Abstract
Complementary DNA clones and a corresponding nuclear gene (lpcr) encoding the NADPH-dependent protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) have been characterized from pea (Pisum sativum L.). The pea lpcr gene encodes a 43 118 Da precursor polypeptide comprised of a transit peptide of 64 amino acids and a mature protein of 336 amino acids. The coding portion of the gene is interrupted by four introns, two of which are located within the transit peptide coding portion of the gene. The deduced primary structure for the pea protein is similar to those reported for Arabidopsis and two monocot species. Northern blot analysis revealed little to no decrease in steady-state levels of mRNA encoding the enzyme in etiolated leaves illuminated with continuous white light for up to 48 h. In contrast, western blot analysis showed that the major immunoreactive species present in whole leaf extracts decreased to nearly undetectable levels during this same 48 h period. These results suggest that pchlide reductase activity in pea is primarily regulated post-transcriptionally, most likely at the level of translation initiation/elongation or protein turnover.
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References
Apel K: The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Phytochrome-induced decrease of translatable mRNA coding for the NADPH-protochlorophyllide oxidoreductase. Eur J Biochem 120: 89–93 (1981).
Apel K, Santel J-J, Redlinger TE, Falk H: The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Isolation and characterization of the NADPH protochlorophyllide oxidoreductase. Eur J Biochem 111: 251–258 (1980).
Benli M, Schulz R, Apel K: Effect of light on the NADPH-protochlorophyllide oxidoreductase of Arabidopsis thaliana. Plant Mol Biol 16: 615–625 (1991).
Cashmore AR: The isolation of polyA+ messenger RNA from higher plants. In: Edelman M, Hallick RB, Chua N-H (eds) Methods in Chloroplast Molecular Biology, pp. 387–392. Elsevier Biomedical Press, Amsterdam (1982).
Darrah PM, Kay SA, Teakle GR, Griffiths WT: Cloning and sequencing of protochlorophyllide reductase. Biochem J 265: 789–798 (1990).
Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).
Forreiter C, vanCleve B, Schmidt A, Apel K: Evidence for a general mechanism of light-dependent control of the NADPH-protochlorophyllide oxidoreductase in higher plants. Planta 183: 126–132 (1991).
Frohman MA: Rapid amplification of cDNA ends (RACE): User-friendly cDNA cloning. Amplifications 5 (September): 11–15 (1990).
Griffiths WT: Protochlorophyll and protochlorophyllide as precursors for chlorophyll synthesis in vitro. FEBS Lett 49: 196–200 (1974).
Gubler U, Hoffman BJ: A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269 (1983).
Hauser I, Dehesh K, Apel K: The proteolytic degradation in vitro of the NADPH-protochlorophyllide oxidoreductase of barley (Hordeum vulgare L.). Arch Biochem Biophys 228: 557–586 (1984).
Huynh TV, Young RA, Davis RW: Constructing and screening cDNA libraries in λgt10 and λgt11. In: Glover DM (ed) DNA Cloning: A Practical Approach, pp. 49–78. IRL Press, Oxford (1985).
Kay SA, Griffiths WT: Light-induced breakdown of NADPH-protochlorophyllide oxidoreductase in vitro. Plant Physiol 72: 229–236 (1983).
Keegstra K, Olsen LJ, Theg SM: Chloroplastic precursors and their transport across the envelope membranes. Annu Rev Plant Physiol Plant Mol Biol 40: 471–501 (1989).
Kittsteiner U, Paulsen H, Schendel R, Rudiger W: Lack of light regulation of NADPH: protochlorophyllide oxido-reductase mRNA in cress seedlings (Lepidium sativum L.). Z Naturforsch 45c: 1077–1079 (1990).
Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Mapleston ER, Griffiths WT: Light modulation of the activity of protochlorophyllide reductase. Biochem J 189: 125–133 (1980).
Mosinger E, Batschaver A, Schafer E, Apel K: Phytochrome control of in vitro transcription of specific genes in isolated nuclei from barley (Hordeum vulgare). Eur J Biochem 147: 137–142 (1985).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain termination inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Schulz R, Steinmuller K, Klaas M, Forreiter C, Rasmussen S, Hiller C, Apel K: Nucleotide sequence of a cDNA coding for the NADPH-protochlorophyllide oxidoreductase (PCR) of barley (Hordeum vulgare L.) and its expression in Escherichia coli. Mol Gen Genet 217: 355–361 (1989).
Spano AJ, Timko MP: Isolation, characterization, and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.). Biochim Biophys Acta 1076: 29–36 (1991).
Speicher DW: Microsequencing with PVDF membranes: Efficient electroblotting, direct protein absorption and sequencer program modifications. In: Hugli TE (ed) Techniques in Protein Chemistry, pp. 24–35. Academic Press, San Diego (1989).
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Spano, A.J., He, Z., Michel, H. et al. Molecular cloning, nuclear gene structure, and developmental expression of NADPH: protochlorophyllide oxidoreductase in pea (Pisum sativum L.). Plant Mol Biol 18, 967–972 (1992). https://doi.org/10.1007/BF00019210
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DOI: https://doi.org/10.1007/BF00019210