Stomatal Action in Plants as Related to DamageFrom Photochemical Oxidants 1 2

W. M. Dugger, Jr., 0. C. Taylor, Eugene Cardiff, & C. Ray ThompsonCitrus Research Center & Agricultural Experiment Station,

University of California, Riverside

A review of the literature indicates that the sto-mates of leaves must be open for smog damage to oc-cur (3, 7, 8, 9, 11). However, there is no direct re-lationship between degree of opening and extent ofinjury, nor has it been proven that injury is prevent-ed when the stomates are closed. Moreover, mostof the earlier work dealt with the reaction product ofozone and gasoline or ozone and an olefin (8,10,16).Such reaction products are unstable and do not nor-mally accumulate in a polluted atmosphere to a levelsufficient to cause plant damage (1, 4). The resultsof this study indicate that under the conditions of theexperiments, stomatal opening is not the primarycontrolling factor in predisposing plants to injuryfrom ozone and peroxyacetyl nitrate (PAN), twophytotoxic products in smog.

Materials and MethodsPinto bean (Phaseoluts vulgaris L.) and petunia

(Petunia hybrida Vilm var. Rosy Morn) plants weregrown in a shaded, carbon filtered greenhouse undera 15 hour photoperiod of 21.5 Klux from Sylvaniavery high output (VHO) fluorescent lamps. Tem-peratures of about 30 C during the day and 22 C dur-ing the night were maintained within the greenhousethrough the use of evaporative coolers.

A large dark-box, located in the greenhouse andfitted with a ventilation fan, was used to maintainplants in the dark. The fumigation chambers usedto expose plants to ozone or PAN were those ofTaylor and English (unpublished material), andhave been used in previous studies (15). Controlledrates of air and pollutant were metered into the 640liter chamber via flow meters at 160 liters/minute.Thus the gas phase in the chamber was replaced onceevery 4 minutes. Ozone was generated electrolyt-ically from compressed oxygen and the concentra-tion determined by continuous monitoring with aKruger ultraviolet photometer designed for ozonemeasurement. PAN was synthesized, concentratedand assayed in a long path infrared spectrometer by

I Received Dec. 8, 1961.- This investigation was supported in part by a re-

search grant (AP-40) from the United States PublicHealth Service, National Institutes of Health.

the methods of Stephens et al. (12). Cylinders ofPAN gas were prepared from the concentrated prod-uct, pressurized with nitrogen gas, and the concentra-tion in the tank determined in the long path infraredspectrometer. Dilutions from these cylinders intothe fumigation chambers were calculated frompressure and volume relationships. The light sys-tem over the plastic covered chambers consisted of apanel of 25 Sylvania VHO lamps with light intensityof 21.5 Klux at plant height. The temperature of thechambers was maintained at 30 C.

Stomatal opening was determined with a Wheat-stone bridge type resistance porometer as describedby Heath and Russell (6). The leaf cup attached tothe porometer was made with a snap action springso that it could be positioned on a leaf and gently re-leased to effect a seal. A gum rubber gasket wassealed around the cup edge and extended above theedge about 1/8 inch. Another gasket was sealed tothe upper arm of the cup. This spring action andgasket made it possible to rapidly change cup positionon a given leaf or to change leaves without using asealing compound or causing injury to the leaves. Itwas possible to make several stomate measurementswithin one minute and the readings were made im-mediately after attaching the cup to a leaf. In a seriesof readings on one leaf the cup was removed fromthe leaf between each measurement.

Plants exposed to ozone or PAN after a darkperiod were given a 15 to 30 minute light period be-fore fumigation. During this period porometermeasurements were made to determine the extent ofstomatal opening. In some experiments stomatalopening was also determined indirectly by measuringgravimetrically the transpirational loss of water.

In several of the experiments apparent photosyn-thesis was determined on pinto bean plants duringthe 30 minute fumigation as another check on thestatus of stomates. A small fraction of the air fromthe inlet and outlet parts of the fumigation chamberwas metered through a differential Liston-BeckerInfrared CO, analyzer, model 1 5A (14). The ap-parent CO2 fixed in photosynthesis during the fumi-gation period was recorded on a strip chart recorder.Because subsequent assays of plant leaf damage fromthe oxidant were desired, the rate of apparent photo-synthesis was expressed on a nine-plant basis ratherthan on the leaf area or dry weight basis.

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PLANT PHYSIOLOGY

Fig. 1. The effect of ozone on bean leaves after various dark periods. Lcft to right, 72 hours (lark, 48 hoursdark, 24 hours dark, 12 hours dark, normal light under 15 hour photoperiod. All the dark treated plants were in thelight 30 minutes prior to the fumigation with 0.70 ppm 0, for 30 minutes.

Evaluation of injury to the leaves of pinto beanan(I petunia plants from ozone or PAN were arbi-trarily determined. Leaf damaged by the oxidantwas estimated according to the following scale:

Group % Leaf area damage0 No injury1 1- 25%2 25- 50%3 50- 75 %4 75-100%

Damage was determined 48 hours after fumiga-tion and each leaf was assigned to one of thesegroups. The percentage of leaf damage was calcu-lated from the average group value. Typical injuryfrom ozone and PAN on pinto bean and petunia hasbeen described by Taylor et al. (13).

ResultsPrevious work in this laboratory has show n that

Plants given an 18 to 24 hour (lark period followedby a 30 minute pre-fumigation light period and 30minutes of fumigation with PAN were not dlamiagecl;whereas a 48 to 72 hour dark period prior to funmi-gation with ozone was necessary to prevent damageefrom this oxidant (15). As shown in figure 1plants given a 12 or 24 hour pre-fumigation (larkperiod and fumigated 30 minutes with 0.7 ppm ozone,were injured to the same extent as plants given thenormal photoperiod (15 hr light, 9 hr (lark). Plantsin the dark for 48 hours were damaged slightly alongthe larger veins and 72 hours of darkness complete-ly protected the plants from ozone damage. Figure2 shows that stomatal opening induced by light isnot influenced by the duration of the preceding dark

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DUGGER ET AL.-STOMATAL ACTION & PHOTOCHEMICAL OXIDANTS

0.5 Ic I * 15 hours pre-dork

L \ 48 hours pre-dork

_ 00

z 1

0o ~~~~~~~~~~~~~~0

o 1.5 -c-

-2.010 20 30 40 50 60

TIME IN LIGHT(minutes)

Fig. 2. The effect of light on stomatal opening inbean leaves after various dark periods. Stomatal re-sistance of one GP unit equals the resistance to airflowof a capillary tube where the length/radius4 = 3.77 X108cm -3.

period. Each point is the average of several deter-minations. Regardless of the duration of the darkperiod preceding fumigation the stomates were wellopened within 30 minutes after the start of the lightperiod. Therefore, if stomatal closure were the con-trolling factor in plant damage the duration of thedark period should not change the degree of injury.

Petunia plants are protected from PAN damageby a prefumigation dark period. Table I shows thatwithin 30 minutes after the beginning of the lightperiod following a 24 hour dark period, the stomateshad opened to about the same degree as those ofcontrol plants. Transpiration rate of dark-treated

Table I

Influence of 24 Hour Pre-Dark Period on Opening ofStomates, Transpiration, & Oxidant Damage

From PAN in Petunia Plants

Time inlight Transpiration Damage

following Stomatal opening rate to leaves24 hr (log G.P. units) (g water loss/

darkness 5 min X 9 plants) ((min)

2 -0.32 ...5 -0.54 ...7 *-- 0.515 -1.0917 ... 1.022 ... 2.525 -1.5227 ... 2.030 Start of fumigation 0Control -1.71 2.5 100

plants also approached the transpiration rate of con-trol plants. However, a 24 hour pre-fumigation darkperiod completely protected petunia plants from theeffect of 1 ppm PAN applied for 30 minutes. Thecontrol plants were severely damaged.

To compare the rate of stomatal closure to the rateof stomatal opening the degree of stomatal resistanceto air flow after transfer to darkness was followedfor 20 hours (fig 3). Although the stomates of beanleaves start the closing process shortly after beingplaced in the dark, the rate of closure was muchslower than the rate of opening. Plants transferredto the dark, after a normal light period, for the 30minute fumigation will be damaged by ozone to thesame extent as plants fumigated in the light. Onthe other hand, as previously reported (15), plantsfumigated with PAN in the dark will not be damaged.

+04

z

-04

a: -08.

-o(r-I. .2

1.. r .

10 20 30 40 50TIME IN OARK (minUtes)

100 -

60 70\~~~~~~~~~ FUN d- -. Oznedmwp IOwdf0.ISMmab res\OXc

- -F~~~~~~~~~~~~~~~~~~~~~~~~~~

6 7 8 9 10 11 12 13AGE OF BEANS IN DAYS FROM SEED

14

0

Fig. 3. The time course of stomatal closure andopening in bean plants.

Fig. 4. The influence of the age of bean plants onthe leaf damage from ozone and PAN and on stomatalopening.

Where apparent photosynthesis and degree ofstomatal opening were determined during ozonefumigation no consistent decrease in the rates ofthese processes was observed. In the experimentsreported in table II there was a 2 % decrease in photo-synthesis and a slight decrease in stomatal openingof the control plants during the 30 minute fumigationperiod; however, plants receiving a 12 to 72 hoursdark period prior to fumigation showed either no

489

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5 15

-.0

0W

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490 PLANT PHYSIOLOGY

Table 11Influence of Ozone on Change in Stomatal Opening,Apparent Photosynthesis, & Damage to Primary

Leaves of Pinto Bean Plants Pre-Exposedto Various Dark Periods*

Change inTime in Change in apparent photo- Leafdark stomates synthesis during area(hr) (AGP units)** fumigation dmg

Control +0.04 - 2.0 7912 0 +12 9224 -0.05 +10 5048 -0.08 +17 2572 -0.32 0 6

* Plants exposed to 30 minutes of light before fumi-gating with 0.7 ppm ozone for 30 minutes. Meas-urements of stomatal resistance and apparent photo-synthesis made during fumigation.

** GP unit of resistance equals the resistance to air flowof a tube where length/radius4 = 3.77 X 108cm-3.A negative value for AGP units means a decrease inresistance caused by stomates opening wider duringthe ozone fumigation.

change or an increase in the rate of apparent photo-synthesis and either no change or increase in stomatalopening during the fumigation. Subsequent evalua-tion of the percentage leaf damage from the ozonefumigation did show the protective action of longdark periocls as well as an increase in leaf injury withplants given a 12 hour dark period.

An important difference, between the effect ofozone and PAN on pinto bean plants, is the age atwhich the primary leaves are susceptible to damage(fig 4). Leaves of young plants (5-8 days) grownunder the conditions of these experiments were muchmore susceptible to PAN clamage than to ozonedamage. Five and six (lay old plants were almostcompletely immune to ozone. On the other hand,the susceptibility of pinto bean leaves to ozone reacheda maximum at about 11 days from planting, and thesusceptibility to PAN at this age was only one-halfas great as in young plants. Primary leaves of olderplants, 14 to 15 days, were insensitive to PAN andless sensitive to ozone. Daily stomatal measurementsover this time period showed a slight increase inopening (a decrease in air flow resistance) from 6to 9 days, but in the youngest leaves large enoughto connect to a porometer cup, the stomates were wellopened (log G.P. Units -1.5) and responsive tolight.

Discussion

In view of the classical concept that the diffusionof gases through stomates is a function of the peri-meter of the stomatal opening and not of the stomatalarea, the damage from air pollutants is thought tobe less dependent on stomatal action than indicatedin previous reports.

Bobrov (2, 3) has described the sequence of mor-phological changes in Poa annua and Avena thatresult from smog damage. The initial conditionsleading to the morphological changes were the pres-ence of functional stomates and internal air space inthe leaves. Young leaves or the young expandingareas of leaves did not have functional stomatestherefore were not damage(l. Figure 4 shows that5 to 8 day old bean plants were insensitive to ozonebut very sensitive to PAN yet the stomates weresufficiently open to allow gas exchange (log of re-sistance to air flow = -1.5 to -1.8). Plants given12 hours of darkness (table II) wvere damage(d to agreater extent than were control plants. Hull andWVent (8) also made this observation. Endive andoat plants pre-treated with a 12 or 24 hour (larkperiod were (lamaged more than control plants whenexpose(d to a short fumigation period of ozonatedgasoline. They concluded that in these dark periodsplants were depleted of carbohydrates and thereforemore susceptible to fumigant damage. They also ex-posed sugar beets and spinach plants to 3 days ofdlarkness with one leaf of each plant dippedl in 7 %sucrose at the start of the dark period. Althoughthere wTas no difference in the response of controland sucrose treated leaves after 1 or 2 days in thedark, plants in the dark for 3 clays dicl show someprotection from the sucrose dip. Their interpreta-tions are in contrast to ours. Within 24 hours ofclarkness the carbohydrate level in plants is not cle-pleted. Unpublished results by Dugger and Humph-reys have shown that black valentine bean leaves loseonly about 50 % of their reducing sugar, 10 %c oftheir sucrose ancl 20 % of their starch within 24hours of darkness. The observation of Hull andWAVent could have been caused by a higher level ofa particular soluble carbohydrate or some product ofcarbohydrate transformation in the 12 hours darkperiod. Storage carbohydrates normally increase inthe light and are converted to soluble forms dluringthe night. Leaves from plants 48 hours in the darkwere injured only along the veins; these areas con-tain soluble carbohydrates in the transport stage fromthe leaf to some more actively growing region.

Injury to plants from PAN is also prevented by aclark period but the nature of this injury is such asto suggest a series of light dependent reactions be-tween PAN and the plant (5). Injury to tobaccoplants from air pollutants has been prevented bycovering portions of the leaves with strips of blackpaper or by covering the lower stomates with lanolin(7). Excluding gas exchange from the leaves willnormally prevent any reaction in which the gas isinvolved. Obviously, stomatal opening is necessarybefore the air pollutants can enter the leaves ofplants, however, the results presented here supportthe thesis that under normal growing conditions andin light, stomates will be open and do not control theentrance of air pollutants into leaf messophyll tissueas previously indicated in the literature.

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DUGGER ET AL.-STOMATAL ACTION & PHOTOCHEMICAL OXIDANTS

SummaryThe role of stomates, as a factor in controlling

the injury to plants from the photochemically pro-duced pollutants, ozone and peroxyacetyl nitrate(PAN), was investigated. Stomatal action wasquantitatively measured by determining the resistanceto air flow through the stomates with a Wheatstonebridge resistance porometer. By a combination ofmeasurements involving apparent photosynthesis,transpiration, and degree of leaf damage producedby the air pollutant oxidants it was shown that: A,Stomates open rapidly in the light, even after a longdark period, and close slowly in the dark; B, Tran-spiration rate from plants transferred from the darkto the light equals the rate of control plants in thelight within 30 minutes; C, The physiological ageof bean plants and not the degree of stomatal open-ing determines the susceptibility to the two oxidants.Five and six day old bean plants are not damaged byozone yet the stomates are functional. Plants of thisage are most susceptible to PAN damage; D, Ap-parent photosynthesis and degree of stomatal openingare not significantly reduced in pinto bean by a 30minute fumigation with ozone. The length of thepre-fumigation dark period determines the extent ofleaf damage from this pollutant.

The nature of ozone damage and the protectiveaction of a long pre-fumigation dark period suggestthat the level of carbohydrates in the leaves has somerole in predisposing plants to damage from thisoxidant.

AcknowledgmentWe wish to acknowledge the assistance of Mr. James

0. Ivie in making the resistant porometer used in this:study.

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2. BOBROV, RUTH A. 1952. The anatomical effect ofair pollution on plants. Proc. 2nd Natl. Air Poll.Symp., Pasadena, Cal.

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13. TAYLOR, 0. C., E. R. STEPHENS, E. F. DARLEY, E. A.CARDIFF. 1960. Effects of air-borne oxidants onleaves of pinto bean & petunia. Proc. Am. Soc.Hort. Sci. 75: 435 444.

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