The Free Proline Content of Water Stressed Maize Roots

Departamento de Ecologia y de Quimica, Facultad de Agronomla Universidad de BuenosAires, Argentina

The Free Proline Content of Water Stressed Maize Roots')

MARTA CARCELLER2) and ADELA FRASCH1NA

With 4 figures

Received April 8, 1980 . Accepted June 5, 1980

Summary

The relation of accumulation of free proline to different stress conditions was investigated in root tips of young plantules of lea mays. The level of proline was found to increaseas a consequence of drought treatments, and decrease to normal values when the seedlingswere rewatered, and root growth reassumed. The accumulation of proline occurred in lightor darkness, and even with restricted sugar supply. Other stress conditions, salinity, lowtemperature and abscisic acid treatment, also determined the accumulation of proline. Theresponse of the two cultivars under study, differed when exposed to stress, and was not related to the degree of reduction in growth rate.

Key words: proline, water stress, lea mays.

Introduction

The content of free proline increases under water stress in plant leaves of manyspecies (BARNETT and NAYLOR, 1966; SINGH et al., 1972), and this accumulation isrelated to the lowering of the water potential (HANSON et al., 1977).

Proline is synthetized de novo from glutamate in leaves exposed to water deficit(BOGGESS et al., 1976). When the turgidity of the tissue is restored, proline disappearsrapidly (STEWART, 1972), except when the leaves are irreversibly damaged.

Increased levels of free proline have also been observed in plants submitted to lowtemperature (DRAPER, 1972; CHU et al., 1974), salinity (BAR NUN and POLJAKOFFMAYBER, 1977) or abscisic acid (ASPINALL et al., 1973). It has been frequentlyassumed that proline is involved in the mechanisms of tolerance of plant tissues tostress conditions, but experimental evidences are still inconclusive.

Few studies concerning the accumulation of proline have been conducted withroots. An increase in proline level is reported in barley (SINGH et al., 1973) and maize

1) This investigation was supported by a grant from the Comisi6n de InvestigacionesCicntificas de la Provo de Buenos Aires, and from the Consejo Nacional de InvestigacionesCiendficas y Tecnicas.

2) Member of the Consejo Nacional de Investigaciones Ciendficas y Tecnicas.

z. Pflanzenphysiol. Bd. 100. S. 43-49. 1980.

44 MARTA CARCELLER and ADELA FRASCHINA

roots (GORING and THIEN, 1978) under water stress, and induced by salinity, in rootsof Tamarix and pea (BAR NUN and POLJAKOFF MAYBER, 1977). We have found maizeroots (CARCELLER and FRASCHINA, in press) a convenient plant material to investigatethe relation of proline content to drought conditions.

Materials and Methods

Preparation of plant material: seedlings of Zea mays were grown at 25 ± 0.5°C, undercontinuous light (1500 ia watt.cm"), as previously described (CARCELLER and FRASCHINA, inpress).

Drought treatments: when the seedlings were 4 days old, they were exposed to a 24hours period at relative humidity 93 Ofo, also under continuous light and 25°C, as previously described (CARCELLER and FRASCHINA, in press).

Determination of proline: method of TROLL and LINDSLEY, 1955, in three replicate samples of 10 root tips, 1 ern long.

Low temperature treatment (10 oq , abscisic acid (10,aM) and NaCI (0.2 M), were applied during periods of 24 hours, under the same conditions of light.

Control seedlings were grown over moisted filter paper.Two hybrids of maize, Abati II INTA, and Dekalb 4F 32 were used in the experiments.

Results

Free proline content, and its relation to the age of the seedlings

The variation of free proline content in the root tip is shown in figure 1. Anincrease in level was found at about 3 days, either when the results were expressed as,ug/tip or as ,ug/mg dry weight.

10

ois. 5r»o

-------..--oc

64 88 112

hOD r s from qe r min atro n

134

Fig. 1: The relationship between the free proline content and the age of the plantules (Dekalb 4F 32). Three replicates samples of ten root tips, 10 mm long, were taken daily.

Z. PJlanzenphysiol. Bd. 100. S. 43-49. 1980.

Proline content and water stress 45

Therefore, throughout the following experiments, care was taken to select roots ofthe same age and length, to be submitted to the stress treatments.

As the water stress rapidly reduces cell expansion, the 1 ern tips from rootssubmitted to drought, contained more cells than the controls, and its dry weight washigher, 1.30 mg./tip compared to 0.75 mgltip in the controls. Therefore, a dry weightbasis was adopted to expre ss the proline content.

Effect of stress treatments on the free proline content

It was intended to clarify if proline accumulation is an specific response to stressconditions, or merely reflects a consequence of the reduction in cell expansion. In theexperiments summarized in table 1, low temperature and salinity, as well as drought,had a severe effect on the root growth of both cultivars of maize, while the effect ofabscisic acid was moderate. The growth response to these treatments was similar inboth hybrids.

Table I: The free proline cont ent and the roo t growth in several treatments . Three replicat e samples often root tips were taken 24 hours after initiat ion of the treatment. Co ntrols were sampled simultaneously.

Treatment

Control (25 ±O.5 °C)NaClO.2 NABA IOuMDroughtCold (l O± O.5°C)

DEKALB 4F 32Pro line Length(JLg mg-I) (mm day-l )

6.2 16.910.4 4.98.0 11.59.1 0.44.8 1.2

ABATIII INTAProline Length(JLg rng-l) (mm day-I)

3.3 14.65.2 2.53.5 7.55.2 0.53.2 0.0

.-----..---./---12 18

hO u ( S

24

Fig. 2: The relationship between th e free proline content and the length of the droughttreatment in the plantules (Dekalb 4F 32). Three replicate samples of ten root tips were taken: 6, 12, 18 and 24 hours after initiation of the drought treatment. Controls were sampled simultaneously. 0 control, • drought.

Z. Pflanzenphysiol . Bd. 100. S. 43-49. 1980.


In the Abati II, there was an increase in proline level under drought or salinity,while no accumulation occurred with abscisic acid or low temperature treatments.

The Dekalb hybrid roots accumulated proline under drought, NaCI or abscisic acidtreatments, but in the low temperature condition the proline content was even lowerthan in the controls.

Time course of proline accumulation under water deficit

The drought treatment applied to the seedlings was severe, and the RWC of theroot tips reached values as low as 600/0. Root growth was rapidly impaired, andceased in about 12 hours after the beginning of the stress period. (Growth was

drought ~4 re w a t e r t n q

50

.>0

300

200 ~L'100 --- --- ---

150 "

100 ~J

100

50

Fig. 3: Changes in the growth rate; dry weight and free proline content as Ofo of the control, in 10 mm root tips of the primary root of the plantules (Dekalb 4F 32) after 24hours of water stress, and 24 hours following rewatering,

Z. Pflanzenphysiol. Bd. 100. S. 43-49. 1980.


1 mm/h in the controls, and in the stressed roots decreased from 0.6 to 0 mm/h

during the drought period.)The level of free proline rised above control values only after 18 hours from the

imposition of drought (figure 2), while the Rwe reached its equilibrium valuesduring the first 6 hours of treatment.

The roots were able to reassume growth when rewatered. Proline disappearedrapidly (figure 3), even before growth or dry weight were again normal.

Effect of carbohydrate reserves on the accumulation of proline

To examine the effect of the elimination of most of the food reserves, anexperiment was performed in which the grains of the young plantules were cut off.Photosynthesis was supposed to be negligible. As it is observed in figure 4, the prolinelevels were very low when the carbohydrate supply was drastically limited, but thedifference between the control and the water stressed root tips is maintained, andsimilar to that of intact plants.

5.0

»,

"0

01

E 2.5

"a.01:0

0'--__--'-"'-L..1-----"--L..<Cl-__

control drought

Fig. 4: The effect of excising the grain on proline accumulation in the plantules (Abati IIINTA). 0 intact plantules, ~ grain excised.

Effect of light and darkness

All experiments were conducted under continuous light, but in one of them theeffect of this condition on the accumulation of proline was investigated.

As can be seen from data presented in table 2, more proline was found in darknessthat in light, but the increase in level determined by water stress was similar in bothconditions.

Z. Pflanzenphysiol. Rd. 100. S. 43-49. 1980.


Table 2: Free proline content in control and water stressed root s, in light (1500u watt ern-2) and darkn ess.

Prolin e (JLg mg-l)Light Darkness

Cont rolDrought

5.37.4

7.39.5

Discussion

The higher levels of fr ee proline found in water stressed roots are quite consistent,but evidently lower than those reported for leaves (McMICHAEL and ELMORE, 1977;SINGH et al., 1972). It is genera lly assumed that a high carbohydrate level , and theprovision of ATP and N ADPH2 fr om photosynthesis are required for accumulationof proline, and that these factors could be limiting the synt hesis of proline in roots.

The amount of stored carbohy dra tes is clearl y important for proline accumulation,but an increase in level occurs even in conditions of low sugar supply, in the maizeroots. It is unlikely th at , in the intact seedlings of our experiments, sugars could belim iting proline synt hesis.

An effect of light, independent from photosynthesis, wa s observed in leaves ofbarley (HANSON and T ULLY, 1979) and in diatoms (SCHOBERT, 1977). Th e effe ct oflight on roots seems to be t he opposite, and more pr oline is accumulated in darkness.

Th e possible interference of tr ansport of proline, fr om or to the shoot, can not berul ed out in our exp erimental conditions, since th e transportation pattern wouldprobably differ under stress conditions, or due to light or darkness.

D rought, and other stress conditions, specificall y alte r the level of proline, in awa y which appears to be ind epend ent of the rate of cell expansion. In one of th emaiz e hybrids, the accumulation of proline in the root tips was different undertreatm ents which det ermined a very simil ar reduction of gro wth. The response of thetw o hybrids investiga ted here, differed when th ey were exposed to the sametr eatment, implying th at the accumulation of proline is not simply a consequence ofmetabolic alterat ions when root grow th is affected.

References

ASPINALL, D. , T . SINGH, and L. PALEG : Aust. J. Bio!. Sci. 26, 319- 327 (1973).BARNARD, R. A. and A. O AKS: Canad ian J. Bot any 48, 1141-1145 (1970).BARNETT, N . M. and A. NAYLOR: P!. Physio!. 41,1222-1230 (1966).BAR-NuN, N. and A. POLJAKOFF-MAYBER: Ann. Bot. 41,173-179 (1977).BOGGESS, S. F., C. STEWART, D. ASPINALL, and L. PALEG: Plant Physio!. 58, 398-401

(1976).CARCELLER, M. and A. FRASCHINA: Turrialba (in press).CHU, T. M., D. ASPINALL, and L. PALEG: Aust . ] . Pl ant Ph ysio!. 1, 87-97 (1974).DRAPER, S. R.: Ph yto chemistr y 11, 639-641 (1972).GORING, H . and Bur H ur THIEN: Biochem. Ph ysio!. Pflanzen 172, 311-314 (1978).H ANSON, A. D., CH. N ELSEN, and E. EVERSO N: Crop Sci. 17, 720- 726 (1977).H ANSON, A. D . and R. TULLY: Planta 145, 45-51 (1979).McM ICHAEL, B. L. and C. ELMORE: Cr op Sci. 17, 905- 908 (1977).



SCHOBERT, B.: Z. Pflanzenphysiol. 85, 457-461 (1977).SINGH, T. N., D. ASPINALL, and L. PALEG: Nature 236, 188-189 (1972).SINGH, T. N., L. PALEG, and D. ASPINALL: Aust, J. BioI. Sci. 26, 45-56 (1973).- - - Aust. J. BioI. Sci. 26, 57-63 (1973).STEWART, C. R.: PI. Physiol. 50, 679-681 (1972).STEWART, C. R., C. MORRIS, and J. THOMPSON: PI. Physiol. 41,1585-1590 (1966).TROLL, W. and J. LINDSLEY: J. BioI. Chern. 215, 655-657 (1955).

M. CARCELLER, Departamento de Ecologia y de Quimica, Facultad de Agronornia, Universidad de Buenos Aires, Argentina.


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The Free Proline Content of Water Stressed Maize Roots