Group Title: Research report (North Florida Research and Education Center (Quincy, Fla.))
Title: Physiological indicators of water stress vs. growth stages in soybean
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 Material Information
Title: Physiological indicators of water stress vs. growth stages in soybean
Series Title: Research report (North Florida Research and Education Center (Quincy, Fla.))
Physical Description: 17 p. : ill. ; 28 cm.
Language: English
Creator: Teare, I. D ( Iwan Dale ), 1931-
North Florida Research and Education Center (Quincy, Fla.)
Publisher: North Florida Research and Education Center
Place of Publication: Quincy Fla
Publication Date: 1992
Subject: Soybean   ( lcsh )
Soil moisture   ( lcsh )
Genre: non-fiction   ( marcgt )
Statement of Responsibility: I.D. Teare ... et al..
General Note: Caption title.
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Bibliographic ID: UF00066098
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 71172217

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I. D. Teare, R. Manam, R. P. Waldren, and D. G. Naylor

Soil water movement and soil water holding capacity as well as the factors

that create plant to air vapor pressure gradients are basically physical in

nature. The living plant however has built in survival mechanisms that can

adjust internal water deficits by using energy derived in the photosynthesis

process to regulate internal water gradients osmotically by active ion uptake

and/or synthesis or degradation to sugars and amino acids (Meyer and Boyer, 1981)

to cause opening and closing of stomata and plant tropisms.

Leaf water potential should be pr dictabl related with physiological

changes that occur in the plant during ntogeny ite E f drou ht stress.

Soybean [Glycine max (L.) Merr.] leaf wa er potea)Vil ha been rel ted to soil

water potential and has shown a linear re aticship where the inter pt exhibits

the greatest change in relation to time o in 8 Afift er po ential range

of -0 to -1.0 MPa (Brady et al., 1974). Since diurna 'e water potential

changes are much slower than diurnal leaf water potential changes, this means

that leaf water potential can be used to predict soil water potentials for a

specific soil and time of day or vice versa.

Stomatal resistance (SR) is suggested by Kramer (1969) as the simplest and

most direct approach to measuring the availability of water to a plant as it is

affected by the soil water potential (Sionit and Kramer, 1976). Brady et al.

(1975) found that soybean stomatal resistance of the adaxial surface of upper

canopy leaves increased rapidly when soil water potentials decrease to near -0.4

MPa (approximately -1.1 MPa leaf water potential).

North Florida Research and Education Center, Quincy FL 32351, Institute of Food

and Agricultural Sciences, Univ. of Fla., Gainesville, FL 32611. Research Report


Stomatal closure is the primary factor associated with inhibition of

photosynthesis during the initial phases of plant water deficit (Boyer, 1970;

Turner et al., 1978). Boyer (74) has shown that soybean stomatal diffusive

resistance increased beginning at a leaf water potential of -1.5 MPa, the point

at which initial effects on photosynthesis were observed. As leaf water

potential decreased stomatal resistance progressively increased. However, Thomas

et al. 1976 has shown different ranges of leaf water potential for stomatal

closure for cotton [Gossvpium hirsutum (L.)] grown in growth chambers and in the

field under well watered and water stressed treatments.

When plants are subjected to intense water stress almost complete

cessation of photosynthetic activity occurs (Ghorasky et al.,1971). Ultimately,

plant growth is reduced as :a result of restricted supply of photosynthate.

Turner et al. (1978) reported that soybean plants did not show a decrease in

photosynthesis rate until, leaf ater potential reached -1.5 to -1.7 MPa.

Leaf water potential was fund to be closely associated with the rate of

nitrogen fixation (Sprent, 1971; Patterson et. al, 1979; Huang et al. 1975).

Huang et al. also reported a close association of decreasing leaf water potential

and rates of photosynthesis and transpiration, but respiration remained

essentially.the same. Nitrate reductase activity (NRA) of field grown soybean

is sensitive to soil water decreases and has been negatively associated with

water stress (Manam, 1977) and may be related NRA changes corn (Zea mays L.) to

decreased enzyme synthesis at low water potential (Morilla et al., 1973).

Nitrate reductase changes were elastic and reversible, at least to a point.

Reduced light (Hageman and Flesher, 1960), and NADH (Losada, et al., 1965) have

also been responsible for reduced NRA.

Free-proline accumulation has been shown on intact plants in relation to

water deficiency by Routley (1966) and Waldren et al. (1974). Stewart (1973)

showed that proline accumulation during wilting was caused by decreased protein

synthesis and that carbohydrates prevent proline oxidation. He also showed that

proline synthesis, or a component thereof, is not sensitive to feedback


A comparison of soil and plant water status in relation to soybean

physiological growth stages that occur during ontogeny of soybean has been

needed. We have studied and characterized leaf water potential in relation with

soil water potential, stomatal closure, nitrate reductase activity, proline

levels, and photosynthesis in relation to soybean ontogeny. Presented here are

combined results of three studies conducted in twelve drainage lysimeters on

field grown soybean plants in conjunction with a rainout shelter to measure

various physiological indicators of water stress in relation to ontogeny and

growth stages under four water regimes.


Three research studies were conducted during the same period at the

Evapotranspiration Research Field 14 km south of Manhattan, Kansas, in twelve

drainage lysimeters where the alluvial, silt-loam soil was excavitated in layers

and replaced in drainage lysimeters to a uniform bulk density of 1.4 g/cm3 (Teare

et al.,1973). Soybean, cv. Calland, were planted 8 June 1972 and 23 May 1973 in

90 cm N-S rows and thinned to 14 plants per lysimeter (linear density of 20.2

plants per meter in the surrounding area) in association with a rainout shelter

(Teare et al, 1973). Additional Calland soybean were planted in the same manner

on the same day around the shelter to provide adequate soybean fetch of the same

physiological age. Rooting depth was measured in the surrounding area using the

trench technique (Mayaki et al., 1976). Mayaki found very little difference

between the rooting depths of irrigated vs. nonirrigated soybean throughout the

growing season.

Soil moisture treatments:

1972: Irrigated to field capacity (full profile, 180 cm) and allowed to dry

to 80, 60, 40, and 0 % available soil moisture (ASM). Then irrigated

to 100, 80, 60, and 20 % ASM. ASM was measured at weekly intervals

throughout the experiment and irrigation added when needed. The full

water profile to a depth of 180 cm at the beginning of the season,

delayed stress in the water deficit treatments.

1973: The procedure was changed in 1973 by keeping the rainout shelter over

the drainage lysimeter until planting, at which time irrigation

amounts were added to bring the upper 60 cm of soil to field capacity

and irrigations, thereafter, were based upon the rooting depth

of soybean surrounding the rainout shelter (Mayaki et al. (1976), the

amount of water in the soil profile to that depth, and the water

stress treatment desired for each of the 12 drainage lysimeters.

Water treatments were allowed to dry to 60, 40, 20, and 0 % ASM, then

irrigated to 80, 60, 40, and 20 % ASM, respectively, whenever needed

as in 1972.

Irrigation dates for the various treatments are shown in table 1. In 1972,

drought stress occurred outside the rainout shelter during 3 weeks of hot, dry

weather in August. In 1973, rainfall prevented drought stress except under the


The water status of the soil (soil water potential) was estimated in the

drainage lysimeters with a combination of gravimetric sampling at 8 and 23 cm

depths (upper 30 cm), and with a neutron attenuation meter (Troxler Model 2601)

at 15-cm intervals from 30 to 152 cm. Soil bulk density was 1.4 g cm"3. Soil

water potential in the soil profile containing roots was determined from soil

moisture release curves for each depth increment and averaged. Field capacity

was taken as 39 % moisture by volume and permanent wilting point as 13.4 % from

the soil moisture release curves and used to calculated ASM for the soil contain


Soybean leaf water potential was measured on the top, fully expanded

central trifoliolate from 1100 to 1200 h using a pressure chamber (Brady et al.,

1974) by inserting the petiole with trifoliolates in the chamber and exerting air

pressure until the xylem sap appeared at the free cut end of the petiole. An

average of two pressures was used as the leaf water potential for each lysimeter.

Stomatal resistance was measured on the adaxial surface of the top fully

expanded central trifoliolate leaf from 1000 to 1100 h with a diffusion porometer

(Kanemasu et al., 1969). Three samples were taken per lysimeter and averaged.

Photosynthesis measurements were taken on fully expanded upper trifoliolate

leaves at 1100 to 1200 h with the portable field technique using 14CO2 described

by Naylor and Teare (1974). Two leaf disc samples (0.35 cm in diameter) were

taken from two leaves for each lysimeter.

Nitrate reductase activity was measured using the technique of Jaworski

(1971) described by Manam et al. (1977) on the top, most fully expanded central

trifoliolate leaf, well exposed to sunlight, and sampled at 1000 to 1100 h each

day throughout the growing season. Samples were twelve 0.35 cm leaf discs from

two leaves of two plants for each lysimeter.

Proline levels were determined colorimetrically as described by Bates et

al. (1974) on fully expanded, upper trifoliolate leaves. Leaf punches were used

to eliminate differences in fresh weight due to water content. Sampling times

were from 1000 to 1400 h. Samples were 0.5 g of leaf material repeated four

times for each lysimeter.

Physiological stages of soybean development are described according to Fehr

and Caveness (1977).


S Soil vs. Leaf Water Potential

The relationships of leaf water potential vs. soil water potential for 1972

and 1973 are shown in figure 1 for ten growth stages. Starting the experiment

at field capacity for the full profile in 1972 was unfortunate for leaf water

potentials did not increase significantly until 19 Aug. The 1973 soil water

applications were calculated and applied only to the estimated rooting depth and

water stress was observed much earlier. Our 1973 data at V5.6 (R2=0.96), V6.4

(R2=0.95), R3.5 (R2=0.93), R3.9 (R2=0.80), R4.5 (R2=0.95) from 1000 to 1100 h seem

to support the linear relationship (R2=0.68, 1300 to 1500 h) published by Brady

et al.(1974), but our 1973 curves for through R1.2 (R2=0.95), R2.4 (R2=0.86, and

R3.2 (R2=0.91) appear to be sigmoidal.

Stomatal Resistance vs. Leaf Water Potential:

Stomatal resistance (SR) has been suggested as the simplest and direct

approach to measuring availability of soil water, however our soybean did not act

simply, figure 2. At R1.4 and R2.4, soybean stomata began closing after -2.0 MPa

leaf water potential, but before 2.4 and 2.7, respectively. At R3.6 stomata

began to close after -2.8 MPa and were closed at -3.5 MPa, at R3.9 and 4.5

stomata began to close after -2.0 and -2.2 MPa, respectively, and were closed at

at -3.0 MPa. This may indicate that growth stage and/or rewatering cycles

(Farquhar, 1982) are influencing stomatal closure. Brady et al. (1975) reported

that soybean stomata in the vegetative growth stage closed at -0.4 MPa soil water

potential or -1.2 MPa leaf water potential (approximated from Figure 1, Brady et

al.,1974) from 1 P.M. to 3 P.M. Zur, et al. (1983) determined the threshold

leaf water potential actuating the stomatal feedback mechanism in the range of -

1.6 to -2.0 MPa. Our data indicate soybean stomata close after -2.0 MPa.

Photosynthesis vs. Leaf Water Potential

Since photosynthesis is inversely related to stomatal closure,

photosynthesis should decrease as leaf water potential decreases. Although we

have comparisons for only three reproductive growth stages (10 and 17 Aug

combined) in 1973 (figure 3), they support the findings of Boyer (1970) and

Turner et al.(1978) that soybean photosynthesis decreased at -1.4 to -1.6 MPa and

-1.6 MPa, respectively. The major significance between growth stages that we

observed is that the intercept changes in relation to each growth stage. The

slopes of the R1.2 and R2.4 curves are not significantly different, but both are

different from the R3.5 to R3.9 (combined) stage of growth.

Nitrate Reductase Activity vs. Leaf Water Potential

Nitrate reductase activity (NRA) in soybean began to decrease when leaf

water potential decreased below -0.7 MPa (figure 4). Characteristic of the

findings of Harper et el. (1972) relating NRA to season, the highest NRA values

(approx. 8 u mole NO2 g' FW h-1) occurred at growth stages V5.6, R1.2, R1.4, and

R2.4-R3.0 From V5.6 to R3.0, the decrease of NRA in relation to water stress

seems to be linear. At stage R3.4 to R4.5, the curve became more curvilinear.

At stage R3.9 the highest unstressed NRA value is 6 u mole NO2/g-1FW h-1 and at

stage R4.5 the highest unstressed value is 4.8 u mole NO2/g9'FW h-1, and at R5.5-

R6.2 the highest unstressed value is 2 u mole NO2/g-1FW h1.

Proline vs. Leaf Water Potential

Figure 5 shows that free proline did not accumulate in the 1972 water

S stressed treatments, but did accumulate in 1973 in the field at -3.0 MPa leaf

water potential at growth stage R4.5. Proline did not accumulate at stage R3.2

or R3.9 when leaf water potential decreased to -3.5 and -3.3 MPa, respectively.

Severe wilting and considerable decrease in growth was observed to occur before

3.0 MPa as well as changes in leaf water potential, stomatal resistance, nitrate

reductase activity, and photosynthesis.


The contributions of E. Brown, Senior Lab Technician; North Fla. Res. and

Educ. Ctr., Univ. of Fla., Quincy, FL for computer processing and illustrating

the data are gratefully acknowledged.


1. Bates, L. S., Waldren, R. P. and I. D. Teare. 1973. Rapid determination

of free proline for water-stress studies. Plant and Soil 39:205-207.

2. Boyer, J. S. 1970.. Differing sensitivity of photosynthesis to low water

potentials in corn and soybean. P1. Physiol. 46:236-239.

3. Boyer, J.S. 1974. Water transport in plants: mechanism of apparent

changes in resistance during absorption. Planta 117:187-207.

4. Brady, R. A., W. L. Powers, L. R. Stone, and S. M. Goltz. 1974. The

relation between soybean leaf water potential to soil water potential.

Agron. J. 66:795-798.

5. Brady, R. A., S. M. Goltz, W. L. Powers, and E. T. Kanemasu. 1975. The

relation of soil water potential to stomatal resistance of soybeans.

Agron. J. 67:97-99.

6. Farquhar, G. D.,and T, D, Sharkey. 1982. Stomatal conductance and

photosynthesis. Ann. Rev. Plant Physiol. 33:317-345.

7. Fehr and Caviness. 1977. Stages of soybean development. SR80, Iowa State


8. Gorasky, S. R., J. W. Pendleton, D. B. Peters, J. F. Boyer, and J. E.

Beverlein. 1971. Internal water stress and apparent photosynthesis with

soybean differing in pubescence. Agron. J. 63:674-676.

9. Hageman, R. H., and D. Flesher. 1960. Nitrate reductase activity in corn

seedlings as affected by light and nitrate content of nutrient media.

Plant Physiol. 35:700-708.

10. Harper, J. E., J. C. Nicholas, and R. H. Hageman. 1972. Seasonal and

canopy variation in nitrate reductase activity of soybean [Glycine max (L.)

Merr.]. Crop Sci. 12:382-386.

11. Huang, C-Y. J. S. Boyer, and L. N. Vanderhoff. 1975. Limitation of

acetylene reduction (nitrogen fixation) by photosynthesis in soybean having

low water potentials. Plant Physiol. 56:228-232.

12. Jaworski, E. G. 1971. Nitrate reductase assay in intact plant tissue.

Biochem, and Biophys. Res. Commun. 43:1274-1279.

13. Kramer, P.J. 1969 Plant and water relationships: A modern synthesis.

McGraw Hill, New York.

14. Kanemasu, E. T., C. B. Tanner, and G. W. Thurtell. 1969. The design,

calibration, and field use of a stomatal diffusion porometer. P1.

Physiol. 44:881-885.

15. Losada, M. A. Paneque, J. M. Ramirez, and F. F. De Campo. 1965. Reduction

in nitrate ammonia in chloroplasts. In Non-heme iron proteins, p. 211-218.

A. San Pietro (ed.) Antioch Press, Yellow Springs, Ohio.

16. Manam, R. 1977. Nitrate reductase activity of soybean in relation to other

indicators of water stress. Oyton 35:189-194.

17. Mayaki, W. C., I. D. Teare, and L. R. Stone. 1976. Top and root growth

of irrigated and nonirrigated soybean. Crop Sci. 16:92-94.

18. Meyer, R.F., and J.S. Boyer. 1981. Osmoregulation, solute distribution,

and growth in soybean seedlings having low water potentials. Planta


19. Morilla, C.A., J.S. Boyer, and R.H. Hageman. Nitrate reductase activity

and polyribosomal content of corn (Zea Mays L.) having low leaf water

potentials, P1. Physiol. 51:817-824.

20. Naylor, D. G. and I. D. Teare. 1974. An improved rapid field method for

measuring photosynthesis with 14CO2. Agron. J. 67:404-406.

21. Patterson, R. P., C. D. Raper, amd H. D. Gross. 1979. Growth and specific

nodule activity of soybean during application and recovery of a leaf

moisture stress. Pi. Physiol. 64:551-556.

22. Routley, D. G. 1966. Proline accumulation in wilted ladino clover leaves.

Crop Sci. 6:358-361.

23. Sionit, N., and P.J. Kramer. 1976. Water potential and stomatal

resistance of sunflower and soybean subjected to water stress during

various growth stages. Plant. Physiol. 58:537-540

24. Sprent, J. I. 1972. Effect of water stress on nitrogen-fixing root

nodules. IV. Effects on the whole plant of Vicia faba and Glycine max.

New Phytol. 71:603-611.

25. Stewart, C. R. 1973. The effect of wilting on proline metabolism in

excised bean leaves in the dark. Plant Physiol. 51:508-511.

26. Teare, I. D., H. Schimmelpfennig, and R. P. Waldren. 1973. Rainout

shelter and drainage lysimeters to quantitatively measure drought stress.

Agron. J. 65:544-547.

27. Thomas, J.C., K.W. Brown, and W.R. Jordon. 1976. Stomatal response to

leaf water potential as affected by preconditioning water stress in the

field. Agron. J. 68:706-708.

28. Turner, N.C., J.E Begg, H.M. Rawson, S.D. English, and A.B. Hearn. 1978.

Agronomic and physiological responses of soybean and sorghum crops to water

deficits III. Components of leaf water potential, leaf conductance, CO2

photosynthesis, and adaption to water deficits. Aust. J. P1 Physiol.


29. Waldren, R. P., I.D. Teare, and S.W. Ehler. 1974. Changes in free proline

concentrations in sorghum and soybean plants under field conditions. Crop

Sci. 14:447-450.

30. Zur, B., J. W. Jones, and K. J. Boote. 1983. Field evaluation of a

water relations model for soybean. I. Validity of some basic assumptions.

Agron. J. 75:272-280.

S Table 1. Soil moisture treatments in relation to irrigation application dates

for drainage lysimeters in conjunction with rainout shelter to study

physiological indicators of moisture stress in 1972 and 1973.

Treatments Irrigation dates
ASM 1972 1973

80% 22, 29 Aug 10, 17, 31 July; 7, 24, 28 Aug

60% 22, 29 Aug 17, 31, July; 28 Aug

40% 8, 15 Aug 17 July; 7, 28 Aug

20% Not irrigated 17, 31 July


Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Leaf water potential of fully expanded, upper triofoliolate soybean
leaves in relation to soil water potential and soybean ontogeny at
different levels of water stress.

Stomatal resistance of fully expanded upper trifoliolate leaves in
relation to leaf water potential and soybean ontogeny at different
levels of water stress.

Photosynthesis of fully expanded, upper triofoliolate leaves (clear
sunny days) vs. leaf water potential and ontogeny of soybean at
different levels of water stress.

Nitrate reductase activity vs. leaf water potential and ontogeny in
fully expanded, upper trifoliolate soybean leaves at different
levels of water stress.

Free proline concentration vs. leaf water potential and ontogeny in
fully expanded, upper soybean trifoliolate leaves at different
levels of water stress.

0 0.6 1 15 2 2.5

3 3.5 0 0.5 1 1.5 2 2.5 3 3.5 4


Figure 1. Leaf water potential of fully expanded, upper triofoliolate soybean

leaves in relation to soil water potential and soybean ontogeny at

different levels of water stress.















0 0.5 1 1.5 2 2.5

3 3.5

0 0.5 1 1.5 2 2.5

3 3.5 4

Stomatal resistance of fully expanded upper trifoliolate leaves in

relation to leaf water potential and soybean ontogeny at different

levels of water stress.

- 19 JULY.72 K-- 26 JULY 72
R1.2 R1.4
-- 6-1UtY-73 o 18-JULY 73

26 JULY 72 -- 11 AUG 72
R2.4 P R3.6
----27UY73 3A-AUG-73


S19 AUG 72 20 AUG 72
R4.0 R4.5
l -7-AUG-73 o 22-AUG73

SI I t 1 1 1 1 1 1

Figure 2.


S-- 16 JULY 73 R 1.2
Y = 22.5 0.51X
o -

** *

CO 2


0 IR 0.84

-0 .*

SY = 40.9 1.03X.

tc 10
= R 0.84

0 0.5 1 1.5 2 2.5 3 3.5 4


Figure 3. Photosynthesis of fully expanded, upper tri foliolate leaves (clear

sunny days) vs. leaf water potential and ontogeny of soybean at

different levels of water stress.
RF -0.78

0 0.5 1 1.5 2 2.6 3 3.5 4

-- 10 JULY 72 V5.6














-9 19 JULY 72 R1.2


2 2
R 0.94 R 0.95

i i I I i I i

-- 26 JULY 72 R1.4 ; 7 AUG 72 R3.0
-e- 18 JULY 73 R1.4 --- 27 JULY 73 R2.4

R 0.91 R- 0.94

11 AUG 72 R3.4 -- 19 AUG 72 R3.9
-- 10 AUG 73 R3.5 -- 17 AUG 73 .R3.9

2 2
R 0.83 R 0.81

S 1 I I I I I i 1

- 26 AUG 72


-- 22 AUG 73 R4.5


- 5 SEPT 72 R6.2
-e- 10 SEPT 73 R5.5

0 0.5 1 15 2 2.5 3 3.5 0 0.5 1 1.5 .2 .2.5 3 3
'LEAF (-MPa) 'LEAF (-MPa)

figuree 4. Nitrate reductase activity vs. leaf water potential and ontogeny in

fully expanded, upper trifoliolate soybean leaves at different

levels of water stress.

,.5 4

- 29 JUNE 73 V5.6

-- 16 JULY 73 R1.2


I t r



I I -











0 0.5 1 1.5 2 2.5

3.5 0

.0.5 1 1.5 2 2.5

3 3.5 4

Figure 5. Free proline concentration vs. leaf water potential and ontogeny in

fully expanded, upper soybean trifoliolate leaves at different

levels of water stress.


-- 17 JULY 73 R12 27 JULY 73 R2.4




0 p ,oop oo o 09 9 ,

-- 3 AUG 73 R3.2 10 AUG 72 R3.4

12 0 10-UG-73----R3.5



0 --o O 00 VD, Xry C ,o

)K 19 AUG 72 R3.9 26 AUG 72 R4.5

12 ---17-AG-73--R9 0 22-AUG-73---R4.5



I I I" 0




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