PHYSIOLOGIA PLANTARUM 101: lSl-192. 1997Printed in Denmark - all rights reserved

Copyright © Physiologia Plantarum 1997

ISSN 0031-9317 ''i'?:

Water and salt stress-induced alterations in proline metabolism ofTriticum durum seedlings

C. Mattioni, N. G. Lacerenza, A. Troccoli, A. M. De Leonardis and N. Di Fonzo

Mattioni, C, Lacerenza, N, G,, Troccoli, A,, De Leonardis, A, M. and Di Fonzo, N,1997, Water and sah stress-induced alterations in proline metabolism of Triticum du-rum seedlings, - Physiol, Plant, 101: 787-792.

Many plants accumulate proline as a non-toxic and protective osmolyte under salineor dry conditions. Its accumulation is caused by both the activation of its biosynthesisand inactivation of its degradation. We report here on the alterations induced by waterand salt stress in the proline metabolism and amino acid content of 5-day-old seed-hngs of Triticum durum cv, Simeto. Most of the amino acids showed an increase withthe induction of either stress, but proline increased more markedly than did otheramino acids. We also measured the activities of two enzymes, A'-pyrroline-5-carbox-ylate (P5C) reductase (EC 1,5,1,2) and proline dehydrogenase (EC 1.5.1.2), which areinvolved in proline biosynthesis and catabolism, respectively. The activity of P5C re-ductase was enhanced during both water and salt stress, while proline dehydrogenasewas inhibited only during salt stress. The results indicate that synthesis de novo is thepredominant mechanism in proline accumulation in durum wheat. Use of a cDNAclone that encodes P5C-reductase from Arabidopsis thaliana, showed no differencesin the gene expression between controls and stressed plants, implying that the increasein enzyme activity is unrelated to the expression of this gene.

Key words - Amino acids, dumm wheat, enzyme activities, proline, Triticum durum,water and salt stress.

C, Mattioni (corresponding author, e-mail isc.fg@isnet.it) et al., Istituto Sperimentaleper la Cerealicoltura, Sez. di Eoggia, S. S. 16 km 675, 1-71100 Eoggia, Italy.

In plants, L-proline is produced from L-glutamic acidIntroduction ^-^ A'-pyrroline-5-carboxylate (P5C) and only two en-Drought and salinity are the most important environ- zymes are involved in this pathway. The activity of themental factors that cause osmotic stress and a reduction enzyme A'-pyrroline-5-carboxylate reductase [L-Pro:in plant growth and crop productivity (Boyer 1982). As NAD(P)-5-oxido P5C reductase, EC 1.5.1.2], whicha counteraction of these effects, the water potential of catalyzes the last step of proline synthesis, is osmoregu-cells may be decreased by the synthesis and accumula- lated (Verbmggen et al. 1993). An increase in activity oftion of compatible osmolytes such as proline, glycine P5C reductase, correlated with proline accumulation,betaine and sugars, allowing additional water to be taken has been reported in Chlorella (Laliberte and Hellebustup. Accumulation of free proline is a widespread stress 1989) and in NaCl-adapted cells of Mesembryanthe-response, well-documented not only for plants, but also mum (Treichel 1986). Similariy, a 6-fold increase in rel-for eubacteria, protozoa, marine invertebrates and algae ative abundance of P5C reductase mRNA was observed(Boyer 1982, Skriver and Mundy 1990). in soybean seedling roots subjected to short-term salin-

De novo synthesis from glutamate is considered the ization (Delauney and Verma 1990). However, in to-predominant mechanism of proline accumulation (Bog- bacco cells adapted to high NaCl, the accumulation ofgess et al. 1976, Rhodes et al. 1986, Voetberg and Sharp proline does not involve induction of this enzyme (La1991), although a decrease in catabolism (Stewart et al. Rosa et al. 1991). The other important factor that con-1977) and enhanced proteolysis (Thompson et al. 1966) trols the level of proline in plants is the catabolic path-may also be implicated. way, which is the reverse of the biosynthesis from

Received 26 March, 1997; revised 22 August, 1997

Physiol, Plant, 101, 1997 787

glutamate through the same intermediate, pyrroline-5- fimol m"' s"', PAR. Temperature and relative humiditycarboxylate (P5C). Two different enzymes are involved were maintained at 25°C and 50%, respectively,in the oxidation of proline to P5C: proline oxidase (EC1.5.99.8), localized in the inner mitochondrial mem- ., /TT J / ^ I - in-7n\ A ^ 1 ^ V. ^^^ RWC, water and osmotic potentialbrane (Huang and Cavalieri 1979) and cytoplasmic pro- ' ^line dehydrogenase (EC 1.5.1.2) (Rena and Splitts- Relative water content (RWC, %) was determined usingtoesser 1975). The latter enzyme's activity occurs in a the method of Barrs and Weatherley (1962), and calcu-bifunctional protein that also shows a P5C reductase ac- lated using the formula: (fresh weight - dry weight)/(tur-tivity (Rena and Splittstoesser 1975). There are few re- gor weight - dry weight) x 100. Turgor weight was de-ports comparing both the anabolic and the catabolic termined after imbibition of the tissue in distilled waterproline pathways in the same material. Veeranjaneyulu for 3 h.and Kumari (1989) and Sudhakar et al. (1993) reported The leaf water potential ("¥) was measured using aan increase in proline content concomitant with the in- pressure chamber (PMS Instmment Co., Corvallis, OR,hibition of proline dehydrogenase and the enhanced USA) according to Scholander et al. (1965). Full-turgorP5C reductase activity in water-stressed mulberry and osmotic potential (*F),°°) was determined with a micro-greengram, respectively. osmometer (Roebhng 16 R. 1, Berhn, Germany). Cut

The aim of the present work was to evaluate whether leaves were placed for 3 h in a test tube containing dis-water and salt stress induce a similar proline accumula- tilled water, then frozen at -20°C and pressed at 0.5 MPation in dumm wheat seedlings and to determine whether for 1 min. The collected sap was used to measure the os-this amino acid is involved in osmoprotection. We mea- motic potential (Rascio et al. 1994).sured P5C reductase and proline dehydrogenase activi-ties to verify whether the accumulation of proline is due -H l •to de novo synthesis and/or reduction of oxidation. Ammo acid analysisMoreover, using a cDNA clone isolated from Arabidop- The samples (10 mg dry weight) were extracted with 0.4sis thaliana and encoding for P5C reductase, we evalu- ml of 4 volumes ethanol: 1 volume water (Chiang andated whether changes in enzyme activity were related to Dandekar 1995). The supernatant was dried in a Speedchanges in the gene expression. Vac (Val Chim, Milano, Italy), and resuspended in 0.2 M

lithium citrate buffer, pH 2.2. The amino acid analysisAbbreviations - RWC, relative water content; *P, leaf water po- was carried out in an Amino Acid Analyzer 3A30 (Carlotential; T f \ full turgor osmotic potential, ErhsL, Milano, Italy), using a 15 x 0.46 cm analytical col-

umn, filled with 3AR/IC/6/10 Li resin and equilibrated, , with 0.2 M lithium citrate buffer, pH 2.8.

Materials and methodsPlant material and growth conditionsCaryopses of durum wheat (Triticum durum Desf.) cv. Enzyme assaySimeto were surface-sterilized for 5 min in NaClO (1% The chilled leaves were placed in a pre-cooled mortaractive chlorine), rinsed and incubated in distilled water and homogenized with 0.1 M Tris-HCl buffer (pH 7.8)at 25°C, in the dark on an agitator (about 90 oscillations containing 1 mM EDTA, 1 mM dithiothreitol and 0.2 gmin"'). After 12 h of imbibition, 30 caryopses were g"' fresh weight PVP The homogenate was filteredspread per petri dish, which was lined with two layers of through cheesecloth and centrifuged at 10000 g for 10water-soaked filter paper. Distilled water (2 ml) was min. The supematant was then used for the enzyme assayadded daily. Water and salt stress were imposed on 5- (Mattioni et al. 1997). The activity of P5C reductase wasday-old seedlings at the first-leaf stage. measured spectrophotometrically by monitoring the de-

crease in absorbance of NADPH at 340 nm. The D,L-P5Cstock solution was prepared by regenerating P5C from its

Stress treatment 2,4-dinitrophenyl-hydrazine hydrochloride double saltTo impose a gradual increase in stress level, sampling (Sigma Chemical Co., St Louis, MO, USA) according towas performed at different times. Water stress was im- Szoke et al. (1992). The reaction mixture contained 0.56posed by uncovering the petri dishes and allowing the mM P5C, 0.1 mM NADPH, potassium phosphate bufferseedlings to desiccate for 8, 10 and 24 h. Unstressed (50 mM, pH 7.2) and 0.02 ml of enzyme in a total volumecontrol seedlings were kept moist. Salt treatment was of 0.5 ml. Proline dehydrogenase was assayed by follow-performed by adding 20 ml of a 200 mM NaCl solution ing the NAD" reduction in 0.5 ml of reaction mediumto each opened dish; 20 ml of distilled water was added containing 15 mM L-proline, 10 mM NAD", 0.2 Mas control. The leaves were harvested after 8, 24, 48 and NajCOg-NaHCOj buffer, pH 10.3, and 0.1 ml of enzyme72 h and used for analysis. according to Rena and Sphttstoesser (1975). The enzyme

All experiments were carried out in a growth chamber activity, defined as the amount of enzyme catalyzing theunder a 16-h photoperiod provided by fiuorescent lamps oxidation/reduction of 1 |imol of NADPH/NAD" min"',(Philips TDL 58 W/3, 36 W) giving an irradiance of 160 was referred to the protein content of each sample and

7 8 8 Physiol, Plant, 101, 1997

Tab, 1. Relative water content (RWC), leaf water potential (¥), full turgor osmotic potential C^],^), total free amino acid and protein contentin leaves of Triticum durum seedlings during water and salt stress. Values are the mean of three independent samples (± SE); *, **, *** =F<0.05, F<0.01, P<0,001; NS, not significant; ND, not determined, ,,,

Dehydration

RWC (%)

^ (MPa)

^'[°° (MPa)

Total free amino acids(^mol g"' DW)

Total protein (mg g"' DW)

NaCl 200 mM

RWC (%)

"¥ (MPa)

^ r (MPa)

Total free amino acids(]umol g ' DW)

Total protein (mg g~' DW)

Control

93.9(0.2)

-0.30(0,03)

-0.65(0.01)

82.2(4.1)

111.9(3.8)

8h

Control

85.9(1,3)

-0.40(0.05)

-0.73(0.01)

93.0(5,6)

119.1(1.0)

8h

Stressed

Stressed

71 5**(4.6)

-1.26***(0.04)

-0.74**(0.01)

122,2***(2.8)

99,1*(2.8)

Control

86.6NS 92,3(0.3) (1.2)

-O,47NS -0.40(0,03) (0.07)

-0,80=*(0,01)

136,1^(18,9)

-0.70(0.03)

122,2(3.3)

117.2NS 87,8(2.1) (17,4)

Control

96.7(0.3)

-0.46(0.04)

-0.59(0.03)

157,9(1.0)

127.6(2.7)

24 h

Stressed

89.2*(0.6)

-O.53NS(0.07)

-0.82NS(0,03)

157,7**(3.9)

102,1 NS(0.8)

10 h

Stressed

73.2**(4.2)

-1.28***(0.10)

-0.70*(0.01)

172.5*(3.8)

111.9*(0,6)

48

Control

91.6(0.1)

-0.57(0,02)

-0.67(0.01)

241.4(6,4)

110.3(0.1)

h

24 h

Control

92.5(2.0)

-0.52(0.02)

-0.67(0.01)

134.6(3.2)110.4(0.9)

Stressed

88,4**(0.8)

-0.80**(0.03)

-1.38=*(0.19)

334.3***(8.4)

91.2=*(4.0)

Control

88.2(0.6)

-0.45(0.02)

-O.61(0.00)

220.7(0.0)

104.4(0.6)

Stressed

33.3**(2.1)

ND

-0.87***(0.01)

403.4***(4.4)

88.7***(1.6)

72 h

Stressed

86.5 NS(0.5)

-0.52*(0.03)

-1.55**(0.10)

403,1**(16.5)

96.4NS(2.0)

wereexpressed as percentage of control. Soluble proteinsdetermined in the supematant according to Bradford(1976) using bovine semm albumin as standard.

RNA extraction and analysis

Total RNA was isolated from leaves by acid guanidin-ium thiocyanate-phenol-chloroform extraction (Cho-meczynski and Sacchi 1987). Total RNA (5 |ig) wassize-fractionated by electrophoresis on 1% agar-ose/formaldehyde gels, visualized by staining withethidium bromide and transferred in 20x SSC to Hy-bond-N membranes (Amersham, Buckinghamshire,UK). The Arabidopsis thaliana cDNA clone AT-P5C1,encoding the P5C reductase (Verbmggen et al. 1993)was kindly provided by Prof. M. Van Montagu, GentUniversiteit, Belgium, and used to examine the expres-sion of the AT-P5C1 gene under salt and water stress inwheat. Prehybridization and hybridization reactionswere carried out at 50°C in a buffer containing 5x SSC,7% (w/v) SDS, 50% (v/v) deionized formamide, 2%(w/v) blocking reagent (Boehringer, Indianapolis, IN,

USA), 50 mM sodium phoshate, pH 7.0, 0.1% (w/v) N-lauryl sarcosine. Filters were prehybridized for 8 h andthen hybridized for 16-20 h with DIG-High Prime La-beled Probe (Boehringer). DIG-labeled DNA was de-tected by an antibody conjugated to alkaline phos-phatase, which catalyzes a color reaction according toBoehringer protocols. Filters were washed twice with 2xSSC, 0.1% SDS at room temperature for 15 min andtwice with 0.5x SSC, 0.1% SDS at 68°C for 15 min.

Statistics

The mean values ± SE are reported in tables and figures.The significance of differences between control andeach treatment was evaluated by the r-test.

ResultsWater relations, proiine and accumulation of free aminoacids

Plants under water stress displayed a more rapid reduc-tion in relative water content and leaf water potential

Physiol, Plant, 101, 1997 789

than that which occurred during salt stress. In contrast,osmotic potential at full turgor was greatly decreased(more negative) by salt treatment, reaching a value of-1.55 MPa after 72 h (Tab. 1). Although it was not pos-sible to determine the water potential after 24 h of dehy-dration, rewatering of the seedlings induced their recov-ery, and the *F after 24 and 48 h was -1.1 and -0.85MPa, respectively (data not shown).

Effects of water and salt stress on the accumulation offree amino acids were also analyzed. Most of the aminoacids showed an increase with the induction of eitherstress (data not shown). Proline was one of the minorfree amino acids in the control, but increased moremarkedly than the others under both treatments (Tab. 2).Increases in proline content were similar at 8 and 10 h ofdehydration, but severe stress caused a marked increase(48-fold as compared with the control). During salt treat-ment proline accumulated gradually, reaching a concen-tration about 7-fold of the control at 72 h.

Table 2 shows the concentrations of the three aminoacids involved in the proline biosynthetic pathway:glutamic acid, omithine and arginine. Glutamic acid in-creased significantly under severe stress (72 h NaCland 24 h dehydration), whereas arginine was alwayshigher during either treatment. Although it was impos-sible to determine ornithine in the controls and in sam-ples subjected to moderate stress, low amounts weredetected during severe stress. (72 h NaCl and 24 h de-hydration).

Total free amino acids and protein content

During salt stress (Tab. 1) no significant differences intotal proteins were found, whereas after 72 h of treat-ment the increase in free amino acids was nearly doublethat of the control. Water stress resulted in a small butsignificant decrease in total proteins, which remainedconstant (about 0.8-fold) during the stress treatment. Incontrast, the free amino acid content increased about2.9-fold after 24 h.

Enzyme activity

Both water and salt stress induced P5C reductase (Fig.1). After 8 h of dehydration the activity was about 200%of control; a similar increase was observed after 48 h ofsalt treatment. Shorter NaCl treatments did not affect en-zyme activity significantly. No changes in proline dehy-drogenase were observed during water stress, while 24 hof NaCl treatment caused a significant reduction in itsactivity.

Expression of AT-P5C1 gene

Figure 2 shows the expression of AT-P5C1 during condi-tions of water and salt stress. RNA hybridization re-vealed one transcript, with no differences being ob-served in the controls, neither in drought- nor salt-stressed tissues. No correlation was found between the

Tab. 2. Concentrations of main free amino acids (^mol g ' dry weight) involved in proline biosynthetic pathway determined in leaves ofTriticum dumm seedlings during water and salt stress. Values are the mean of three independent samples (± SE); *, **, *** = P<0,05,P<0,01, P<0,001; NS, not significant; ND, not determined.

Dehydration

Glutamic acid

Arginine

Omithine

Prohne

NaCl 200 mM

Glutamic acid

Arginine

Omithine

Proline

Control

2,43(0.27)

0.47(0.01)

ND

0,93(0.06)

8h

Control

2.33(0.26)

0,44(0.03)

ND

1.21(0.08)

8h

Stressed

2.00NS(0.12)

0,87**(0.02)

ND

1.35**(0.03)

Stressed

3,98*(0.42)

0.69*(0.09)

ND

2.06**(0.24)

Control

4.44(1.09)

0.90(0,05)

ND

1.35(0,10)

lOh

Control

2,20(0.10)

1,34(0.15)

ND

1.60(0.00)

24 h

Stressed

5.5 INS(0.97)

1.30**(0.04)

ND

(0.49)

Stressed

1.50NS(0.30)

1.98*(0.01)

ND

2.50*(0.25)

48

Control

6.91(0,76)

2.56(0.13)

ND

4.11(0.33)

24 h

Control

2,00(0,20)

2.05(0.14)

ND

1.00(0,05)

h

Stressed Control

6.20NS 1.20(0.62) (0,00)

4.36** 4.45(0.24) (0.27)

ND ND

21.99*** 2,65(1,28) (0,11)

Stressed

5,00**(0.00)3.88**(0.12)0.18**(0.01)48.66***(0.89)

72 h

Stressed

(0,34)10.30*(0.10)

0.41***(0.00)

18.8**(0,90)

790 Physiol, Plant, 101, 1997

Fig. 1 • Effects of water and saltstress on pyrroline-5-carboxylate reductase andproline dehydrogenase.Enzyme activty is expressed aspercent of control (100%). Allthe values are the means ofthree replicates (± SE).

250

10 24 8

Duration of stress (h)

24 48 72

changes in the P5C reductase activity and the expressionof the AT-P5C1 gene.

Discussion

The accumulation of compatible solutes may help tomaintain the relatively high water content necessary forgrowth and cellular function. Although a general in-crease in all amino acids was observed in T. durum underwater and salt stress, only the proline content was highenough for this amino acid to be considered the princi-pal solute in osmoprotection. To evaluate whether accu-mulation of proline is an active process brought about bythe onset of the stress, we measured the activity of P5Creductase and proline dehydrogenase, enzymes involvedin proline biosynthesis and catabolism, and we com-pared the values of total proteins and total free amino ac-ids to determine the possible role of proteolysis. P5C re-ductase activity increased under both stresses, but onlyduring NaCl treatment could an inhibition of proline de-hydrogenase activity be observed. This could have beendue to the interaction of the salt with the -SH groups ofprotein.

Duration of stress treatment (h)

0 8 10 24 0 8 10 24 48 72

B

Dehydration NaCl 200 mM

Fig. 2. Expression of the AT-P5C1 gene in response to water andsalt stress (A) in Triticum durum cv. Simeto plants. Total RNAswere visualizated by staining with ethidium bromide (B).

It has been shown that ammonium sulphate and heavymetals inhibit proline dehydrogenase but have no effecton P5C reductase, suggesting that -SH groups are not re-quired for the activity of either enzyme (Rena andSplittstoesser 1975). During the onset of both stresses asignificant increase in total free amino acids was ob-served, but only water stress resulted in a decrease in to-tal protein, which remained constant during the induc-tion of the stress, and which therefore cannot be relatedto the increase in amino acids. Hence, these resultswould suggest a reduction in protein synthesis ratherthan the initiation of proteolysis, as previously shown inBrassica napus by Good and Zaplachinski (1994).Therefore, in durum wheat, de novo synthesis seems tobe the predominant mechanism of proline accumulation,but a reduction in cytoplasmic proline oxidation and inprotein synthesis might also be involved in salt and wa-ter stress, respectively. Long exposure to NaCl resultedin lower proline and osmotic potential values than thoseobserved under shorter (24 h) periods of dehydration.Osmotic potential may also be influenced by accumula-tion of salt. It has been demonstrated that with the waterdeficit resulting from a saline environment, plant cellsaccumulate three kinds of osmotica: small organic sol-utes, glycine-rich proteins, and a controlled amount ofsalt (Chiang et al. 1995). Furthermore, the accumulationof proline during water deficit may have other functions,such as enzyme protection and stabilization of biologicalmembranes; the degradation of proline may improve theenergy status of cells recovering from water deficit (Sa-radhi and Saradhi 1991). Although the level of P5C re-ductase activity was found to increase about 2-fold inleaves of stressed plants, no change was observed in theexpression of the AT-P5C1, implying that the increase inenzyme activity is not related to changes in the expres-sion of this gene.

In summary, we have shown that in seedlings of Triti-cum durum proline accumulation under water and saltstress is an active process that requires the activation ofbiosynthetic and catabolic enzymes.

Acknowledgments - We thank Drs R. Coccia and C. Foppoli,Dip. Scienze Biochimiche, Univ. La Sapienza, Roma, for theamino acid analysis. Prof. M. Van Montagu, Univ. Gent, for pro-

Physiol, Plant. 101, 1997 791

viding the P5C1 clone, and Dr J, Napier, IACR-Long Ashton,for his comments on the manuscript. This work was supportedby the Italian Ministry of Agriculture 'Progetto Finalizzato suUeBiotecnologie Vegetali' DM, 123/7240/96,

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Edited by C. H. Bomman

792 Physiol, Plant, 101, 1997