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Chlorophyllase in Piper betle L. has a role in chlorophyll homeostasis and senescence dependent chlorophyll breakdown

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Abstract

Total chlorophyll content and chlorophyllase (chlorophyll-chlorophyllido hydrolase EC 3.1.1.14) activity in fresh leaves of Piper betle L. landrace KS was, respectively, twofold higher and eight fold lower than KV, showing negative correlation between chlorophyll and chlorophyllase activity. Specific chlorophyllase activity was nearly eightfold more in KV than KS. ORF of 918 nt was found in cloned putative chlorophyllase cDNAs from KV and KS. The gene was present as single copy in both the landraces. The encoded polypeptide of 306 amino acids differed only at two positions between the KV and KS; 203 (cysteine to tyrosine) and 301 (glutamine to glycine). Difference in chlorophyllase gene expression between KV and KS was evident in fresh and excised leaves. Up regulation of chlorophyllase gene by ABA and down regulation by BAP was observed in both the landraces; however, there was quantitative difference between KV and KS. Data suggests that chlorophyllase in P. betle is involved in chlorophyll homeostasis and chlorophyll loss during post harvest senescence.

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Abbreviations

Chl:

Chlorophyll

Chlase:

Chlorophyllase

KV:

Kapoori Vellaikodi

KS:

Khasi Shillong

PT:

Post treatment

PAR:

Photosynthetically active radiation

Chlid:

Chlorophyllide

nt:

Nucleotide

References

  1. Hendry GAF, Houghton JD, Brown SB (1987) The degradation of chlorophyll-a biological enigma. New Phytol 107:255–302

    Article  CAS  Google Scholar 

  2. Kumar N, Gupta S, Tripathi AN (2006) Gender-specific responses of Piper betle (L.) to low temperature stress: changes in chlorophyllase activity. Biol Plant 50:705–708

    Article  CAS  Google Scholar 

  3. Tsuchiya T, Ohta H, Masuda T, Mikami B, Kita N, Shioi Y, Takamiya KI (1997) Purification and characterization of two isozymes of chlorophyllase from mature leaves of Chenopodium album. Plant Cell Physiol 38:1026–1031

    Article  CAS  Google Scholar 

  4. Hortensteiner S (2006) Chlorophyll degradation during senescence. Annu Rev Plant Biol 57:55–77

    Article  PubMed  CAS  Google Scholar 

  5. Berghold J, Eichmüller C, Hörtensteiner S, Kräutler B (2004) Chlorophyll breakdown in tobacco: on the structure of two nonfluorescent chlorophyll catabolites. Chem Biodivers 1:657–668

    Article  PubMed  CAS  Google Scholar 

  6. Shemer TA, Harpaz-Saad S, Belausov E, Lovat N, Krokhin O, Spicer V et al (2008) Citrus chlorophyllase dynamics at ethylene-induced fruit color-break: a study of chlorophyllase expression, posttranslational processing kinetics, and in situ intracellular localization. Plant Physiol 148:108–118

    Article  CAS  Google Scholar 

  7. Schenk N, Schelbert S, Kanwischer M, Goldschmidt EE, Dormann P, Hortensteiner S (2007) The chlorophyllases AtCLH1 andAtCLH2 are not essential for senescence-related chlorophyll breakdown in Arabidopsis thaliana. FEBS Lett 27:5517–5525

    Article  Google Scholar 

  8. Arkus KAJ, Cahoon EB, Jez JM (2005) Mechanistic analysis of wheat chlorophyllase. Arch Biochem Biophys 438:146–155

    Article  PubMed  CAS  Google Scholar 

  9. Jacob-Wilk D, Holland D, Goldschmidt EE, Riov J, Eyal Y (1999) Chlorophyll breakdown by chlorophyllase: isolation and functional expression of the Chlase1 gene from ethylene- treated Citrus fruit and its regulation during development. Plant J 20:653–661

    Article  PubMed  CAS  Google Scholar 

  10. Tsuchiya T, Ohta H, Okawa K, Iwamatsu A, Shiomada H, Masuda T, Takamiya KI (1999) Cloning of chlorophyllase gene the key enzyme in chlorophyll degradation; finding of a lipase motif and induction by methyl jasmonate. Proc Nat Acad Sci (USA) 96:15362–15367

    Article  CAS  Google Scholar 

  11. Tsuchiya T, Suzuki T, Yamada T, Shimada H, Masuda T, Ohta H, Takamiya K (2003) Chlorophyllase as a serine hydrolase: identification of a putative triad. Plant Cell Physiol 44:96–101

    Article  PubMed  CAS  Google Scholar 

  12. Harpaz-Saad S, Azoulay T, Arazi T, Ben-Yaakov E, Mett A, Shiboleth YM, Hortensteiner S et al (2007) Chlorophyllase is a rate-limiting enzyme in chlorophyll catabolism and is post-translationally regulated. Plant Cell 19:1007–1022

    Article  PubMed  CAS  Google Scholar 

  13. Trebitsh T, Goldschmidt EE, Riov J (1993) Ethylene induces de novo synthesis of chlorophyllase, a chlorophyll degrading enzyme, in Citrus fruit peel. Proc Nat Acad Sci (USA) 90:9441–9445

    Article  CAS  Google Scholar 

  14. Schelbert S, Aubry S, Burla B, Agne B, Kessler F, Krupinska K, Hortensteiner S (2009) Pheophytin pheophorbide hydrolase (Pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. Plant Cell 21:767–785

    Article  PubMed  CAS  Google Scholar 

  15. Lichtenthaler HK (1987) Chlorophyll and carotenoids. In: L Packer, R Douce (eds) Pigments of photosynthetic biomembranes, Methods Enzymol 148, pp 350–382

  16. Tang L, Okazawa A, Itoh Y, Fukusaki EI, Kobayashi A (2004) Expression of chlorophyllase is not induced during autumnal yellowing in Ginkgo biloba. Z Naturforsch CJ Biosci 59:415–420

    CAS  Google Scholar 

  17. Todorov DT, Karanov EN, Smith AR, Hall MA (2003) Chlorophyllase activity and chlorophyll content in wild type and eti 5 mutant of Arabidopsis thaliana subjected to low and high temperatures. Biol Plant 46:633–636

    Article  CAS  Google Scholar 

  18. Beisel KG, Jahnke S, Hofmann D, Koppchen S, Ulrich MS (2010) Continuous turnover of carotenes and chlorophyll a in mature leaves of Arabidopsis revealed by 14CO2 pulse-chase labeling. Plant Physiol 152:2188–2199

    Article  PubMed  CAS  Google Scholar 

  19. Vavilin D, Vermaas W (2007) Continuous chlorophyll degradation accompanied by chlorophyllide and phytol reutilization for chlorophyll synthesis in Synechocystis sp. PCC6803. BBA 1767:920–929

    Article  PubMed  CAS  Google Scholar 

  20. Iriyama K, Ogura N, Takamiya A (1974) A simple method for extraction and partial purification of chlorophyll from plant material, using dioxane. J Biochem 76:901–904

    PubMed  CAS  Google Scholar 

  21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  22. Asif MH, Dhawan P, Nath P (2000) A simple procedure for the isolation of high quality RNA from ripening banana fruit. Plant Mol Biol Rep 18:109–115

    Article  CAS  Google Scholar 

  23. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21

    Article  CAS  Google Scholar 

  24. Sambrook J, Fritsch EF, Manniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold spring harbor

    Google Scholar 

  25. Gupta SM, Srivastava S, Sane AP, Nath P (2006) Differential expression of genes during banana fruit development, ripening and 1-MCP treatment: presence of distinct fruit specific, ethylene induced and ethylene repressed expression. Postharvest Biol Technol 42:16–22

    Article  CAS  Google Scholar 

  26. Kariola T, Brader G, Li J, Palva ET (2005) Chlorophyllase 1, a damage control enzyme, affects the balance between defense pathways in plants. Plant Cell 17:282–294

    Article  PubMed  CAS  Google Scholar 

  27. Benedetti CE, Arruda P (2002) Altering the expression of the chlorophyllase gene ATH-COR1 in Arabidopsis caused in the chlorophyll to chlorophyllide ratio. Plant Physiol 128:1255–1263

    Article  PubMed  CAS  Google Scholar 

  28. Wang HC, Huang XM, Hu GB, Yang ZY, Huang HB (2005) A comparative study of chlorophyll loss and its related mechanism during fruit maturation in the pericarp of fast- and slow-degreening litchi pericarp. Scientia Hort 106:247–257

    Article  CAS  Google Scholar 

  29. Costa ML, Civello PM, Chaves AR, Martinez GA (2005) Effect of ethephon and 6-benzylaminopurine on chlorophyll degrading enzymes and peroxidase-linked chlorophyll bleaching during post-harvest senescence of broccoli (Brassica Oleracea L.) at 20°C. Postharvest Biol Technol 35:191–199

    Article  CAS  Google Scholar 

  30. Smart CM (1994) Gene expression during leaf senescence. New Phytol 126:419–448

    Article  CAS  Google Scholar 

  31. Gupta S, Gupta SM, Kumar N (2011) Role of chlorophyllase in chlorophyll homeostasis and post harvest breakdown in Piper betle L. leaf. Ind J Biochem Biophys 48:353–360

    Google Scholar 

  32. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing Phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

  33. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791

    Article  Google Scholar 

  34. Zuckerkandl E, Pauling L (1965) Evolutionary divergence and convergence in proteins. In: Bryson V, Vogel HJ (eds) Evolving genes and proteins. Academic Press, New York, pp 97–166

    Google Scholar 

  35. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We are thankful to Department of Science and Technology (DST), New Delhi for partial support of the studies by Grant No. SP/SO/A42/95. SG worked as project fellow in DST project and later as CSIR Senior Research Fellow. Dr S.A. Ranade for drawing tree view and helpful discussions.

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Author notes

  1. Supriya Gupta

    Present address: Infusion College of Science & Technology, Haldwani, 263 139, India

Authors and Affiliations

  1. Betel Vine Biotechnology Lab, National Botanical Research Institute, Lucknow, 226 001, India

    Supriya Gupta & Nikhil Kumar

  2. Plant Gene Expression Lab, National Botanical Research Institute, Lucknow, 226 001, India

    Sanjay Mohan Gupta & Aniruddha P. Sane

  3. Molecular Biology & Genetic Engineering Laboratory, Defence Institute of Bio-energy Research, P.O. Arjunpur, Goraparao, Haldwani, 263 139, India

    Sanjay Mohan Gupta

Authors
  1. Supriya Gupta
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  4. Nikhil Kumar
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Corresponding author

Correspondence to Sanjay Mohan Gupta.

Additional information

Database accession numbers: Kapoori Vellaikodi: Accession No. AY327108, Khasi Shillong: Accession No. DQ911625. Genomic sequence of KS: Accession No. DQ444457.

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Gupta, S., Gupta, S.M., Sane, A.P. et al. Chlorophyllase in Piper betle L. has a role in chlorophyll homeostasis and senescence dependent chlorophyll breakdown. Mol Biol Rep 39, 7133–7142 (2012). https://doi.org/10.1007/s11033-012-1545-8

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  • DOI: https://doi.org/10.1007/s11033-012-1545-8

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