A new porometer based upon the electrical

A New Porometer Based upon the Electrical Current Produced by Guard Cells Author(s): D. J. F. BOWLING Source: Journal of Experimental Botany, Vol. 40, No. 221 (December 1989), pp. 1407-1411 Published by: Oxford University Press Stable URL: http://www.jstor.org/stable/23691992 Accessed: 26-05-2015 11:34 UTC

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Journal

of Experimental

Vol.

Botany,

40, No.

221,

December

pp. 1407-1411,

1989

upon the Electrical by Guard Cells

A New Porometer

Based

Current Produced D. J. F. BOWLING of Plant

Department

26 June

Received

and

Soit Science,

University

St. Machar

of Aberdeen,

Drive,

Aberdeen

AB9

UK

2UD,

1989

ABSTRACT when Stomatal guard cells extrude protons An instrument opening. degree of stomatal The performance of the new of porometer.

the stomata has

been

open.

This

developed has been

gives rise to an electrical to measure this leaf surface

current current

which

is proportional to the is, in effect, a new type available diffusion

which

with that of a commercially porometer compared for all the species conductance was observed between leaf surface current and stomatal relationship in particular, its small over the diffusion porometer, It is concluded that the instrument has several advantages investigated. in the field. suitable for use and simplicity of operation, it making especially and

porometer

Key

words:

a close

Leaves,

stomata,

electrical

currents,

size

porometry.

INTRODUCTION There

are two basic

of measuring stomatal and porometry. In the direct

methods

opening, direct observation method stomatal aperture is measured under the microscope using a micrometer eyepiece. This gives a value for the aperture in micrometres but is tedious and timeat a time need to be

as at least 30 stomata

consuming measured to

obtain a statistically significant result. size of the pore provides only limited the Furthermore, information about the diffusion capacity of the stomata. Porometers

have

been

developed

to

overcome

the

disad-

vantages of the direct method. They measure gas transfer through the open stomata and can be divided into two types, viscous flow and diffusion. The firstviscous flow porometer was introduced in 1911 by Darwin and Pertz. In this device air was pulled through the leaf by reduced pressure created by a water column. A modification of their instrument incorporating a double manometer system was described by Gregory and Pearse

(1964) and Van Bavel, Nakayama, and Ehrler (1965). This instrument measures the diffusion of water vapour out of the stomatal pores using a relative humidity sensor. The humidity sensor is housed in a leaf cup which can be

clamped onto the leaf. The sensor and the air in the cup is dried to a pre-determined humidity level by passing dry air through the leaf cup and after that the time required for transpiration to bring the humidity up to some pre determined indication

point is measured.

of

transpiration

rate

This time-interval is an and

leaf

resistance

to

water vapour transfer. The device is calibrated using a plate with perforations of known diffusion resistance and a flow of water vapour saturated air. Versions suitable for use in the field have been developed by Byrne, Rose, and Slatyer (1970) and Stiles, Monteith, and Bull (1970). The latter instrument is commercially available as the Delta-T Devices Automatic Porometer Mark 3. Another diffusion is the Li-cor 1600 porometer commercially available

in 1934. This was followed in 1951 by the Wheatstone bridge porometer of Heath and Russell in which flow

which works on a steady-state null balance principle, Water vapour diffusion porometers have proved to be the most useful instruments available up to the present

needle

time and have largely replaced the viscous flow type. They have the advantage that, because they measure total gas exchange, i.e. leaf conductance, the results relate directly to the phenomenon being studied. Furthermore, measure ments by diffusion porometers can be used to calculate stomatal aperture, provided certain anatomical character

from a split air-stream is balanced between the leaf and a valve by adjustment with a single manometer. These instruments were relatively complicated and un-

wieldy and so this led to the development of more simple, portable, viscous flow porometers for use in the field (Bierhuizen, Slatyer, and Rose, 1965; Weatherley, 1966). The diffusion porometer was introduced by Wallihan ©

Oxford

University

Press

1989

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1408

New Porometer

Bowling—A

istics are known (Jarvis, Rose, and Begg, 1967). The main of diffusion porometers is that they are disadvantage in their construction, containing components complicated such

as

means

RH

thermistors, that

are

they

leaf

and

to build,

expensive

care in their operation. between

timers

sensors,

Also

porometer

and

pumps.

bulky

and

a good

seal

This require

is required

cup.

The purpose of this paper is to introduce a new type of The

porometer.

new

instrument

is

based

on

a

principle

entirely different from those of the viscous flow and diffusion types already described and avoids many of their It is based on the discovery that a small disadvantages. electrical current is produced in the leaf epidermis which is proportional to the stomatal aperture (Bowling, Edwards, and Gow, 1986). The current appears to be due to a flow of protons

from

emanating

the

cells

guard

but

not

Fig. 2. Circuit of the current-voltage

used in the instrument.

converter

from

the other cells in the epidermis (Edwards, Smith, and Bowling 1988). Evidence suggests that it is due to the activity of a proton efflux pump which plays a fundamen tal role in the mechanism of the stomatal opening (Zeiger, of this current should, therefore, 1983). Measurement an

provide

indirect

MATERIALS In

place diffusion current used

measure

AND

of

the

in some

moistened

used

cup

in

viscous

flow

and

was used to collect the pad surface. It consisted of a filter of the type automatic Medical Electronics, (Gilson pipettes

Villiers-le-Bel, A diameter.

France) sintered

a

aperture.

METHODS

conventional

porometers from the leaf

of piece electrode

of stomatal

contact

held

in a

tubing made contact

with

of tubing was held

length electrode

Ag/AgCl fitted inside

the

first

the bottom

with

tap water and 0-5 cm2 of contact area. It could

so

that

of suitable in

another

the

tip of the of the filter. The filter, to the leaf provided

adpressed be fitted into a clip made from a modified hair grip for 'hands-ofT of fixing to the leaf. A diagram the contact in Fig. 1. As long as the contact pad is shown pad was saturated it did not matter whether distilled water or tap water For not

was

used

the same

as

the currents

reason

the degree to be critical.

appear The current

picked

being measured of pad pressure

up from the contact converter. A simple

were

so

using a current-voltage and an operational was required amplifier (CA3140) together with a resistor an output of 1 mV nA_1 (1 Mohm) provided was connected to a digital panel meter (RS (Fig. 2). The output

and the completed in Components, Corby, England 258-041) strument housed in a hand held case (RS The instru 507-983). ment was powered batteries 591 by two 9 V rechargeable (RS pad was con cable and the

—silicone tubing -rubber tubing

2mm connector pin Fig.

1. Diagram porometer.

to show the construction

of the contact

1. The new porometer

complete

with its contact

pad and earthing

circuit

connected to a completed by a similar length of cable rod. A photograph of the complete instrument is earthing in Plate shown 1. brass

The the size

with dimensions instrument, of a hand-held multimeter.

of 15 x 8 x 5 cm, was about only one operational

It had

an on-off switch. The meter was set to read zero when control, the electrodes were disconnected. With the contact pad on the leaf and the brass earthing rod inserted into the soil close to the roots

of the plant the maximum was 2 V (2 ju.A). reading possible the reading 1 ¡j.A. There was a small rarely exceeded in the instrument case where the electrodes and compartment In practice cables

could

be stored

Calibration T

Mark

when

not in use.

of the instrument

3 diffusion

was

carried

(Delta-T porometer of four species, two

out against

a Delta

Devices,

Cambridge, two dicotyledonous,

Leaves England). at 18-24 °C monocotyledonous, growing in pots in a greenhouse were sampled at different times of the day for a period at random of 5 d. Readings from the same

-filter

sintered Ag/AgCI

rod.

small.

on the leaf did

pad was measured circuit was all that

and weighed less than 300 g. The contact 089) nected to the instrument by a length of flexible

Plate

pad used in the

from both porometers were taken consecutively part of the leaf with that from the Delta-T instrument taken first. Current was plotted being (jj.A cm-2) stomatal conductance was also against (cm s~ '). The instrument calibrated in one stomatal of the species against aperture A {Commelina communis). then the epidermis stripped

of 30 stomata measured apertures number of similar measurements were at random

at different

times

was taken and reading of the leaf and the

porometer from that

in one

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part under

taken

day.

A microscope. from leaves chosen

the

Bowling-—A New Porometer

1409

RESULTS The relationship between current and stomatal conduc tance for three species, Commelina communis, Tradescan tia virginiana and Pelargonium zonale is shown in Fig. 3. There was good correlation between the data (r=0-86) and the line fitted by regression analysis had a slope of 0-64. This indicated that the current could be used as a measure of stomatal

conductance

and that the relation

ship was linear. Also, all three species investigated ap peared to behave in a similar manner. Measurements were also carried out on Vicia faba and the results shown in Fig. 4. It can be seen that a similar relationship was obtained but with a slightly higher correlation coefficient (0-95) and a higher slope (0-69). The measurements in

Figs 3 and 4 were taken throughout the day between dawn and dusk and so the relationship between current and conductance appeared to be independent of light levels. In steady-state conditions, therefore, there appeared to be a linear relationship between leaf current and stomatal conductance. It was necessary to find out if this relation conditions were ship remained when environmental changing rapidly. A potted plant of Commelina was taken from the greenhouse at midday and placed in a dark cupboard in the laboratory for 3 h. Current and conduc tance measurements were made for 10 min before the plant was taken out of the dark and placed on the bench in bright sunlight. Measurements were continued with

readings being taken 2 min before those for current on the same part of the leaf. Different leaves of conductance

0.6

0.4

0.2

Stomatal Fig. 4. Calibration

of the porometer

1.0

0.8

conductance

1

cm s

for Vicia faba.

similar age were used for each pair of readings. The results are shown in Fig. 5. The scatter of the points appeared to be due to sampling error. The behaviour of the stomata varied markedly from place to place over the leaf surface and it was not easy to sample exactly the same location each time. The stomata began to open almost immediately after the plant was transferred to the light as shown by the rise in stomatal conductance.

Leaf

current

rose

at

the

approximately

same

rate indicating that it was just as responsive to the changing conditions as the stomatal conductance. The relationship between current and stomatal conduc tance during stomatal closure was also investigated. Figure 6 shows the results from an experiment in which two Commelina plants were left in sunlight at 20 °C in the laboratory.

Current

measurements

were

made

on

one

plant

and conductance

followed on the other. In order to reduce error the same part of the leaf was tested at 1 min sampling or 2 min intervals. After a short time the plants were transferred to a dark cupboard. There was rapid stomatal closure as shown by the fall in leaf conductance and it can be seen that the current responded

to stomatal

closure

equally rapidly. Alternating periods of light and dark produced results similar to those shown in Figs 5 and 6. The relationship between leaf surface current and sto matal aperture was investigated in Commelina. Current measurements were made at random on the leaves of a 0.2

0.4

0.6

Stomatal

conductance

0.8 cm s

1.0 -1

Fig. 3. Calibration of the porometer conductance, against stomatal Closed for three species. measured circles, by diffusion porometer, Commelina; closed triangles, Tradescantia\ closed squares, Pelargonium.

plant standing in the laboratory in sunlight. The epidermis was removed and stomatal immediately apertures measured. Figure 7 shows that there was a linear relation ship between current and aperture, with each increase by a micrometre in aperture being accompanied by an incre ment in current of approximately 01 /aA cm-2.

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1410

New

Bowling—A

Porometer light

40

50 Time minutes

measured by a diffusion porometer (open circles) and current output measured Fig. 5. Stomatal conductance the lower surface of the leaves of a plant of Commelina communis transferred from darkness to light.

10

12

14

16

18

20

22

by the new porometer

(closed

circles) of

24

Time minutes Fig. 6. The trend in current output (closed circles) and conductance Current and (open circles) for the lower leaf surface of Commelina. conductance were measured on two separate plants which were trans ferred from light to dark.

DISCUSSION The results establish that the leaf epidermis current pro a

vides

reliable

measure between

relationships tance

and

current

of the

and

stomatal

Therefore,

would

diffusion

as

opening.

Current

measurement sion

is very

a

of

sensitive

to

be

valid

that

as

the

gaseous during over

advantage

diffusion in that gaseous changes

in

the

diffu

through

temperature

changes of relative humidity in the porometer cup, whereas current is likely to change only by the direct effect of leaf temperature on the metabolism of the guard cells. The

new

porometer

provides

a

way

of

measuring

stomatal activity depending on neither mass flow nor diffusion. It avoids the problem of changing internal tissue resistance

because

inherent

with

viscous

flow

porometers

aperture

pm

Fig. 7. The relationship between current output and storaatal for the lower surface of leaves of Commelina communis.

aperture

activity.

conductance

has

Stomatal

linear

conduc

indicate

as

stomatal

measurement

of gaseous

stomatal

aperture to

appear

measure

The

opening. and

attribute of stomatal

current is a fundamental it

stomatal current

and,

it follows guard cell activity directly, it measures

stomatal which The

conductance measure

per

the

instrument

se

unlike

had

diffusion

of the

conductance several

other

porometers

leaf.

advantages

over

the

diffusion and viscous flow types. First, it was completely portable because of its simple design. Second, the instru ment could be set up and a reading obtained within 30 s without the need for any preliminary adjustment. There was, surprisingly, very little outside interference with the current

readings

and

there

was

usually

very

little

drift,

with the reading often reaching a steady value well within 30 s. Third, the instrument did not require to be calibrated before

each

series

of measurements.

Fourth,

by incorpora

tion of a potentiometer into the circuit it was possible to

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1411

New Porometer

Bowling—A make the instrument read directly in units of stomatal conductance.

its small size and simplicity in use. These features make it very suitable for measurements in the field. It is envisaged

It was interesting to find that the slope of the calibra tion curve was approximately the same for all the four species examined. Some more limited data were also obtained for Phaseolus vulgaris, Zea mays and Helianthus

that its underlying principle could provide a valuable new method for following the behaviour of stomata both in the laboratory and in the field.

annuus which indicated

that, they too, behaved in the same way. This consistency in behaviour occurred, pre sumably, because stomata occupy a surprisingly con

ACKNOWLEDGEMENTS The author wishes to thank Dr M. C. Edwards

for his

stant proportion of the surface area of the leaf. Meidner and Mansfield (1968) presented data which indicate that

help and encouragement during the early stages of this work and Mr Harry Cobb for assembling the instrument.

the pores of moderately open stomata of a large number of species occupy approximately 1% of the surface of the leaf. The curves in Figs 3, 4 and 6 did not pass through the origin after extrapolation indicating a residual current

LITERATURE

when the stomata were closed. This appeared to be due to electrical activity at the interface between the earthing electrode and the soil. In preliminary work platinum, copper, stainless steel and iron earthing electrodes were tried but they gave larger residual currents than the brass electrode which was eventually adopted. However, it is possible that there is also a component of the residual current which originates at the leaf surface. Edwards et al. (1988) observed that proton efflux by the guard cells of Commelina preceded stomatal opening. It would be expected, therefore, that on transferring leaves of Commelina to the light, there should be a lag between the rise in current and the increase in diffusive conduc tance as the stomata open. This lag is not apparent in the results shown in Fig. 5. The reason for this could be statistical as Edwards et al. (1988) looked at individual stomata whilst porometers sense large numbers of sto mata

at each

reading. Also the lag could have been disguised by the scatter of the results due to sampling error. On the other hand, as the diffusion porometer measures leaf conductance rather than stomatal conduc tance, it was conceivable risen

coincidently

that leaf conductance

as the leaf

surface

current

might have

rose

when

the

light was switched on. The experiments described by Figs 5 and 6 where there were rapid changes in stomatal aperture show neither an initial surge of current during stomatal opening (Fig. 5) nor an initial trough on stoma

tal closure (Fig. 6). This indicated that the relationship between conductivity and current observed under steady state conditions (Figs 3 and 4) held also during rapid changes of stomatal aperture.

To summarize, the strong points of the instrument are

CITED R.

Bierhuizen,

F.,

for

porometer

R.

Slatyer,

46, Botany, Experimental D. J. F., Edwards, currents

C.

Rose,

field

1965.

W.,

A

Journal

operation.

of

182-91. M.

Bowling,

Electrical

and

O.,

and

laboratory

and

C.,

at the leaf surface

N. A.

Gow,

of Commelina

1986.

R.,

communis

and their relationship to stomatal activity. Ibid. 37, 876-82. Byrne,

G.

F.,

C.

Rose,

diffusion

aspirated

and

F.,

D.

Pertz,

the aperture

estimating

F.

1911.

M.,

of stomata.

Society, Series B, 84, 136-54.

Edwards,

M.

Guard

cells

C.,

Smith,

extrude

G.

R.

Slatyer, Agricultural

porometer.

39-44.

Darwin,

and

W.,

and

N.,

protons

prior

1970.

An

Meteorology,

7,

O.,

a new

On

D.

Bowling, to

method

of

of the Royal

Proceedings

J. F.,

1988.

stomatal

and pH study using fluorescence microscopy Journal of Experimental 39, 1541-7. Botany, F. G., and H. 1934. Pearse, L., Gregory,

opening—A micro-electrodes. The

resistance

and its application to the study of stomatal move porometer ment. Proceedings Series B, 114,477-93. of the Royal Society, O. V. S., and Russell, Heath, J., 1951. The Wheatstone bridge Journal of Experimental porometer. P. J., Rose, C. W., and Begg,

2, 111-16. Botany, J. E., 1967. An experimen of viscous and diffusion resis

Jarvis,

tal and

theoretical

tances

to gas

comparison

flow

leaves. through amphistomatous Agricul 103-17. 4, Meteorology, T. A., 1968. and Meidner, Mansfield, H., Physiology of stomata. London. McGraw-Hill, tural

Stiles, W., resistance

Monteith, porometer

7,617-38. Van

C. H. Bavel, 1965. Measuring 40,

Physiology, E.

Wallihan, hygrometer

86-90.

Weatherley, Phytologist, Zeiger, Review

E.,

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T. A., 1970. A diffusive J. L., and Bull, for field use. Journal of Applied Ecology, Nakayama,

transpiration 535-40.

F. S., and Ehrler, of leaves. resistance

1964. Modification and F., for estimating relative stomatal P. E., 1966. 65, 376-87.

1983.

of Plant

The

A porometer

biology

Physiology,

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of an

aperture.

L., Plant

electric Ibid.

39,

for use in the field. New

of stomatal 34, 441-75.

use

W.

guard

cells.

Annual