Historic, archived document Do not assume content reflects current scientific knowledge, policies, or practices. 1 ^ ^ t^eS. RESEARCH IN PLANT TRANSPIRATION: 1963 Production Research Report No. 89 L I m fi. JUL 8 1966 U. S. DEFiliilkfeT OF BELTSVILLE BRANCH Agricultural Research Service U.S. DEPARTMENT OF AGRICULTURE in cooperation with Georgia Agricultural Experiment Stations and Meteorology Department U.S. Army Electronics Research and Development Activity CONTENTS Page Introduction 1 Controlled environment studies 1 Growth chambers 1 Comparative bean and tomato growth and fruiting under two fluorescent lamp sources 3 Transpiration, leaf temperature, and stomatal activity of certain plants as affected by CO2 concentration of the air and soil moisture tension 5 Guard cell action 13 Protoplasmic streaming and guard cell operation 13 Effects of certain chemicals on transpiration 15 Atrazine 15 Hexadecanol-octadecanol 20 Summary 23 Literature cited 24 V WASHINGTON, D.C. ISSUED June 1966 For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C., 20402 Price 20 cents RESEARCH IN PLANT TRANSPIRATION: 1963 By James E. Pallas, Jr., research plant physiologist, and Anson R. Bertrand, soil scientist. Agricultural Research Service INTRODUCTION The transfer of water from the soil to the atmos- phere by evaporation from leaf surfaces accounts for a large part of the loss of water from land. The quantity of water delivered to the atmosphere by evaporation from leaves is large and much of it may be an unnecessary loss when viewed from the standpoint of plant production. This report presents results from a portion of the continuing etfort by the Agricultural Research Service re- search group at the Southern Piedmont Conserva- tion Research Center to discern factors affecting transpiration and to develop methods for control of transpirational loss of water by plants. A report by USAEPG (4^)^ of research con- ducted in 1960 described the principal character- istics and capabilities of a controlled environment growth room with high light intensity where many of the studies reported here were conducted. The report of 1961 studies in USDA Production Research Report No. 70 (26) presented prediction equations to account for approximately 80 percent of the observed variations in transpiration rates. This report also emphasized the dependence of transpiration rate on soil moisture availability and the extraordinary ability of guard cells to remain operative under adverse conditions. The pathway of ^uard cell starch accumulation was partially elucidated, and results with several com- pounds having potential as antitranspirants were recorded. Further studies in 1962 were reported in USDA Production Research Report No. 87 (23). The 1962 report includes a description of light dis- tribution in the growth room and studies of transpiration rates using grain sorghum and corn with various combinations of light, temperature, humidity, and soil moisture tension. Also re- ported was further experimentation with foliarly applied chemicals for control of transpiration by plants. Results of studies conducted in 1963 are reported herein. This report includes a discussion of the characteristics and capabilities of small portable growth chambers where several of the experiments were conducted. Results of studies with bean and tomato plants under two light sources illustrate the effects of light quality on growth and development of plants. This report calls attention to the effects of CO2 concentration of the air and soil moisture tension on transpiration, leaf temperature, and stomatal activity of several agronomic crops. Some insight into the relation of protoplasmic streaming and guard cell action is provided. Also included are negative results obtained with soil- applied Atrazine ^ and soil and foliar applications of hexadecanol-octadecanol for decreasing tran- spiration and increasing water use efficiency by plants. CONTROLLED ENVIRONMENT STUDIES Growth Chambers Discrepancies found in the literature frequently can be traced to lack of detailed reporting. This research report contains detailed descriptions of facilities and techniques because the authors be- lieve such treatment is necessary to avoid mis- understanding. An experiment has real value only if it can be reproduced. This is not always possible, even with the best equipment available today. Much remains to be done in the science of artificial cli- mate control. Italic numbers in parentheses refer to Literature cited, p. 24. The first annual report of research in plant transpiration at the Southern Piedmont Conserva- tion Research Center mentioned the use of small environment chambers (45, p. 17) as precondition- ing units for plant growth. Their use has also been mentioned in subsequent reports (26,23). It is the purpose of this section to discuss their capabilities and give information for assessment of their limitations. All growth chambers are limited to some degree in the uniformity of environmental conditions that " Trade names and company names are used in this publication solely to provide specific information. Men- tion of a trade name does not constitute a guarantee or warranty, and does not signify that the product is ap- proved to the exclusion of comparable products. 1 2 PROD. RES. RPT. 89, U.S. DEPARTMENT OF AGRICULTURE can be maintained. Precision of control mecha- nisms is usually the limiting factor. The basic unit of our growth chambers is a U.S. Army walk- in refrigerator (fig. 1) from which the top has been removed and replaced with 1- to d-mil poly- ethylene or 6-mil Mylar sheeting. Polyethylene allows CO2 passage, but its transmission of visible light decreases with age. Mylar does not age ap- preciably but allows no CO2 passage to make up deficiencies caused by photosynthesis in the cham- ber. Inside dimensions are 68 by 72 by 64i/^ inches and outside dimensions, 76 by 79 by 78 inches. The compressor unit for cooling is mounted on the rear wall. The original gasoline engine has been replaced by a 1.5-hp. electric motor that drives the compressor on demand and a 0.5-hp. motor that drives the 20-inch-diameter squirrel cage fan located behind the condenser cooling coils (fig. 2). This fan runs continuously, insur- ing constant air circulation in the chamber. Four- foot-wide tables with adjustable legs have been placed in the middle of the chamber. The typical wind pattern 1 foot above the table and 2 feet from the lamp is shown in figure 3. The data were obtained with a Hastings air meter. Model B15A, with a directional probe, type N-7. Areas with high velocity ( >300 ft. /min.) are not used for ex- perimental plants. BN 26187 Figure 1. — Front and side view of a growth chamber in- cluding overhead light bank. Bank projects over chamber, excluding the use of the burned and blackened fluorescent tube ends. BN 26189 Figure 2. — Inside view of growth chamber. Condenser cooling coil on rear wall with wind deflector above. Humidifier on shelf in left corner; dehumidifler on floor under table. Cooled air, which is expeiled at top of condenser unit, flows into cooling coils below bench height. Edges of cone-t.vpe electric heater coils are barely visible above air deflector. The chambers are capable of maintaining tem- peratures ranging from 10° to 40° C. ±2° con- stantly or with diurnal cycling. Humidity is con- trollable during the photoperiod within the limit of 30 to 90 percent ±10 percent. This is accom- plished with a Walton Model SW2 ® humidifier wired into a Honeywell H63A humidity controller. Dehumidification is obtained by use of self-con- tained portable dehumidifiers capable of extract- ing 14 quarts in 24 hours at 80° F., 60 percent relative humidity. A thermostatically activated safety switch interrupts all power to the chamber and triggers an alarm system if internal tempera- tures exceed or go below the preset limits. Light is supplied from a bank of cool white VHO fluorescent lamps supplemented with 23 incandes- cents, if necessary. Incandescents used normally vary from 15 to 75 watts, depending on the species’ needs. The fluorescent lights are adequate for ® Walton, Inc., Irvington, N.J. RESEARCH IN PLANT TRANSPIRATION! 19 63 3 WIND VELOCITY IN FEET/MINUTE 5' 8" » 1 390 1 r3^ COOLING COILS t AND FAN 9" „ 28" U [3301 3901 130 1 240 QD LroJ 390 130 120 H H _100_ ” 1 OH QD F] ~ F] 140 H FI FI s 160 1 130 H F~1 FI ISO 190 fn^ nn HR TsFI 74^ Figitre 3. — ^Wind distribution of selected points at 1-foot intervals, 1 foot above bench height in growth chambers. All values are in feet per minute. normal plant growth of some species without the supplemental mean descents. Figure 4 shows the light distribution of this system at 100 centimeters below the bank or 16 cm. above bench height. Polyethylene sheeting was used in the foregoing light determinations; with the use of Mylar sheet- ing total radiant energy decreased, whereas visible light increased. The data were gathered from lamps with approximately 500 hours’ service. Light output from fluorescent lamps decreases con- siderably with age; therefore, to help offset this, bulbs are replaced after logging 1,500 hours. Excellent growth of the species listed below has been attained in the chambers when soil is ade- quately fertilized and photoperiods, temperature, and humidities are optimum for individual species’ needs. The following are species that were grown suc- cessfully in growth chambers made from U.S. Army walk-in refrigerators ; Dicotyledons Gossypium hirsutum (cotton) Rumex patientia (dock) Phaseolus vulgaris (field bean) Monocotyledons Zea mays ( corn ) Sorghum vulgare (sorghum) Cynodon dactylon (bermudagrass) Dicotyledons — Con. Vicia faba (horsebean) Vinca major (periwinkle) Rheum rhaponticum (rhubarb) Glycine max (soybean)^ Lycopersicon esculentum (tomato) Brassica rapa (turnip) Monocotyledons — Con. Poa pratensis (Kentucky bluegrass) Poa trivalis (Roughstalk bluegrass) Festuca elatior (tall fescue) Lolium multifiorum (annual ryegrass) Zebrina pendula (wandering- Jew) Comparative Bean and Tomato Growth and Fruiting Under Two Fluorescent Lamp Sources With the present trend toward controlled en- vironment research, the biologist is frequently con- fronted with the problem of having to choose a light source for the culture of his plants. Unfor- 5‘ 8" 0. .71 b. 46 c 1650 d.l425 K- COOLING COILS AND FAN 28"- cn \k a. .71 b. .51 C.I800 d.l700 0. .74 0 72 b .5 1 b. 50 C.I975 0.1975 d.l700 d.1800 a. .73 0 .74 b .50 b. .50 0.1975 0.1875 d.)600 d 1750 a. .62 b .43 C.I625 d.l550 4' -H 1 DOOR r 0. INCANDESCENT S FLUORESCENT- CAL./CM^/MIN. b. FLUORESCENT - CAL./CM2/MI N. c. INCANDESCENT a FLUORESCENT - FOOT CANDLES d. FLUORESCENT - FOOT CANDLES Figure 4. — Light distribution at selected points 16 centi- meters above bench height or 1 meter from the nearest source. ^ Will not grow successfully without adding supple- mental incandescent lights to the fluorescents. 4 PROD. RES. RPT. 89, U.S. DEPARTMENT OF AGRICULTURE tunately, little data are presently recorded in the literature to help him reach his decision. Measur- ing growth rates of plants under a series of light intensities for each light source {17) serves a very useful purpose of evaluating growth ; however, the effect of lights on flowering and fruiting should also be considered. In some instances physiologi- cal abnormalities traceable to light quality may be most easily detected in flowering and fruit set of any one species. For several years in our research on plant tran- spiration we have used light banks containing cool white VHO fluorescent lamps (F96T12/CW/ VHO)® over environmental chambers for the suc- cessful culture of a number of plants (see list on p. 3 of this report) . With some species, such as soybeaUj incandescent lighting to supplement the cool white VHO’s has been found to be absolutely necessary for optimum growth ; other plants, such as field bean and tomato plants, have not required supplementary lighting. Gro-Lux lamps such as the F20T12/GEO have been widely acclaimed as superior light sources for growth of “shade” plants (gloxinia, African violet, etc.). Lamps with similar emissive char- acteristics are also available in high-output form, e.g., F96T/GRO/VHO. The question arose whether F96T/GRO/VHO lamps are a better light source than the VHO cool white presently employed for the culture of field crops that nor- mally grow under light of high intensity. A com- parison was made therefore between the growth, flowering, and fruiting of bean and tomato plants grown under VHO cool white and VHO Gro-Lux. The study was conducted in a single growth chamber using two test species : Lycopersicon esculentum var. Rutgers, and PJiaseolus vulgaris var. Red Kidney. Plants were grown in asphalted metal containers with 178 pounds of Cecil sandy loam limed to pH 6.5 and fertilized with 4,000 pounds of 6-12-12 (120 p.p.m. nitrogen (N), 100 p.p.m. phosphorus (P), and 200 p.p.m. potassium (K) ) per acre. This procedure insured a uniform aboveground environment and minimized edaphic influences. The test population consisted of four plants of each species under each light source. The soil moisture was monitored throughout the stud- ies by the neutron probe method (4^) and was maintained above 50 percent available to minimize any moisture tension effect on growth, flowering, or fruiting. The chamber was divided by a large polyethylene sheet hung between the two light sources (fig. 5), which effectively intercepted the light between the two sections but not air movement. New ballasts and lamps were used. The light source above each section consisted of either six- “ Sylvania Lighting Products Division, Salem, Mass. teen 8-foot VHO cool white lamps or sixteen 8-foot VHO Gro-Lux lamps. No supplemental incan- descent light was used. The photoperiod was 14 hours with 25° C. day and 20° C. night temper- atures. The net radiant energy from the two light sources was equal at the beginning of the experi- ment, being 0.68 cal. cm.-^ min.-) as measured 6 inches above the soil surface with a Beckman- Whitley Net Radiometer (model N 188-01). Under both light sources the initial date of flow- ering for each species was the same — 29 days after planting for beans and 47 days after planting for tomatoes. Table 1 summarizes the data obtained. The differences in growth or fruiting of bean plants, although appearing to favor VHO cool white, were not significantly different at the 5 -percent level under the two light sources. The growth, flowering, and fruiting of tomato plants were definitely inferior under F96T/GRO/VHO lamps to those under VHO cool white; all differences were highly significant. Table 1. — Average yield of red kidney heanplcmts and Rutgers tomato plants grown under light sources indicated Yield per plant F96T/ GRO/ VHO r96T12 CW/VHO Plant Red kidney beans: * Fruit No_- Fruit, fresh weight g-- Plant, fresh weight g-- Plant, dry weight g-- Rutgers tomatoes: ^ Flower No.. Fruit No-- Fruit, fresh weight g-- Plant, fresh weight g-- Plant, dry weight g-- 8 26 65 10 7 0. 25 13 495 46 11 32 67 12 12 3 37 850 92 1 55 days old. 2 82 days old. Plants grown under F96T/GRO/VHO showed excessive internode elongation when compared with field-grown plants or plants grown under VHO cool white, indicating an improper spectral balance for the culture of those test species. Unfortunately, man has not yet produced an artificial light source comparable to the sun in in- tensity and quality. Therefore, considerably more of his attention could be directed toward the neces- sary research and development of such light sources. The effort put forth in developing lamps RESEARCH IN PLANT TRANSPIRATION: 19 63 5 Figure 5. — Part of test plants as seen inside growth chamber ; plants grown under F96T13/CW/VHO on left of plastic barrier (see text for description), plants grown under F96T/GRO/VHO on right. Excessive intemode elongation can be noted on both bean and tomato plants growing under the F96T/GRO/VHO. Access tube for neutron probe can be seen in container in front on left. such as the Gro-Lux series can certainly be con- sidered as in the right direction; however, ad- ditional efforts would also be most welcome by bi- ologists. More thorough descriptions of presently available light sources would be of some help in their evaluation. In addition to the output data frequently available from the commercial light companies on imaged lamps, there are other meas- urable characteristics not normally available but also of primary importance to the environmentolo- gist in deciding what to use — for instance {a) the spectral emission of lamp X as affected by ambient temperatures; and (6) the changes to be antici- pated in spectral emission of lamp X with usage, such as those related to differential deterioration rates of the lamps’ respective phosphors. A new model tube of Gro-Lux (F96T12/GRO/ VHO/WS) was being tested at the time this manuscript went to press to measure to what de- gree the bulb has been improved for bean or tomato growth. Transpiration, Leaf Temperature, and Stomatal Activity of Certain Plants as Affected by CO2 Concentration of the Air and Soil Moisture Tension Earlier investigations {23) on transpiration from corn plants indicated that a relatively high percentage of the stomata remained closed under conditions expected to foster high stomatal ac- tivity. This finding posed the question of why stomatal activity was low under what were consid- ered optimum environmental conditions. Field ob- servations (see pp. 18-19) indicated considerably more open stomata could be expected. Further ex- perimentation showed that the CO2 concentration of the air in a plant’s environment can drastically affect stomatal opening. Thus, the high CO2 con- centrations in the growth room (as noted in 23^ figs. 21 and 22) were responsible for low stomatal activity. Since many of the stomatal responses recorded in the aforementioned studies were within the range of external CO2 changes in the environ- 6 PROD. RES. RPT. 89, U.S. DEPARTMENT OF AGRICULTURE ment, the influence of CO2 content in air needed assessing to answer the following : (a) Does cuticular transpiration of crop plants assume proportions not heretofore recognized? (5) How do changes in cuticular and stomatal transpiration affect the heat budget of the plant ? (c) Of what importance to stomatal operation is soil moisture tension as opposed to CO2 concentra- tion of the ,air? Answers to these questions are paramount to man’s controlling the moisture loss from plants and increasing his economy of water use. Our knowledge of the total quantity of water transpired by plants that can be ascribed to cutic- ular transpiration seems to be somewhat clouded by the experimental methods employed by various researchers. Many attempts in the past to esti- mate cuticular transpiration appear to have had limitations that could result in low cuticular values. The inadequacies of such methods have never been completely discussed or defined. Prob- ably the most serious error in early studies was the use of hypostomatic leaves. Transpiration from such leaves has been measured before and after coating the underside of the leaves with white vaseline or cocoa butter or both. (See Stalfelt (39) for extensive literature citations.) Cuticular transpiration was considered to be that which was recorded in the coated condition. In a little dif- ferent experimental approach Fusser (6) has caused the absorption of transpired water from both hypostomatic leaf surfaces simultaneously and independently, but this upset the microen- vironment. It would appear that such techniques employing hypostomatic leaves cannot be directly compared with nonhypostomatic leaves because of several possible dissimilarities. First, differences in cu- ticle thickness on upper and lower surfaces were not ascertained. Also, the cuticular chemical com- position of the two surfaces may be different enough so as to differentially affect the magnitude of transpiration. Comparative use of transpira- tion values from the two surfaces also obviously fails to consider the plumbing interior of the leaf and its possible effect on the water available for cuticular transpiration from either or both sur- faces. Kamp (12) has postulated that the epi- dermal cells are more of a factor in controlling cuticular water loss than any differences in cutic- ular thickness. We must then ask ourselves the question : Wliat is the normal route or routes for water molecules to follow as they pass to the gaseous phase — ^through and from the upper epi- dermis and lower epidermis, or through and from bundle parenchyma, mesophyll cells, or intercellu- lar spaces and out the stomata? It is obvious that no one answer will usually suffice, considering all aspects of the plant in question and its environ- mental status. The other method employed to some extent for assessment of the magnitude of cuticular trans- piration is the weighing method. Stalfelt (38), Pisek and Berger (28), Hygen (9, 10, 11), and others consider the rates of water loss of severed leaves (transpiration decline) as being directly re- lated to stomatal aperture and, thus, by following the transpirational loss with time, an assessment of the magnitude of cuticular and stomatal transpi- ration can be made. As transpiration continues, water content of severed leaves also drops. Since the leaf (severed or not) has a limited reservoir, free energy of its water decreases with time ; there- fore, both stomatal and cuticular and, finally, any wholly cuticular transpiration should show de- cline. Hygen (10) considered transpiration thus, but Williams and Amer (Jp6) have disagreed with his assumption that transpiration from a leaf can be treated as though the vapor pressure at the evaporating surface falls in direct proportion to the water content. They consider such a postu- late generally invalid since transpiration from the experimental subject. Pelargonium, appeared to them to be independent of leaf-water content. In turn, Williams and Amer’s work must be ques- tioned as to the relevancy of their Pelargonium data compared with the species Hygen used, which did not include Pelargonium. Rather high cuticular transpiration values are recorded in the literature. Pisek and Berger (28) have compared cuticular transpiration of a num- ber of diverse species with evaporation from blot- ter paper under average room conditions. Quite extreme values were obtained from species repre- senting different habitats. Transpiration of Opuntia camanchica was as little as 0.03 percent of the evaporation from blotter paper, whereas Impatiens noti tangare transpired at a rate greater than 50 percent of blotter paper evaporation. Hygen (10) obtained cuticular transpiration val- ues ranging from 10 to 20 percent for some meso- phytes and values of 25 to 40 percent for what he describes as plants growing without moisture stress. Our approach to the problem was to change the CO2 concentration in growth chambers to effec- tively open or close stomata. We then determined transpiration when stomates were closed versus transpiration when stomates were open. Trans- piration was determined by weight changes ob- served per unit of time. Simultaneously, leaf temperatures were assessed by thermocouples. Finally, the stomata were opened by lowering CO2 concentrations, and stomatal activity was recorded as soil-moisture tension increased. Our hypothesis was that cuticular transpiration of crop plants is of considerable magnitude. Materials and Methods Five species were used — corn (Zea mays L.), cotton (Gossypium Mrsutum L.), sorghum (Sor- ghum vulgare Pers.), soybean (Glycine max RESEARCH IN PLANT TRANSPIRATION! 19 63 7 Merr.), and tomato {Lycopersicon esculentum Mill.)— a total of 17 lines, hybrids, and varieties (table 2). All are important agriculturally ex- cept the three single-cross corn lines and Smooth Leaf Empire cotton. The corn lines were in- cluded because they have shown distinctly different relationships between yield and soil moisture in field studies. Smooth Leaf Empire cotton was included because it contains a Da gene thought to increase cuticle thickness, eliminate pubescence, and decrease evaporation through the upper leaf surface. Table 2. — Transpiration and percentage stomata open on upper and lower leaf surfajces at CO 2 con- centrations -^250 p.p.m. and IfiO to 500 p.p.m. and percent reduction in transpiration resulting from the higher G O2 concentrations <250 p.p m. CO2 400 to 500 p.p.m. 0 p Transpira- Crop, age of plants, and “variety” Transpira- tion Standard deviation Stomata open Lowest CO2 value maintained Transpira- tion Standard deviation Stomata open tion reduction Corn (21 days) : Dixie 82 - Gldm^U hr. 14. 44 0.76 Percent 100 P.-p.m. 20 G/dm2/4 hr. 4.75 0.77 Percent 0 Percent 68 MP339 X MP311 12. 90 . 15 100 20 4. 14 .56 0 68 MP305 X T101_ 12. 57 . 60 100 20 4. 34 1. 60 0 66 MP305 X MP307. 11.85 . 46 100 20 3.91 .94 0 68 Sorghum (29 days) : RS-610 11. 75 1.30 100 30 3.85 2.2 0 68 NK-210 11. 50 1. 30 100 30 4.92 1.7 0 58 Amak-R 11. 00 .86 100 30 4. 34 1.3 0 61 Tomatoes (43 days) : Marglobe 10.43 1.30 87 115 8. 24 1. 10 0 21 Rutgers 9.35 1.03 83 115 6.32 .48 0 33 Marion.. .. 9. 29 1.03 85 115 5. 97 . 51 0 36 Soybeans (36 days) : Hampton 10. 27 2.50 91 35 6. 87 1.7 0 34 Hardee 8.90 1.03 91 35 4. 27 1. 4 0 53 Beinville. .... 8.83 .62 92 35 5.28 1.5 0 41 Cotton (56 days) : Smooth Leaf Empire.. . 7. 15 .62 87 40 4. 75 . 59 0 34 Auburn 7. 03 .73 92 40 5. 05 .69 3 29 Empire .. 3.49 . 64 84 40 2.33 .83 2 34 Carolina Queen. 3. 11 .47 85 40 2.34 . 70 2 25 General groioth conditions. — A standard ferti- lized, Krilium-treated soil was prepared as pre- viously described (23) and used for plant growth. All seeds were pregerminated in vermiculite; uni- form populations were selected on the basis of radicle length and were transplanted 24 to 32 hours after sowing. Single plants were grown in 3,600 grams of Cecil sandy clay loam in tarred 92-ounce juice containers. During the preexperimental periods, soil moisture availability was kept above 50 percent but did not exceed 0.05 bar. Such soil moisture control was expected to minimize its effect on plant growth and related processes. Moisture desorption curves were developed (30) and used for relationships of water availability with matric suction. Experiments were per- formed only after fully expanded leaves had developed but before self-shading or moisture drawdown limited physical description. All pop- ulations were grown and tested in controlled en- vironment chambers. Growth and test conditions were: 14 hours light (VHO cool white fiuores- cents approximately 0.5 to 0.6 cal. cm."^ min.'^), temperature 25° C., relative humidity 50 to 70 per- cent; and 10 hours dark, temperature 20° C., and relative humidity 85 to 95 percent. CO 2 control of stomata. — Preliminary ex- perimentation indicated that various carbon di- oxide concentrations could effectively open or close the stomata of crop plants. The response was not consistent at any one CO2 concentration from species to species. Neither did all stomata on a leaf behave exactly the same at the same concen- tration of CO2. Apertures changed with changes in CO2 concentration ; however, even more striking than changes in aperture was the finding that in a given microscopic field on a single leaf a large number of stomata can appear closed while others appear open. Figure 6 shows the CO2 concentrations in air as they change stomatal condition of the several varieties sampled under the conditions stated. This research was first attempted in small leaf 8 PROD. RES. RPT. 89, U.S. DEPARTMENT OF AGRICULTURE Figtibe 6. — Influence of changing CO2 concentration in air on 10 selected stomata contained in a microscopic fleld. Light intensity approximately 0.5 cal. cm."^ min.'\ temperature 25 ° O., relative humidity 50 to 70 i)ercent. chambers but was transferred to growth chambers when it was found that the leaf was frequently in delicate equilibrium with the rest of the plant. Differences between leaf chamber environment and plant environment were found to cause abnormal stomatal activity in the leaf chamber. Face masks (fig. 7) for expiring exhaled air high in CO2 were required to maintain low con- centrations of CO2 in the growth chamber. Both control and monitoring of CO2 were accomplished with a Liston-Beckman infrared gas analyzer, model 15A. Measurements of observed cuticular and sto- matal transpiration. — Estimates of cuticular and stomatal transpiration of the species were made by weight differences on several consecutive days. All weights except plant weights and soil moisture weights remained constant. To estimate plant weight, six representative plants of each variety ® were sacrificed the evening before the first experi- mental run. During the experimental period soil moisture availability was kept high (less than 0.3 bar) to minimize its effect on stomatal operation or transpiration. Plants were watered to 0.05 bar soil moisture tension the evening preceding each experimental day. Five replicates were the min- ° “Variety” is used in a loose sense hereafter to indicate lines, hybrids, or varieties. imum used in any study. All varieties of a species were tested simultaneously ; however, the different species were run separately. Stomatal and cutic- ular transpiration were measured daily over a 4- hour period under low CO2, with the stomata open. Observed cuticular transpiration was also meas- ured daily over a 4-hour period under high CO2, with stomata visibly closed, or in some instances nearly closed. When stomata were visibly closed, the difference between the two measurements was considered to be cuticular transpiration. Studies of soil moisture tension effects on sto- matal opening under low CO2 were started as soon after stomatal and observed cuticular transpira- tion measurements as practicable. F or these stud- ies the plants were watered to 0.05 bar after lights were out ; then, the next morning the CO2 was low- ered to cause the stomata to open and was main- tained low during the daylight hours of each ex- periment. The percentage of stomata open was followed microscopically during daylight hours (usually for several days) as soil moisture tension increased and the stomata shut. The leaf areas in all studies were determined as reported for corn and sorghum (23) and cotton (£), and for tomatoes and soybeans by cutting out the shadow-cast replicas of leaves from ozalid paper and relating their weight to actual weight per unit area. Stomatal monitoring. — The condition of the stomata on upper and lower leaf surfaces was as- sessed hourly by use of a special microscope (25) . Records were kept on the number of stomata open on the upper and lower epidermis; 40 individual stomatal counts, 2 leaves per plant, 2 plants per variety were the minimum. After the counts were made and recorded, all the plants were scanned hourly to see if stomatal reaction was uniform. Leaf temperature. — The temperature of upper and lower leaf surfaces was continually sensed by BN 26188 Figure 7. — Monitoring stomatal activity to determine ef- fects of CO2 concentration or soil moisture tension. Face mask was used to maintain low CO2. RESEARCH IN PLANT TRANSPIRATION: 19 63 9 small thermocouples {23) placed in intimate con- tact with the cuticle and held by small pieces of masking tape. Four thermocouples per plant, with one plant representing each variety, were used. Results T ranspiratian. — The observed cuticular tran- spiration values obtained in these studies (table 2) appear quite high when compared with the aver- age value of 90 percent so often quoted as typical of transpiration through stomata {13, 15) . If we accept low cuticular values as normal, then a rather large error must exist in our analysis. Either a low number of stomata open under the low CO2 or incomplete closure of stomata under high CO2 could effectively increase the proportion of transpiration considered cuticular in this study. The recent report of Tiim and Loomis {4-1), as well, as the older work of Stalfelt {38) and Hygen and Midgaard {11), indicates that the degree of opening as reflected in size of the stomatal pore above several microns may be of minor importance in determining the magnitude of transpiration. Most important is whether the stomata are open at all. Table 2 summarizes the observed stomatal condition of the species during the experimental periods. Stomata of the crop plants exclusive of cotton were observed to be closed under the high CO2; however, none of the dicotyledonous plants had all stomata visibly open under low CO2 (see table 2). The variations in transpiration as ex- pressed by the rather high standard deviations associated with the transpiration measurements are noteworthy; possibly they indicate the sto- matal condition of our plants was poorly defined because we were unable to ascertain complete closure or because the sampling of stomata was inadequate. Successive runs with several other populations of corn plants indicated that the listed values of transpiration under the same experi- mental conditions were reproducible. The stand- ard deviations in table 2 were used to set limits for the realistic ranges of transpiration described in table 3. For those species indicating complete visual stomatal closure the question arises whether a hermetic seal existed at the interface of the guard cells knowing the resolution at the magnification used (/'>-'2 /i) was insufficient to detect complete closure. For the present we do not have a com- pletely satisfactory method for measuring either the stomatal seal or improving our microscopic potential. Hygen’s approach of studying the change in transpiration with time as related to sto- matal closure {9, 10, 11) is probably the best ap- proach presently available, but his method has been limited to severed plant parts. In our future Table 3.- — Range of transpiration after stomatal closure expressed as percentages of total tran- spiration [Values based on standard deviations of table 2] Crop and “variety” “Observed” cuticular Cuticular -1- stomatal Range Low High Low High Corn: GIdm? GIdw? Gj dwl Gldm^ Percent Dixie 82 3. 99 5. 51 13. 67 15. 21 26-40 MP339XMP311-- 3. 99 4. 29 12. 34 13. 56 30-35 MP305XT10I 3. 74 4. 94 10. 97 14. 17 26-45 MP305XMP307_- 3. 45 4. 37 10. 91 12. 79 27-40 Sorghum : RS-6I0 2. 55 5. 15 9. 59 13. 95 18-53 NK-2I0 3. 62 6. 22 9. 80 13. 20 27-63 Amak-RI2 3. 48 5. 20 9. 70 12. 30 28-54 Tomatoes: Marglobe - 6. 94 9. 54 9. 33 11. 53 60-90 Rutgers- 5. 29 7. 35 8. 87 9. 83 54-92 Marion 4. 94 7. 00 8. 78 9. 80 50-80 Soybeans: Hampton 4. 37 9. 37 8. 57 11. 97 37-89 Hardee 3. 24 5. 30 7. 50 10. 30 32-71 Beinville- 4. 66 5. 90 7. 33 10. 33 45-81 Cotton: Smooth Leaf Empire _ 4. 13 5. 37 6. 56 7. 74 53-82 Auburn 56. 4. 32 5. 78 6. 34 7. 72 56-91 Empire 1. 69 2. 97 2. 66 4. 32 39-90 Carolina Queen 1. 87 2. 81 2. 41 3. 81 49-85 studies his method will be employed with whole plants to further clarify transpiration-stomatal- CO2 interactions. Leaf temperature. — Changes in leaf temperature with changes in stomatal condition are reflected in table 4. In general, leaf temperature increased several degrees for com, sorghum, tomato, and soybean plants when stomatal transpiration was minimized. Cotton leaf temperature did not change significantly. Optimum soil moisture in this phase of the study probably had some bearing on the leaf temperatures recorded. Soil moisture tension and stomatal operation.— Figure 8 depicts how stomata opened by low con- centrations of CO2 respond to increases in soil moisture tension. The responses observed differ among species and among some varieties of the same species. Discussion If the stomata observed in our studies were com- pletely closed, the data indicate that high cuticular rates may also occur in several of the crop plants we tested. The values must be interpreted with due caution. They are presently indicative of transpirational changes brought about by changes in CO2 levels for the conditions of growth and ex- perimentation employed. Our hypothesis still re- 10 PROD. RES. RPT. 8 9, U.S. DEPARTMENT OF AGRICULTURE Table 4. — Radiation impinging {R), temperature {LT) and energy dissipated as latent heat {El) of five species of leaves at high and low concen- trations of C 0.1^ in the atmosphere [Latent heat exchange as based on average transpiration values in table 2] Crop and variety R» High CO2 Low CO2 AT AEl LT El LT El Corn: Dixie 82 Call 0. 50 °C. 28. 7 Call 0. 12 °C. 25. 3 Call 0. 35 3. 4 0. 23 MP339X MP311 . 50 29. 2 . 10 25. 9 . 31 3. 3 . 21 MP305X TlOl... - . 50 28. 1 . 11 24. 8 . 30 3. 3 . 19 MP305X MP307 . 50 27. 6 . 10 24. 0 . 29 3. 6 . 19 Sorghum : RS-610 . 50 30. 4 . 09 26. 4 . 28 4. 0 . 19 NK-210 . 50 29. 8 . 12 25. 7 . 28 4. 1 . 16 Amak-R12-- . 50 28. 6 . 11 26. 5 . 27 2. 1 . 16 Tomatoes: Marglobe . 50 29. 1 . 20 25. 6 . 25 3. 5 . 05 Rutgers . 50 25. 4 . 15 24. 0 . 23 1. 4 . 08 Marion . 50 29. 2 . 14 26. 8 . 22 2. 4 . 08 Soybeans: Hampton . 60 30. 6 . 17 25. 5 . 25 5. 1 . 08 Hardee. . _ . 60 29. 8 . 10 25. 1 . 22 4. 7 . 12 Beinville . 60 28. 9 . 13 24. 6 . 21 4. 3 . 08 Cotton: Smooth Leaf Empire . 58 26. 5 . 12 24. 8 . 17 1. 7 . 05 Auburn 56... . 58 25. 2 . 12 24. 7 . 17 . 5 . 05 Empire . 58 24. 7 . 06 23. 9 . 08 . 8 . 02 Carolina Queen . . 58 23. 9 . 06 24. 6 . 08 -. 7 . 01 1 Measured with a Beckman- Whitley Model H 188-01 radiometer. ^ Cal. cm. min.“‘. mains to be proved or disproved, since the state of stomatal opening below 2/.i was not defined. However, in light of some of the earlier dis- cussion, it is obviously imperative that we reas- sess our physical description of gaseous transfer by leaves. The stomatal pore has too long been emphasized as the portal of entry and exit of water and CO2 molecules. In studies of impedance (rather than the term resistance because of vectors involved) to water movement from any leaf we should consider both the stomatal and cuticular routes. Both the air layer adjacent to evaporative cell surfaces and the evaporative cell surfaces themselves are important components in the va- porization process ; however, pathways leading to these evaporative surfaces are equally important. Wylie’s {J^t) and Armacost’s {!) work indicated that vein extensions and the epidermis supplement water transfer by the mesophyll and in the leaf may be the primary route of water transfer from veins to evaporative surfaces. The work of Kob- erts et al. {31) also suggests that the route of water movement in itself involves such vein extensions along pectinaceous paths. Our knowledge in this area is scant. In the plant there exists an imped- ance to water movement up to and including the leaf surface and stomatal pore. Such impedance in the leaf is quite complex ; it includes such factors as the cell wall matrix, the free energy status of water available for transpiration, and the chemical and physical constitution and state of cuticle and suberin, and their underlying layers offering resistance to flow. Also, the involvement of the protoplasm, especially ectodesmata {6) in the movement of water eventually transpired, is of some presently undefined importance ; albeit deal- ing with protoplasmic water in itself, its availa- bility is ill defined. The efficiency of flow (diffu- sion of water vapor) from mesophyll walls through the stomatal pore is probably not as simple in all species as Bange {3) describes for Zehrina pendula Schuizl. If the intercellular air were saturated with water vapor at all times and in all places, the vapor transfer phenomenon from mesophyll cells through stomatal pores might be treated simply; however, we have no assurance that this is the case. Dynamic changes in the plant’s environment alone will affect the microconditions, both external and internal to the leaf. Thus, we can interpret the hourly fluctuations in stomatal opening {36, 37) induced by environmental demand, as reflecting the inability of water to move from the soil to the plant to continuously meet such demand. From this and our previous work, the authors recognize that the major barrier to an accurate assessment of the impedance at the leaf surface when stomata are not fully opened involves the lack of constancy in stomatal response. Through- out these studies {26, 23) we have continued to re- port stomatal operation as the percentage of stomata open. When some stomata can be com- pletely open and some completely closed on the same leaf, (see p. 36 and fig. 28 of 23), or when 50 percent of the stomata assayed appear closed, or when their activity is oscillatory, then average stomatal aperture is somewhat ambiguous. There is also relevance in these studies to CO2 passage through the leaf. The CO2, which is es- sential for photosynthesis, must solubilize when diffusing through aqueous and possibly lipoidal pathways before entry into the chloroplasts. CO2 probably solubilizes under certain conditions via the cuticle, and, thence, diffuses to palisade and mesophyll cells. Such diffusion would be comple- mentary to diffusion into substomatal chambers and, thence, to palisade or mesophyll cells. Under RESEARCH IN PLANT TRANSPIRATION! 19 63 11 lN33y3d ‘N3d0 VIVIMOIS 795-862 0—66 2 Figure 8. — Stomatal activity under low CO2 concentrations in air as related to increasing soil moisture tension— (A) sorghum, (B) tomatoes, (C) cotton, and (D) corn. 12 PROD. RES. RPT. 89, U.S. DEPARTMENT OF AGRICULTURE those conditions -where stomata are completely closed it would be the only pathway of CO2 diffu- sion. Involvement of this route coidd help explain why under enriched CO2 supply (7, ii, ^6) plants ma}^ increase their assimilation rate even when stomata appear to close and transpiration mark- edly decreases. Differences in diffusive resistance of the cuticle between species could also explain the variation in efficiencies found between species uti- lizing the same concentration of CO2 (S) for photosynthesis. Table 4 summarizes leaf temperature changes of the individual varieties as well as energy dis- sipated by transpiration. Such data are almost nonexistent (£9) . Six degrees centigrade (RS610) above the ambient temperature of 25° C. was the highest recorded in these studies. In general, leaf temperatures increased several degrees for corn, sorghum, tomato, and soybean plants when stomatal transpiration was reduced. Recorded leaf temperatures of the corn leaves are essentially the same as those already reported (^S) for corn grown under similar environmental conditions and total radiant energy, but under incandescent lights. The most striking difference found among the species listed in table 4 concerns the leaf temper- ature of cotton plants. Although cotton leaves had the lowest transpiration of all the species tested, and thus the least energy dissipated as latent heat, they had a remarkable tendency to remain either below ambient temperature or at the most a degree and a half above. Thus, the transpiration of cot- ton leaves does not account in any large measure for the dissipation of impinging radiation. Cot- ton’s low leaf temperature may result from either or both a high capacity of the leaves for convective and reradiative loss or low long-wave interception, such as would result from a high ability for re- flection or transmittance, or both. Figure 8 shows that when the CO2 of the at- mosphere was kept low to effectively open the stomata, increasing soil moisture stress eventually reached a point that offset photoactive opening (^6) and brought about complete visible closure. For corn, sorghum, and tomatoes the first response was near 0.3 bar moisture tension. Other than this initial effect, the slope and intercepts of the curves are quite different. The stomata of the dif- ferent tomato varieties and sorghum hybrids re- sponded within species in a remarkably uniform manner. Tomato stomata closed at a soil moisture tension several bars lower than sorghum stomata. Corn stomata closed at significantly lower soil moisture tensions than any of the other species. The data also indicate that variation existed in the stomatal response between the variety and lines. There may be an important correlation be- tween the field observation that Dixie 82 is drought resistant and the data in figure 8 indicating that its stomata close at lower soil moisture tensions than the other lines tested. Analysis of the extent of root systems in these studies, as indicated by their fresh and dry weight, showed Dixie 82 had a much smaller root system. It is possible that a smaller root system, especially in the shallow soils found in the southern Piedmont, would be more conservative of soil moisture. Such a root system could not absorb water at the same rate (to meet evaporative demand) as a more extensive root system. The ultimate effect would be an earlier closure of stomata, as seen in figure 8, and from there on a more conservative use of soil water by prolonging the period of availability. Complete stomatal closure of Dixie 82 was not observed until soil moisture tension reached the same value as that attained with the other varieties. In periods of high soil moisture availability Dixie 82 would develop at maximum efficiency. This hypothesis is opposite from what one would expect for the adaptation of corn plants to a deep soil profile in which a larger root system extending to greater depths could tap a greater soil water reservoir. At the outset of the studies on soil moisture tension cotton stomata were never completely opened by low CO2. Recent experimentation has shown complete opening is possible at higher radiant energy values and different spectral quality. The curves in figure 8 show that Empire and Carolina Queen, and also Auburn 56 and Smooth Leaf Empire, tend to parallel each other in their soil moisture stomatal response. Their cuticular and stomatal transpiration (table 2) also show the same tendencies for grouping. Further experi- mentation is necessary to prove or disprove that such differences are relevant to soil moisture con- servation. The high rate of transpiration per unit of leaf surface of Smooth Leaf Empire (table 2) does introduce an element of uncertainty that its D2 gene improves its drought resistance as stated earlier. In these studies the ability of low CO2 to main- tain stomata in an open position has been shown to be of secondary importance to that of increasing soil moisture tension and is in agreement with Stalfelt’s recent findings (40) . Soil moisture ten- sions greater than 1 bar have a drastic effect on guard cell activity, as indicated by our studies on corn and sorghum (36). The number of stomata open continuously decreased as the soil moisture tension increased, with the magnitude of the effect of this increase determined by air vapor pressure difference and radiation level. RESEARCH IN PLANT TRANSPIRATION! 19 63 13 GUARD CELL ACTION Protoplasmic Streaming and Guard Cell Operation That guard cell cytoplasm undergoes cyclosis has been reported by both Weber (^4) and Sinke (^^). This protoplasmic activity has been used by several workers to ascertain whether guard cells were alive or dead ; however, very little effort has been put forth to characterize the streaming phenomenon. Weber states that streaming is characteristic of closed stomata (in Yicia faba) ; with opening, the phenomenon is reduced to a lo- cal sliding motion, finally, ceasing, or on rare oc- casions exhibiting local jerky sliding motions in the fully opened stomate. In cursory examinations cyclosis in guard cells of several species (including Vida faba) was de- tected; however, its association, as depicted by Weber, with the closed condition was not absolute. In attempts to document cyclosis in guard cells of Vida faba on movie film, working shortly after dark gave us consistently a few subjects to record. Our studies on cyclosis were initiated to find out if streaming is correlated with the peculiari- ties of guard cell activity, such as stomatal open- ing and closing, or is just another indication of a living cell. Only the first experiment will be dis- cussed here; others continue to be performed and their descriptions will follow in subsequent reports. Procedure The first series of experiments was designed to find out what, if any, correlation exists between guard cell streaming and time of day. A vigor- ously growing population of Vida faba, (horse- bean), Rheum rhaponticum (rhubarb). Cyclamen indicum (cyclamen), and Antirrhinum majus (snapdragon) in individual 6-inch pots were transferred from the greenhouse to a growth chamber a week before the study of streaming and guard cell operation. In the growth chamber a 12-hour photoperiod and 20° day and 15° night temperatures were standard. Light was provided by cool white fluorescents (0.5 cal. cm.“^ min."^) from 6 a.m. to 6 p.m. Beginning the day that the plants were placed in the growth chamber,’ daily observations were made microscopically to determine whether or not stomata were ojien. At least 10 stomata of two selected (tagged) mature leaves of each species were observed each time. Later, when both open- ing and streaming were being assessed, similar areas on untagged leaves were observed and if the stomata were essentially in the same condition as those monitored on tagged leaves, the areas ob- served on the untagged leaves were stripped as quickly as possible, placed in water, and checked for streaming under oil immersion. This method of consistently observing the same tagged leaves provided a check on changes in stomatal activity that might occur during growth. Checks were made on all species just before and immediately after “lights on” in the morning, at noon, and in the evening before and right after darkness. The first experimental period lasted 9 days. The light was then reduced to 0.05 cal. cm.‘^ min.'^ for 6 days of observation. Results and Discussion For several days preceding and including the day of transfer from greenhouse to growth cham- ber, March 26, 1963, the sky was overcast, there were occasional showers, and, in general, light intensity was low. A low number of open stomata were found the first few days after the plants were transferred to the growth chamber. As can be seen in the March 28 observations contained in table 5, more Vida faba stomata are open during the day than during the night. A similar cycle, but at an almost imperceptible level of activity, occurred during the same period on snapdragon and cyclamen leaves. After 7 days in the growth chamber, the number of stomata opening increased considerably (compare, in table 5, 3-28 with 4-4 and then 4-10) . Vida faba stomata apparently reached photoactive saturation, with opening maintained both day and night. Cyclamen and snapdragon also showed considerable increase in the number of stomata visibly open both day and night with increased number of days in the growth chamber. The tendency for rhubarb to show con- siderable night opening (4—10) is not consistent with our previous report {23) ; however, the ac- tivity at the lower light level (4—17) is. The dis- crepancy may be because of the different light sources used (incandescent in the earlier study versus fluorescent in these) or differences in CO2 availability between the two studies. Infrared gas analysis has shown there is a tendency for CO2 to remain at external levels or lower in growth chambers that contain a photosynthesizing popu- lation. As already explained (see p. 5), growth room studies have been affected by CO2 concentra- tions usually exceeding outside air values, thus causing in some species less stomatal activity than would be normal. The tendency for carryover of open stomata into the dark periods was related to the light in- tensity to which the plants had been subjected. It was deduced that the photoactive phase of open- ing was light saturated. To test this hypothesis, lights were reduced to 0.05 cal. cm."" min."^ on April 15. This resulted in a drastic reduction in the percentage of stomata open by April 17 during both the night and day (table 5). More definite 14 PROD. RES. RPT. 8 9, U.S. DEPARTMENT OF AGRICULTURE Table 5. — Percentage of stomata open in growth chamber ^ [1963] 5:30 Light 6:30 Mid- Species and date a.m. 6:00 a.m. 6:30 a.m. 9:30 a.m. 10:30 a.m. Noon 5:30 p.m. p.m. night Vida faba: 3-28- -- Pet. 0 Pet. 30 Pet. 90 Pet. Pet. Pet. 100 Pet. 90 Pet. 0 Pet. 4-4- - - - 90 90 100 100 100 100 90 4-10- 90 90 90 100 80 80 4-17--- -- - -- -- 0 10 30 40 20 0 Antirrhinum majus: 3-28- -------- 0 0 0 20 5 0 4-4 -- 10 20 60 70 80 80 70 4-10- - 20 70 100 80 40 0 4-17- - - -- --- 0 0 0 10 10 0 Cyclamen indicum: 3-28 - - - -- 0 5 10 5 5 0 4-4 - 0 30 30 50 60 50 4-10 10 70 80 40 30 0 4-17- - - - 0 10 10 20 20 0 Rheum rhaponticum: 4-10 100 100 100 100 100 0 4-17- -- 10 10 80 80 70 0 > Conditions in growth chamber are described on page 13. patterns reappeared of opening in light and clos- ing in dark. These patterns somewhat repeated those found after the original transfer from the greenhouse to the growth chamber, but in con- trast to the earlier findings, stomata remained open after dark. Very little cyclosis in guard cells was found any time during these studies. Streaming appeared to take place only when the stomata were closed and then mainly during daylight hours. At the higher radiant energy value the number of guard cells showing streaming at times appeared inversely related to the number of stomata open ; but at the lower energy level, streaming was observed in 10 percent of the Vida faba cells only at midnight. No streaming was observed at any time with the other three species. This study strengthened our belief that if cyclosis was associated with guard cell movement, it was not as readily apparent as Weber {bit.) had indicated. More detailed hour- by-hour studies are contemplated on guard cell cyclosis and opening movements of stomata. During these studies it was discovered that fre- quently stomata observed as closed in situ opened when the epidermis was peeled and placed on water. This finding has considerable relevance to our cellular studies and will be discussed in future reports. From the observed stomatal response to changes in light quantity, we can hypothesize a relation- ship between cool-weatlier crops and their re- sponse to radiant energy. These studies indicate that both heat and drought tolerance of the test species are correlated with their stomatal response and plastid number. Table 5 shows that the species Vida faba and Rheum rhaponticum had the highest percentage of stomata ojien at both high- and low-light values. The plastid number and size of guard cells in these two species appeared to be significantly greater than in Cyclamen indicum and Antirrhinum, majus. Of the four species studied, the snap- dragon has the smallest number of plastids per guard cell and is probably the most heat- and drought-tolerant. Rhubarb and cyclamen are somewhat intermediate between cool- and warm- weather crops ; horsebean is thought to be a cool- weather crop intolerant of warm weather. Any classification of plant species into warm- and cool- season crops is very general and will have exceptions. Effective radiation levels are normally greater during warm seasons than during cool seasons be- cause of the angle of incident radiation and day length. In these studies guard cells of the cool- season crops evidently became saturated by light, thus the photoactive and hydroactive balance, which a plant must maintain with its environment, was upset. These studies indicate that a cool- season plant such as Vida faba grown under an intermediate temperature and high total daily light (360 cal. cm.“^ day) ceases to haA^e normal stomatal reactions. Under natural conditions this combination of temperature and light could be lethal for Vida faba. With all stomata open night and day, larger quantities of water would be RESEARCH IN PLANT TRANSPIRATION: 19 63 15 transpired, thus upsetting the total water balance of the plant and certainly reducing potential water recharge of tissues. Much more research is necessary to prove or dis- prove the importance of light saturation of guard cells in any plant’s ecological relationship ; how- ever, it is evident that normal metabolic processes (including stomatal action) may be altered by changing the temperature and light environment of the plant. Experimenters should give more consideration to environmental conditions when they are using controlled facilities. The optimum normal environment of the plant in the field should serve as a base for experimental conditions. EFFECTS OF CERTAIN CHEMICALS ON TRANSPIRATION Atrazine In agricultural production at least 50 percent of the water that passes from the soil to the atmos- phere is transpired. However, transpiration does not appear to be a biologically efficient pi’ocess since less than 5 percent of the soil water absorbed is incorporated in the constitution of the plant. The potential, therefore, is thought to exist of markedly reducing transpiration by physical bar- riers such as wax, latex, or plastic coatings; by enzymatic control of guard cells; or by plant breeding, incorporating those morphological, physiological, and biochemical properties respon- sible for efficient soil moisture usage. Published results from greenhouse work with Atrazine suggest that the compound as presently applied for weed control may also increase effi- ciency of water use. Smith and Buckholtz {36) reported reductions in transpiration of 40 percent by corn and 65 percent by soybean plants 6 hours after additions of 20 p.p.m. of Atrazine to their nutrient solutions. Reduction in transpiration was found in soil-grown plants as well as those grown in solution. Foliar applications were also effective. Wills and Davis {Ip?) found a reduc- tion in transpiration rate of whole plants and ex- cised shoots of corn, cotton, and soybeans after 10 or 25 p.p.m. of Atrazine was added to the culture solution. The effect of the Atrazine was con- sidered to be that of closing stomata. With such basic information at hand, it appeared a test for Atrazine effects should be made under field condi- tions. The field plan included an evaluation of water usage, yield, and stomatal operation of Atrazine- and non-Atrazine-treated corn under irrigated and simulated dryland conditions. Experimental Procedure T reatments. — Six treatments involving two soil moisture tension regimes, two corn varieties, and Atrazine versus no Atrazine were employed. They consisted of a modified split-split plot ex- perimental design with moisture regime as the main plot, corn variety as the split plot, and Atra- zine as the split-split plot replicated four times. All data were treated by factorial analysis of variance by computers under the direction of the University of Georgia Statistical Laboratory personnel. An access tube was placed in the middle of each plot, facilitating assessment by the neutron probe method {^3) of changes in available soil moisture during the growing season. Half of the plots re- ceived supplementary irrigation during the grow- ing season. Water applied at any one time varied from 1 to 2 inches and was considered sufficient to lower the soil moisture tension in the top 24 inches to approximately one third atmosphere. The other plots were covered with plastic to prevent soil moisture recharge subsequent to saturation of the profile after planting ; thus a droughty condi- tion was assured. Cultural 'practice. — Before planting, 84 pounds per acre of N, 73 pounds per acre of P, and 139 pounds per acre of K were broadcast and har- rowed in. Atrazine at the rate of 6 pounds per acre (SOW) was applied as a broadcast spray to subplots immediately after planting. After spraying, one-half inch of water was applied to all plots, and 4 days later 1 inch more was added to impi'ove the action of Atrazine. Subplots were 25 by 25 feet on Cecil sandy loam. Eight rows of Zea mays, either Dixie 82 (a double-cross hybrid) or Hastings open pollinated, were planted in 40-inch rows in each plot May 17, 1962. Thir- teen days after planting, seedlings were thinned to one plant per foot of row length. Two days later the soil on all nonirrigated plots was covered completely with transparent plastic and the plas- tic covered with one-quarter inch of soil so that ground reflectivity of the irrigated and nonirri- gated plots was comparable. Noncropped plastic- covered and fallowed plots 50 by 50 feet were maintained so that subsurface water changes could be assessed as the season progressed. Data recorded. — Soil moisture content in the increments of the profile (depths) as indicated in table 6 was determined once each week until 2 weeks before tasseling, after which it was meas- ured twice weekly. Moisture determinations were also made the second day following irrigation or rain. Figure 9 shows the representative soil moisture desorption curves developed using the pressure membrane and plate technique {30). Variations of water content shown in table 6 are primarily due to differences in soil texture and possibly soil structure. 16 PROD. RES. RPT. 89, U.S. DEPARTMENT OF AGRICULTURE Table 6. — Moisture content of flastic-covered and irrigated soil at specified depths at beginning {6/Jf./62) of measuring period'^ Measured at depths of — Treatment and species of plant 0 to 9 inches 9 to 15 inches 15 to 23 inches 23 to 33 inches 33 to 45 inches 45 to 57 inches PLASTIC COVERED Dixie 82: Inches Inches Inches Inches Inches Inches With Atrazine. _ 1. 42 1. 51 2. 60 3. 56 4. 19 4. 23 No Atrazine . __ 1. 52 1. 58 2. 75 3. 44 3. 66 3. 85 Hastings open pollinated: With Atrazine _ _ . 1. 64 1. 52 2. 42 3. 51 4. 36 4. 32 No Atrazine _ ... 1. 59 1. 39 2. 26 3. 54 4. 21 4. 14 IRRIGATED Dixie 82: With Atrazine _ 1. 55 1. 57 2. 68 3. 64 4. 19 4. 16 No Atrazine __ 1. 49 1. 49 2. 68 3. 73 4. 34 4. 20 Hastings open pollinated: With Atrazine.. . .. 1. 58 1. 68 2. 73 3. 64 4. 08 4. 17 No Atrazine. _ _ 1. 67 1. 78 2. 89 3. 53 3. 98 3. 95 * No significant difference exists between moisture contents listed at any one depth. Figure 9. — Soil moisture desorption curves for indicated levels of soil profile. Each point represents four pres- sure membrane or plate determinations. A composite sample of soil was used from four different locations at the test site for each determination. Vertical lines in graphs represent standard error of the mean. Rainfall and irrigation were recorded, thus allowing for an assessment of total water input for each plot. Stomatal activity was recorded as the percentage of stomata open and was monitored on selected cloud-free or nearly cloud-free days throughout the growing season with the stomata viewer (^5). The percentage of stomata open on upper and lower surfaces of the third and fourth youngest leaf midway between the tip and base was checked on the same two representative plants from each subplot throughout the season. These leaves were chosen because they were not shaded and previous experimentation {23) indicated all stomata on such leaves are operable. Corn grain and aboveground dry matter yields were measured at the end of the season. When it was noted that drought was affecting the initia- tion of silking, counts of the number of plants silking, as related to treatment and age of the population, were made during the latter part of the season. Results and Discussion The heterogeneity of the soil layers made it impossible to relate neutron probe measurements to small changes in available water or soil mois- ture tension. Such heterogeneity is exemjilified in the lack of overlapping of most of the moisture desorption curves (fig. 9) . Large variations were also found in bulk density values determined at the various levels. This was both surprising and discouraging, since the area selected for the studies was considered to be one of the most geo- physically uniform available for such experimen- tation in the Piedmont. Interpretations of water use were based, therefore, on moisture content changes both in the overall soil profile and in indi- vidual segments of the profile. An analysis of variance on average water con- tent of replications at the beginning of neutron probe measurements (table 6) indicated that at the age of 18 days all corn plants were under RESEARCH IN PLANT TRANSPIRATION: 1963 17 similar soil moisture conditions at each level of the soil profile sampled. Again, at the end of the season (table 7), no significant differences were found in the soil moisture remaining at any one level of measurement between any treatments. As expected, the irrigated corn consumed significantly more water than the nonirrigated corn (table 8). Irrigated plots received 13.71 inches more mois- ture than the plastic-covered plots — 8.96 inches of rainfall and 4.75 inches of irrigation water. Table 7. — Moisture content of plastic-covered a/nd irrigated soil at specified depths at end of season {9/3/62) 1 Measured at depths of — Treatment and species of plant 0 to 9 inches 9 to 15 inches 15 to 23 inches 23 to 33 inches 33 to 45 inches 45 to 57 inches PLASTIC COVERED Dixie 82; With Atrazine - _ Inches 0. 74 Inches 1. 14 Inches 2. 23 Inches 3. 19 Inches 3. 80 Inches 3. 78 No Atrazine _ _ __ . 82 1. 18 2. 34 2. 97 3. 13 3. 40 Hastings open pollinated: With Atrazine _ . 89 1. 11 1. 95 2. 94 3. 83 3. 77 No Atrazine . . 72 . 89 1. 70 2. 96 3. 58 3. 52 IRRIGATED Dixie 82: With Atrazine _ . 76 1. 15 2. 17 3. 12 3. 74 3. 85 No Atrazine __ _ . 75 1. 08 2. 18 3. 23 3. 93 3. 75 Hastings open pollinated: With Atrazine. _ .. ... . . No Atrazine _ . 83 . 92 1. 26 1. 38 2. 19 2. 37 3. 12 2. 92 3. 58 3. 35 3. 68 3. 31 ‘ No significant difference exists between moisture contents listed at any one depth. Table 8. — Seasonal water use on irrigated and plastic-covered plots, yield oj corn, and efficiency of water use as shown hy the transpiration coefficient Treatment and species of plant Water used from depths of — Total water used, 0 to 57 in. -h irrig. & rainfall Yield of corn Transpiration coefficient * 0-9 in. 9-15 in. 15-23 in. 23-33 in. 33-45 in. 45-57 in. Total Per plot per plot Grain Dry matter PLASTIC COVERED Dixie 82: With Atrazine Inches 0. 68 Inches 0. 37 Inches 0. 37 Inches 0. 37 Inches 0. 39 Inches 0. 45 Inches 2. 63 Pounds 8, 548 Pounds 24 356 74 No Atrazine _ __ _ . 70 . 40 . 41 . 47 . 53 . 45 2. 96 9,620 28 343 85 Hastings open pollinated: 94 With Atrazine . 75 . 41 . 47 . 57 . 53 . 55 3. 28 10, 660 12 888 No Atrazine.. .... . 87 . 50 . 56 . 58 . 63 . 62 3. 76 12, 220 15 814 99 IRRIGATED ^ Dixie 82: With Atrazine . . . 79 . 42 . 51 . 52 . 45 . 31 16. 71 54, 308 84 647 172 No Atrazine ... . 74 . 41 . 50 . 50 . 41 . 45 16. 72 54, 340 83 654 171 Hastings open pollinated: 948 222 With Atrazine. . 75 . 42 . 54 . 52 . 50 . 49 16. 93 55, 023 58 No Atrazine. .. . 75 . 40 . 52 . 61 . 63 . 64 17. 29 56, 095 65 863 222 * Pounds of water to produce 1 pound of grain or above- ground dry matter. 2 On irrigated plots rainfall and irrigation were con- sidered to be 100 percent effective and, therefore, added to moisture content changes, which introduces the un- defined error of soil evaporative loss and deep percolation. Because the fallowed plot was always directly subject to total incoming radiation, its changes were not considered to be indicative of evaporation from the soil surface under cropped conditions. 18 PROD. RES. RPT. 89, U.S. DEPARTMENT OF AGRICULTURE Corn in the Atrazine-treated plots did not show a significant increase in yield (table 9) over non- Atrazine-treated plots under equal moisture avail- ability nor more efficient use of water (table 8). A very significant increase in yield and, thus, water use efficiency by the hybrid was found, as deter- mined by pounds of water used per pound of grain yield. Differences in net production, as measured in tons of dry matter per acre (table 9), are only sigaiificant between irrigated and droughty conditions. Although the percentage of stomata open during any of these studies was not significantly affected by the Atrazine treatment, highly significant dif- ferences between activity on irrigated and non- irrigated plots were found. The change in sto- matal activity throughout the day and season was also very pronounced. In general, the greatest number of open stomata was found in early morn- ing (fig. 10). The decrease in number of visibly open stomata is probably associated with increased plant water stress bringing about hydroactive ’’ closure as the day progressed. The decrease in stomatal activity became more pronounced on the plastic-covered plots as the season progressed and the soil became increasingly drier. Figure 11 shows the increase in stomatal activity on June 27 as related to activity on June 26. A 2-inch rain occurred late in the afternoon of the 26th; therefore, the increased percentage of stomata open the following day is attributed to ’’ See page 18 of {26) for discu.ssion of this term. soil moisture recharge. A very low level of ac- tivity was noted the same day on the plastic- covered plots where soil moisture was not re- charged. Subsequent observations indicated that there was a permanent cessation of stomatal open- ing on plants in the plastic-covered plots. Not all of the progressive decrease in stomatal activity (fig. 12) as the season advanced could be Table 9. — Yields of co