oxygenase in barley leaves

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PI.M © 1998 by Gustav Fischer Verlag, Jena

. Effect of Salicylic acid on the Synthesis of Ribulose-1,5bisphosphate Carboxylase/Oxygenase in Barley Leaves TANIA

V.

PANCHEVA

and

LOSANKA

P.

POPOVA

Bulgarian Academy of Sciences, Institute of Plant Physiology, Acad. G. Bonchev str., bl. 21, 1113 Sofia, Bulgaria Received April 1, 1997 . Accepted June 3, 1997

Summary

Hordeum vulgare L. (cv. Alfo) was grown for 7 days over a range of salicylic acid (SA) concentrations (100 ~mol/L-l mmol/L), and the effect on the level of ribulose-l,5-bisphosphate carboxylase/oxygenase (RubisCO) was examined. The treated plants showed a decrease in the level of total soluble protein, in particular in the level of RubisCO. In the presence of 1 mmol/L SA the level of total soluble protein on a fresh weight basis was about 68 % of the control, whereas the level of RubisCO was about 50 % that of control plants. The percentage of inhibition on the small subunit was higher, as a result of which the small/large subunit ratio was lower for the experimental variants. When 7-day-old barley seedlings were , supplied with SA through the transpiration stream for 24 h no changes in the levels of total soluble protein and RubisCO were observed. The influence of SA on the synthesis of RubisCO was followed by in vivo labelling with a 14C-amino acid mixure (14C_AAM). Results demonstrated that SA inhibited both the synthesis of total soluble protein and, more pronounced, the synthesis of RubisCO.

Key words: Hordeum vulgare L., cv. A/fo, protein synthesis, RubisCO synthesis, Salicylic acid. Abbreviations: 14C_AAM = 14C-amino acid mixture; C j = intercellular CO 2 concentration; DTT = dithiothreitol; EDTA = ethylenediaminotetraacetic acid; PMSF = phenylmethylsulfonyl fluoride; PVP = polyvinylpyrrolidone; RubisCO = ribulose-l,5-bisphosphate carboxylase/oxygenase; LSU = large subunit of RubisCO; SSU = small subunit of RubisCO; SA = salicylic acid; TCA = trichloroacetic acid. Introduction

Several physiological and biochemical effects of salicylic acid (SA) or its derivate acetyl SA applied to plants have been known for a long time. In various species these compounds are known to inhibit growth and dry weight accumulation (Khurana and Maheshwari, 1980; Schettel and Balke, 1983), phosphate and potassium uptake (Harper and Balke, 1981), and transpiration rate (Larque-Saavedra, 1978, 1979). There is also mounting evidence that SA is a signal molecule in the sequence of metabolic events leading to the expression of systemic resistance to plant pathogens (see review by Yalpini and Raskin, 1993). It has been demonstrated that exogenous application of SA induced synthesis of pathogenesis-related proteins and other compounds and resulted in an increased resistance of 'the treated plants to ]. Plant Physiol WJl 152. pp. 381-386 (1998)

virus and fungi (Ward et al., 1991; Enyedi et al., 1992; Ukness et al., 1992). The increased values of proline content and titratable acidity in barley plants treated with SA show that this compound could provoke alterations very often associated with plant responses to stressful conditions (Pancheva et al., 1996). In recent investigations we have demonstrated that longterm treatment (7 days) of barley seedlings with SA decreased the rate of photosynthesis and the carboxylating activity of RubisCO, and increased both the COrcompensation point and stomatal resistance. The short-term treatment with SA (for minutes to 2 h) did not affect either the rate of photosynthesis or the capacity of biochemical machinery as compared with control plants (Pancheva et al., 1996). An explanation for these changes could in part be due to stomatal closure and reduced supply of CO2 • However, the Ci values were not

382

TANIA V. PANCHEVA and LoSANKA P. POPOVA

declined in SA-treated plants. This implies that stomatal closure did not restrict CO 2 entry into the leaf enough to reduce internal CO 2 level, and the reduction in photosynthesis probably was non-stomatal. This may be due to reductions in either substrate concentrations (C0 2 or RuBP) or in the activity of the enzymes, which produce ATP or reducing equivalents. Another possibility for these effects is an alteration in proteins, especially in the synthesis of some photosynthetic proteins, including RubisCO. The aim of this study was to check the validity of the assumption that the inhibition of photosynthesis by SA was partially due to the reduced level of RubisCO.

Materials and Methods

Plantmatmm Seeds of barley (Hortkum vulgart L., cv. Alfo) were germinated for 2 days in two layers of moist filter paper in moist vermiculite at 25 'C in the dark. Seedlings were grown in Petri dishes containing 40 mL distilled water or equal amounts of water solutions from the required SA concentrations (100 J.Ul1oI/L, 500 ~mol/L and 1 mmoll L), pH 4.6-4.8. Stock of SA (Sigma Chern. Co.) was prepared in a small volume of ethanol (final concentration 1 %), diluted to its final concentration in water and kept refrigerated until use. The s0lutions were changed every 24 h. During the experimental period, the seedlings grew in a growth chamber. The growing conditions were: 25 ± 2 ·C, 60 ± 5 % relative humidity and 12-h photoperiod. «White» fluorescent lamps provided 160~molm-2s-1 PAR In another set of experiments (for short-term effect of SA) the plants were grown in plastic pots (fiUed with soil). The growing conditions were the same as for the long-tertn treatment. All measurements were done on the second fully expanded leaves of 7-day-old seedlings. Well-watered plants were excised with their stems, submerged in water and placed in beakers containing distilled water (control), or 100 ~mol/L, 500 ~mol/L and 1 mmol/L SA, pH 4.64.8. Samples were taken 6 h, 12 h and 24 h afrer treatment with the respective solutionS. Each measurement was done independendy.

Prottin t:araaion The second-leaves of 7-day-old seedlings were harvested. One g of leaf tissue without the major veins was ground in a mortar on ice with 5 mL extraction buffer containing 10 % glycerol, 50 mmol/L HEPES-NaOH, 5 mmol/L MgCh, 10 mmol/L NaCl, 1 mmol/L OTT and 1 mmol/L PMSF, pH 7.5. The homogenate was filtered through four layers of cheesecloth and centrifuged at 10,000 gn for 20 min at 4 .c.

SDS-PAGE gtl tkctrophortSis One-dimensional gel electrophoresis was performed according to the procedure of Laemmli (1970) on 12 % (w/v) acrylamide slab gels (1.5 mm thick), containing 0.1 % (w/v) SOS and 375 mmol/L TrisHCl, pH 8.7. Samples of soluble fractions were solubilized in a sample buffer containing 62.5 mmol/L Tris-HCl, pH 6.8, 2 % (w/v) SOS, 2% (v/v) Ji-mercaptoethanol, 5mmol/L EOTA, and 10% (vI v) glycerol. Samples of soluble fractions containing 100 ~ protein were boiled for 3 min in sample buffer and loaded on the gels. Following electrophoresis, the gels were stained with a solution containing 0.2 % (w/v) Coomassie Brilliant Blue R250 (Sigma), methanol/acetic acid/water (4/115, v/v) and destained in methanol/acetic acid/water (411/5, v/v). The dried gels were scanned at 560 nm using

a Shimadzu CS-930, TLS. Molecular weights were estimated from a standard plot using a-lactalbumin (14.4ku, (kDa», soybean trypsin inhibitor (20.1 ku), carbonic anhydrase (30 ku), ovalbumin (43 ku), bovinelserum albumin (67 ku) and phosphorylase b (94 ku), (Pharmacia).

RubisCO prottin dettrmination The amounts of total soluble protein and RubisCO protein were quantified by the method of Lowry et al. (1951) with BSA as a standard. The stained large and small subunit of RubisCO bands were cut off the gels, cut into thin strips and transferred to 1.5 mL 30 % H 20 2 , followed by heating at 65 ·C for 4-6 h until the gels were fully dissolved. The concentration of RubisCO protein was determined as described earlier (Popova and Vaklinova, 1988). Careful quantitative analysis, of the gels by comparison with a known amount of purified RubisCO protein enabled quantification of the relative amount of RubisCO subunits and served as an additional control.

Prottin synthtsis. Incubation conditions In vivo protein synthesis was examined by labelling newly synthesized proteins with a 14C-amino acid mixture 4C-AAM), (Chemapol, Czechoslovakia) according to Nivison and Stocking (1983). Two sets of experiments were carried out. One with long-term treated plants (grown for 7 days in SA solutions), and another with short-term treated plants (6 h, 12 h and 24 h) with SA. Thirty leaf disks (4 mm d) were cut from the middle part of the leaf and were transferred to 5 mL incubation solutions containing 1 ~C 14C_AAM (sp. activity 1295MBq/mg C atom). During incubation the temperature was 25 ·C, the light intensity was 100 W m -2 and gende shaking kept the solutions mixed. The incubation period lasted 4 h. SA at the required concentrations was present in all variants. The labelled barley leaf disks were rinsed with ice-cold double-distilled H 20 to remove excess label, then with 10 mmol/L Tris buffer, pH 7.4, and were blotted.

e

Dttnmination of radioactivity in ntw/y synthtsiud prottins Blotted leaf disks were homogenized with 1.5 mL ice-cold 15 mmol/L Tris-HCI buffer, containing 5 mmol/L EOTA, 5 mmol/L OTT, 5mmoi/L PMSF, 3.3mglmL PVP and 0.1 % (v/v) p-mercaptoethanol, pH 8.0. Fifty ~L of non-centrifuged homogenates were pipetted to Whatman 7 Chromatography paper for 14C_AAM uptake determination. Further, the homogenates were centrifuged at 10,000 g" for 30 min at 4 "c. The incorporation of radiolabel into polypeptides was determined by spotting 25 ~L samples onto 1 cm2 pieces of Whatman 7. The squares were air-dried and treated with boiling 10 % (w/v) trichloroacetic acid (TCA) for 5 min and then kept on ice for 30 min. After the squares were washed with doubledistilled H 20, 5 % boiling TCA, ethanol, ethanol and ethyl ether, and bleached with drops of 30 % H 20 2 , they were air-dried and placed in scintillation vials containing 5 mL of scintillation cocktail. The radioactivity of TCA-precipitated polypeptides was measured with a LKB scintillation counter.

Results and Discussion

Plants cultivated for 7 days on SA solutions in concentrations of 100 J.l.mollL, 500 J.lmollL, and 1 mmollL had a lower soluble protein content compared with control plants (Table 1). The highest decrease was observed when 1 mM SA was ap-

383

Salicylic acid effect on RubisCO

plied, protein content being about 68 % that of the control on a fresh weight basis. RubisCO is the most abundant protein in nature, comprising more than 50 % of the soluble protein in leaves. The data from our experiments indicated that the percentage content of RubisCO in barley leaves was about 50 % of the total soluble protein (from 474 to 555 Jlglmg soluble protein) in the controls. These data are close to those given in the literature for barley (Peterson and Huffaker, 1975; Friedrich and Huffaker, 1980). In the SA-treated plants the RubisCO level on a soluble protein basis was considerably lower than in the controls. This effect was clearly observed at all SA concentrations examined, although it was the highest at 1 mM SA. The differences in RubisCO content were still greater when the data are expressed per gram fresh weight, since the total soluble protein per gram fresh weight also decreased with increased SA concentrations. Treatment of barley seedlings for 24 h with SA did not affect either the level of total soluble protein or the level of RubisCO protein (Table 1). Table 2 shows the data for the levels of the large and small subunits of RubisCO in the control and SA-treated plants. In

plants cultivated for 7 days on SA solutions (long-term treatment), the amount of protein decreased in both subunits, but the percentage of inhibition was higher for the small subunit, as a result of which the SSU/LSU ratio was lower for the experimental variants. Short-term treatment of barley plants (24 h) with SA did not affect the protein levels of both subunits of RubisCO, and the SSU/LSU ratio remained constant for the controls and experimental variants. This dependence was illustrated also in Fig. 1. Polypeptide patterns of the SDS-extractable proteins from the soluble fractions (supernatants after 10,000 gJ are given in Fig. 1. About 36 polypeptide bands with molecular masses from 107 to 10.6 ku were resolved. Among them, the polypeptides with molecular masses of 55, 47, 43.5, 27-24, and 15 ku were the most prominent in the controls. As compared with the control, long-term treated variants showed the following changes (Fig. 1 A): (a) Increasing SA concentrations led to a drastic reduction in the level of 55 and 15 ku polypeptides corresponding, respectively, to the LSU and SSU of RubisCO. The positions of both subunits of RubisCO were identified by running purified barley RubisCO as a marker. (b) One polypeptide with a molecular mass of 10.6 ku,

Table 1: Total soluble protein and RubisCO protein in batley leaves after treatment with salicylic acid. For long-term treatment plants were

grown for 7 days on SA solutions. For short-term treatment well-watered plants were excised with their stems and placed in beakers containing distilled water (control) or the respective concentration of SA for 24 h. Leaf soluble protein was determined by the method of Lowry et al.; RubisCO protein was also determined by Lowry et al., following the procedure described under «Materials and Methods». Data ate means ±SE (n = 4).

SA [f.lmollL]

[mg (g FW)-l]

[%]

If.lg (mg sol. pr.)-l]

[%]

[f.lg (g FW)-l]

[%]

long-term treatment (7-d) H 20 (control) 100 f.lmol/L 500 f.lmol/L 1 mmollL

12.5±0.6 11.3± 1.0 9.8±1.1 8.5±1.0

100.0 90.3 78.5 68.1

474.7±64.7 342.7±31.2 250.9±47.5 219.5±28.9

100.0 74.2 52.8 50.4

5938.5±680 3869.1±470 2463.8±350 1870.1±270

100.0 65.2 41.5 31.5

short-term treatment (24 h) H 20 (control) 100 f.lmollL 500 f.lmollL 1 mmollL

12.7±0.7 12.5±0.9 12.1±0.9 12.8±1.0

100.0 97.9 94.4 100.3

555.0±39.8 519.1±37.6 533.2±30.5 546.3±41.2

100.0 97.1 96.0 98.4

7070.7±610 6478.4±590 6466.5±630 6987.2±520

100.0 91.6 91.5 98.8

Soluble protein

RubisCO protein

RubisCO protein

Table 2: Effect of Salicylic acid on the amount of latge and small RubisCO subunit in batley laves. Values ate means ± SE for four experiments.

SA lJ,LmollL]

Small subunit

Large subunit

[f.lg (mg sol. pr.)-l]

[%]

[f.lg (mg sol. pr.)-l]

[%]

Small/Large subunits ratio

long-term treatment (7-d) H 20 (control) 100 f.lmollL 500 f.lmollL 1 mmollL

313.3±15.2 254.l±27.1 196.6±19.7 188.7±20.6

100.0 81.1 62.8 60.2

161.5±27.4 98.3±17.8 54.3± 12.2 50.8±17.1

100.0 60.9 33.6 31.5

0.52 0.37 0.28 0.27

short-term treatment (24 h) H 20 (control) 100 J.lffiol/L 500 f.lmollL 1 mmollL

363.5±20.7 354.1±21.6 348.5±18.9 359.9±26.2

100.0 97.4 95.9 99.0

191.5± 19.7 193.l±21.2 185.l±15.6 186.4±18.7

100.0 100.9 96.7 97.4

0.53 0.55 0.53 0.52

384

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TANIA V. PANCHEVA and LOSANKA P. POPOVA

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43-

b

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d

tI. 1 n': r.

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-lSU

a

-!

30-

2014-

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,-

ll'

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B

Fig. I: Polypeptide profiles (12 % SDS-PAGE) of soluble proteins of barley leaves. A. long-term treated plants with SA (7-d); B, shortterm treated plants (24 h). a, control (H 20); b, lOOllmollL; c, 500llmollL SA;, d, 1 mmol/L SA. All lanes were loaded with lOOllg protein. Arrows indicate the main polypeptide differences berween controls and SA-treated plants. Positions of molecular weight markers in kD are indicated at the left.

slightly discernible in the control, was greatly increased in SA-treated seedlings. (c) Among the high molecular polypeptides the relative share of 87, 93.6 and 99.6ku bands was increased in SA-treated plants. (d) Some other bands with molecular masses of 24, 25, 27 ku, and 17.2-18.9 ku, well expressed in the control, decreased in SA-treated plants. Among the results obtained, the inhibitory effect of SA on the level of RubisCO and its two subunits can be underscored, the inhibition appearing stronger with respect to SSU. Another polypeptide of interest is the 10.6 ku protein, whose level increased in SA-treated plants. Treatment of barley seedlings with SA for 24 h did not affect the level of the large and small subunits of RubisCO. The 10.6 ku polypeptide, well present in long-term treated plants, was not expressed after this mode of treatment (Fig. IB). Coomassie blue-stained gels were scanned at 560 nm to quantify separated polypeptides. Clear differences in the densitometer scans were observed between the control and SAtreated variants. From the areas under the peaks, the 55 ku fraction (LSU) was estimated to represent approximately 33.4 % of the total soluble protein of the control and to decline to 19.3 % with 1 mmollL SA. For SSU, this percentage was 17.5 % for the control and 3.8 % for 1 mmollL SA. The relative share of the band with a molecular mass of 10.6 ku increased from 0.4 % for the control to 5.1 % for 1 mmollL SA (Fig. 2). It is well established that the level of RubisCO in vivo is balanced by its synthesis and degradation. For most plants about 90 % of the RubisCO level is reached in the period

c

e

--------"'11..... Fig. 2: Densitometer scans of soluble proteins from barley leaves treated with SA for 7-d. a, control (H 20); b, lOOllmollL SA; c, 500llmollL SA, d, 1 mmollL SA; e, molecular weight markers. The major polypeptide differences among control and treated plants are indicated in kD with arrows. The horizontal arrow denotes the direction of band migration.

when the leaf is fully formed, and then the gradual destruction of the protein molecules begins (Mae et al., 1983). Un-

Salicylic acid effect on RubisCO

385

e

Table 3: Effect of salicylic acid on the uptake and incorporation of 14C_amino acid mixture 4C-AAM) into leaf protein (TCA-insoluble fraction) and on the synthesis of large and small subunits of RubisCO in barley leaves treated for 7 days with SA. Values are means ±SE for three experiments .. Rate of 14C_AAM uptake [cpmxlO- 4/ [%]

SM [~mol/L]

14C_AAM incorporation into protein

30 leaf disks]

H 20 (control) 100 ~ol/L 500 ~mol/L 1 mmol/L

35.2±2.3 29.9± 1.6 21.1 ± 1.8 16.2±2.3

100.0 85.0 60.0 45.0

[cpmxlO- 4/ 30 leaf disks]

[%]

11.9± 1.8 9.5±1.0 6.3±0.6 4.7±0.5

100.0 79.6 53.2 39.7

Large subunit [cpmxlO- 4/

[%]

30 leaf disks] 1.92±0.07 1.24±0.13 0.77±0.10 0.54±0.08

Small subunit [cpmxlO- 4/

[%]

30 leaf disks] 100.0 65.2 40.0 27.9

0.99±0.18 0.59±0.09 0.35±0.07 0.23±0.06

100.0 59.1 34.8 23.3

e

Table 4: Short-term effects of salicylic acid on the uptake and incorporation of 14C_amino acid mixture 4C-AAM) into leaf protein (TCA-

insoluble fraction) and on the synthesis of large and small subunits of RubisCO protein in barley leaves. Well-watered plants were excised with their stems and placed in beakers containing distilled water (control) of the respective concentration of SA. Measurements were performed 6 h, 12 h and 24 h mer exposure of the detached seedlings in SA solutions. Data given are the mean of rwo independent experiments with rwo replications each. SA ~mol/L]

Rate of 14C_AAM uptake [cpmx 10- 4/ [%]

14C_AAM incorporation into protein [cpmx 10-4/ [%]

Large subunit [cpmx 10- 4/

30 leaf disks]

30 leaf disks]

30 leaf disks]

[%]

Small subunit [cpmx 10-4/

[%]

30 leaf disks]

incubation time 6 h H 20 (control) 100 ~mol/L 500 ~mol/L 1 mmol/L

44.3±2.6 34.0±2.7 22.8± 1.6 19.2±1.8

100.0 76.7 51.4 43.4

16.8±1.2 15.5± 1.1 12.4±0.7 9.2±1.4

100.0 92.1 74.1 55.0

2.68±0.17 1.97±0.12 0.81±0.09 0.41±0.07

100.0 73.4 30.2 15.3

l.28±0.19 1.00±0.14 0.36±0.05 0.30±0.05

100.0 77.6 27.9 23.3

incubation time 12h H 20 (control) 100 ~mol/L 500 ~mol/L 1 mmol/L

36.3±3.4 30.4±1.6 23.1±1.4 19.4± 1.5

100.0 83.9 64.5 53.4

13.8± 1.6 12.4± 1.6 8.7±1.2 7.1±1.1

100.0 90.1 62.9 51.5

2.75±0.21 1.32±0.17 1.12±0.1l 0.51±0.07

100.0 47.8 40.7 18.6

1.15±0.9 0.72±0.09 0.54±0.08 0.31±0.04

100.0 62.9 47.1 26.5

incubation time 24 h H 20 (control) 100 ~mol/L 500 ~mol/L 1 mmol/L

35.2±2.3 31.7±3.7 26.3±2.5 20.2±1.2

100.0 90.2 74.7 57.5

10.8± 1.5 9.8±0.7 6.6±0.7 5.5±0.8

100.0 90.3 61.2 51.0

2.22±0.23 0.78±0.12 0.62±0.08 0.53±0.05

100.0 36.2 28.7 24.4

0.92±0.09 0.37±0.07 0.35±0.07 0.19±0.04

100.0 40.4 38.1 20.8

der our experimental conditions the leaves of7-day-old seed- synthesis of large and small subunits of RubisCO (Table 3). lings were young and fully structured, and the processes of The decrease was smaller in LSU than in SSU of RubisCO. synthesis are considered predominant over those of destruc- The inhibition was approximately over two-fold higher at tion. This view was confirmed from the experiments in which 500 Jlmoi/L SA and four-fold at 1 mmollL SA on a leaf area we investigated the de novo protein synthesis, and in particu- basis. lar, the synthesis of RubisCO and its subunits. It should also be emphasized that the data presented in TaData on 14C_AAM uptake and its incorporation in leaf ble 3 concerning the de novo synthesis of protein, and espeprotein are given in Table 3. Treatment with SA caused an in- cially the synthesis of LSU and SSU of RubisCO, are in good hibition in the rate of 14C_AAM uptake, the degree of inhibi- agreement with those from Tables I and 2, where the effects tion being increased with the increase of SA concentrations. of SA on the total soluble protein content, the RubisCO conThese results also showed that there was a significant reduc- tent and the content of its two subunits are presented. Thus, tion of 14C_AAM incorporation in protein too (TCA-insolu- the observed reduction in RubisCO content appears to be ble fraction). due to the inhibition of its synthesis. Treatment of barley seedlings with SA for a short time (6 h, It should be noted that we do not know the amino acid composition of 14C_AAM and its distribution in the chloro- 12 h and 24 h) also caused an inhibition in the rate of 14C_ plasts and cytoplasm; consequendy, we can not take the val- AAM uptake and protein synthesis (Table 4). The inhibition of total protein synthesis increased with increasing SA conues of incorporation as the absolute rate of protein synthesis. The results from investigations carried out indicated that centrations. The most prominent effect was with 1 mmollL long-term treatment of barley seedlings with SA inhibited the SA, an approximately two-fold decrease as compared with the

386

TANIA V. PANCHEVA and LoSANKA P. POPOVA

controls. It is also shown that at all concentrations investigated SA inhibited protein synthesis, however, without the establishment of a definite dependence of the duration of treatment. The same was also true separately for the synthesis ofLSU and SSU of RubisCO. In summary, we demonstrated that treatment of barley seedlings with SA caused a significant decrease in the level of total soluble protein, particularly in the level of RubisCO. Radioactive labelling experiments showed that SA inhibited RubisCO synthesis, the effect being more strongly expressed with the synthesis ofSSU of RubisCO. The decrease in the amount of RubisCO is accelerated by various stress treatments, such as NaCl-salinity (Miteva et al., 1992), nitrogen starvation (Garcia-Ferris and Moreno, 1994) or after exogenous treatment of plants with some stressrelated phytohormone, such as abscisic acid (Popova, 1989) or jasmonic acid (Popova and Vaklinova, 1988). All of these factors lead to a decrease in photosynthetic ability and activity of RuBP carboxylase. The observed inhibition of photosynthesis and the activity of RubisCO by SA is mainly, if not entirely, caused by a decrease in the RubisCO content. We believe that the observed effect of SA on RubisCO synthesis is not the sole reason for the low activity of the enzyme and low rate of photosynthesis. It can be assumed that, like other stress factors; the exogenous SA application diminishes chloroplast photosynthetic activity as a result of effects on the thylakoid membranes and light-induced reactions connected with them, and in this way it may indirectly participate in regulating the activity of RubisCO. Acknowledgements

This research was supponed by a grant from the National Fund for Scientific Investigations (K 623), Bulgaria. The skilful technical assistance of Mrs. Zhivka Stoinova is gratefully acknowledged. References ENYEDI, A., N. YALPINI, P. SILVERMAN, and I. RAsKIN: Localization, conjugation, and function of salicylic acid in tobacco during the hypersensitive reaction to tobacco mosaic virus. Proc. Nat!. Acad. Sci. USA. 89,2480-2484 (1992). FRIEDRICH, J. w. and R C. HUFFAKER: Photosynthesis, leaf resistances, and ribulose-l,5-bisphosphate carboxylase degradation in senescing barley leaves. Plant PhysioI. 65, 1103-1107 (1980). GARCIA-FERRIS, C. and J. MORENO: Oxidative modification and breakdown of ribulose-l,5-bisphosphate carboxylase/oxygenase induced in Euglmagracilis. Planta 193, 208-215 (1994). '

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