Plant Molecular Biology

, 71:425 | Cite as

Expression of chlorophyll synthase is also involved in feedback-control of chlorophyll biosynthesis

  • Nikolai Shalygo
  • Olaf Czarnecki
  • Enrico Peter
  • Bernhard Grimm


At the last step of the chlorophyll biosynthetic pathway chlorophyll synthase (CHLG) esterifies chlorophyllide a and b with phytyl or geranyl-geranyl pyrophosphate in chloroplasts. Transgenic tobacco plants expressing CHLG RNA in sense and antisense orientation were examined for the effects of excessive and reduced ectopic CHLG expression, respectively, on the chlorophyll biosynthetic pathway and the expression of chlorophyll-binding proteins. Reduced chlorophyll synthase activity does not result in accumulation of chlorophyllide and caused reduced ALA formation and Mg and ferrochelatase activity, while CHLG overexpression correlated with enhanced ALA synthesizing capacity and more chelatase activities. The transcript levels of genes expressing proteins of chlorophyll biosynthesis and chlorophyll-binding proteins were down-regulated in response to reduced CHLG expression. Thus, reduced expression and activity of chlorophyll synthase caused a feedback-controlled inactivation of the initial and rate limiting step of the pathway leading to down regulation of the metabolic flow, while overexpression can mediate a stimulation of the pathway. Chlorophyll synthase is proposed to be important for the co-regulation of the entire pathway and the coordination of synthesis of chlorophyll and the chlorophyll-binding proteins.


Chlorophyll CHLG Porphyrins Feedback regulation Light harvesting chlorophyll binding protein ALA biosynthesis 



5-aminolevulinic acid






Mg protoporphyrin monomethylester (oxidative) cyclase

Mg chelatase

Mg protoporphyrin IX chelatase


Mg protoporphyrin IX


Mg protoporphyrin monomethyl ester




Protoporphyrin IX


Zn chlorophyll a


Zn chlorophyllide a



This work was supported in the DFG-Sonderforschungsbereich (SFB) 492 by grants to B.G. (TP A8/B9).

Supplementary material

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Supplementary material 1 (DOC 33 kb)
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Supplementary material 2 (JPG 1702 kb)
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Supplementary material 3 (JPG 2174 kb)


  1. Alawady AE, Grimm B (2005) Tobacco Mg protoporphyrin IX methyltransferase is involved in inverse activation of Mg porphyrin and protoheme synthesis. Plant J 41:282–290CrossRefPubMedGoogle Scholar
  2. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ, Gapped BLAST and PSI-BLAST (1997) A new generation of protein database search programs. Nucleic Acids Res. 25:3389–3402CrossRefPubMedGoogle Scholar
  3. Beale SI (1999) Enzymes of chlorophyll biosynthesis. Photosynth Res 60:43–73CrossRefGoogle Scholar
  4. Bollivar DW, Beale SI (1996) The chlorophyll biosynthetic enzyme Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase (characterization and partial purification from Chlamydomonas reinhardtii and Synechocystis sp. PCC 6803. Plant Physiol 112:105–114PubMedGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  6. Castelfranco PA, Jones OT (1975) Protoheme turnover and chlorophyll synthesis in greening barley tissue. Plant Physiol 55:485–490CrossRefPubMedGoogle Scholar
  7. Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81:1991–1995CrossRefPubMedGoogle Scholar
  8. Domanskii V, Rassadina V, Gus-Mayer S, Wanner G, Schoch S, Rüdiger W (2003) Characterization of two phases of chl formation during greening of etiolated barley leaves. Planta 216:475–483PubMedGoogle Scholar
  9. Eberhard S, Finazzi G, Wollman FA (2008) The dynamics of photosynthesis. Annu Rev Genet 42:463–515CrossRefPubMedGoogle Scholar
  10. Eckhardt U, Grimm B, Hörtensteiner S (2004) Recent advances in chl biosynthesis and breakdown in higher plants. Plant Mol Biol 56:1–14CrossRefPubMedGoogle Scholar
  11. Frigerio S, Campoli C, Zorzan S, Fantoni LI, Crosatti C, Drepper F, Haehnel W, Cattivelli L, Morosinotto T, Bassi R (2007) Photosynthetic antenna size in higher plants is controlled by the plastoquinone redox state at the post-transcriptional rather than transcriptional level. J Biol Chem 282:29457–29469CrossRefPubMedGoogle Scholar
  12. Fuhrhop J-H, Smith KM (1975) Laboratory methods. In: Smith KM (ed) Porphyrins and metalloporphyrins. Elsevier, Amsterdam, pp 757–869Google Scholar
  13. Gaubier P, Wu HJ, Laudié M, Delseny M, Grellet F (1995) A chlorophyll synthetase gene from Arabidopsis thaliana. Mol Gen Genet 249:58–64CrossRefPubMedGoogle Scholar
  14. Grimm B, Porra R, Rüdiger W, Scheer H (2005) Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications, In: Advances in Photosynthesis and Respiration, Vol 25. Springer, Dordrecht, the NetherlandsGoogle Scholar
  15. Heddad M, Norén H, Reiser V, Dunaeva M, Andersson B, Adamska I (2006) Differential expression and localization of early light-induced proteins in Arabidopsis. Plant Physiol 142:75–87CrossRefPubMedGoogle Scholar
  16. Hedtke B, Alawady A, Chen S, Börnke F, Grimm B (2007) HEMA RNAi silencing reveals a control mechanism of ALA biosynthesis on Mg chelatase and Fe chelatase. Plant Mol Biol 64:733–742CrossRefPubMedGoogle Scholar
  17. Höfgen R, Willmitzer L (1990) Biochemical and genetic analysis of different patatin isoforms in various organs of potato (Solanum tuberosum). Plant Sci 66:221–230CrossRefGoogle Scholar
  18. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231Google Scholar
  19. Hutin C, Nussaume L, Moise N, Moya I, Kloppstech K, Havaux M (2003) Early light-induced proteins protect Arabidopsis from photooxidative stress. Proc Natl Acad Sci USA 100:4921–4926CrossRefPubMedGoogle Scholar
  20. Ikegami A, Yoshimura N, Motohashi K, Takahashi S, Romano PG, Hisabori T, Takamiya K, Masuda T (2007) The CHLI1 subunit of Arabidopsis thaliana magnesium chelatase is a target protein of the chloroplast thioredoxin. J Biol Chem 282:19282–19291CrossRefPubMedGoogle Scholar
  21. Kim J, Eichacker LA, Rüdiger W, Mullet JE (1994) Chlorophyll regulates accumulation of the plastid-encoded chlorophyll proteins P700 and D1 by increasing apoprotein stability. Plant Physiol 104:907–916CrossRefPubMedGoogle Scholar
  22. Kimura M, Yamamoto YY, Seki M, Sakurai T, Sato M, Abe T, Yoshida S, Manabe K, Shinozaki K, Matsui M (2003) Identification of Arabidopsis genes regulated by high light-stress using cDNA microarray. Photochem Photobiol 77:226–233CrossRefPubMedGoogle Scholar
  23. Koski VM, Smith JH (1948) The isolation and spectral absorption properties of protochlorophyll from etiolated barley seedlings. J Am Chem Soc 70:3558–3562CrossRefPubMedGoogle Scholar
  24. Kruse E, Mock HP, Grimm B (1995) Reduction of coproporphyrinogen oxidase level by antisence system. EMBO J 14:3712–3720PubMedGoogle Scholar
  25. Masuda T, Fujita Y (2008) Regulation and evolution of chl metabolism. Photochem Photobiol Sci 10:1131–1149CrossRefGoogle Scholar
  26. Matsumoto F, Obayashi T, Sasaki-Sekimoto Y, Ohta H, Takamiya K, Masuda T (2004) Gene expression profiling of the tetrapyrrole metabolic pathway in Arabidopsis with a mini-array system. Plant Physiol 135:2379–2391CrossRefPubMedGoogle Scholar
  27. Mauzerall D, Granick S (1956) The occurrence and determination of delta-amino-levulinic acid and porphobilinogen in urine. J Biol Chem 219:435–446PubMedGoogle Scholar
  28. Mock HP, Grimm B (1997) Reduction of uroporphyrin decarboxylase by antisense RNA expression affects activities of other enzymes involved in tetrapyrrole biosynthesis and lead to light-dependent necrosis. Plant Physiol 113:1101–1112PubMedGoogle Scholar
  29. Moulin M, Smith AG (2005) Regulation of tetrapyrrole biosynthesis in higher plants. Biochem Soc Trans 33:737–742CrossRefPubMedGoogle Scholar
  30. Papenbrock J, Mock HP, Tanaka R, Kruse E, Grimm B (2000) Role of magnesium chelatase activity in the early steps of the tetrapyrrole biosynthetic pathway. Plant Physiol 122:1161–1169CrossRefPubMedGoogle Scholar
  31. Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975:384–394CrossRefGoogle Scholar
  32. Rüdiger W, Benz J, Guthoff C (1980) Detection and partial characterization of activity of chlorophyll synthetase in etioplast membranes. Eur J Biochem 109:193–200CrossRefPubMedGoogle Scholar
  33. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  34. Smith AG (1988) Cellular localization of two porphyrin-synthesis enzymes in Pisum sativum (pea) and Arum (cuckoo-pint) species. Biochem J 249:423–428PubMedGoogle Scholar
  35. Soll J, Schulz G, Rüdiger W, Benz J (1983) Hydrogenation of geranylgeraniol: two pathway exist in spinach chloroplasts. Plant Physiol 71:849–854CrossRefPubMedGoogle Scholar
  36. Tanaka R, Tanaka A (2007) Tetrapyrrole biosynthesis in higher plants. Annu Rev Plant Biol 58:321–346CrossRefPubMedGoogle Scholar
  37. Tzvetkova-Chevolleau T, Franck F, Alawady AE, Dall’Osto L, Carrière F, Bassi R, Grimm B, Nussaume L, Havaux M (2007) The light stress-induced protein ELIP2 is a regulator of chlorophyll synthesis in Arabidopsis thaliana. Plant J 50:795–809CrossRefPubMedGoogle Scholar
  38. Vandepoele K, Quimbaya M, Casneuf T, De Veylder L, Van de Peer Y (2009) Unraveling transcriptional control in Arabidopsis using cis-regulatory elements and coexpression networks. Plant Physiol 150:535–546CrossRefPubMedGoogle Scholar
  39. Weinstein JD, Beale SI (1984) Biosynthesis of protoheme and heme a precursors solely from glutamate in the unicellular red alga Cyanidium caldarium. Plant Physiol 74:146–151CrossRefPubMedGoogle Scholar
  40. Willows RD (2003) Biosynthesis of chlorophylls from protoporphyrin IX. Nat Prod Rep 20:327–341CrossRefPubMedGoogle Scholar
  41. Wu Z, Zhang X, He B, Diao L et al (2007) A chlorophyll-deficient rice mutant with impaired chlorophyllide estererification in chlorophyll biosynthesis. Plant Physiol 145:29–40CrossRefPubMedGoogle Scholar
  42. Yaronskaya E, Ziemann V, Walter G, Averina N, Boerner T, Grimm B (2003) Metabolic control of the tetrapyrrole biosynthetic pathway for porphyrin distribution in the barley mutant albostrians. Plant J 35:512–522CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Nikolai Shalygo
    • 1
    • 2
  • Olaf Czarnecki
    • 1
  • Enrico Peter
    • 1
  • Bernhard Grimm
    • 1
  1. 1.Institut für Biologie/PflanzenphysiologieHumboldt-Universität zu BerlinBerlinGermany
  2. 2.Institute of Biophysics and Cell Engineering of NAS of BelarusMinskBelarus

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