CHAPTER V*

PROTEIN ALTERATIONS IN TALL FESCUE IN RESPONSE TO DROUGHT

STRESS AND ABSCISIC ACID

* This chapter will be submitted to Crop Sci.

135

ABSTRACT

Drought stress is a major factor limiting growth of

turfgrass. The study investigated physiological changes

associated with synthesis of dehydrin and heat shock protein

synthesis in response to drought stress and effects of

Abscisic acid (ABA) application on drought tolerance in two

cultivars, 'Southeast' and 'Rebel Jr.' of tall fescue

(Festuca arundinacea L.). Grasses were subjected to three

treatments in growth chambers: well-watered control, drought

stress, and drought stress following ABA treatment. Turf

quality and leaf relative water content (RWC) decreased and

electrolyte leakage (EL) increased during drought stress for

both cultivars. The ABA-treated plants maintained higher

turf quality and RWC and lower EL than untreated plants

under drought stress conditions. Levels of 20- and 29-kDa

polypeptides increased during drought stress, and a 35-kDa

polypeptide was noted in both cultivars only when subjected

to drought stress either with or without ABA treatment.

Immunoblot analysis indicated that dehydrin-like proteins of

about 23-60 kDa were induced by progressive water deficit in

both cultivars. The 53-kDa and 40-kDa dehydrins accumulated

only at 10 d of drought-stressed plants in Southeast and

Rebel Jr., respectively. The 23- and 27-kDa dehydrins were

present at 10 d in drought-stressed and ABA-treated plants

at 10 d of both cultivars, but were more pronounced in the

drought-stressed plants without ABA application. A

cytosolic-heat shock protein (Hsc 70) was detected in all

136

treatments including well-watered plants of both cultivars,

but its levels were higher in drought-stressed and ABA-

treated plants. No single dehydrin was induced by ABA

treatment under drought stress, suggesting that ABA-enhanced

drought tolerance in tall fescue might not be related to

regulation of dehydrin during drought stress.

137

ABBREVIATIONS:

RWC, relative water content; ABA, abscisic acid; EL,

electrolyte leakage; PMSF, phenylmethylsulfonyl fluoride;

SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel

electrophoresis; Hsc, heat shock cognates

138

Numerous physiological and biochemical changes occur in

response to drought stress in various plant species. The

alteration of protein synthesis or degradation is one of the

fundamental metabolic processes that may influence drought

tolerance (Chendler and Robertson, 1994; Ouvrard et al.,

1996). Both quantitative and qualitative changes of proteins

are detected during water stress (Riccardi et al., 1998).

Evidence is increasing in favor of the relationship between

accumulation of drought-induced protein and physiological

adaptations to water limitation (Bray, 1993; Han and

Kermode, 1996; Riccardi et al., 1998).

One family of proteins that accumulate in a wide range

of species under dehydration stress are dehydrin proteins

[late embryogenesis abundant (LEA) D11 family], which range

in size from 9 to 200 kDa (Close, 1996). They are

hydrophilic, and heat stable and may protect other proteins

and help maintain physiological integrity of cells (Bray,

1993; Close et al., 1993). Arora et ale (1998) reported that

the accumulation of dehydrin proteins (25-60 kDa) in

geranium (Pelargonium x hortorum) leaf induced by water

stress was associated with increased heat tolerance. Drought

regulation of dehydrin gene expression has been found in

both drought-tolerant and drought-susceptible cultivars

(Wood and Goldsbrough, 1997; Cellier et al., 1998).

Dehydrin synthesis in response to abscisic acid (ABA)

also has been observed (Cellier et al., 1998; Giordani et

aI, 1999). A correlation occurred between dehydrin gene

139

transcript level and endogenous ABA content in maize (Zea

mays L.) (Mao et al., 1995). Exogenous application of ABA to

several plant species induced a number of dehydrin-like

proteins during dehydration or under drought stress

(Bradford and Chandler, 1992; Han and Kermode, 1996; Pelah

et al., 1997). But the pathways of expression of dehydrins

were found to be ABA-independent or only dehydration-

dependent (Espelund et al., 1995; Whitsitt et al., 1997).

The family of heat-shock proteins, such as Hs70, is

also stress related proteins (Vierling, 1991). Hs70 is an

evolutionarily conserved family of 70-kDa proteins, which

are considered as molecular chaperones (Anderson et al.,

1994; Ellis and Van der Vies, 1991.) and presumed to playa

role in protein folding and transport (Giorini and Galili et

al., 1991). Heat shock cognates, such as cytosolic Hsc 70,

are constitutive and not induced strongly by heat shock

(Lindquist and Craig, 1988). They also accumulate during

water stress (Arora et al., 1998).

Although the alterations of proteins such as dehydrins

under stress conditions have been investigated widely in

many plant species, reports on identifying and understanding

drought stress-related proteins in cool-season turfgrasses

are limited. For example, little is known about the pattern

of changes in accumulations of dehydrin and Hsc 70 under

drought stress and the effects of ABA on their expressions

in cool-season turgfgrasses. Knowledge of protein

alterations under drought stress would help identify

140

physiological traits that could be incorporated into

breeding programs to improve drought tolerance of cool-

season turfgrasses. Therefore, the objective of this study

was to determine changes in soluble protein, dehydrin, and

Hsc 70 in response to drought stress in two tall fescue

cultivars and a possible role of ABA in drought tolerance.

141

MATERIALS AND METHODS

Plant materials and growth conditions

Seeds of tall fescue (cv. Southeast and Rebel Jr.) were

sown in pots (20 cm in diameter and 23 cm deep) containing

topsoil (fine, montmorillontic, mesic, aquic arquidolls) and

a starter fertilizer with N-P-K (9-13-7) (Voluntary

Purchasing Groups Inc. Bonham, TX) in a greenhouse at Kansas

State University. After germination, plants were fertilized

with liquid N-P-K fertilizer (20-10-20) (Scotts-Sierra

Horticultural Products Compo Marysville, OH) on alternate

days. Plants were grown in the greenhouse for 60 d and then

transferred to growth chambers with a temperature regime of

20°C/15°C (day/night), relative humidity of 65 %, a 14-h

photoperiod, and a photosynthetically active radiation of

400 ~mol m-2S-l. Plants were well-watered and maintained at

the above conditions in the growth chambers for 15 d to

allow adaptation before drought stress was imposed.

Water stress and ABA treatments

Water stress was imposed by withholding irrigation for

10 d from plants treated or untreated with ABA. Control

plants were maintained well watered by irrigating every

other day without ABA application. For ABA treatment, 40 mL

of ABA (100~M) solution were sprayed uniformly on foliage

using a spray bottle at 10:00 am once daily for a 3 d

142

before drought stress initiated. Plants untreated with ABA

were sprayed with 40 mL of deionized water. Treatments were

arranged in a completely randomized design with four

replicates.

Measurements of physiological parameters

Turf quality was rated visually as an integral of grass

color, uniformity, and density on the scale of 0 (the worst)

to 9 (The best) (Turgeon, 1999). The minimum acceptable

level was 6.

Leaf relative water content (RWC) was determined

according to the method of Barrs and Weatherley (1962) based

on the following calculation: RWC = (FW-DW)/(SW-DW)X100,

where FW is leaf fresh weight, DW is dry weight of leaves

after drying at 85°C for 3 d, and SW is the turgid weight of

leaves after soaking in water for 4 h at room temperature

(approximately 20°C) .

Electrolyte leakage (EL) of leaves was measured

according to the method of Blum and Ebercon (1981) and

Marcum (1998) with modifications. Leaves were excised and

cut into 2-cm segments. After being rinsed 3 times with

distilled deionized H20, 10-15 leaf segments were placed in

a test tube containing 10 mL distilled deionized H20. Test

tubes were shaken on a shaker for about 17-18 h, and the

initial conductivity (C1) was measured with a conductivity

meter (Model 32, Yellow Springs Instrument Inc., Yellow

143

Springs, Ohio). Leaf samples then were killed in an

autoclave at 121°C for 15 min, and the conductivity of

killed tissue was measured after tubes cooled down to room

temperature (C2). The relative electrolyte leakage was

calculated as (C1 / C2) *100.

Protein extraction

Total soluble protein was extracted from leaves

according to the method of Arora et al. (1992) with a few

modifications. Leaf tissue (0.5 g fresh weight) was ground

with liquid N2 to fine powder. Protein was extracted in a 2

mL of borate buffer (50 roM sodium borate, 50 roM ascorbic

acid, 1% ~-mercaptoethanol, 1 mM phenylmethylsulfonyl

fluoride, [PMSF], pH 9.0). Samples then were centrifuged at

26,000 g at 4°C for 1 h, and supernatant was collected.

Protein content was determined by the method of Bradford

(1976) .

SDS-PAGE and immunoblots

Samples for SDS-PAGE were prepared by the method of

Wetzel et al. (1989). The volume of extract contained equal

amount of protein (200 ~g) diluted to 1 mL with deionized

water. The diluted protein samples were precipitated by

adding 100 ~L of trichloroacetic acid (final concentration

of about 10%, v/v). The precipitate was collected by

144

centrifugation at 16,000 g for 15min at 4°C, washed with

acetone and dried. The pellet then was dissolved in 100 ~l

of SDS-PAGE sample buffer (65 mM Tris-HCl, 10 % glycerol, 2

% SDS, pH 6.8, 5 % ~-mercaptoethanol) (Laemmli, 1970).

Proteins were separated by discontinous SDS-PAGE with a

PROTEAN II electrophoresis unit (Bio-Rad, La Jolla, CA, USA)

using a 4 % stacking gel and 12.5 % running gel. Gels were

stained over night with Colloidal Coomassie Blue G-250

(Neuhoff et al., 1998).

For immunoblotting, 15 ~g SDS unstained gels were

electroblotted onto nitrocellulose membrane with 0.45-~m

pores (Mini Trans-Blot Electrophoretic Transfer Cell, Bio-

Rad) for 1.25 h at 100V in Towbin buffer (Towbin et al.,

1979). Gel membranes were blocked with 3 % (w/v) nonfat milk

in Tris-buffer saline plus Tween 20 (TBST). The membranes

were probed overnight with 1:1000 dilution of antidehydrin

antiserum (kindly provided by Dr. T. J. Close, Univ. of

California, Riverside, CA, USA) and with 1:1000 dilution of

antiheat-shock protein (anti-Hsc70) polyclonal antibody

(StressGen Biotech. Corp. Victoria, Canada). After three

washes in TBST, membranes were incubated with the secondary

goat antirabbit IgG (dilution 1:10000) conjugated with

alkaline phosphatase for 1 h at room temperature. The bands

were detected with a premixed BCIP/NBT substrate solution

(Sigma, St. Louis, MO, USA). The SDS-PAGE and immunoblot

analyses of protein were repeated three times.

145

Analysis of variance was based on the general linear

model procedure of the Statistics Analysis System (SAS) (SAS

Institute Inc., Cary, NC). Effects of drought stress and ABA

treatment were analyzed by comparing responses with their

respective controls at a given time of treatment. The least

significance difference (LSD) at a 0.05 probability level

was used to detect the differences between treatment means.

146

RESULTS

Physiological responses to drought stress

The turf quality rating decreased to below 6 at 10 d of

drought stress in both cultivars, but plants treated with

ABA maintained higher quality than untreated plants (Table

1). Leaf RWC decreased during drought stress. For Southeast,

RWCs of ABA-treated plants were about 28 % and 12 % higher

than those of untreated plants at 5 and 10 d of stress,

respectively. For Rebel Jr., ABA-treated plants had 27 % and

15 % higher RWCs than untreated plants at 5 and 10 d,

respectively. The EL of both cultivars increased during

drought stress (Table 1). For Southeast, ABA-treated plants

had 4 % lower EL at 5 d and 19 % lower EL at 10 d than

untreated plants. For Rebel Jr., the ABA-treated plants had

29 % and 12 % lower ELs than untreated plants at 5 and 10 d,

respectively.

For Southeast, total soluble protein content increased

at 10 d in drought-stressed and ABA-treated plants, but ABA-

treated plants had protein content similar to that of

untreated plants. For Rebel Jr., protein content decreased

in drought-stressed plants at 10 d, and ABA-treated plants

had higher protein content than untreated plants (Table 1) .

Protein changes

The SDS-PAGE analysis of soluble protein from leaves

revealed that several polypeptides of 20-, 29- and 35-kDa

147

accumulated or their band intensities increased during

drought stress in both cultivars (Fig. lA and IB) .

Specifically, an accumulation of the 35-kDa polypeptide was

noted only in drought-stressed plants with or without ABA

treatment for both cultivars. The level of the 29-kDa

polypeptide was higher in drought-stressed and ABA-treated

plants than in well-watered plants for both cultivars. The

20-kDa polypeptide was barely detected in well-watered

plants but clearly present in drought-stressed plants with

or without ABA treatment for both cultivars. For Southeast,

its intensity was higher in ABA-treated plants than

untreated plants.

Immunoblots indicated that dehydrins of about 23-, 27-,

40-, 42-, 48-, 53-, and 60-kDa were induced by drought

stress, and the accumulations of all them generally were

increased with progressive water deficit in both cultivars

(Fig. 2A and 2B). The 23- and 27-kDa dehydrins strongly

accumulated in drought-stressed plants with or without ABA

treatment at 10 d in both cultivars, but especially in the

drought-stressed plants without ABA treatment. The 23-kDa

was only slightly visible for Southeast and was not detected

in Rebel Jr. for ABA-treated plants at 5 d of stress.

Dehydrins of 53 kDa and a 40-kDa accumulated only at 10 d of

drought stress with or without ABA treatment for Southeast

and Rebel Jr., respectively.

The immunoblot analysis showed that Hsc 70 was present

in all treatments for both cultivars including well-watered

148

plants, but the levels were higher in drought-stressed

plants with or without ABA treatment than in well-watered

plants, especially at 10 d of treatment (Fig. 3A and 3B) .

149

DISCUSSION

Turf quality and RWC decreased and EL increased with

progressive drying to a similar extent for both cultivars,

suggesting that growth, water relations, and cell membrane

permeability for both Southeast and Rebel Jr. suffered from

drought injury, although tall fescue is considered to be

able to avoid drought by developing a deep root system

(Sheffer et al., 1987). The ABA treatment enhanced drought

tolerance, as indicated by higher RWC and lower EL in both

cultivars treated with ABA than those without ABA. The

mechanisms by which drought stress and exogenous ABA affect

drought tolerance are numerous and complex, which may

include the induction of some polypeptides and dehydrin-like

proteins (Pruvot et al., 1996; Han et al., 1997).

Drought-induced polypeptides have been observed in many

studies (Bewley et al., 1983; Perez-Molphe-Balch et aI,

1996; Arora et al., 1998; Riccardi et al., 1998), and they

are assumed to playa role in water stress tolerance. Our

results in tall fescue also indicated that accumulation of a

35-kDa polypeptide was responsive to drought stress, and the

other two polypeptides of 20 and 29-kDa were intensified in

drought-stressed plants for both cultivars. But no

relationship between protein changes and drought tolerance

was apparent in this study, similar to the results reported

by Perez-Molphe-Balch et al. (1996). However, ABA-treated

plants of Southeast had a higher level of a 20-kDa

polypeptide than untreated plants at 10 d of stress,

150

suggesting that this protein may play a role in ABA-enhanced

drought tolerance. Because ABA-mediated protein changes were

found only in one cultivar, the functional role of drought-

responsive proteins and the regulation of ABA in tall fescue

need to be investigated further. Pruvot et ale (1996)

reported that a drought-induced increase in the synthesis of

a chloroplastic protein of 34-kDa likely was regulated by

ABA application.

Recently, drought-induced dehydrin proteins have been

found in many species (Wechsberg et al., 1994; Han et al.,

1997; Pelah et al., 1997; Arora et al., 1998). Drought-

induced expressions of dehydrin genes were identified in

both drought-tolerant and-sensitive cultivars (Wood and

Goldsbrough, 1997) or to a higher level in tolerant

cultivars (Labhilili et al., 1995) or in sensitive cultivars

(Volaire et al., 1998). In this study, dehydrin-like

proteins ranging from 23 to 60 kDa, especially 23- and 27-

kDa, present, and their intensities increased with

progressive water deficit when leaf RWC dropped to about 47

% in drought-stressed plants in both cultivars of tall

fescue. Also, a 53- and 40-kDa dehydrins accumulated only at

10 d of stress in drought-stressed plants in Southeast and

Rebel Jr., respectively. The results indicated that the

accumulation of dehydrin was induced strongly by severe

drought stress. Wechsberg et ale (1994) also found that the

accumulation of 18-, 28-, 31-kDa dehydrin-like proteins in

the seeds of Ranunculus sceleratus depended on stages of

151

water stress. Accumulation of dehydrin protein could protect

cells from further dehydration during drought stress (Han

and Kermode, 1996; Cellier et al., 1998).

Dehydrins have been found to be induced by ABA in other

species (Cellier et al., 1998; Giordani et al., 1999)

However, in the present study, no specific dehydrins were

induced by an application of 100 ~M ABA. Dehydrins of 53-kDa

and 40-kDa did not occur in ABA-treated plants when leaf RWC

was 88-90 %, but appeared when leaf RWC dropped to about 60

%. Yamaguchi-Shinozaki and Shinozaki (1994) reported that

ABA-independent and the ABA-dependent signal transduction

pathways might exist between stress and dehydrin gene

expression. Giordani et al. (1999) suggested that these two

pathways of regulating dehydrin transcript accumulation

might have cumulative effects. Our results suggested that

the enhancement of drought tolerance by ABA application, as

manifested by higher turf quality during drought stress, was

not related to the induction of dehydrins in tall fescue.

Other mechanisms could be involved in this positive effect

of ABA, including the regulation of stomatal closure for

water conservation (Davis, 1978), and membrane integrity

(Rajasekaran and Blake, 1999), because ABA-treated plants

maintained higher RWC and lower EL.

The Hsc70 can accumulate under water stress (Arora et

a., 1998) and cold acclimation (Wisniewski et al., 1996).

Our results of immunoblots indicated that Hsc 70 was not

inducible under drought stress, because it also accumulated

152

in well-watered plants. But the level of Hsc 70 was higher

in drought-stressed plants than well-watered plants. Arora

et ale (1998) found that Hsc 70 could be detected only in

water-stressed plants. Therefore, the accumulation of Hsc 70

under drought stress may vary in plant species.

In summary, drought stress induced changes in protein

synthesis in tall fescue. Accumulations of dehydrins were

detected in drought-stressed and ABA-treated plants of both

cultivars, which could protect plants from further

dehydration damage. The increased drought tolerance

resulting from ABA application might not be related to

accumulation of higher level of dehydrins or to a unique

dehydrin protein.

153

REFERENCES

Anderson, J.V., and Q. Li, D.W. Haskell, and C.L. Guy. 1994.

Structural organization of spinach endoplasmic

reticulum-70-kilodalton heat-shock cognate gene and

expression of 70-kilodalton heat-shock genes during

cold acclimation. Plant Physiol. 104: 1359-1370.

Arora, R., D.S. Pitchay, and B.C. Bearce. 1998. Water-

stress-induced heat tolerance in geranium leaf tissues:

A possible linkage through stress proteins? Physiol.

Plant. 103: 24-34.

Arora, R., M.E. Wisniewski, and R. Scorza. 1992. Cold

acclimation in genetically related (sibling) deciduous

and evergreen peach [Prunus persica (L.) Batsch]. I.

Seasonal changes in cold hardiness and polypeptides of

bark and xylem tissues. Plant Physiol. 99: 1562-1568.

Barrs, H.D., and P.E. Weatherley. 1962. A re-examination of

the relative turgidity techniques for estimating water

deficits in leaves. Aust. J. BioI. Sci. 15: 413-428.

Bewley, J.D., K.M. Larsen, and J.E.T. Papp. 1983. Water-

stress-induced changes in the pattern of protein

synthesis in maize seedling mesocotyls: A comparison

with the effects of heat shock. J. Exp. Bot. 34: 1126-

1133.Blum, A., and A. Ebercon. 1981. Cell membrane stability as

measurement of drought and heat tolerance in wheat.

Crop Sci. 21: 43-47.

154

Bradford, M.M. 1976. A rapid and sensitive method for the

quantitation of microgram quantities of protein

utilizing the principle of protein-dye binding. Anal.

Biochem. 72: 248-254.

Bradford, K.J., and P.M. Chandler. 1992. Expression of

dehydrin-like proteins in embryos and seedlings of

Zizania palustris and Oryza sativa during dehydration.

Plant Physiol. 99: 488-494.

Bray, E.A. 1993. Molecular responses to water deficit. Plant

Physiol. 103: 1035-1040.

Cellier, F., G. Conejero, J.C. Breitler, and F. Casse. 1998.

Molecular and physiological responses to water deficit

in drought-tolerant and drought-sensitive lines of

sunflowers. Accumulation of dehydrin transcripts

correlates with tolerance. Plant Physiol. 116: 319-328.

Chandler, P.M., and M. Robertson. 1994. Gene expression

regulated by abscisic acid and its relation to stress

tolerance. Annu. Rev. Plant Physiol. Plant Mol. BioI.

45: 113-141.

Close, T.J. 1996. Dehydrins: Emergence of a biochemical role

of a family of plant dehydration proteins. Physiol

Plant. 97: 795-803.

Close, T.J., R.D. Fenton, A. Yang, R. Asghar, D.A. DeMason,

D.E. Crone, N.C. Meyer, and F. Moonan. 1993. Dehydrin:

The protein. In: Close, T.J. and E.A. Bray (ed.). Plant

responses to cellular dehydrin during environmental

155

stress. ppl04-114. American Society of Plant

Physiologists. Rockville, MD. ISBN 0-943088-26-7.

Davis, W.J. 1978. Some effects of abscisic acid and water

stress on stomata of Vicia faba J. Exp. Bot. 29: 175-

182.

Ellis, R. J., and S.M. Van der Vies. 1991. Molecular

chaperones. Annu. Rev. Biochem. 60: 321-347.

Espelund, M., J.A. de Bedout, W.H. Jr. Outlaw, and K.S.

Jakobsen. 1995. Environmental and hormonal regulation

of barley late-embryogenesis-abundant (Lea) mRNA is via

different signal transduction pathways. Plant Cell

Environ. 18: 943-949.

Giordani, T., L. Natali, D.A. Ercole, C. Pugliesi, M.

Fambrini, P. Vernieri, C. Vitagliano, and A. Cavallini.

1999. Expression of a dehydrin gene during embryo

development and drought stress in ABA-deficient mutants

of sunflower (Helianthus annuus L.) Plant Mol. BioI.

39: 739-748.

Giorini, S., and G. Galili. 1991. Characterization of HSP-70

cognate proteins from wheat. Theor. Appl. Genet. 82:

615-620.

Han, B., P. Berjak, N. Pammenter, J. Farrant, and A.R.

Kermode. 1997. The recalcitrant plant species,

Castanospermum australe and Trichilia dregeana, differ

in their ability to produce dehydrin-related

polypeptides during seed maturation and in response to

156

ABA or water-deficit-related stresses. J. EXp. Bot. 48:

1717-1726.

Han, B., and A.R. Kermode. 1996. Dehydrin-like proteins in

castor bean seeds and seedlings are differentially

produced in responses to ABA and water-deficit-related

stresses. J. Exp. Bot. 47: 933-939.

Labhilili, M., P. Joudrier, and M.F. Gautier. 1995.

Characterization of cDNAs encoding Triticum durum

dehydrins and their expression patterns in cultivars

that differ in drought tolerance. Plant Sci. 112: 219-

230.

Laemmli, U.K. 1970. Cleavage of structure proteins during

the assembly of the head of bacteriophage T4. Nature

227: 680-685.

Lindquist, S.C., and E.A. Craig. 1988. The heat-shock-

proteins. Annu. Rev. Genet. 22: 631-677.

Mao, Z., R. Paiva, and A.L. Kriz. 1995. Dehydrin gene

expression in normal and viviparous embryos of Zea mays

during seed development and germination. Plant Physiol.

Biochem. 33: 649-653.

Marcum, K.B. 1998. Cell membrane theromostability and whole-

plant heat tolerance of Kentucky bluegrass. Crop Sci.

38: 1214-1218.

Neuhoff, V., Arold, D. Taube, and W. Eirhardt. 1988.

Improved staining of proteins in polyacrylamide gels

including isoelectric focusing gels with clear

background at nanogram sensitivity using Coomassie

157

Brilliant Blue G-250 and R-250. Electrophoresis 9: 255-

262.

Ouvrard, 0., F. Cellier, K. Ferrare, D. Tousch, T. Lamaze,

J.M. Dupuis, and F. Casse-Delbart. 1996. Identification

and expression of water stress- and abscisic acid-

regulated genes in a drought-tolerant sunflower

genotype. Plant Mol. BioI. 31: 819-829.

Pelah, D., O. Shoseyov, A. Altman, and D. Bartels. 1997.

Water-stress responses in aspen (Populus tremula) :

Different accumulations of dehydrin, sucrose synthase,

GAPDH homologues, and soluble sugars. J. Plant Physiol.

151: 96-100.

Perez-Molphe-Balch, E., M. Gidekel, M. Segura-Nieto, L.

Herrera-Estrella, and N. Ochoa-Alejo. 1996. Effects of

water stress on plant growth and root proteins in three

cultivars of rice (Oryza sativa) with different level

of drought tolerance. Physiol. Plant. 96: 284-290.

Pruvot, G., J. Massimino, G. Peltier, and P. Rey. 1996.

Effects of low temperature, high salinity and exogenous

ABA on the synthesis of two chloroplastic drought-

induced proteins in Solanum tuberosum. Physiol. Plant.

97: 123-131.

Rajasekaran, L.R., and T.J. Blake. 1999. New plant growth

regulators protect photosynthesis and enhance growth

under drought of jack pine seedlings. J. Plant Growth

Reg. 18: 175-181.

158

Riccardi, F., P. Gazeau, D.V. Vienne, and M. Zivy. 1998.

Protein changes in responses to progressive water

deficit in maize. Plant Physiol. 117: 1253-1263.

Sheffer K.M., J.H. Dunn, and D.D. Minner. 1987. Summer

drought responses and rooting depth of three cool-

season turfgrasses. HortScience 22: 296-297.

Towbin, H., T. Staehlin, and J. Gordon. 1979.

Electrophoretic transfer of proteins from

polyacrylamide gels into nitrocellulose sheets:

procedure and some applications. Proc. Natl. Acad. Sci.

USA. 76: 4350-4354.

Turgeon, A.J. Turfgrass management. 1999. p97. 5th ed.

Prentice Hall, Englewood Cliffs, NJ.

Vierling, E. 1991. The roles of heat-shock-proteins in

plants. Annu. Rev. Plant Physiol Plant Mol. BioI. 42:

579-620.

Volaire, F., H. Thomas, N. Bertagne, E. Bourgeois, M.F.

Gautier, and F. Lelievre. 1998. Survival and recovery

of perennial forage grasses under prolonged

Mediterranean drought: Water status, solute

accumulation, abscisic acid concentration and

accumulation of dehydrin transcripts in bases of

immature leaves. New Phytol. 140: 451-460.

Wechsberg, G.E., C.M. Bray, and R.J. Probert. 1994.

Expression of dehydrin-like protein in orthodox seeds

of Ranunculus scleratus during development and water

stress. Seed Sci. Res. 4: 241-246.

159

Wetzel, S., C. Demmers, and J.S. Greenwood. 1989. Seasonally

fluctuating bark proteins are a potential form of

nitrogen storage in three temperate hardwoods. Planta

178: 275-281.

Whitsitt, M.S., R.G. Collis, and J.E. Mullet. 1997.

Modulation of dehydration tolerance in soybean

seedlings. Dehydrin MatI is induced by dehydration but

not by abscisic acid. Plant Physiol. 114: 917-925.

Wisniewski, M., T.J. Close, T. Artlip, and R. Arora. 1996.

Seasonal patterns of dehydrins and 70-kDa heat-shock

proteins in bark tissue of eight species of woody

plants. Physiol. Plant 96: 496-505.

Wood, A.J., and P.B. Goldsbrough. 1997. Characterization and

expression of dehydrins in water-stressed Sorghum

bicolor. Physiol. Plant 99: 144-152.

Yamaguchi-Shinozaki, K., and K. Shinozaki. 1994. A novel

cis-acting element in an Arabidopsis gene is involved

in responsiveness to drought, low temperature, or high

salt stress. Plant Cell 6: 251-264.

160

Table 5-1. Effect of drought stress (DS) and ABA (ABA+DS) on

turf quality (TQ) I leaf relative water content (RWC) I

electrolyte leakage (EL) I and total soluble protein content

(SPC) in tall fescue. CL, well-watered plants.

Cultivar Day ofStress treatment TQ RWC EL SPC

% % mg/g FW

0 CL 8.5a* 97a 3.3c 4.6b

Southeast 5 DS 6.5c 63c 28b 5.0abABA+DS 7.5b 90b 3.7c 5.6ab

10 DS 4.5d 47d 44a 6.8aABA+DS 6.5c 59c 25b 5.9a

o CL 8.5a 95a 2.8c 6.5a

Rebel Jr. 5 DSABA+DS

6.3c

7.5b61c

88b30b3.1c

6.2ab6.1ab

10 DSABA+DS

4.5d6.3c

46d62c

45a33b

5.5b6.7a

*Means followed by the same letters within a column at a

given day of drought treatment were not significantly

different based on LSD test (P=0.05).

161

Fig 5-1. SDS-PAGE profiles of soluble protein from tall

fescue leaves under drought stress for Southeast (A) and

Rebel Jr. (B). MW, molecular marker; Lanes 1-5, well-watered

plants, 5 d of drought stress, 10 d of drought stress, ABA

treatment at 5 d of drought, ABA treatment at 10 d of

drought. Equal amounts of protein (30 ~g) were loaded in

each lane. Arrows indicate protein changes in response to

drought and ABA treatment.

162

A

B

MW 1 3 54220711881

52.5

36.2

29.9

20.7

7.1

?Pl81

52.5

36.2

29.9

20.7

7.1

163

Fig. 5-2. Immunoblots of soluble protein from tall fescue

leaves under drought stress and probed with dehydrin

antibody for Southeast (A) and Rebel Jr. (B). MW, molecular

marker; Lanes 1-5, well-watered plants, 5 d of drought

stress, 10 d of drought stress, ABA treatment at 5 d of

drought, ABA treatment at 10 d of drought. All lanes were

loaded with 15 ~g protein. Arrows indicate dehydrin-like

proteins in response to drought and ABA treatment.

164

5 4 3 2 1 MWA -207

-118- 81

-52.56053484240 -36.2

27 -29.9

23-20.7

B

-8160 ----+

-52.55348 =t42 ----+40 ----+

-36.2

- 29.927 ----+

23 ----+

-20.7

165

Fig. 5-3. Immunoblots of soluble protein from tall fescue

leaves under drought stress and probed with Hsc 70 antibody

for Southeast (A) and Rebel Jr. (B). MW, molecular marker;

Lanes 1-5, well-watered plants, 5 d of drought stress, 10 d

of drought stress, ABA treatment at 5 d drought, ABA

treatment at 10 d drought. All lanes were loaded with 15 ~g

protein. Arrows indicate Hsc 70 in response to drought and

ABA treatment.

166

A 5 4 3 2 1 MW

B

70 ----+ _.

167

- 52.5

- 36.2

29.9

- 20.7

- 7.1

-207- 118- 81

- 52.5

- 36.2

- 29.9

- 20.7

- 7.1