Abstract
The expression patterns of the two distinct subfamilies of genes (designated porA and porB) encoding the light- dependent NADPH:protochlorophyllide oxidoreductases (PORs) in loblolly pine (Pinus taeda L.) were examined. Transcripts arising from both gene subfamilies were shown to be present at high levels in the cotyledons of dark-grown pine seedlings and to a lesser extent in their stems. Exposure of dark-grown seedlings to light resulted in increased levels of both porA and porB transcripts, as well as increased levels of mRNAs encoding other photosynthesis-related gene products, suggesting that they are under a common mode of regulation. Relative levels of the porA and porB transcripts were similar in seedling cotyledons and primary needles of two-month-old pine trees, whereas only porB transcripts were present at a significant level in mature secondary needles of two-year-old trees. Immunoblot analysis showed that the 37 kDa PORA protein was most abundant in dark- grown tissues, decreased dramatically upon exposure to light, but could still be detected at low levels in light-grown seedlings. In comparison, levels of the 38 kDa PORB protein were not significantly changed upon transfer of dark-grown tissues to light. While both PORA and PORB were detected in cotyledons and primary needles, only PORB could be detected in mature needles. Transcripts derived from the three plastid genes, chlL, chlN, and chlB, encoding subunits of the light-independent protochlorophyllide reductase were detected in the cotyledons and stems of dark-grown seedlings, and in mature needles. The highest levels of chlL, chlN, and chlB transcripts were detected within the top one-third of the stem and decreased gradually towards the stem/root transition zone. Correspondingly, the highest levels of light-independent chlorophyll formation took place near the top of the hypocotyl. A similar pattern of expression was observed for other photosynthesis-related gene products, including porA and porB. Our results suggest that many aspects of the light-dependent, tissue-specific and developmental regulation of POR expression first described in angiosperms were already established in the less evolutionarily advanced gymnosperms. However, unlike angiosperms, light is not the dominant regulatory factor controlling porA expression in these species.
Similar content being viewed by others
References
Alosi MC, Neale DB, Kinlaw CS: Expression of cab genes in Douglas-fir is not strongly regulated by light. Plant Physiol 93: 829–832(1990).
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).
Argüello-Astorga GR, Herrera-Estrella LR: Ancestral multipartite units in light-responsive plant promoters have structural features correlating with specific phototransduction pathways. Plant Physiol 1112: 1151–1166(1996).
Armstrong GA, Runge S, Frick G, Sperling U, Apel K: Identification of NADPH:protochlorophyllide oxidoreductases A and B: a branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana. Plant Physiol 108: 1505–1517(1995).
Batschaier A, Apel K: The inverse control by phytochrome of the expression of two nuclear genes in barley (Hordeum vulgare L.). Eur J Biochem 143: 593–597(1984).
Baier CE, Bollivar DW, Suzuki JY: Genetic analysis of photopigment biosynthesis in eubacteria: A guiding light for algae and plants. J Bact 175: 3919–3925(1993).
Chang S, Puryear J, Cairney J: A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11: 113–116(1993).
Chinn E, Silverthorne J, Hohtola A: Light-regulated and organ-specific expression of types 1, 2, and 3 light-harvesting complex b mRNAs in Ginkgo biloba. Plant Physiol 107: 593–602(1995).
Drumm-Herrel H, Mohr H: Regulation by light of chlorophyll synthesis in the cotyledons of Scots pine (Pinus sylvestris) seedlings. Physiol Plant 91: 300–306(1994).
Forreiter C, Apel K: Light-independent and light-dependent protochlorophyllide-reducing activities and two distinct NADPH-protochlorophyllide oxidoreductase polypeptides in mountain pine (Pinus mugo). Planta 190: 536–545(1993).
Fujita Y: Protochlorophyllide reduction: a key step in the greening of plants. Plant Cell Physiol 37: 411–421(1996).
Gray-Mitsumune M, Yoo BY, Charest PJ: Chloroplastdependent and light-dependent expression of the tobacco rbcS promoter-GUS chimeric gene in black spruce. Can J For Res 26: 909–917(1996).
Greenwood MS: Phase change in loblolly pine: shoot development as a function of age. Plant Physiol 61: 518–522 (1984).
Griffiths WT, Kay SA, Oliver RP: The presence and photoregulation of protochlorophyllide reductase in green tissues. Plant Mol Biol 4:13–22(1985).
He Z-H, Li J, Sundqvist C, Timko, MP: Leaf developmental age controls expression of genes encoding enzymes of chlorophyll and heme biosynthesis in pea (Pisum sativum L.). Plant Physiol 106: 537–546(1994).
Holtorf H, Reinbothe S, Reinbothe C, Bereza B, Apel K: Two routes of chlorophyllide synthesis that are differentially regulated by light in barley (Hordeum vulgare L.). Proc Natl Acad Sci USA 92: 3254–3258(1995).
Holtorf H, Apel K: The regulation of NADPH protochlorophyllide oxidoreductases A and B in green barley plants kept under a diurnal light/dark cycle. Planta 199: 289–295(1996).
Hutchison KW, Greenwood MS: Molecular approaches to gene expression during conifer development and maturation. For Ecol Mngt 43: 273–286(1991).
Jansson S, Gustafsson P: Type I and Type II genes for the chlorophyll a/b-binding protein in the gymnosperm Pinus sylvestris (Scots pine): cDNA cloning and sequence analysis. Plant Mol Biol 14: 287–296(1990).
Kay SA, Griffiths WT: Light-induced breakdown of NADPHprotochlorophyllide oxidoreductase in vitro. Plant Physiol 72:229- 236 (1983).
Kuroda H, Masuda T, Ohta H, Shioi Y, Takamiya K: Lightenhanced gene expression of NADPH-protochlorophyllide oxidoreductase in cucumber. Biochem Biophys Res Comm 210: 310–316(1995).
Kuroda H, Masuda T, Ohta H, Shioi Y, Takamiya K: Effects of light, developmental age and phytohormones on the expression of the gene encoding NADPH-protochlorophyllide oxidoreductase in Cucumis sativus. Plant Physiol Biochem 34: 17–22(1996).
Lebedev N, van Cleve B, Armstrong G, Apel K: Chlorophyll synthesis in a deetiolated (det340) mutant of Arabidopsis without NADPH-protochlorophyllide (Pchlide) oxidoreductase (POR) A and photoactive PChlide-F655. Plant Cell 7: 2081–2090(1995).
Lidholm J, Gustafsson P: Homologues of the green algal gidA gene and the liverwort frxC gene are present on the chloroplast genomes of conifers. Plant Mol Biol 17: 787–798(1991).
Lidholm J, Gustafsson P: The chloroplast genome of the gymnosperm Pinus contorta: a physical map and a complete collection of overlapping clones. Curr Genet 20: 161–166 (1991).
Mapleston ER, Griffiths WT: Light modulation of the activity of the protochlorophyllide oxidoreductase. Biochem J 189: 125–133(1980).
Mösinger E, Batschaier 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).
Mukai Y, Yamamoto N, Koshiba T: Light-independent and tissue-specific accumulation of light-harvesting chlorophyll a/b binding protein and ribulose biphosphate carboxylase in dark-grown pine seedlings. Plant Cell Physiol 32: 1303–1306 (1991).
Mukai Y, Tazaki K, Fujii T, Yamamoto N: Light-independent expression of three photosynthetic genes, cab, rbcS, and rbcL, in coniferous plants. Plant Cell Physiol 33: 859–866(1992).
Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sano T, Sano S, Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota S, Inokuchi H, Ozeki H: Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322: 572–574(1986).
Peer W, Silverthorne J, Peters JL: Developmental and lightregulated expression of individual members of the lightharvesting complex b gene family in Pinus palustris. Plant Physiol 111: 627–634(1996).
Raven PH, Evert RF, Eichhorn SE: Biology of Plants. Worth Publishers, New York (1992).
Reinbothe S, Reinbothe C: The regulation of enzymes involved in chlorophyll biosynthesis. Eur J Biochem 237: 323–343 (1996).
Reinbothe S, Reinbothe C: Regulation of chlorophyll biosynthesis in angiosperms. Plant Physiol 111: 1–7(1996).
Reinbothe S, Runge S, Reinbothe C, van Cleve B, Apel K: Substrate-dependent transport of the NADPH:protochlorophyllide oxidoreductase into isolated plastids. Plant Cell 7: 161–172(1995).
Reinbothe S, Reinbothe C, Runge S, Apel K: Enzymatic product formation impairs both the chloroplast receptorbinding function as well as translocation competence of the NADPH:protochlorophyllide oxidoreductase, a nuclearencoded plastid precursor protein. J Cell Biol 129: 299–308 (1995).
Reinbothe S, Reinbothe C, Lebedev N, Apel K: PORA and PORB, two light-dependent protochlorophyllide-reducing enzymes of angiosperm chlorophyll biosynthesis. Plant Cell 8: 763–769(1996).
Reinbothe S, Reinbothe C, Neumann D, Apel K: A plastid enzyme arrested in the step of precursor translocation in vivo. Proc Natl Acad Sci USA 93: 12026–12030(1996).
Reinbothe S, Reinbothe C, Apel K, Lebedev N: Evolution of chlorophyll biosynthesis: the challenge to survive photooxidation. Cell 86: 703–705(1996).
Runge S, Sperling U, Frick G, Apel K, Armstrong GA: Distinct roles for light-dependent NADPH:protochlorophyllidc oxidoreductases (POR) A and B during greening in higher plants. Plant J 9: 513–523(1996).
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Santel H-J, Apel K: The protochlorophyllide holochrome of barley (Hordeum vulgare L.). The effect of light on the NADPH:protochlorophyllide oxidoreductase. Eur J Biochem 120: 95–103(1981).
Selstam E, Widell A: Characterization of prolamellar bodies, from dark-grown seedlings of Scots pine, containing lightand NADPH-dependent protochlorophyllide oxidoreductase. Physiol Plant 67: 345–352(1986).
Selstam E, Widell A, Johansson B-Å: A comparison of prolamellar bodies from wheat, Scots pine, and Jeffrey pine. Pigment spectra and properties of protochlorophyllide oxidoreductase. Physiol Plant 70: 209–214(1987).
Skinner JS, Timko MP: Loblolly pine (Pinus taeda L.) contains multiple expressed genes encoding light-dependent NADPH:protochlorophyllide oxidoreductase (POR) Plant Cell Physiol 39: 795–806(1998).
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).
Spano AJ, He Z-H, Timko MP: NADPH:protochlorophyllide oxidoreductases in white pine (Pinus strobus) and loblolly pine (P. taeda). Evidence for light and developmental regulation of expression and conservation in gene organization and protein structure between angiosperms and gymnosperms. Mol Gen Genet 236: 86–95(1992).
Spano AJ, He Z-H, Michel H, Hunt DF, Timko MP:Molecular cloning, nuclear gene structure, and developmental expression of NADPH:protochlorophyllide oxidoreductases in pea (Pisum sativum L.). Plant Mol Biol 18: 967–972(1992).
Sperling U, Franck F, van Cleve B, Frick G, Apel K, Armstrong GA: Etioplast differentiation in Arabidopsis: Both PORA and PORB restore the prolamellar body and photoactive protochlorophyllide-F655 to the cop1 photomorphogenic mutant. Plant Cell 10: 283–296(1998).
Stabel P, Sundå s A, Engström P: Cytokin treatment of embryos inhibits the synthesis of chloroplast proteins in Norway spruce. Planta 183: 520–527(1991).
Takeo S, Nakao N, Suzuki T, Tanaka K, Yamamoto I, and Satoh T: Light-dependent expression of protochlorophyllide oxidoreductase gene in the liverwort, Marchantia paleacea var. diptera. Plant Cell Physiol 39: 665–669(1998).
Terzaghi WB, Cashmore AR: Light-regulated transcription. Annu Rev Plant Physiol Plant Mol Biol 46: 445–474(1995).
Thummler F, Dittrich P: Phytochrome represents a small gene family in Picea abies: six partial sequences (Accession No. U38363 to U38368) encoding at least three different phytochrome isotypes. Plant Physiol 109: 1498 (1995).
Wakasugi T, Tsudzuki J, Ito S, Shibata M, Sugiura M: A physical map and clone bank of the black pine (Pinus thunbergii) chloroplast genome. Plant Mol Biol Rep 12: 227–241(1994).
Yamamoto N, Mukai Y, Matsuoka M, Kano-Murakami Y, Tanaka Y, Ohashi Y, Ozeki Y, Odani K: Light-independent expression of cab and rbcS genes in dark-grown pine seedlings. Plant Physiol 95: 379–383(1991).
Yamamoto N, Kojima K, Matsuoka M: The presence of two types of gene that encode the chlorophyll a/b-binding protein (LHCPII) and their light-independent expression in pine (Pinus thunbergii). Plant Cell Physiol 34: 457–463(1993).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Skinner, J.S., Timko, M.P. Differential expression of genes encoding the light-dependent and light-independent enzymes for protochlorophyllide reduction during development in loblolly pine. Plant Mol Biol 39, 577–592 (1999). https://doi.org/10.1023/A:1006144630071
Issue Date:
DOI: https://doi.org/10.1023/A:1006144630071