Abstract
One approach towards understanding the transduction pathways of phytochromes is the selection of mutants impaired in various steps. We report here the construction of an inducible counter-selection system for such mutants employing the enzyme nitrate reductase. This enzyme can convert the benign substrate analogue chlorate to the toxic product chlorite, resulting in severe growth inhibition. An Arabidopsis thaliana nitrate reductase gene (Nia1 * 2) was placed under the regulation of an Arabidopsis thaliana light-harvesting chlorophyll a/b protein (Lhcb1 * 3) promoter that is phytochrome-responsive. The chimeric Lhcb::Nia gene was transformed into A. thaliana. Homozygous transformant lines were selected and grown in the absence of nitrate and the presence of L-glutamine, conditions that substantially inhibited the expression of the endogenous nitrate reductase genes. In darkness seedlings of the trans-formed lines were resistant to chlorate; however, when seedlings were grown with intermittent red light, increased sensitivity to chlorate was observed. This sensitivity was correlated with an increase in both Nia1 * 2 RNA levels and nitrate reductase activity. The resistant seedlings were clearly distinguishable from the sensitive ones based on hypocotyl length, with no overlap in this parameter between the two populations. Thus, this system should allow for the selection of mutants that are impaired in phytochrome regulation of the transcription of Lhcb genes.
Similar content being viewed by others
References
Deng X-W: Fresh view of light signal transduction in plants. Cell 76: 423–426 (1994).
Tobin EM, Kehoe DM: Phytochrome regulated gene expression. Sem Cell Biol 5: 335–346 (1994).
Chory J: Out of darkness: mutants reveal pathways controlling light-regulated development in plants. Trends Genet 9: 167–172 (1993).
Karlin-Neumann GA, Brusslan JA, Tobin EM: Phytochrome control of the tms2 gene in transgenic Arabidopsis: a strategy for selecting mutants in the signal transduction pathway. Plant Cell 3: 573–582 (1991).
Brusslan JA, Karlin-Neumann GA, Huang L, Tobin EM: An Arabidopsis mutant with a reduced level of cab140 mRNA is a result of cosuppression. Plant Cell 5: 667–677 (1993).
Hoff T, Stummann BM, Henningsen KW: Structure, function and regulation of nitrate reductase in higher plants. Physiol Plant 84: 616–624 (1992).
Goksoyr J: On the effect of chlorate upon nitrate reduction of plants II: the effect upon the nitrate-reducing system in Escherichia coli. Physiol Plant 5: 228–240 (1952).
Oostindier-Braaksma FJ, Feenstra WJ: Isolation and characterization of chlorate-resistant mutants of Arabidopsis thaliana. Mutation Res 19: 175–185 (1973).
Wilkinson JQ, Crawford NM: Identification of the Arabidopsis CHL3 gene as the nitrate reductase structural gene NIA2. Plant Cell 3: 461–471 (1991).
Wilkinson JQ, Crawford NM: Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2. Mol Gen Genet 239: 289–297 (1993).
Tsay Y-F, Schroeder JI, Feldmann KA, Crawford NM: The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter. Cell 72: 705–713 (1993).
Nussaume L, Vincentz M, Caboche M: Constitutive nitrate reductase: a dominant conditional marker for plant genetics. Plant J 1: 267–274 (1991).
Murashige T, Skoog F: A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15: 473–497 (1962).
Tobin EM: Phytochrome mediated regulation of messenger RNAs for the small subunit of ribulose 1,5-bisphosphate carboxylase and the light harvesting chlorophyll a/b protein in Lemna gibba. Plant Mol Biol 1: 35–51 (1981).
Jansson S, Pichersky E, Bassi R, Green BR, Ikeuchi M, Melis A, Simpson DJ, Spangfort M, Staehlin LA, Thornber JP: A nomenclature for the genes encoding the chlorophyll a/b binding proteins of higher plants. Plant Mol Biol Rep 10: 242–253 (1992).
Deblaere R, Reynaerts A, Hofte H, Hernalsteens J-P, Leemans J, Van Montagu M: Vectors for cloning in plant cells. Meth Enzymol 153: 277–292 (1987).
Rogers SG, Klee H, Horsch RB, Fraley RT: Use of cointegrating Ti plasmid vectors. In: Gelvin SB, Schilperoort RA, Verma DPS (eds) Plant Molecular Biology Manual, pp. A2/1–12. Kluwer Academic Publishers, Dordrecht (1988).
Valvekens D, Van Montagu M, Lusbettens MV: Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci USA 85: 5536–5540.
Brusslan JA, Tobin EM: Light-independent developmental regulation of cab gene expression in Arabidopsis thaliana seedlings. Proc Natl Acad Sci USA 89: 7791–7795 (1992).
Crawford NM, Smith M, Bellissimo D, Davies RW: Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metallo flavoprotein with three functional domains. Proc Natl Acad Sci USA 85: 5006–5010 (1988).
Smarrelli J, Malone MJ, Watters MT, Curtis LT: Transcriptional control of the inducible nitrate reductase iso-form from sobeans. Biochem Biophys Res Comm 146: 1160–1165 (1987).
Cheng C-L, Acedo GN, Dewdney J, Goodman HM, Conkling MA: Differential expression of two Arabidopsis nitrate reductase genes. Plant Physiol 96: 275–279 (1991).
Rajasekhar VK, Gowri G, Campbell WH: Phytochrome-mediated light regulation of nitrate reductase expression in squash cotyledons. Plant Physiol 88: 242–244 (1988).
Melzer JM, Kleinhofs A, Warner RL: Nitrate reductase regulation: effects of nitrate and light on nitrate reductase mRNA accumulation. Mol Gen Genet 217: 341–346 (1989).
Solomonson LP, Barber MJ: Assimilatory nitrate reductase: functional properties and regulation. Annu Rev Plant Physiol Plant Mol Biol 41: 225–253 (1990).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Heimer, Y.M., Brusslan, J.A., Kenigsbuch, D. et al. A chimeric Lhcb::Nia gene: an inducible counter selection system for mutants in the phytochrome signal transduction pathway. Plant Mol Biol 27, 129–136 (1995). https://doi.org/10.1007/BF00019184
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00019184