Rev.Fac. Agronomia - UNLPam Vol. 14 N°]/26300 Santa Rosa - ARGENTINA - 2003 ISSN 0326-6184

Molecular and physiological responses to water deflcit in wheat

{Triticum aesHvum L.)

Respuestas fisiologicas y moleculares al deficit hidrico en trigo (Triticum

aestivumL.)

Recibido:26/02/03 Aceptado:11/12/03

Pereyra, M. & M. del C. Torroba'

Abstract

Physiological and molecuiar responses of five wheat genotypes: one Canadian cuttivar,with low temperature toierance, and 4 experimental lines, which demonstrated in the field highyield under drought stress were studied under water stress. We determined leaf relative watercontent and gene expression of rob 5 and dehydrins in response to low water availability. Onegenotype showed a higher RWC with less leaf Iresh weight and water soil content. There were notdifferences between genotypes in expression genes but, we found differences in gsne expressionbetween both tested genes. Rob 5 expressed in control as well as in stressed plants, whiledehydrins only expressed in stressed plants and its expression increased as the water contentof leaves decreased.

Key words: water stress . Rob 5, dehydrin, Triticum aestivum L

Resumen

Las respuestas fisiolbgicas y moleculares de cinco genotipos de trigo: un cultivar cana-diense. tolerante a las bajas temperaturas y 4 lineas experimentales argentinas, que habianmostrado, un alto rendimiento a campo bajo condiciones de deficiencia hidnca, fueron estudia-das en condiciones de baja disponibilidad de agua. Se determine el contenido relativo de agua enlas hojas y la expresion de 2 genes, rob 5 y dehidrinas, en respuesta a la baja disponibilidad deagua. Uno de los genotipos mostrd mayor CRA con un menor contenido de agua en las hojas oen el suelo. No hubo diferencias genoti'picas en !a expresi6n de los genes sin embargo, fuediferente la expresion de estos, en respuesta al estr6s hidrico, dado que el gen Rob 5 se expresdtanto en las plantas control como en las estresadas, mientras que el de las dehydrinas solo seexpreso en las plantas bajo estres, y su expresion aument6 en funcidn de la disminuci6n delcontenido relativo de agua de las hojas.

Palabras clave: sequia, Rob 5. dehidrinas, Triticum aestivum L.

1 Facultad de Agrooomia Facullad de Agronomfa UNLPam CC. 300, Santa Rosa, La Pampa, Argen-tina, g-mail: Derevra@agro unlnam.edu.ar

Introduction

Drought is a major factor limitingcrop productivity vi'orld-wide andcultivating plants with increasedresistance to this stress appears critical tokeep yields at a sufficient level. Thescreening of such cultivars based on theirproductivity, is a long and tedious taskthat would undoubtedly be accelerated iftraits that could be reliably related to waterwere identified.

Whole plants respond to droughtthrough morphological, physiological,and metabolic modifications occurring inall plant organs. At the cellular level plantresponses to water deficit may result incell damage, whereas other responses maycorrespond to adaptative processes.Although a large number of drought-induced genes have been identified in awide range of plant species, a molecularbasis for plant tolerance to water stressremains far from being completelyunderstood (Ingram and Barteis, 1996).

Water deficit is intrinsic to mostabiotic forms of stress- not only duringdrtjught, but also at tow temperature andwhen the soil contains highconcentrations of ions. Changes in geneexpression are involved in physiologicalresponses to water stress, but the methodby which specific genes might play a partin stress is unknown. Such lack of progressis mainly due to the multigenic nature ofsensitive and tolerant phenotypes. Inaddition, different plant species have iivariety of mechanisms that have evolvedin a family specific or order specificmanner to confer tolerance. The stressresponse can also vary depending on thedevelopmental stage during which a plantis subject to stress.

Breeding programs to improve saltand drought tolerance are particularly

important in countries that would benefitmost from stress-tolerant crops.Conventional breeding approaches alonewill be valuable, but it would be morebeneficial to include as molecular markersthe genes for the mechanisms associatedwith stress tolerance.

Among the water-stress inducedproteins so far identified, dehydrins, theD-U subgroup of late-embryogenesis-abundant (LEA) proteins { Dure et al.1989; Wood and Goldsbrough, 1997) arefrequently observed, and more than 65plant dehydrin sequences are available(Close, 1997). Dehydrins are a group ofproteins that accumulate during coldacclimation (Robertson et al. 1994 b;Danyluk et al. 1994). water stress ( Closeet al. 1989) and heat tolerance (Robertsoner al. 1994 a). Dehydrins are heat stable,highly hydrophylic proteins that arethought to help stabilize other proteinsand membranes during periods of cellulardehydration {Close et al. 1993a). Theseproteins display particular structuralfeatures such as the highly conserved Lys-rich domain predicted to be involved inhydrophobic interaction leading tomacromolecule stabilization. Very Uttle isknown about dehydrin functions in plants(Cellier et al. 1998). Studies haveestablished correlation between droughtadaptation and dehydrin accumulation inwheat and poplar (Labhilili et al. 1995;Pelah et al. 1997) and sunflower ( Cellieret al. 1998). Rob 5 is a gene that wasstudied in wheat in response to cold stress,it was observed that its expression increasein response to low temperatures (personalcommunication).

In most of the published studiesgene expression was described as afunction of time after the stress was appliedrather than as a function of parametersdescribing the plant water status.

14

Therefore, it is difficult to determine fromthese data precise relationship betweenplant physiological responses to drought-induced gene expression (Cellier ^t ai1998)

The present work was conductedin order to determine plant physiologicalresponses and the expression of rob5 anddehydrin gene in four Argentinean expe-rimental wheat lines that showed a goodyield growth in a field under water stressand a Canadian cultivar that had a goodperformance under low temperatures.

Materials and Methods

Seedling growth cgnditions

Seeds of four Argentinean experi-mental lines supplied by Ing. Agr. RubenMiranda (Dpto. of Agronomy, UNSur, Ar-gentina): 425-94 (line 1), 873-97 (line 2),890-97 (line 3); 898-97 (line 4) and aCanadian cultivar, AC domain, (cultivar5) were sown on 18/1/2002, and seedlingswere grown in a greenhouse under 18 h oflight, 2O/23°C (night/day) and the potswere filled with composite soil (terralite:soil, 1:3). We carried out two kind ofexperiments with the object to generate adifferent type of stress: Experiment I,leaves of well watered plants weredehydrated on the bench. We took samplesto determine fresh weight, relative watercontent (RWC) and part of the materialwas frozen for immunoblot analysis atdifferent times, at the start of theexperiment, two and five hours later, 20days old seedlings were used in theexperiment.

Experiment II: On 25 February wewatered all the pots, then, we separatedthose into two groups referred to as con-trol watered during al! the experiment andstressed plants were subject to progressive

drought by withholding water. Controland stressed leaves were harvested after10.14 and 20 days of stress, first, secondand third sampling. Theri the rest ofstressed plants were rewatered, and 72hours later were harvested, fourthsampling time. At each sampling time werecollected all the leaves from individualplants of each line, for physiologicalmeasurements and frozen separately forsubsequent inmunoblot analysis with rob5 and dehydrin gene.

Leaf Relative water content

RWC were estimated using thefollowing formula: RWC= (FW - DW) /TW - DW) X 100, where FW= weight offreshly collected material. TW = weightafter rehydration for 24 hours at 4° C inthe dark and DW = weight after drying at60 " C for 24 hours.

Soil water content

Each pot was weighed daily at 10AM and gravimetric soil water contentwas measured as grams of water per gramof oven - dried soil.

Protein isolation. SPS-PAGEelectrophoresis and Western Blotting

Leaves of control and stressedplants after the harvest were rapidly frozenin liquid N,. Then, they werehomogenated, 500 mg fresh weight in 1.5ml of buffer of Sodium borate, pH 8.0,containing sodium borate 0.95 gr, L-ascorbic acid 0.088 gr, 0.5 M EDTA 100ml, b mereaptoethano 1 500 ml, e - amino -caproic acid 0,333 gr. 20 % SDS 2.5 ml ina final volume of 50 ml. Then, leaves weredisrupted in a motorized groundhomogenizes The 13000 rpm ( 90 min)supernatant fraction was collected forprotein assay. SDS-PAGE electrophoresisand immunoblot analysis.

15

Electrophoresis on denaturingpolyacrylamide gels was performed by themethod ofLaemmli (1970) with a 4% (w/v) stacking gel and a 12.5 % (w/v)resolving gel. Equal mass of protein (60mg) were loaded on the gel. We did twoequal gels, one to stain with coomasie blueand the other for western blotting. Forimmunological detection, proteins werefixed and elctrotransferred tonitrocellulose in a buffer containing 25mM Gly at a constant current of 90 volts.Biots were blocked with 2% dry milk inTris-buffered saline, incubated with rabbitanti - peptide antibodies followed by a goatanti-rabbit IgG alkaline phosphataseconjugate, The primary antibody wasantyrob 5 ( sumninistrated by Plant andstres laboratory. University ofSaskatchewan), or against a syntheticpeptide (EKKGIMDKIKELPG) thai ishighly conserved in the C- termini of group2 LEA/ dehydrin proteins ( Courtesy of T.J. Close). The secondary antibody wasdetected using 4-nitroblue-tetrazoliumchloride and 5-bromo 4-chloro 3-indoIyl-phosphiite.

Results and Discussion

Physiological Characterization ofdrought stress

Experiment I

When leaves of well wateredseedlings were dehydrated on the bench,apparently line 2 showed a lower lost ofwater in function of dehydration time, thenit had the highest RWC after five hours ofdehydration (Eigure 1) and interestedly,when we related RWC in function of leaffresh weight we found that apparentlygenotype 2 maintained a higher RWC withlower fresh weight (% control) (Figure2). Water loss of excised leaves is atechnique that was suggested for droughtscreening (Clarke and McCaig, 1982).Winter etal. (1988) showed in wheat, thatwater loss of excised leaves was correlatedwith an index of drought susceptibility (S),(least loss = lowest S), then we can saythat under these conditions, genotype 2 ismore tolerant to drought conditions.

100

80 •

60-

40-

20-

0

Dehydration time (hours)

-Genotype 1

-Genotype 4

-Genotype 2

-Gsriotype 5

-Genotype 3

Figure 1. Leaf RWC (%) in function of dhydration time. Determinations in 20 daysold seedlings, were made at the start of the experiment (time 0), two (time 1)and five (time 2) hours later.

16

80 -

60 •

40 -

20 -

n -

XX

XX •

*

m*X

20 40 60 80 100

Leaf fresh weight (% control)

120

• Genotype 1 • Genotype 2 Genotype 3 x Genotype 4 * Genotype 5

Figure 2. Variation ot KWC (%) in tunction ot leaves tresh weight (% control). Leavesof well watered plants were dehydrated on the bench during five hours.Values are the means from four repetitions.

Experiment II

During the development of thewater stress, soil water loss increasedprogressively and differently in pots of thedifferent genotypes. At the end of theexperiment line 2 showed the lowest valueof soil water content (P < 0,05) (Table 1).

Leaf relative water content wasdifferent between lines after withholdingwater. Changes in RWC during waterstress development are shown in Table 2.

At the time water was withheld (day 0)the five genotypes displayed similarvalues, but. at the end of the experimentthe relative water content was 79, 54, 66,62 and 67% for lines 1. 2. 3, 4 and 5respectively. Then line one had highest leafRWC under water stress while line 2 thelowest value ( P < 0.05) (Table 2) Line Ilost less water from the soil as well as fromits leaves during water stress, while lines2 lost the highest amount of water fromthe soil and reached the lowest RWC.

Table 1. Soil water content percent (%) at 10 (first sampling date), 14 (second sampling date) and 20 (third sampling date) days after withholding water, infive wheat lines

(%) Soil water contentControl Stressed plants

Genotypes Firstsampling date

Secondsampling date

Thirdsampling date

Genotype 1Genotype 2Genotype 3Genotype 4GenotvDe 5

4351544952

4036404338

3014232225

16811910

17

Table 2. RWC {%) in leaves at 10 (first sampling date), 14 (second sampling date)and 20 (third sampling date) days after withholding water, in five gentypesof wheat

Genotypes

Genotype 1Genotype 2Genotype 3Genotype 4GenotVDe 5

{%) Relative

Firstsampling

9193959693

water contentstressed plants

Seconddate samplinq date

8683888880

Thirdsamplina date

7954666267

However, while the soil moisturecontents after 14 days of drought werenotably different ( 30 and 14 % for lines 1and 2 respectively), at that sampling timethe difference of leaf RWC between lineswas minimal (86 and 83 % for lines 1 and2 respectively); then, when we associatedRWC of leaf in function of gravimetricsoil water content, genotype 2 showed ahighest RWC at a specific gravimetric soilwater content fFieure .̂ V Therefore

genotype 2 had the highest relative leafwater content in both types of experimentsat the lowest leaf and soil water contents.The RWC differences according togenotype in wheat cultivars under waterstress were also observed by Schonfeld etal. 1988; and Siddique et ai 2000, andthe fonner authors suggest the use of RWCas a selection criterion for droughtresistance in wheat (Schonfeld e/a/. 1988).

60Gravimetric soil water content

40 30 20 10

• Genotipo 1 • Genotipo 2 A Genotipo 3 x Genotipo 4 x Genotipo i

Figure 3. Soil and leaf water status of five genotypes plants dxiring progressive droughtinitiated by withholding water. Leaf relative water content as a function ofgravimetric soil water content ( grams of water per gram of dry soil). Leafrelative water content and gravimetric soil water content values are themeans from four repetitions.

Molecular Characterization of drouehtstress

The protein patterns obtained fromstressed leaves were not obviouslydifferent from those of control leaves(results are not shown). The occurrence ofrob5 and dehydrin antibody was followedin leaves of control and stressed plants inthe different sampling dales. In allgenotypes, the inmunoblots rob 5 (Figu-re 4 and 5) showed the presence of bandsin control and stressed leaves, and did notincrease its expression under water stress.TheRob5 imnmnoblot showed a band ofabout 42 - 45 kD polypeptides. This genewas present in the five genotypes in con-trol and stressed leaves, having similarresponse in all the genotypes in differentdrought times. An increase in rob 5expression gene was found under coldtreatment in wheat (personalcommunication, Russell Trischuk). Theseproteins were observed in Bromus inermisas the most abundant set of heat - stablepolypeptides in the cell fraction isolatedfrom ABA - treated cells (Robertson, et al.1994). However under water stresstreatments we did not observe an increasein the concentration of these proteins.

On the contrary, the dehydrinsshowed a different behavior, as the bandsdid not appear in control leaves but theydid in stressed leaves in all genotypes (Fi-gure 6) and the increased expression washigher in leaves with lower RWC (Figure6). Western Blots showed that dehydrinsare proteins of about 25 kD, and althoughthey were detected in plant leaves thathave not been watered during 10 days.the band was stronger in those which hadnot been watered during 20 and 27 days(Figure 6).

Western blot did not showdifferences in dehydrin patterns between

genotypes., showing an increased band inall lines in the second harvest and thisband did not disappear in rewatered plants(Figure 6). Different authors observed anincrease of dehydrin concentration asresponse to water stress that could beassociated with difference in drought to-lerante (Cellier et al. 1998; Lopez et al.2003). This autor also showed that wheatcultivar less susceptible to droughtexpressed dehydrins with a higher leafwater potential. However, in ourexperiment, accumulation betweengenotypes was observed, and thisresponse could not be related to thedifferent RWC of genotype 2 under lowwater availability.

Since the tested genotypesbehaved in a similar way under water stress,no difference in relation to induced geneexpression could be established. Howeverwe work with two genes which expressionunder water stress was different. Someauthors suggest that there are two groupsof genes, a group that is expressed underwater stress and another with ABA (Skriver and Mundy, 1990). It is possiblethat rob5 responds to ABA in contrast tothe dehydrin gene.

Acknowledgments

We thank Ing. Agr. Ruben Miran-da (Dpto. de Agronomi'a, Universidad Na-cional del Sur) for suministrating the seedsof Argentinean experimental lines. Thisresearch was supported Canadian - LatinAmerica and the Caribbean ResearchExchange grant. Specially, we also thankDr. Lawrence Gusta for facilitating hislaboratory and equipment, and also for hisscientific support (Plant ScienceDepartment, University of Saskatchewan,Canada).

o •£

r/1 — Cij ^ •"> M - .

(U t/1 Q

—1 •'::^ ! -

\ /

cp

;ave

s

—Eo

l i :•o

lat

ISO

X)

^Rob

x :~̂

T3_n

ofc

l o g

3 U

.H UJ

(N

• : • } '

o X

01

£

> D, • —

^ E b

ca:

o c

w Oo

CQ u•3

3en

C 4JE

p oX) f^

— i«.

-it 5)CJ

gig nf'Bi""*'

L)

o Sr2 ''J

O

a;

CQ

C

"H.

3OC

20

Genotype 1

R S2 S3

Genotype 2

C S2 S3

Genotype S

Figure 6. Western-Blot analysis of dehydrin antibody for genotype 1. 2 and 5 on controlplants (C) and stressed plants, second sampling date 14 days after

withholding water (S2). 20 days after withholding water (S3) and rewateredplants after 27 days of withholding water (R). Experiment il. Arrowindicates dehydrin hand

Bibliography

Cellier, F..G. Conejero, J.C. BreJtIer&F. Casse,1998. Molecular and physiologicalresponses to water deficit in drought-tolerant and drought - sensitive lines ofsunflower. Plant Physiol. 116:319-328.

Clarke, J.M. &T.N. Mccaig. 1982. Evaluation oftechniques for screening no differencein dehydrin for drought resistance inwheat. Crop Sci. 22: 503-506.

Close, T.J,,A.A.Kortt& P.M. Chandler. 1989. AcDNA- based comparison ofdehydration - induced proteins(dehydrins) in barley and corn. PlantMoi. Biol. 13:95-108.

Close, T.J., RD. Fenton & F. Moonan. 1993a. Aview of plant dehydrins usingantibodies specific to the carboxy ter-minal peptide. Plant MoL BioL 23: 279-286.

Close, T.J. 1996. Dehydrins: emergence of abiochemical role of a family of plantdehydration proteins. Physiol. Plant. 97:795-803.

Close, T.J, 1997. Dehydrins: A commonalty inthe response o{ plants to dehydrationand low temperature. Physiol. Plant.100:291-296.

Danyluk, J., M- Houde, E. Rassart S F Sarhan.1994. Differential expresion of a geneencoding an acidic dehydrin in chillingsensitive and freezing tolerantGramineae species. FEBS Lett 344:20-24.

Dure, L,, M. Crouch, J. Harada, T.D. Ho, J.Mundy, R. Quatrano, T. Thomas & Z.R.Sung, 1989. Common amino acidsequence domains among the LEAproteins of higher plants. Plant Moi. Biol.12:475-486.

Ingram, J. & D. Bariels. 1996. The molecularbasis of dehydration tolerance in plants.Annu. Rev. Plant Physiol. Plant Moi. Biol.47: 377-403.

Laemmli, U.K. 1970- Cleavage of structuralproteins during the assembly of the

21

head of bacteriopbage T4. Nature 227:680-685.

Labhilili, M., P Joudrier & M,F, Gautier. 1995.Characterization of cDNAs encodingTriticum durum dehydrins and theirexpression patterns in cultivars thatdiffer in drought tolerance. Plant Sci.112:219-230,

Lopez, C.G., G.M. Banowetz, C.J, Peterson &WE, Kronstad. 2003. Dehydrinexpression and drought tolerance inseven wheat cultivars. Crop Sci. 43:577- 582.

Petah, D., W, Wang, A. Aitman, 0. Shoseyov &D, Bartels, 1997. Differentialaccumulation of water stress - relatedproteins, sucrose synthase and solu-ble sugars in Populus species that differin their water stress response. Physiol.Plant, 99:153-159.

Robertson. A.J., M. Ishikawa, L.V, Gusta & S.L. Mackenzie, 1994 (a). Abscisic acid -induced heat tolerance in Bromusinermis Leyss cell - suspensioncultures. Plant Pbysiol. 105:181-190.

Robertson, A.J-, A, Weninger, R.W. Wilen, P.Fu, & L,V. Gusta. 1994b. Comparison

of dehydrin gene expression andfreezing tolerance in Bromus ir)ermisand Secaie cereale grown in controlledenvironments, hydroponics, and thefield. Plant Physiol. 106:1213-1216.

Schonfeld, M.A., R.C. Johnson. B.F. Carver &.D.W. Mornhlnweg. 1988. Water relationsin winter wheat as drought resistanceindicators. Crop Sci. 28: 526-531.

Siddique, M.R.B., A, Hamid & M.S, Islam. 2000,Drought stress effects on waterrelations of wheat. Bot. Bull. Acad. Sin.41: 35-39,

Skriver. K, & J, Mundy. 1990. Gene expressionin response to abscisic acid andosmotic stress. Plant cell 2: 503-512.

Winter, S,R,, J.T. Musick & K,B, Porter, 1988.Evaluation of screening techniques forbreeding drought - resistant winterwheat. Crop Sci. 28:512-516.

Wood, AJ. & P.B. Goidsbrough, 1997.Characterization and expression ofdehydrins in water - stressed Sorghumbicolor. Physiol. Plant 99:144-152.

22