JPlantPhysiol. Vol. 132.pp. 86-89{1988}

Introduction

Appearance of N-malonyl-D-tryptophan in Excised Leaves during Wilting. 1. The Content of Tryptophan and N-malonyl­D-tryptophan as Affected by Water Deficit

N. I. REKOSLAVSKAYA, T. A. MARKOVA, and K. Z. GAMBURG

Institute of Plant Physiology and Biochemistry, Siberian Branch of the Academy of Sciences of the USSR, 664033 Irkutsk, USSR

Received May 7, 1987 . Accepted August 18, 1987

Summary

The leaves of 33 plant species were excized and allowed to wilt for 3 days. 10-SO-fold increase of L-Try content was observed in all plants studied. N-malonylated and N-acetylated D-Try appeared in drought­stressed leaves of 20 species, the remaining 13 species failed to exhibit the same response. N-acylation of infiltrated D-Try proceeded in turgid leaves of plants both forming and not forming MTry under wilting. Accumulation of L-T ry ceased after 3 days of tomato leaf wilting, whereas the content of MT ry increased during the whole 7 days of wilting and finally exceeded that of L-Try. Even small water loss from tomato leaves was sufficient for maximal increase of L-Try content. The rate of MTry accumulation enhanced concomitantly with an increase of the severity of drought stress. Recovery of excized tomato leaves from wilting did not result in a decrease of L-Try and MTry contents. It is concluded that many, but not all plants may respond to drought stress by D-Try formation followed by its N-acylation. L-Try is proposed to be a possible precursor of D-Try.

Key words: Leaf, Nmalonyl-D-tryptophan, tryptophan, wilting, drought stress.

Abbreviations: Try - tryptophan, MTry - N-malonyl-D-tryptophan, AcTry - N-acetyl-D-tryp­tophan, IAA - indole-3-acetic acid.

Materials and Methods

It is known that some plants contain not only L-amino acids but also D-amino acids, especially D-alanine and D-Try (Robinson, 1976). However, functions of D-amino acids and factors controlling their accumulation in plants remain ob­scure.

Cultivated and wild plants of 33 species from 12 families were used in the experiments. Cultivated plants were grown in a phyto­trone, plastic greenhouses, and in the field. Wild plants were harvested from natural habitats near Irkutsk. The major experi­ments were performed with leaves of tomato (cv. Moskovski osenny) grown for two months in a phytotrone at 25/18 °C (day/ night) under 16h day. The content of amino acids and especially that of proline

significantly increases under water deficit influence (Stewart and Larher, 1980; Hanson and Hitz, 1982). We have found MTry in tomato leaves (Rekoslavskaya and Gamburg, 1983) and observed variation of its content depending on wilting before extraction.

The aim of this paper has been to investigate the influence of wilting induced by water deficit on Try and MTry con­tent in plant leaves.

© 1988 by Gustav FIscher Verlag, Stuttgart

Fully expanded turgid leaves of adult plants were excised and al­lowed to lose 30 - 35 % of the initial fresh weight and then trans­ferred to a wet chamber for 3 days, where further change of leaf weight did not occur. The freshly excised leaves were used as a control. In some experiments excised tomato leaves were immersed in water by their petioles after 3 days of wilting and maintained for 7 days.

The leaves were fixed and extracted 3 times with boiling 80 % ethanol containing 0.02 % sodium diethyldithiocarbamate. Com-

JPlantPhysiol. Vol. 132.pp. 86-89{1988}

Introduction

Appearance of N-malonyl-D-tryptophan in Excised Leaves during Wilting. 1. The Content of Tryptophan and N-malonyl­D-tryptophan as Affected by Water Deficit

N. I. REKOSLAVSKAYA, T. A. MARKOVA, and K. Z. GAMBURG

Institute of Plant Physiology and Biochemistry, Siberian Branch of the Academy of Sciences of the USSR, 664033 Irkutsk, USSR

Received May 7, 1987 . Accepted August 18, 1987

Summary

The leaves of 33 plant species were excized and allowed to wilt for 3 days. 10-SO-fold increase of L-Try content was observed in all plants studied. N-malonylated and N-acetylated D-Try appeared in drought­stressed leaves of 20 species, the remaining 13 species failed to exhibit the same response. N-acylation of infiltrated D-Try proceeded in turgid leaves of plants both forming and not forming MTry under wilting. Accumulation of L-T ry ceased after 3 days of tomato leaf wilting, whereas the content of MT ry increased during the whole 7 days of wilting and finally exceeded that of L-Try. Even small water loss from tomato leaves was sufficient for maximal increase of L-Try content. The rate of MTry accumulation enhanced concomitantly with an increase of the severity of drought stress. Recovery of excized tomato leaves from wilting did not result in a decrease of L-Try and MTry contents. It is concluded that many, but not all plants may respond to drought stress by D-Try formation followed by its N-acylation. L-Try is proposed to be a possible precursor of D-Try.

Key words: Leaf, Nmalonyl-D-tryptophan, tryptophan, wilting, drought stress.

Abbreviations: Try - tryptophan, MTry - N-malonyl-D-tryptophan, AcTry - N-acetyl-D-tryp­tophan, IAA - indole-3-acetic acid.

Materials and Methods

It is known that some plants contain not only L-amino acids but also D-amino acids, especially D-alanine and D-Try (Robinson, 1976). However, functions of D-amino acids and factors controlling their accumulation in plants remain ob­scure.

Cultivated and wild plants of 33 species from 12 families were used in the experiments. Cultivated plants were grown in a phyto­trone, plastic greenhouses, and in the field. Wild plants were harvested from natural habitats near Irkutsk. The major experi­ments were performed with leaves of tomato (cv. Moskovski osenny) grown for two months in a phytotrone at 25/18 °C (day/ night) under 16h day. The content of amino acids and especially that of proline

significantly increases under water deficit influence (Stewart and Larher, 1980; Hanson and Hitz, 1982). We have found MTry in tomato leaves (Rekoslavskaya and Gamburg, 1983) and observed variation of its content depending on wilting before extraction.

The aim of this paper has been to investigate the influence of wilting induced by water deficit on Try and MTry con­tent in plant leaves.

© 1988 by Gustav FIscher Verlag, Stuttgart

Fully expanded turgid leaves of adult plants were excised and al­lowed to lose 30 - 35 % of the initial fresh weight and then trans­ferred to a wet chamber for 3 days, where further change of leaf weight did not occur. The freshly excised leaves were used as a control. In some experiments excised tomato leaves were immersed in water by their petioles after 3 days of wilting and maintained for 7 days.

The leaves were fixed and extracted 3 times with boiling 80 % ethanol containing 0.02 % sodium diethyldithiocarbamate. Com-

bined ethanolic extract was reduced to the aqueous phase under vac­uum. Lipids and pigments were removed by peroxide-free diethyl ether at pH 9. The aqueous phase was adjusted to pH 2 using H3P04, MTry and other derivatives of Try were extracted 10 -15 times with diethyl ether. The acid ether fraction was evaporated and the dry residue was dissolved in 1-2 ml ethanol. Chromatography was performed on aluminium foil plates covered with thin layer of silica gel (<<Silufob, Kavalier, Czechoslovakia) using chloroform­ethyl-acetate-80 % formic acid (5: 4: 1) as the solvent system. Indo­Iylic substances were visualized with Ehrlich spray reagent (Sprince, 1960). For quantitative determination of MTry the acid ether frac­tion was chromatographed on thin layer of cellulose MN 300 (Di.iren, West Germany) with n-butanol-25 % ammonia-water (10: 1: 1). The zone corresponding to MTry was eluted with 80 % ethanol and colorimetric determination of MTry was made with Ehrlich reagent.

The aqueous phase remaining after MTry extraction was adjusted to pH 6 -7 and evaporated to dryness. The residue was dissolved in 0.067 M phosphate buffer, pH 8 and chromatographed on a polyvin­ylpyrrolidone (Polyclar AT, Serva) column (9 x 400 mm) with the same buffer. The fraction of eluate from 20 to 30 ml was used for colorimetric determination of Try with Ehrlich reagent. The losses of Try and MTry were about 35-40%. No corrections for these losses are made in the Tables and Figures.

Try eluted from the polyvinylpyrrolidone column was addition­ally purified on a column of Dowex SOW (H+ -form) with 2 N am­monia as eluent and then on a thin layer of silica gel with methyl­acetate-iso-propanol-25 % ammonia (9: 7: 4). The zone with Rf 0.4 was eluted with 80 % ethanol, the eluate evaporated and the residue dissolved in Tris-HCl buffer, pH 8.4. The purified preparation of Try was treated with L-amino acid oxidase from snake venom (pro­duction of the Institute of Chemistry, Academy of Sciences of the Estonian SSR). The reaction mixture containing 1ltmole of isolated Try and 1 a.u. of the enzyme in 1-2 ml of the same buffer was in­cubated at 37°C for 30 min. The reaction products were separated on a thin layer of silica gel and visualized by treatment with Ehrlich and ninhydrin reagents. L-Try was found to be completely de­stroyed and D-Try remained unchanged under these conditions.

The results were expressed on the basis of the initial fresh (before wilting) weight. Experiments were repeated twice or more often with similar results.

Results

As shown in Table 1, water deficit induced 1- 2 order in­crease of Try content in leaves of all plants used in the ex­periments. The Try content in turgid leaves was consistent with that observed by other investigators (Allen and Baker, 1980; Tarr and Arditti, 1981) and varied from less than 10 to 40 nmoles x g-l fresh weight. MTry was not found in turgid leaves while it appeared in wilted ones. In some cases the content of MTry was greater than that of Try. Wilted leaves of Fabaceae plants contained the greatest amounts of MTry. However, some plants (Asteraceae, Brassicaceae, Plantagi· naceae, maize, pepper) failed to form MTry under wilting.

It was observed that some drought-stressed plants accu­mulated not only MTry but also other derivatives of Try (Fig. 1). The compound with Rf0.75 was of the same chro­matographic mobility in several solvent systems and of the same coloration with Salkowski, Ehrlich and Prohazka rea­gents as AcTry. This compound predominated in wilted leaves of clover and beet.

N-malonyl-D-tryptophan and drought stress 87

Table 1: The effect of drought stress on Try and MTry content in plant leaves (nmoles x g-l fro wt.).

Families Species Control Drought stress

Try Try MTry

Fabaceae Melilotus albus Medik. -*) 680 1300

Vicia faba L. 980 1280

Medicago sativa L. 490 1110

Trifolium repens L. 370 280

Phaseolus vulgaris L. 510 810

Pisum sativum L. 39 530 340

Apiaceae Heracleum sibiricum L. 310 530

Anethum graveolens L. 630 390

Daucus sativus (Hoffm.) Roehl. 220 110

Chenopodiaceae Chenopodium album L. 410 970

Beta vulgaris L. (beetroot) 410 440

(leaf beet) 940 580

Solanaceae Solanum tuberosum L. 11 440 1660

L ycopersicon esculentum Mill. 22 550 550

Solanum melongena L. <10 270 <10

Capsicum annuum L. 36 590 0

Cucurbitaceae Cucumis sativus L. <10 490 350

Citrullus lanatus (Thunb.) Matsum. <10 400 280

Brassicaceae Brassica capitata (L.) Pers. <10 200 0

Brassica botrytis (L.) Mill. <10 160 0

Brassica pekinensis (Lour.) Rupr. 10 490 0

Asteraceae Cichorium intybus L. <10 270 0

Lactuca sativa L. <10 250 0

Taraxacum officinale L. 0

Poaceae Zea mays L. 10 800 0

Triticum aestivum L. 14 590 300

Rosaceae Malus baccata (L.) Borkh. 390 410

Rosa canina L. 360 <10

Rubus saxatilis L. 240 <10

Plantaginaceae Plantago major L. 300 0

Plantago media L. 440 0

Saxifragaceae Ribes nigrum L. 300 <10

Onocleaceae Matteuccia struthwpteris (L.) Tod. <10 130 <10

*) not determined

Infiltration of D-Try in excised turgid leaves of plants both forming and not forming MTry and AcTry under drought stress resulted in its transformation to N-acylated derivatives (Table 2). It may be assumed that the ability to N-acylation is characteristic of the leaves of all plants even without the wilt­ing, and that water deficit appears to induce the formation of D-Try, but not its N-acylation.

The experiments with L-amino acid oxidase showed that Try isolated from drought-stressed leaves of tomato, potato, beet, sweet clover (forming MTry) was completely de­stroyed. No ninhydrin-positive compounds were found after the oxidase treatment and only indole-pyruvic acid was ob­served as Ehrlich-positive compound. It may be proposed that the amount of free D-Try (if any is present in the leaves) does not exceed 0.01 % of the amount of L-Try in terms of the sensitivity of ninhydrin reagent. It is probable that a rate constant of N-acylation of D-Try in wilted leaves is signifi­cantly greater than that of D-Try formation resulting in the prevention of accumulation of measurable amounts of free D-Try.

The accumulation of Try and MTry as dependent on the duration of the exposure of tomato leaves to wilting is pre-

bined ethanolic extract was reduced to the aqueous phase under vac­uum. Lipids and pigments were removed by peroxide-free diethyl ether at pH 9. The aqueous phase was adjusted to pH 2 using H3P04, MTry and other derivatives of Try were extracted 10 -15 times with diethyl ether. The acid ether fraction was evaporated and the dry residue was dissolved in 1-2 ml ethanol. Chromatography was performed on aluminium foil plates covered with thin layer of silica gel (<<Silufob, Kavalier, Czechoslovakia) using chloroform­ethyl-acetate-80 % formic acid (5: 4: 1) as the solvent system. Indo­Iylic substances were visualized with Ehrlich spray reagent (Sprince, 1960). For quantitative determination of MTry the acid ether frac­tion was chromatographed on thin layer of cellulose MN 300 (Di.iren, West Germany) with n-butanol-25 % ammonia-water (10: 1: 1). The zone corresponding to MTry was eluted with 80 % ethanol and colorimetric determination of MTry was made with Ehrlich reagent.

The aqueous phase remaining after MTry extraction was adjusted to pH 6 -7 and evaporated to dryness. The residue was dissolved in 0.067 M phosphate buffer, pH 8 and chromatographed on a polyvin­ylpyrrolidone (Polyclar AT, Serva) column (9 x 400 mm) with the same buffer. The fraction of eluate from 20 to 30 ml was used for colorimetric determination of Try with Ehrlich reagent. The losses of Try and MTry were about 35-40%. No corrections for these losses are made in the Tables and Figures.

Try eluted from the polyvinylpyrrolidone column was addition­ally purified on a column of Dowex SOW (H+ -form) with 2 N am­monia as eluent and then on a thin layer of silica gel with methyl­acetate-iso-propanol-25 % ammonia (9: 7: 4). The zone with Rf 0.4 was eluted with 80 % ethanol, the eluate evaporated and the residue dissolved in Tris-HCl buffer, pH 8.4. The purified preparation of Try was treated with L-amino acid oxidase from snake venom (pro­duction of the Institute of Chemistry, Academy of Sciences of the Estonian SSR). The reaction mixture containing 1ltmole of isolated Try and 1 a.u. of the enzyme in 1-2 ml of the same buffer was in­cubated at 37°C for 30 min. The reaction products were separated on a thin layer of silica gel and visualized by treatment with Ehrlich and ninhydrin reagents. L-Try was found to be completely de­stroyed and D-Try remained unchanged under these conditions.

The results were expressed on the basis of the initial fresh (before wilting) weight. Experiments were repeated twice or more often with similar results.

Results

As shown in Table 1, water deficit induced 1- 2 order in­crease of Try content in leaves of all plants used in the ex­periments. The Try content in turgid leaves was consistent with that observed by other investigators (Allen and Baker, 1980; Tarr and Arditti, 1981) and varied from less than 10 to 40 nmoles x g-l fresh weight. MTry was not found in turgid leaves while it appeared in wilted ones. In some cases the content of MTry was greater than that of Try. Wilted leaves of Fabaceae plants contained the greatest amounts of MTry. However, some plants (Asteraceae, Brassicaceae, Plantagi· naceae, maize, pepper) failed to form MTry under wilting.

It was observed that some drought-stressed plants accu­mulated not only MTry but also other derivatives of Try (Fig. 1). The compound with Rf0.75 was of the same chro­matographic mobility in several solvent systems and of the same coloration with Salkowski, Ehrlich and Prohazka rea­gents as AcTry. This compound predominated in wilted leaves of clover and beet.

N-malonyl-D-tryptophan and drought stress 87

Table 1: The effect of drought stress on Try and MTry content in plant leaves (nmoles x g-l fro wt.).

Families Species Control Drought stress

Try Try MTry

Fabaceae Melilotus albus Medik. -*) 680 1300

Vicia faba L. 980 1280

Medicago sativa L. 490 1110

Trifolium repens L. 370 280

Phaseolus vulgaris L. 510 810

Pisum sativum L. 39 530 340

Apiaceae Heracleum sibiricum L. 310 530

Anethum graveolens L. 630 390

Daucus sativus (Hoffm.) Roehl. 220 110

Chenopodiaceae Chenopodium album L. 410 970

Beta vulgaris L. (beetroot) 410 440

(leaf beet) 940 580

Solanaceae Solanum tuberosum L. 11 440 1660

L ycopersicon esculentum Mill. 22 550 550

Solanum melongena L. <10 270 <10

Capsicum annuum L. 36 590 0

Cucurbitaceae Cucumis sativus L. <10 490 350

Citrullus lanatus (Thunb.) Matsum. <10 400 280

Brassicaceae Brassica capitata (L.) Pers. <10 200 0

Brassica botrytis (L.) Mill. <10 160 0

Brassica pekinensis (Lour.) Rupr. 10 490 0

Asteraceae Cichorium intybus L. <10 270 0

Lactuca sativa L. <10 250 0

Taraxacum officinale L. 0

Poaceae Zea mays L. 10 800 0

Triticum aestivum L. 14 590 300

Rosaceae Malus baccata (L.) Borkh. 390 410

Rosa canina L. 360 <10

Rubus saxatilis L. 240 <10

Plantaginaceae Plantago major L. 300 0

Plantago media L. 440 0

Saxifragaceae Ribes nigrum L. 300 <10

Onocleaceae Matteuccia struthwpteris (L.) Tod. <10 130 <10

*) not determined

Infiltration of D-Try in excised turgid leaves of plants both forming and not forming MTry and AcTry under drought stress resulted in its transformation to N-acylated derivatives (Table 2). It may be assumed that the ability to N-acylation is characteristic of the leaves of all plants even without the wilt­ing, and that water deficit appears to induce the formation of D-Try, but not its N-acylation.

The experiments with L-amino acid oxidase showed that Try isolated from drought-stressed leaves of tomato, potato, beet, sweet clover (forming MTry) was completely de­stroyed. No ninhydrin-positive compounds were found after the oxidase treatment and only indole-pyruvic acid was ob­served as Ehrlich-positive compound. It may be proposed that the amount of free D-Try (if any is present in the leaves) does not exceed 0.01 % of the amount of L-Try in terms of the sensitivity of ninhydrin reagent. It is probable that a rate constant of N-acylation of D-Try in wilted leaves is signifi­cantly greater than that of D-Try formation resulting in the prevention of accumulation of measurable amounts of free D-Try.

The accumulation of Try and MTry as dependent on the duration of the exposure of tomato leaves to wilting is pre-

88 N. 1. REKOSLAVSKAYA, T. A. MARKOVA, and K. Z. GAMBURG

: •• v

"

<::) 0 ~ C)

A B C D

, " ..... ~' ::' ',;, .

C)

E

5

• 4

2

• F

Fig. 1: The schematic presentation of the chromatogram of acid ether fractions of ethanolic extracts from drought-stressed plant leaves. A - Anethum graveolens L., B - Beta vulgaris L., C - Tnfo. lium repens L., D - Matteuccia struthiopteris (L.) Tod., E - Hera· cleum sibincum L., F - tracers. 1 - Try, 3 - MTry, 4 - AcTry, 2 and 5 - unknown Ehrlich-positive compounds. TLC, «Silufob> pla­tes, chloroform-ethylacetate-85 % formic acid (5: 4: 1), Ehrlich spray reagent (Sprince, 1960).

Table 2: The formation of MTry and AcTry in plant leaves as induc­ed by drought stress or infiltration with D-Try (200 mg x I-I).

Plants 3 days of 3 days after wilting D-Try

MTry

Capsicum annuum L. 0 Cichorium tntybus L. 0 Brassica pekinensis (Lour.) Rupr. 0 T rifoltum repens L. + Matteuccia struthiopteris (L.) T od. + *) + - ~100nmolesxg-1 fr.wt.

AcTry

o o o +++ +

+ + - between 120 and 200 nmoles x g-I fr.wt. + + + - between 250 and 500 nmoles x g-I fr.wt.

infiltration MTry AcTry

++*) ++ + + + 0 +++ + ++ +++

sented in Table 3. The leaves were allowed to lose 30 % of the initial fresh weight and then placed in a wet chamber for the 7 days. The content of Try increased only for the first 2-3 days, whereas the rate of accumulation of MTry was nearly constant for the whole 7 days of the experiment. As a result, the MTry content exceeded that of Try at the end of the ex­periment.

Table 4 shows the effect of different initial dehydration of tomato leaves on Try and MTry accumulation. It is evident that even small loss of water from the leaves produced the maximal increase of Try content. More severe wilting re­sulted in the diminishing of Try content. On the contrary,

Table 3: Time course of Try and MTry accumulation in drought-stressed tomato leaves (nmoles x g-I fr.wt.).

Time, Try MTry days 1st expo 2nd expo 1st expo 2nd expo

0 29 10 66 1 343 191 148 131 3 529 431 331 379 6 569 196 662 703 7 627 284 1241 772

Table 4: The effect of 3-day's drought stress of different severity on Try and MTry content in tomato leaves (nmoles x g-I fr.wt.).

Water loss, % of Try MTry the initial fr. wt. 1st expo 2nd expo 1st expo 2nd expo

0*) <10 <10 7 117 10 392 412 183 724 20 245 328 290 1152 30 328 270 1014 40 353 186 776 1352 50 147 186 1603 1283

*) - freshly excized leaves

Table 5: The contents of Try and MTry as influenced by drought­stress and by following rewatering (nmoles x g-I fr.wt.).

Variants Try MTry 1st expo 2nd expo 1st expo 2nd expo

before wilting 98 17 n.d.*) n.d. 3 days of wilting 431 300 428 359 7 days after wilting 588 642 497 421

*) - not detected

the accumulation of MTry was enhanced by water loss up to 50 % of the initial fresh weight. The rate of MTry accumula­tion seems to correlate with the severity of drought stress.

Accumulation of MTry and Try induced by drought stress was hindered, but did not stop when excized tomato leaves regained their turgor due to rewatering (Table 5).

Discussion

Our results unequivocally show that water deficit is one of the factors inducing the appearance of D-amino acids (Try) in plants. It is important that plants may be divided into two groups: one - forming and other - not forming N-acyl-D­Try under drought stress, whereas L-Try content increases in plants of both groups. This fact concerns species, genera, and families. The representatives of both groups were found in dicot and monocot species. However, it is not known yet, whether this difference may exist at the intraspecific level (i.e. between races, cultivars, or mutants). It is an interesting field for further investigation.

We found some plants to form AcTry in addition to MTry. The ability to acetylate D-amino acids was previously reported only for fungi and algae, but not for vascular plants

88 N. 1. REKOSLAVSKAYA, 1. A. MARKOVA, and K. Z. GAMBURG

. :.~ ".

5 .4 •• J

2

• a 0 C) C) C)

A B C D E F

Fig. 1: The schematic presentation of the chromatogram of acid ether fractions of ethanolic extracts from drought-stressed plant leaves. A - Anethum graveolens L., B - Beta vulgaris L., C - Tnfo. lium repens L., D - Matteuccia struthiopteris (L.) Tod., E - Hera· cleum sibincum L., F - tracers. 1 - Try, 3 - MTry, 4 - AcTry, 2 and 5 - unknown Ehrlich-positive compounds. TLC, «Silufob> pla­tes, chloroform-ethylacetate-85 % formic acid (5: 4: 1), Ehrlich spray reagent (Sprince, 1960).

Table 2: The formation of MTry and AcTry in plant leaves as induc­ed by drought stress or infiltration with D-Try (200 mg x I-I).

Plants 3 days of 3 days after wilting D-Try

MTry

Capsicum annuum L. 0 Cichorium tntybus L. 0 Brassica pekinensis (Lour.) Rupr. 0 T rifoltum repens L. + Matteuccia struthiopteris (L.) T od. + *) + - ~100nmolesxg-1 fr.wt.

AcTry

o o o +++ +

+ + - between 120 and 200 nmoles x g-I fr.wt. + + + - between 250 and 500 nmoles x g-I fr.wt.

infiltration MTry AcTry

++*) ++ + + + 0 +++ + ++ +++

sented in Table 3. The leaves were allowed to lose 30 % of the initial fresh weight and then placed in a wet chamber for the 7 days. The content of Try increased only for the first 2-3 days, whereas the rate of accumulation of MTry was nearly constant for the whole 7 days of the experiment. As a result, the MTry content exceeded that of Try at the end of the ex­periment.

Table 4 shows the effect of different initial dehydration of tomato leaves on Try and MTry accumulation. It is evident that even small loss of water from the leaves produced the maximal increase of Try content. More severe wilting re­sulted in the diminishing of Try content. On the contrary,

Table 3: Time course of Try and MTry accumulation in drought-stressed tomato leaves (nmoles x g-I fr.wt.) .

Time, Try MTry days 1st expo 2nd expo 1st expo 2nd expo

0 29 10 66 1 343 191 148 131 3 529 431 331 379 6 569 196 662 703 7 627 284 1241 772

Table 4: The effect of 3-day's drought stress of different severity on Try and MTry content in tomato leaves (nmoles x g-I fr.wt.).

Water loss, % of Try MTry the initial fr. wt. 1st expo 2nd expo 1st expo 2nd expo

0*) <10 <10 7 117 10 392 412 183 724 20 245 328 290 1152 30 328 270 1014 40 353 186 776 1352 50 147 186 1603 1283

*) - freshly excized leaves

Table 5: The contents of Try and MTry as influenced by drought­stress and by following rewatering (nmoles x g-I fr.wt.).

Variants Try MTry 1st expo 2nd expo 1st expo 2nd expo

before wilting 98 17 n.d.*) n.d. 3 days of wilting 431 300 428 359 7 days after wilting 588 642 497 421

*) - not detected

the accumulation of MTry was enhanced by water loss up to 50 % of the initial fresh weight. The rate of MTry accumula­tion seems to correlate with the severity of drought stress.

Accumulation of MTry and Try induced by drought stress was hindered, but did not stop when excized tomato leaves regained their turgor due to rewatering (Table 5).

Discussion

Our results unequivocally show that water deficit is one of the factors inducing the appearance of D-amino acids (Try) in plants. It is important that plants may be divided into two groups: one - forming and other - not forming N-acyl-D­Try under drought stress, whereas L-Try content increases in plants of both groups. This fact concerns species, genera, and families. The representatives of both groups were found in dicot and monocot species. However, it is not known yet, whether this difference may exist at the intraspecific level (i.e. between races, cultivars, or mutants). It is an interesting field for further investigation.

We found some plants to form AcTry in addition to MTry. The ability to acetylate D-amino acids was previously reported only for fungi and algae, but not for vascular plants

(Zenk and Scherf, 1964; Pokorny et al., 1970). Our results suggest that this ability spreads wider and involves higher plants. We failed to find any plants forming only AcTry as the synthesis of AcTry usually proceeded concomitantly with the MTry synthesis. The acetylation and malonylation are not the only ways of D-Try derivatization in plants be­cause other unidentified derivatives of D-Try were also ob­served in the chromatograms (Fig. 1).

Experiments with D-Try infiltration showed that the in­duction of the synthesis of N-malonyl-transferase (see Ma­tern et aI., 1984) cannot be responsible for MTry appearance in drought-stressed leaves. Exogenous D-Try was mal­onylated even in turgid leaves of plants both forming and not forming MTry under wilting. Therefore it was con­cluded that the crucial biochemical event necessary for MTry appearance under drought stress is the induction of D­Try synthesis.

As shown in Tables 3 and 4 the accumulation of L-T ry pre­ceeded the accumulation of MTry, and the amount of L-Try diminished with the MTry increase. These data suggest that L-Try may be a precursor of D-Try in wilted leaves. One of the possible ways of transformation of L-Try to D-Try is ra­cemization. The enzyme, racemase of D-Try, was found by Miura and Mills (1971) in cultured tobacco cells. It may be argued that water deficit induces the synthesis of this en­zyme. As a result, D-Try appears and is acylated. Further study will show, whether Try only appears in D-form under water deficit.

The physiological significance of MTry appearance during wilting is unknown. It has been shown in our previous com­munications (Rekoslavskaya and Gamburg, 1984; Rekoslav­skaya, 1986) that exogenous MTry is transformed to IAA in cultured soybean cells and tomato petioles. It is possible that MTry synthesized endogenously during wilting may be used as an IAA precursor after the restoration of leaf turgescence. However, we did not observe any decrease of MTry content in rewatered tomato leaves (Table 5). Furthermore the usual level of IAA in plant tissues is significantly lower (100-1000 times) than that of MTry observed in wilted leaves. There-

N-malonyl-D-tryptophan and drought stress 89

fore the consumption of MTry for IAA synthesis may not significantly change the MTry content in the leaves. Further experiments with labelled MTry can solve this problem.

References

ALLEN, J. R. F. and D. A. BAKER: Free tryptophan and indole-3-ac­etic acid levels in the leaves and vascular pathways of RIcinus communis L. Planta 148, 69-74 (1980).

HANSON, A. D. and W. D. HITZ: Metabolic responses of mesophytes to plant water deficits. Ann. Rev. Plant Physiol. 33, 163 - 203 (1982).

MATERN, U., C. FESER, and W. HELLER: N-malonyltransferases from peanut. Arch. Biochem. Biophys. 235, 218-227 (1984).

MIURA, G. A. and S. E. MILLS: The conversion of D-tryptophan to L-tryptophan in cell cultures of tobacco. Plant Physiol. 47, 483 -487 (1971).

POKORNY, M., E. MARCENKO, and D. KEGLEVIC: Comparative studies of L- and D-methionine metabolism in lower and higher plants. Phytochem. 9, 2175-2188 (1970).

REKOSLAVSKAYA, N. 1.: Possible role of N-malonyl-D-tryptophan as an auxin precursor. BioI. Plantarum 28,62-67 (1986).

REKOSLAVSKAYA, N. 1. and K. Z. GAMBURG: Do tomato plants con­tain endogenous indoleacetylaspartic acid? BioI. Plantarum 25, 166-172 (1983).

- - N-malonyl-D-tryptophan as a possible auxin precursor. Fi­siol. rast. (Moscow) 31, 617 -627 (1984).

ROBINSON, T.: D-amino acids in higher plants. Life Sci. 19, 1097 -1102 (1976).

SPRINCE, H.: A modified Ehrlich benzaldehyde reagent for the detec­tion of indoles on paper chromatograms. J. Chromatogr. 3, 97 -98 (1960).

STEWART, G. R. and F. LARHER: Accumulation of amino acids and re­lated compounds in relation to environmental stress. In: MIFLIN, B. J. (ed.): Amino Acids and Derivatives, Vol. 5, 609-630. Aca­demic Press, New York (1980).

T ARR, J. B. and J. ARDITTI: Analysis of tryptophan and its meta­bolites by reverse phase high-pressure liquid chromatography. New Phytol. 88, 621-626 (1981).

ZENK, M. H. and H. SCHERF: Verbreitung der D-Tryptophan-Kon­jugations-Mechanismen im Pflanzenreich. Plant a 62, 350-354 (1964).

(Zenk and Scherf, 1964; Pokorny et al., 1970). Our results suggest that this ability spreads wider and involves higher plants. We failed to find any plants forming only AcTry as the synthesis of AcTry usually proceeded concomitantly with the MTry synthesis. The acetylation and malonylation are not the only ways of D-Try derivatization in plants be­cause other unidentified derivatives of D-Try were also ob­served in the chromatograms (Fig. 1).

Experiments with D-Try infiltration showed that the in­duction of the synthesis of N-malonyl-transferase (see Ma­tern et aI., 1984) cannot be responsible for MTry appearance in drought-stressed leaves. Exogenous D-Try was mal­onylated even in turgid leaves of plants both forming and not forming MTry under wilting. Therefore it was con­cluded that the crucial biochemical event necessary for MTry appearance under drought stress is the induction of D­Try synthesis.

As shown in Tables 3 and 4 the accumulation of L-T ry pre­ceeded the accumulation of MTry, and the amount of L-Try diminished with the MTry increase. These data suggest that L-Try may be a precursor of D-Try in wilted leaves. One of the possible ways of transformation of L-Try to D-Try is ra­cemization. The enzyme, racemase of D-Try, was found by Miura and Mills (1971) in cultured tobacco cells. It may be argued that water deficit induces the synthesis of this en­zyme. As a result, D-Try appears and is acylated. Further study will show, whether Try only appears in D-form under water deficit.

The physiological significance of MTry appearance during wilting is unknown. It has been shown in our previous com­munications (Rekoslavskaya and Gamburg, 1984; Rekoslav­skaya, 1986) that exogenous MTry is transformed to IAA in cultured soybean cells and tomato petioles. It is possible that MTry synthesized endogenously during wilting may be used as an IAA precursor after the restoration of leaf turgescence. However, we did not observe any decrease of MTry content in rewatered tomato leaves (Table 5). Furthermore the usual level of IAA in plant tissues is significantly lower (100-1000 times) than that of MTry observed in wilted leaves. There-

N-malonyl-D-tryptophan and drought stress 89

fore the consumption of MTry for IAA synthesis may not significantly change the MTry content in the leaves. Further experiments with labelled MTry can solve this problem.

References

ALLEN, J. R. F. and D. A. BAKER: Free tryptophan and indole-3-ac­etic acid levels in the leaves and vascular pathways of RIcinus communis L. Planta 148, 69-74 (1980).

HANSON, A. D. and W. D. HITZ: Metabolic responses of mesophytes to plant water deficits. Ann. Rev. Plant Physiol. 33, 163 - 203 (1982).

MATERN, U., C. FESER, and W. HELLER: N-malonyltransferases from peanut. Arch. Biochem. Biophys. 235, 218-227 (1984).

MIURA, G. A. and S. E. MILLS: The conversion of D-tryptophan to L-tryptophan in cell cultures of tobacco. Plant Physiol. 47, 483 -487 (1971).

POKORNY, M., E. MARCENKO, and D. KEGLEVIC: Comparative studies of L- and D-methionine metabolism in lower and higher plants. Phytochem. 9, 2175-2188 (1970).

REKOSLAVSKAYA, N. 1.: Possible role of N-malonyl-D-tryptophan as an auxin precursor. BioI. Plantarum 28,62-67 (1986).

REKOSLAVSKAYA, N. 1. and K. Z. GAMBURG: Do tomato plants con­tain endogenous indoleacetylaspartic acid? BioI. Plantarum 25, 166-172 (1983).

- - N-malonyl-D-tryptophan as a possible auxin precursor. Fi­siol. rast. (Moscow) 31, 617 -627 (1984).

ROBINSON, T.: D-amino acids in higher plants. Life Sci. 19, 1097 -1102 (1976).

SPRINCE, H.: A modified Ehrlich benzaldehyde reagent for the detec­tion of indoles on paper chromatograms. J. Chromatogr. 3, 97 -98 (1960).

STEWART, G. R. and F. LARHER: Accumulation of amino acids and re­lated compounds in relation to environmental stress. In: MIFLIN, B. J. (ed.): Amino Acids and Derivatives, Vol. 5, 609-630. Aca­demic Press, New York (1980).

T ARR, J. B. and J. ARDITTI: Analysis of tryptophan and its meta­bolites by reverse phase high-pressure liquid chromatography. New Phytol. 88, 621-626 (1981).

ZENK, M. H. and H. SCHERF: Verbreitung der D-Tryptophan-Kon­jugations-Mechanismen im Pflanzenreich. Plant a 62, 350-354 (1964).