Chlorophyll biosynthesis is inhibited by iron deficiency in- duced by a number of metals (1)-cobalt and manganese in particular. The mechanism is considered to ...
Proc. Natl. Acad. Sci. USA Vol. 81, pp. 476-478, January 1984 Cell Biology
Mn2+ and Co2+ toxicity in chlorophyll biosynthesis (tetrapyrrole/protoporphyrin/Mg protoporphyrin/Anacystis)
K. CSATORDAY*, Z. GOMBOS*, AND B. SZALONTAIt *Institute of Plant Physiology and tInstitute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, P.O. Box 521,
Hungary, H-6701
Communicated by Winslow R. Briggs, September 28, 1983
oxide-free ether and then twice with 5 ml of ether. The combined ether extracts were concentrated under N2. The water/ acetone residue was extracted three times with 5 ml of ether. This final ether phase was concentrated under N2. All procedures were carried out in the dark. Chromatography. The pigments were separated by chromatography on thin layers (20 x 20 cm) of silica gel (Merck). The plates were developed in benzene/ethyl acetate/ethanol, 8:2:2 (vol/vol) at 40C in the dark. Pigment bands were located by their red fluorescence under UV light. The bands were scraped from the plates, pigments were eluted from the silica gel with methanol/acetone (4:1), and the fluorescence spectra were recorded. The extracted pigments were identified by comparing them to porphyrin standards (Porphyrin Products, Logan, UT) both during chromatography and fluorescence measurements. 5-aminolevulinic acid (Sigma) was added to the algae.
The metal ion-induced inhibition of tetrapyrABSTRACT role biosynthesis was studied in the cyanobacterium Anacystis nidulans. The accumulation of protoporphyrin and Mg protoporphyrin due to the effect of Co2' and Mn2+ treatment, respectively, pointed to two different sites of inhibition.
Chlorophyll biosynthesis is inhibited by iron deficiency induced by a number of metals (1)-cobalt and manganese in particular. The mechanism is considered to be competition for an active site where iron is necessary for functional integrity. The photosynthetic pigment apparatus of blue-green algae contains both chlorophyll and phycobiliproteins. The chromophore of the latter is also a tetrapyrrole, albeit a linear one (2). After nitrate starvation the blue-green algae are capable of regenerating their pigment apparatus, and in the process, several fluorescent tetrapyrroles, intermediates in tetrapyrrole biosynthesis, are detectable (3). In the present paper we show that manganese and cobalt, both inhibitors of chlorophyll biosynthesis, are acting at different stages of tetrapyrrole biosynthesis, and we shall identify the steps in biosynthesis where inhibition occurs.
RESULTS AND DISCUSSION The effect of increasing concentrations of Mn2+ on the fluorescence spectrum of a slowly growing culture ofA. nidulans regenerating after nitrate starvation is shown in Fig. 1. The high-intensity fluorescence band at 596 nm belongs to Mg protoporphyrin as determined by chromatography and fluorimetry (not shown), whereas the long wavelength part of the spectrum represents phycobiliprotein and chlorophyll fluorescence at 650-660 and 680 nm, respectively. Pure Mg protoporphyrin fluorescence has a considerable contribution at 650 nm as well (0.3 times the intensity measured at 596 nm). The concentration of Mn2+ eliciting the fluorescence spectrum (curve 5) is the highest one shown in the given experiment; higher concentrations retard cell growth as well and attain similar features at successively later times. Because of the effect of mild Co + treatment in nitratestarved cultures, accumulation of protoporphyrin IX occurs with concurrent retardation of both chlorophyll and phycobiliprotein synthesis. At a later stage, the protoporphyrin is transformed into the phycocyanobilin chromophore and chlorophyll, and, consequently, phycobiliprotein fluorescence at 650-660 nm and chlorophyll fluorescence at 680 nm increase at the expense of protoporphyrin fluorescence at 636 nm (Fig. 2). Higher concentrations of Co2+ retard cell growth and prevent further transformation of protoporphyrin. In work with Chlorella mutants unable to synthesize chlorophyll, Granick (8) has shown that they accumulate protoporphyrin monomethyl ester and some Mg-protoporphyrin monomethyl ester, thus indicating that these are intermediates in chlorophyll biosynthesis. In view of the generally accepted scheme of chlorophyll biosynthesis (9), the emergence of Mg protoporphyrin and protoporphyrin IX in our experiments indicates that a block occurs in the biosynthetic route. The evolution of the Mg-protoporphyrin fluorescence band occurs rapidly. The maximum intensity is attained
METHODS Anacystis nidulans (strain IU 625) was maintained on agar slants enriched with medium C of Kratz and Myers (4) and illuminated by fluorescent tubes (3,000 lux). When maintained in liquid medium C, the cells were grown in glass tubes at 38°C with an illumination of 10,000 lux and were supplied with 95% air/5% CO2. Nitrate starvation was induced by suspending the algal cells in nitrate-deficient culture medium. For regeneration experiments, the cells were collected and resuspended in whole medium C. Fluorescence measurements were carried out using a Perkin Elmer MPF 3 spectrofluorimeter. Both excitation and observation slits were set at 6-nm bandwidth. Fluorescence spectra were not corrected for the spectral sensitivity of the apparatus because only relative changes in fluorescence are discussed. Absorption spectra were recorded by a UNICAM SP 1800 UV spectrophotometer, using the opal glass method of Shibata et al. (5). Phycocyanin/chlorophyll ratios were calculated from the in vivo absorption spectra (6). Pigment Extraction. A 3-ml algal suspension was mixed with 15 ml of acetone/0.1 M NH4 OH, (9:1 (vol/vol) at 0°C and centrifuged at 39,000 x g for 10 min (7). The supernatant was extracted once with 10 ml and twice with 5 ml of nhexane. The pooled hexane extracts were back-extracted with 5 ml of fresh 75% aqueous acetone fraction. The pigments in the acetone extract were transferred to ether before chromatography. This was achieved by adding 1/17 of its volume of saturated NaCl and 1/70 of its volume of 0.5 M KH2PO4. The mixture was extracted once with 10 ml of perThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Wavelength, nm FIG. 1. Fluorescence spectra of nitrate-starved A. nidulans 6 hr after resuspension in complete growth medium supplemented with various concentrations of MnCl2. Curves 1-5: MnCl2 concentrations of 9 uM, 45 AuM, 90 ALM, 140 uM, and 180 ,LM, respectively. The excitation wavelength was 425 nm.
within 6 hr of manganese treatment (Fig. 3). The block in chlorophyll biosynthesis does not affect the other branch of the tetrapyrrole biosynthetic pathway. Thus, the phycocyanobilin chromophores are freely synthesized, as evidenced by the relative increase in phycobiliprotein content when manganese is present. The addition of the common tetrapyrrole precursor 5-aminolevulinic acid to the cell suspension results in no change in the ratio of phycocyanin to chlorophyll, whereas in the presence of manganese, an excess amount of phycobiliprotein is synthesized as a function of 5-aminolevulinic acid concentration (Fig. 4). Fig. 5 shows the absorption spectra of nitrate-starved cultures of A. nidulans 13 hr after resuspension in complete culture medium supplied with a toxic amount of manganese in one case and with a normal amount in the other. The retardation of chlorophyll synthesis is clearly accompanied by an enhancement of phycocyanin production.
0
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Time, hr FIG. 3. Change in the relative fluorescence intensity of the 596nm band in nitrate-starved Anacystis as a function of time since re-
suspension in complete medium supplemented with 180 ,uM MnCI2. The excitation wavelength was at 425 nm.
After cobalt treatment, however, the cells of A. nidulans accumulate protoporphyrin IX, and at the same time both chlorophyll and phycobiliprotein syntheses are blocked. In Cyanidium caldarium, a rhodophyte, protoporphyrin accumulation was observed in the dark when the cells were growing heterotrophically, and phycobiliprotein formation occurred only in the light (10). On the other hand, in adult rat hepatocytes, Co2+ effectively prevents association of heme to apocytochrome P-450 (11). In Anacystis, the accumulation of protoporphyrin indicates that its association with an apoprotein, a light-dependent process, is prevented by Co2+. The site of inhibition of chlorophyll biosynthesis by Co2+, therefore, is at a step preceding the insertion of magnesium
U
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Wavelength, nm FIG. 2. Time-dependent changes in the fluorescence spectrum of a nitrate-starved culture of Anacystis resuspended in complete
growth medium with the extra microelement Co(NO3)2-6H2O added (1.7 ,uM). Numbers indicate days since resuspension. Excitation was at 396 nm to observe phycobiliprotein fluorescence simultaneously with protoporphyrin fluorescence at 636 nm. The Soret band of the latter is at 410 nm.
5-Aminolevulinic acid, mM FIG. 4. The phycobiliprotein/chlorophyll ratio in nitrate-starved Anacystis 24 hr after resuspension in complete medium is shown as a function of 5-aminolevulinic acid concentration without (0) and with (x) 270 AM MnCl2.
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FIG. 5. Absorption spectra of nitrate-starved Anacystis 13 hr after resuspension in complete medium (----) and with 270 ,uM MnCl2 added to the growth medium (-). Both cultures contained 30 mM 5-aminolevulinic acid.
into the protoporphyrin ring, most probably after protoporphyrin formation (Fig. 2). Iron- and oxygen-deficient plants were found to accumulate Mg protoporphyrin and Mg protoporphyrin monomethyl ester (12), and we concluded that this is consistent with the concept of an iron- and oxygen-requiring step (13) between these pigments and protochlorophyllide. Addition of the tetrapyrrole precursor 5-aminolevulinic acid resulted in an enhanced level of accumulated Mg protoporphyrin. This observation and our data on Mn + treatment may well have a common source. A number of metals are capable of inducing iron deficiency and consequently inhibit chlorophyll synthesis in plants (1). Among these, manganese seems to be rather effective in eliciting symptoms of iron deficiency in plants and is the cause of the manganese toxicity observed. Our data support the contention that the mode of action of manganese is competition with iron by blocking the access of iron ions to some functional site involved in the magnesium branch of the tetrapyrrole synthesis pathway, thereby causing symptoms of iron deficiency. On the basis of Figs. 1 and 2, it is evident that manganese toxicity is manifested through the emergence of Mg protoporphyrin, and thus the site of action can be determined as the step after the insertion of
magnesium into the protoporphyrin ring as deduced from the accumulation of Mg protoporphyrin in A. nidulans. 1. Hewitt, E. J. & Nicholas, F. D. (1%3) in Metabolic inhibitors, eds. Hochster, R. M. & Quastel, J. H. (Academic, New York), pp. 311-436. 2. 6Carra, P. 0. & OhEocha, C. 0. (1976) in Chemistry and Biochemistry of Plant Pigments, ed. Goodwin, T. W. (Academic, London), Vol. 1, pp. 328-376. 3. Csatorday, K. (1978) Biochim. Biophys. Acta 504, 341-343. 4. Kratz, W. A. & Myers, J. (1954) Am. J. Bot. 42, 282-287. 5. Shibata, K., Benson, A. A. & Calvin, M. (1954) Biochim. Biophys. Acta 15, 461-470. 6. Goedheer, J. C. (1976) Photosynthetica 10, 411-422. 7. Rebeiz, C. A. & Castelfranco, P. A. (1971) Plant Physiol. 47, 24-32. 8. Granick, S. (1961) J. Biol. Chem. 236, 1168-1172. 9. Bogorad, L. (1976) in Chemistry and Biochemistry of Plant Pigments, ed. Goodwin, T. W. (Academic, London), pp. 64148. 10. Guzelian, P. S. & Bissell, D. M. (1976) J. Biol. Chem. 251, 4421-4427. 11. Csatorday, K., MacColl, R. & Berns, D. S. (1981) Proc. Natl. Acad. Sci. USA 78, 1700-1702. 12. Spiller, C. S., Castelfranco, A. M. & Castelfranco, P. A. (1982) Plant Physiol. 69, 107-111. 13. Jones, 0. T. G. (1963) Biochem. J. 86, 429-432.
