMENT TECHNIQUES
Various techniques have been used to monitor gas exchange and aperture size of
stomates. Gas exchange can be measured in terms of photosynthetic CO2 uptake, trans-
pirational water vapor loss, and in terms of SO2 uptake. Infrared gas analysis can
assess changes in both CO and H 0 gas exchange. Transpirational loss of water vapor
has been ascertained by tie measurement of weight changes of the plant of interest
or by means of various types of porometer. First, the earliest models measured the
flow of air forced through the'leaf blade. This was dependent on the distribution
of stomates on both sides of the leaf. One big disadvantage with the pressure-flow
system is that the pressures used will per se induce a stomatal response. A later
type of porometer diffusivee resistance porometer) measured the diffusion of a gas
such as nitrous oxide through the leaf blade, eliminating the pressure problem but
still confining its use to leaves with stomates on both surfaces. Currently, diffu-
sive resistance porometers measure water vapor diffused out through the stomates
and leaf surface by means of a humidity sensor. This improvement was more practical
since it relies on the biological function of the plant and avoids stomatal reaction
during the measurement process due to short sensor contact. Most important, the
measurement of water vapor diffusion is a more accurate assessment of the gas ex-
change phenomenon. When the stomatal pores are closed or only partially opened,
the resistance to the exit of water vapor (or entry of SO2) is greater than if they
are open. Less fumigant enters the leaf when the path of diffusion is restricted.
The measurement of the actual size of the aperture is difficult and may be de-
ceiving in terms of estimation of the degree of openness and relation to gas exchange.
Direct microscopic examination is difficult on attached leaves so the leaves usually
are detached, the epidermis removed and examined. This method is cumbersome and not
reliable due to the changes induced by peeling the epidermis from the lamina. A
less drastic method involves making impressions of the leaf surface with silicone
rubber. It causes little injury but may affect stomatal aperture during the solidi-
fication and imprint development process. Another indirect method is the observa-
tion of the time for liquids of various viscosities to infiltrate the leaf blade,
the time for absorption of a fluid of specific viscosity being used as an indicator
of aperture size. This method is not good for hairy leaves or needle-shaped leaves,
is destructive, and requires many observations. All of these techniques to measure
aperture size are not only time consuming and frequently destructive, but also are
not strictly related to gas exchange. For instance, Schramel (9) has substantiated
that the diffusive resistance of water vapor could be high, but not related to aper-
ture size under conditions of water stress. Stomates opened in response to SO in
water-stressed plants, but the diffusive resistance did not change (did not become
lower); rather it stayed high due to internal water relations. So an open stomate
does not mean more water efflux in water stressed conditions. Also, it may be that
SO in such a low H 0 condition would enter the leaf, but would not be adsorbed
effectively by the tissue due to the dehydrated condition, hence causing less injury.
Water stress does, in fact, afford protection from SO damage in many cases, and is
assumed to be principally due to stomatal closure. But when SO causes stomatal
opening, then the inhibition of SO, adsorption by water stress may further explain
the protective effect of drought; since SO2 cannot enter the dehydrated cells, even
with passage through the stomates. The geometry of the stomate may complicate the
measurement of aperture size. Stomatal opening may be such that the guard cells,
themselves, narrow the aperture as seen from the surface by extending in depth (7).
This geometry, the three-dimensional character of the pores, limits the extent to
which surface measurements accurately access openness of the stomate, as defined by
effective gas exchange.
Diffusive resistance of gases remains the best tool to date.
RESEARCH AT AREC-BRADENTON, SIGNIFICANCE, AND FUTURE NEEDS
Prospects for SO2 air pollution problems are increasing in Florida as fossil
fuel use patterns are being adapted to permit the use of more high-sulfur content
fuels. Federal standards are being modified in expectation of an increased petro-
leum shortage. Currently, sulfur dioxide is not a serious problem in Florida, but
research is needed to evaluate present conditions in the state and determine the
impact of projected increases in sulfur dioxide emissions. Part of a comprehensive
airborne sulfur dioxide project at AREC-Bradenton deals with the physiological im-
pact on vegetation. Particular emphasis will be placed on stomatal physiology.
The participation of stomates in response to SO2 exposure as it may affect suscepti-
bility or resistance of specific species is a pertinent segment cr overall reaction
of a plant to SO. The stomatal response can be an effective avoidance mechanism,
if they close, o a serious threat to continued existence, if they open. Stomatal
physiology, sulfur metabolism, and enzymatic properties of various species when
combined with information on injury resulting from SO exposure, will lead to a
more comprehensive knowledge of what plants are susceptible or tolerant to SO2 and
Facilities at AREC-Bradenton include seven fumigation greenhouses, each 9' x 12'
in size with an air volume of about 800 cubic feet. They were designed to provide
steady state sulfur dioxide levels. Container grown bean, poinsettia, and potato
plants have been fumigated under 65% of natural light and prevailing temperature and
humidity conditions. Stable atmosphere of SO is maintained by metering the flow of
a 1.5% source gas above a fan which draws outside air through evaporative cooling
pads and into the chambers. The chambers are equilibrated for at least 15 minutes
with the fumigant before plants are placed on benches inside.
Poinsettia and bean responded to a wide range of levels of SO2 by closing their
stomates. Potato stomates were nonresponsive to SO under conditions of low or high
humidity. Brief summaries of observations made on these plant materials follows.
Poinsettia plants from a growth regulator study were damaged by fumigation for 2
hours at 3 ppm. Injury was light, about 5% of green leaves and less than 1% of
bract area being destroyed. Observation of stomatal response indicated that at
fumigation at 2 ppm SO one plant out of the five monitored responded to SO with
stomatal closure. At 3and 4 ppm, all five plants had increased stomatal closure.
In addition, in cases where closure was evident there was a cyclic response to SO2
exposure. The degree of stomatal closure increased to a peak at 20 minutes from
the start of fumigation and decreased to initial levels (re-opening) at 30-40 min-
utes. Two fumigation trials (3 ppm-2 hours and 4 ppm-1 hour) showed indications
of a second cycle beginning at the end of the first hour of fumigation. In the
case of 3 ppm for 2 hours the second cycle peaked between 65-90 minutes and declined
by 120 minutes. Environmental conditions were not monitored in these four prelim-
inary studies; the magnitude of changes in stomatal aperture was variable, probably
dependent on environmental circumstances. Growth retardants used on these plants
may have affected the stomatal response. Stomatal closure as observed may afford
protection from SO by inhibiting the fumigant's entry into the plant. The cycling
phenomenon may reflect the plant's cyclical response to changing gas and water levels
within the leaf (S02, CO2, and H20).
Various concentration-duration levels of SO2 fumigation of Harvester bean were
employed to dispense similar total dosages. For example, 0.5 ppm-8 hours versus
1 ppm-4 hours versus 4 ppm-1 hour all represented the same total dosage. In one
experiment, 0.5, 1, 2, and 4 ppm SO were administered for 8, 4, 2, and 1 hours,
respectively. Scorch symptoms appeared on all 6 plants exposed to 4 ppm SO for
an hour. Heaviest damage was on the primary and first trifoliate leaves. the
second trifoliate leaves had little damage; they were the newest growth on the
plants examined. At 2 ppm for 2 hours only a single plant sustained scorch injury
and only on the primary leaves. Other concentrations of SO0 caused no damage. Only
two plants from each fumigation treatment were examined for stomatal response.
Because of the nature of fumigation time, all concentration responses can only be
compared for the first hour. Again as in poinsettia, the stomates closed in response
to fumigation. A correlation of concentration of SO was observed relative to sto-
matal opening, namely that increasingly higher levels of SO produced increasing
stomatal closure as measured by diffusive resistance (Figure 1). Plants exposed to
higher concentrations of SO for short time intervals had less total accumulated
sulfur than plants exposed to lower SO2 concentrations for longer time periods.
They were ranked as follows: Control 4 ppm-1 hour 2 ppm-2 hours 1 ppm-4 hours
0.5 ppm-8 hours or sulfur contents of .29 .33 .36 .40 .47% dry weight, respec-
Stomatal clou'jre may not have been as protective to beans at 4 ppm as at 2 ppm,
since injury was more severe at 4 ppm and very slight at 2 ppm. Stomatal closure ap-
peared' to have a definite effect on total sulfur accumulation related to the mag-
nitude of stomatal closure. The burst of SO taken into the plant during its
response may be critical to injury sustained depending on chemical and biological
detoxification rate capacity. Subjecting bean to 4 ppm may be too severe within
the first 10 minutes of fumigation before stomates are closed. The response time
is critical since individual plants slow to respond to 4 ppm SO2 will be injured
while faster response seems to afford protection.
In the second experiment, beans were fumigated at 0, 0.5, and 2 ppm SO for
one and one-half hours. Again there was an increase in stomatal closure. The
closure was greater at 2 ppm than for the control or 0.5 ppm SO plants which
remained unchanged. These results are similar to those with po nsettia. Addition-
ally, there was a cycling of the stomatal closure as seen for poinsettia. The
cycling was very apparent at 2 ppm SO and not obvious at 0.5 ppm SO2. The environ-
ment seems to play a large part in determining the cycling response of the stomates.
Recent work on 'Atlantic' and 'Sebago' potato cultivars indicates that stomates
are nonresponsive under conditions of high temperatures (600-750) and low or high
relative humidity (45-30% or 100-80%) at 0.5, 1, and 2 ppm SO Initial indications
are that 'Sebago' is more sensitive to SO2 than 'Atlantic,' w ile stomatal responses
There is a wide range of susceptibility-tolerance categories for the response
of plants to SO based on the assessment of injuries. More information on how
species respond to fumigation can be used in land-use-planning for locating fossil
fuel burning facilities, new agricultural and horticultural production sites, and
in selection of plant materials for use within the vicinity of SO, emissions. Tol-
erant species near a source scrub the air of SO to minimize pollution problems.
Also, breeding programs may benefit from tolerances of different varieties.
The physiological, metabolic, and anatomical characteristics of SO suscepti-
bility have not been fully explored and, when determined, should be useful tools
in assessing the impact of airborne SO2 on Florida's unique agricultural and native
1. Biscoe, P. V., M. H. Unsworth, and H. R. Pinckney. 1973. The effects of low
concentrations of sulfur dioxide on stomatal behavior. New Phytol. 72:1299-
2. Black, C. R., and V. J. Black. 1979. The effects of low concentrations of
sulphur dioxide on stomatal conductance and epidermal cell survival in field
bean (Vicia faba L.). Jour. Exp. Bot. 30:291-298.
3. Kondo, N., and K. Sugahara. 1978. Changes in transpiration rate of SO2-resis-
tant and sensitive plants with SO fumigation and the participation of abscisic
acid. Plant and Cell Physiol. 19 365-373.
4. Majernik, 0. 1971. A physiological study of the effects of SO pollution,
phenylmercuric acetate sprays, and parasitic infection on stomatal behaviour
and ageing in barley. Phytopath. Z. 72:255-268.
5. Mansfield, T. A., and 0. Majernik. 1970. Can stomata play a part in protection
plants against air pollutants? Environ. Pollut. 1:149-154.
6. McLaughlin, S. B., D. S. Shriner, R. K. McConathy, and L. K. Mann. 1979. The
effects of SO2 dosage kinetics and exposure frequency on photosynthesis and
transpiration of kidney beans (Phaseolus vulgaris L.). Environ. Exp. Bot.
7. Meidner, H., and C. Willmer. 1976. In: H. Smith, ed., Commentaries in Plant
Science. Pergaman Press. N.Y.C. pp 137-151.
8. Rist, D. L., and D. D. Davis. 1979. The influence of exposure temperature
and relative humidity on the response of pinto bean foliage to sulfur di-
oxide. Phytopathology 69:231-235.
9. Schramel, M. 1975. Influence of sulphur dioxide on stomatal apertures and
diffusive resistance of leaves in various species of cultivated plants under
optimum soil moisture and drought conditions. Bull. Acad. Polon. Sci. 22:
10. Woltz, S. S. and Teresa
on Florida vegetation.
K. Howe. 1979. Effects of airborne sulfur dioxide
Bradenton AREC Res. Rept. GC1979-17.
O 30 60.
Stomatal response of Bean (Phaseolus
vulgaris) to SO .