altered Î´-aminolevulinic acid metabolism by lead and mercury in germinating seedlings of bajra...

Altered o-aminolevulinic Acid Metabolism by Lead and Mercury in Germinating Seedlings of Bajra (Pennisetum typhoideum)

D. D. K. PRASAD and A. R. K. PRASAD

Department of Biochemistry, School of Biological and Earth Sciences, Sri Venkateswara University, Tirupati-517 502, Andhra Pradesh, India

Received June 13, 86 . Accepted August 25, 86

Summary

Germinating seeds of Bajra (Pennisetum typhoideum) were treated with different concentrations of lead and mercury. Measurements of o..aminolevulinic acid levels, o..aminolevulinic acid dehydratase activity and total chlorophyll content were performed. It was found that both the metals have no effect on o..aminolevulinic acid levels but inhibit the o..aminolevulinic acid dehydratase activity and chlorophyll biosynthesis. Aminolevulinic acid dehydratase activity was localized in the chloroplast fraction of the seedlings. Sensitivity of the enzyme to variations in pH and temperature and to amino acids such as arginine, lysine, and cystine were studied.

Key words: Delta aminolevulinic acid, Delta aminolevulinic acid dehydratase, Porphobilinogen, Heavy metals, Chlorophyll.

Introduction

The heavy metals lead and mercury have become major environmental contaminants. When present in elevated levels in water and soil they are readily taken up by plants and accumulated in different parts of the plants, thereby inducing impaired metabolism and reduced growth Uastrow and Koeppe, 1980; Ernst, 1980; Weigel and Jager, 1980 a, b; Van Bruwaene et aI., 1984). Earlier reports have shown that photosynthesis in higher plants is highly sensitive to heavy metals like cadmium (Weigel, 1985) and that cadmium inhibits the formation of chlorophyll by reacting with protochlorophyllide reductase and synthesis of aminolevulinic acid (Stobart et al., 1985).

The formation of o-aminolevulinic acid (ALA) is the first identified step in tetrapyrrole biosynthesis leading to the formation of heme, chlorophyll, bilins, vitamin BJ2,

and other specialized plant products (Granick and Sassa, 1971). Succinate and glycine are the precursors for the formation of ALA by ALA synthetase in bacteria (Kikuchi et aI., 1958; Tait, 1972), yeast (Porra et aI., 1972) and higher animals (Gibson et aI., 1958; Marver et aI., 1966; Bottomley and Smithee, 1968). On the other hand the absence of ALA synthetase in plants indicate the existence of an alternate pathway for the formation of ALA from glutamic acid via dioxovaleric acid (Beale, 1971 and Beale et al., 1975). ALA synthetic ability has been ascribed a central role in the regula-

Abbreviations: ALA = o..aminolevulinic acid; ALA dehydratase = o..aminolevulinic acid dehydratase; PBG = porphobilinogen; DTT = dithiothreitol.

J Plant. Physiol. Vol. 127. pp. 241-249 {1987}

242 D. D. K. PRASAD and A. R. K. PRASAD

tion of chlorophyll biosynthesis in higher plants (Bogorad, 1976 and Beale, 1978). It has also been suggested that chlorophyll synthesis may be regulated not only by ALA synthetic ability but also by o..aminolevulinic acid dehydratase (ALA dehydratase) acitivity (Naito et al., 1980).

The present investigations were performed in order to elucidate the possible sites of action of heavy metals lead and mercury on the initial steps of chlorophyll biosynthesis.

Materials and Methods

Plant material

Seeds of Bajra (Pennisetum typhoideum, L.) were procured from A. P. State Seed Corporation and healthy seeds were washed thoroughly with tap water, allowed to imbibe in water for 5 h, and spread over moist filter paper in Petri dishes. Controls were maintained with distilled water and other lots of seeds were treated separately with metal solutions of different concentrations (50 JLM, 100 JLM, and 250 JLM) of lead and mercury (added as lead acetate and mercuric chloride). Seedlings were maintained under natural daylight for 5 days at day and night temperatures of 300 ±2°C. Seedlings were removed at regular intervals and used for ALA estimation, assay of ALA dehydratase activity and chlorophyll measurements. The results presented are the average of four separate experiments.

Determination of ALA

ALA was estimated by the method of Mauzerall and Granick (1956). ALA from seedlings was extracted in 10 % trichloroacetic acid and condensed with ethyl acetoacetate in the presence of 0.2M phosphate buffer (pH6.8). Ehrlich reagent was added to the condensate (1: 1) and optical densities were measured after 15 min at 553 nm. ALA contents were expressed as p. molesl g fresh weight.

Chromatography of ALA

Chromatography of ALA was done according to the method of Miller et al. (1979). ALA was extracted with 10 % trichloroacetic acid and then transfered to ethyl acetate and vacuum dried. The dried residues were dissolved in 1 to 2 drops of methanol and spotted on Whatmann No.3 MM filter paper and developed in methyl acetate/isopropanol/25 % NH40H (45: 35 : 20) solution and sprayed with Ehrlich reagent and compared with authentic ALA run in the same way.

Assay of ALA dehydratase activity

ALA dehydratase activity was assayed by the method of Schneider (1970). 1 ml of buffer extract was incubated with 0.27ml of 1 mg/ml ALA, 1.35ml of 0.05M Tris-HCI buffer (pH 8.2) with 0.1 M DTT and 0.08 ml of 0.02 M magnesium chloride for 2.5 h at 37°C. The reaction was arrested with 0.3 ml of 3 M trichloroacetic acid and centrifuged at 2,000 x g. The supernatant was mixed with (1: 1) modified Ehrlich reagent (Mauzerall and Granick, 1956) and absorbance was measured at 555 nm after 10 min against zero time blank. ALA dehydratase activity was expressed as n moles of porphobilinogen (PBG) formedl mg protein/2.5 h at 37°C. Protein estimations were done by the method of Lowry et al. (1951) using BSA as standard.

Chlorophyll measurements were carried out spectrophotometrically by the method of Arnon (1949).

Mitochondria and chloroplasts were isolated from 4-day-old seedlings. Chloroplasts were isolated by the method of Walker (1980) and were ruptured by sonication. Mitochondria were isolated by the method of Bonner (1967) and were lysed osmotically by treatment with 0.05 M Tris-HCI buffer (pH 8.2).


Altered o.-ALA metabolism by lead and mercury 243

0--<> CONTROL ....... lEAD 50,uM

100 ......... lEADIOO,uM - ______ l EA D 2500 M s:: '" 0-0 QI

~ 80 s:: II' QI .. u.

(50 ~ A-A :::

I#~ >-s:: a. 40 0 .. .9 s:: u o~ '0 20

'" ~ ~I 0 24 48 72 96 120

TIME IN HOURS

Fig. 1: Effect of lead on chlorophyll content in germinating seedlings of Bajra.

Results

Effect of lead and mercury treatment on chlorophyll content

Figs. 1 and 2 show the change in chlorophyll content caused by treatment with lead and mercury respectively. The results indicate that the chlorophyll content increased with age up to the 4th day and remained unchanged on day 5 in both control and treated seedlings. Further, it was observed that chlorophyll content was dose-dependent and decreased with increasing metal concentrations.

ALA synthetic ability

Our results show that ALA levels of the germinating seedlings were unaltered by the metals at the concentrations tested (Table 1). Further, the estimated ALA was confirmed by paper chromatography with authentic commercial ALA.

ALA dehydratase activity

In both control and treated seedlings ALA dehydratase activity increased with age up to the 4th day and decreased on the 5th day (Figs. 3 and 4). Further, it was observed that inhibition of ALA dehydratase activity was dose-dependent and a maximum of 50 % inhibition caused by lead and mercury was observed at the level of 250 I'M. Since maximum activity of the enzyme was found on the 4th day of germination, all other experiments were carried out on the 4th day.

Subcellular distribution studies of the enzyme ALA dehydratase indicated that the activity was localized in the chloroplasts (Table 2). Other experiments on pH optima

J. Plant. Physio!. Vo!' 127. pp. 241-249 (1987)


_ 100 s: C\ ·iii ~ s: III (II .. u.

C\ '::::->. s: Co

E ~ s: U ... 0

C\

~

80

60

40

20

0

o--<l CONTROL ___ MERCURY 50A.lM

.......... MERCURY IOO,uM ___ MERCURY 250,uM 0----0

A-A

tf-. o~

24 48 72 96 120

TIME IN HOURS

Fig. 2: Effect of mercury on chlorophyll content in germinating seedlings of Bajra.

Table 1: Effect of lead and mercury on O-aminolevulinic acid levels in germinating seedlings of Bajra.

Treatment JL moles of ALA/g fresh weight*)

Germination time in hours

0 12 24 48 72 96 120

Control 1.40±0.08 1.60±0.23 2.30±0.42 3.3S±0.36 2.88±0.23 2.6S±0.14 2.39±0.17 Lead

SOJLM 1.30±0.14 1.60±0.14 2.20±0.03 3.S7±0.23 2.61±0.36 2.S4±0.14 2.24±0.18 100JLM 1.30±0.1S l.S0±0.31 2.20±0.26 3.21±0.19 2.76±0.11 2.S0±0.11 2.26±0.22 2S0JLM 1.20±0.07 l.S0±0.20 2.00±0.28 3.3S±0.14 2.48±0.OS 2.46±0.11 2.24±0.24

Mercury SOJLM 1.20±0.OS 1.60±0.23 2.30±0.18 3.34±0.22 2.76±0.33 2.60±0.28 2.30±0.2S

100JLM 1.30±0.07 l.S0±0.03 2.20±0.20 3.36±0.30 2.80±0.09 2.40±0.07 2.21±0.18 2S0JLM 1.10±0.08 l.S0±0.04 2.20±0.18 3.04±0.16 2.46±0.30 2.36±0.24 2.20±0.22

*) Values are average of four separate experiments ± standard deviation.

confirmed that the enzyme was specific for Tris-HCI buffer (O.05M) at pH8.2 and much less activity was observed with regard to phosphate buffer and no activity with acetate buffer. Furthermore, the enzyme was thermolabile and total loss of activity was observed when incubated at 45°C for 20 min.

In Fig. 5 the effect of lead and mercury on ALA dehydratase in vitro was shown. This is the first report on germinating seedlings, where we could demonstrate that lead and mercury inhibited ALA dehydratase activity even in vitro in the presence of a thiol activating agent. In view of the possible changes in the pH optima of the en-


Altered a-ALA metabolism by lead and mercury 245

'& CD Q. ..... 0--0 CONTROL

° ___ LEAD 50ftlM II' 100 01 _ LEAD 100ftlM

"0 ___ LEAD 250JJM E <:

> 80 I-~

~ -<: >.-_01 1-"-Ue 60 «0. we> VIE « 13· · I- 40 « a: ---=O~ • ...---·~ Cl > ~:/ . :r: w 20 ~. Cl I « ...J «

0 24 48 72 96 120

TIME IN HOURS

Fig. 3: Effect of lead on ALA dehydratase activity in germinating seedlings of Bajra.

~ CD Q. 0--0 CONTROL

'0 .......... MERCURY 50,uM

II' 100 ...- MERCURY 100 .oM

01 ___ MERCURY 250 ,uM "0 E

..= 80

/.~ > I-~ -" >.--01 1-"-UO 60 «a. we> VIE « I- 40 ~.---~~ « a: Cl

.. ____ I

> • • :r: w 20 ~~ Cl

I

« ...J «

0 24 48 72 96 120

TIME IN HOURS

Fig. 4: Effect of mercury on ALA dehydratase activity in germinating seedlings of Bajra.

zyme ALA dehydratase by heavy metals which might result in a decreased activity profile (Wigfield and Farant, 1979), the effect of basic amino acids like arginine and lysine were studied. It was observed that arginine and lysine have no effect on enzyme activity when present in the incubation mixture with or without mercury,

J. Plant. Physiol. Vol. 127. pp. 241-249 {1987}


Table 2: Subcellular distribution of o..aminolevulinic acid dehydratase in 4-day-old seedlings of Bajra.

----------------------------~~------------------

ALA dehydratase activity Fraction

Chloroplast Mitochondria Supernatant

*) N.D. = Not detected.

Protein mg/ml

4.22±0.27a)

1.60±0.osa) 5.14±0.S7a

)

n moles of PBGI mg proteinl2.5 h at 37°C

131.6 ± 5.39a) N.D.*) N.D.*)

a) = indicates standard deviation of the mean, n = 4.

III C!I "0 E

-= 80 > I-~ 5.!: i=2 uE! <to. Wtl' <fiE ;!~ 40 <tCD 0:0.. 0 .... >0 [5 20 o <t ..J <t

o

_ LEAD

tr--A MERCURY

50 100 150 200 250

CONCENTRATION IN ).J moles

Fig. 5: In vitro effect of lead and mercury on ALA dehydratase activity in Bajra seedlings. Buffer extracts of the enzyme from 4-day-old seedlings were preincubated with different concentrations (50/LM, 100 JLM, and 250 JLM) of lead and mercury for 30 min at 37°C. Aliquots were taken after incubation and enzyme activity was assayed as described in the text with proper controls.

while cystine showed more inhibition in combination with mercury. Further, the expression of maximum activity in presence of DTT indicated the requirement of thiol groups for ALA dehydratase activity (Table3).

Discussion

The present results show that chlorophyll synthesis is inhibited by heavy metals as already indicated by Stobart et al. (1985) for cadmium. It was observed that lead and mercury interact with ALA dehydratase activity thus altering the levels of chlorophyll. Earlier reports show that the biosynthesis of all tetrapyrrole compounds proceed through metal sensitive enzyme ALA dehydratase which requires the presence of sulfhydryl groups regardless of the source (Nandi and Shemin, 1968).


Altered &-AlA metabolism by lead and mercury 247

Table 3: In vitro effect of amino acids, mercury, and DTT on AlA dehydratase activity in 4-day-old seedlings of Bajra.

Treatment

Control ( - DTT) Control ( + DTT) Control + Arg. (100,M)

+ Lys. (100,M) + Cyst. (100,M) + Mercury (Hg) (100 JLM) + Mercury (250 JLM) + Arg. (100JLM) + Hg (100JLM) + Arg. (100 JLM) + Hg (250,M) + Lys. (100,M)+Hg(100,M) + Lys. (100JLM)+Hg (250,M) + Cyst. (lOO,M)+Hg (100,M) + Cyst. (100 ,M)+ Hg (250,M)

ALA dehydratase activity

n moles of PBG/ mg protein/2.5 h at 37°C

32.43 58.30 32.27 32.36 32.43 30.27 20.69 30.28 20.69 30.28 20.69 27.30 17.10

Enzyme extract was preincubated with known concentrations of amino acids and metals for 30 min and the enzyme activity was assayed as described in the text.

Interestingly, both the heavy metals have no effect on ALA levels which is an intermediate for chlorophyll synthesis and the substrate for ALA dehydratase. However, it was observed that increasing metal concentrations further inhibited the ALA dehydratase activity resulting in decreased formation of PBG which is required for chlorophyll synthesis. The decreased total chlorophyll content indicates the positive correlation between decreased ALA dehydratase activity and chlorophyll synthesis (Shibata and Ochiai, 1976).

Subcellular localization studies revealed that the enzyme was located exclusively in chloroplasts and no activity was detected in mitochondria and cytosol fractions. Present results indicate that the enzyme is not membrane-bound as we could detect much less activity in intact chloroplasts. There was an enormous increase in the activity when the isolated chloroplasts were lysed. The release of ALA dehydratase into lysate suggest that the enzyme might be in soluble form in the stroma or loosely bound to lamella.

Present findings show that basic amino acids like arginine and lysine failed to alter the in vitro effect of mercury on ALA dehydratase activity, while the amino acid cystine in combination with mercury acts as a potent inhibitor of ALA dehydratase but its actual mechanism is not clear.

The increase in relative levels of chlorophyll and ALA dehydratase activity was observed up to the 4th day of germination and the chlorophyll content remained constant on the 5th day, indicating that the chloroplasts are almost fully developed by the 4th day under the conditions employed. Interestingly, after the chlorophyll reached a constant the ALA dehydratase activity decreased, indicating the positive interrelationship between ALA dehydratase and the chlorophyll content.

]. Plant. Physiol. Vol. 127.pp. 241-249 (1987)


Acknowledgements

One of the authors D. D. K. Prasad is thankful to the University Grants Commission, New Delhi, for providing financial assistance in the form of a Junior Research Fellowship.

References

ARNON, D. 1.: Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant PhysioI. 24, 1-15 {1949}.

BEALE, S. I.: Studies on the biosynthesis and metabolism of O-aminolevulinic acid in Chlorella. Plant PhysioI. 48, 316-319 {1971}.

- Aminolevulinic acid in plants. Ann. Rev. Plant PhysioI. 29, 95-120 {1978}. BEALE, S. 1., S. P. GOUGH, and S. GRANICK: Biosynthesis of O-aminolevulinic acid from intact

carbon skeleton of glutamic acid in greening barley. Proc. Nat. Acad. Sci., USA. 72, 2719-2723 {1975}.

BOGORAD, L.: Chlorophyll biosynthesis. In: T. W. GOODWIN {Ed.}. In: Chemistry and biochemistry of plant pigments. Academic Press, New York, London 2nd Ed., 1, 64 -148 {1976}.

BONNER, W. D.: A general method for the preparation of plant mitochondria. In methods in EnzymoI. 10, 126-133 {1967}.

BOTIOMLEY, S. S. and G. A. SMITHEE: Characterization and measurement of O-aminolevulinic acid synthetase in bone marrow cell mitochondria. Biochem. Biophys. Acta. 159, 27-37 {1968}.

ERNST, W. H. 0.: Biochemical aspects of cadmium in plants. In: J. O. NRIGAU {Ed.}: Cadmium in environment, Part I, 639-653, Wiely Inter Science, New York, Chichester {1980}.

GIBSON, K. D., W. D. LAVERE, and A. NEUBERGER: Initial stages in the biosynthesis of O-aminolevulinic acid from glycine and succinyl Co A by particles from chicken erythrocytes. Biochem. J. 61, 618-629 {1958}.

GRANICK, S. and S. SASSA: In metabolic regulation {Ed.}: H. J. VOGEL. Academic Press, New York, Vol. V, 77-141 (1971).

JASTROW, J. D. and D. E. KOEPPE: Uptake and effects of cadmium in higher plants. In: J. O. NRIGAU (Ed.): Cadmium in environment, Part I, 608-637, Wieley Inter Science, New York, Chichester (1980).

KIKUCHI, G., A. KUMAR, P. TALMAGE, and D. SHEMIN: The enzymatic synthesis of O-aminolevulinic acid. J. BioI. Chern. 233, 1214-1219 {1958}.

LoWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the Folin-Phenol reagent. J. BioI. Chern. 193,265-275 {1951}.

MARVER, H. S., A. COLLINS, D. D. TSCHUDY, and M. RECHCIGL Or}: O-aminolevulinic acid synthetase. ii. Induction in rat liver. J. BioI. Chern. 241, 4323-4329 {1966}.

MAUZERALL, D. and S. GRANICK: The occurrence and accumulation of /l-aminolevulinic acid and prophobilinogen in urine. J. BioI. Chern. 219, 435-446 {1956}.

MiLLER, G. W., A. DENNY,J. K. WOOD, and G. W. WELKIE: Light induced aminolevulinic acid in dark grown barley seedlings. Plant Cell PhysioI. 20, 131-143 {1979}.

NAITO, K., T. EBATO, Y. ENDo, and S. SHIMIZU: Effect of Benzyladenine on /l-aminolevulinic acid synthetic ability and O-aminolevulinic acid dehydratase. Differential responses to Benzyladenine according to leaf age. Z. PflanzenphysioI. 96, 95-102 {1980}.

NANDI, D. L. and D. SHEMIN: /l-aminolevulinic acid dehydratase of Rhodopseudomonas sphe· roides. J. BioI. Chern. 243, 1236-1242 {1968}.

PORRA, R. J., E. A. IRVING, and A. M. TENNICK: The detection of O-aminolevulinic acid synthetase in anaerobically grown Torulopsis utilis. Arch. Biochem. Biophys. 149, 563-565 {1972}.

SCHNEIDER, H. A. W.: Activities and Properties of O-aminolevulinic acid dehydratase in greening tissue cultures of Nicotiana tabacum, c.v. Samsum. Z. PflanzenphysioI. 62, {2} 133-145 {1970}.

J. Plant. Physiol. Vol. 127. pp. 241-249 (1987)

Altered O-ALA metabolism by lead and mercury 249

SHIBATA, H. and H. OCHIAI: Studies of O-aminolevulinic acid dehydratase in radish cotyledons during chloroplast development. Plant Cell Physiol. 17, 281-288 (1976).

STOBART, A. K., W. T. GRIFFITHS, I. A. BUKHARI, and R. P. SHERWOOD: The effect of Cd+ 2 on the biosynthesis of chlorophyll in leaves of barley. Physiol. Plant. 63, 293 -298 (1985).

TAIT, G. H.: O-aminolevulinic acid synthetase of Micrococcus denitrificans. Biochem. J. 128, 32 (1972).

VAN BRUWAENE, R., R. KiRCHMANN, and R. WPENS: Cadmium contamination in agriculture and zootechnology. Experientia, 40, 43-52 (1984).

WALKER, D. A.: Preparation of higher plant chloroplasts. In methods in Enzymol. 69, 94-104 (1980).

WEIGEL, H. J. and J. J. JAGER: Different effects of cadmium in vitro and in vivo on enzyme activities in bean plants (Phaseolus vulgaris, L, c.v. Sankt Andreas). Z. Pflanzenphysio!. 97, 103 -113 (1980, a).

Subcellular distribution and chemical form of cadmium in bean plant. Plant Physio!. 65, 480-482 (1980, b).

WEIGEL, H. J.: Inhibition of photosynthetic reactions of isolated intact chloroplasts by cadmium. J. Plant Physio!. 119, 179-189 (1985).

WIGFIELD, C. D. andJ. P. FARANT: Factors influencing the pH activity relationship of O-aminolevulinic acid dehydratase from human blood and their relevance to blood lead assay. J. Anal. Toxico!. 3, 161-168 (1979).


altered Î´-aminolevulinic acid metabolism by lead and mercury in germinating seedlings of bajra...

Documents