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^ ^

t^eS.

RESEARCH

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

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.

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

WIND VELOCITY IN FEET/MINUTE

5' 8" »

1 390

1 r3^

COOLING COILS t

AND FAN 9"

„ 28" U

[3301

3901

130

1 240

LroJ

390

130

120

_100_

” 1

140

160 1

130

F~1

ISO

190

fn^

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"-

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

-H

DOOR

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.

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--

0. 25
13
495
46

850

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

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-

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

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.

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.

G/dm2/4 hr.
4.75

0.77

Percent

MP339 X MP311

12. 90

. 15

100

4. 14

.56

MP305 X T101_

12. 57

. 60

100

4. 34

1. 60

MP305 X MP307.

11.85

. 46

100

3.91

.94

Sorghum (29 days) :

RS-610

11. 75

1.30

100

3.85

2.2

NK-210

11. 50

1. 30

100

4.92

1.7

Amak-R

11. 00

.86

100

4. 34

1.3

Tomatoes (43 days) :

Marglobe

10.43

1.30

115

8. 24

1. 10

Rutgers

9.35

1.03

115

6.32

.48

Marion.. ..

9. 29

1.03

115

5. 97

. 51

Soybeans (36 days) :

Hampton

10. 27

2.50

6. 87

1.7

Hardee

8.90

1.03

4. 27

1. 4

Beinville. ....

8.83

.62

5.28

1.5

Cotton (56 days) :

Smooth Leaf Empire.. .

7. 15

.62

4. 75

. 59

Auburn

7. 03

.73

5. 05

.69

Empire ..

3.49

. 64

2.33

.83

Carolina Queen.

3. 11

.47

2.34

. 70

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

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

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-

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

AEl

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

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.

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

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

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.