Relocating a gene for herbicide tolerance: A chloroplast gene is converted into a nuclear gene

Abstract

The chloroplast gene psbA codes for the photosynthetic quinone-binding membrane protein Q which is the target of the herbicide atrazine. This gene has been converted into a nuclear gene. The psbA gene from an atrazine-resistant biotype of Amaranthus hybridus has been modified by fusing its coding region to transcriptionregulation and transit-peptide-encoding sequences of a bona fide nuclear gene. The constructs were introduced into the nuclear genome of tobacco by using the Agrobacteium tumorinducing (Ti) plasmid system, and the protein product of nuclear psbA has been identified in the photosynthetic membranes of chloroplasts. Recovery of atrazine-tolerant transgenic plants shows that the product of the transplanted gene functions in photosynthesis. These experiments show that it is possible to modify chloroplast-gene-specified functions via nuclear-genome transformation and also raise evolutionary questions.A major puzzle of eukaryotic cell biology is: Why are genes for some organelle proteins located in the nucleus, while genes for others are in the organelles (1)? One commonly entertained view is that proteins coded for by chloroplast and mitochondrial genes (e.g., photosynthetic membrane proteins) have properties that would block their passage through the organelle's outer membrane system. We have tested this proposition as well as the possibility ofgenetically altering functions normally encoded by chloroplast genes by determining whether a modified chloroplast gene for a photosynthetic membrane protein introduced into the nuclei of tobacco plants can serve as the source of a protein that functions in photosynthetic electron transport.In photosynthesis, plastoquinone molecules from the large pool present in the thylakoid membrane become associated with the quinone-binding protein, QB1 of photosystem II, where they are reduced. Once reduced they leave the binding site to be replaced by an oxidized quinone molecule (2). Triazine herbicides compete with the plastoquinone for binding and, thus, interrupt photosynthetic electron flow (3). In situ in thylakoid membranes, the QB protein of atrazinesensitive plants, including Amaranthus hybridus, binds azidoatrazine; QB protein from an atrazine-resistant biotype does not bind the herbicide because of a single amino acid residue substitution (4). Thus, if this mutation in the QB protein, encoded by the chloroplast psbA gene, is the basis of atrazine resistance, the functional integration of this protein into the photosynthetic apparatus of a plant that is normally sensitive would confer atrazine tolerance.Plants transformed with the bacterial neomycin phosphotransferase gene spliced behind the promoter and transit peptide-encoding sequences of the small subunit (SSU) of the gene for ribulose bisphosphate carboxylase/oxygenase can transport the protein product into chloroplasts (5). We have spliced the coding region of the psbA gene isolated from the chloroplast DNA of the atrazine-resistant biotype of Amaranthus to the transcriptional-control and transitpep...

A Serine-to-Threonine Substitution in the Triazine Herbicide-Binding Protein in Potato Cells Results in Atrazine Resistance without Impairing Productivity

“…In other atrazine-resistant higher plants that have been examined, the Ser at position 264 is altered to either Gly (Blyden and Gray, 1986;Betrini et al, 1987;Barros and Dyer, 1988;Mazur and Falco, 1989) or Asn (Pay et al, 1988). Definitive confirmation that point mutations to the 264th codon in the psbA gene result in atrazine resistance has been provided in cyanobacteria (Golden and Haselkorn, 1985;Ohad and Hirschberg, 1992) and tobacco (Cheung et al, 1988). Anacystis nudulans was transformed with a psbA gene mutated at the 264th codon and exhibited resistance to atrazine (Golden and Haselkorn, 1985).…”

“…Anacystis nudulans was transformed with a psbA gene mutated at the 264th codon and exhibited resistance to atrazine (Golden and Haselkorn, 1985). In tobacco, a chimeric gene constructed with a nuclear promoter, mutant psbA gene, and chloroplast transit peptide-encoding sequence was incorporated into the nuclear genome of atrazine-susceptible tobacco, with recovery of atrazine-tolerant transgenic plants (Cheung et al, 1988). Atrazine resistance in two weed biotypes (Pfister et al, 1981;Barros and Dyer, 1988) was linked to the inability of [azido-14 C]atrazine to bind to the Q B protein, and this altered binding was based on the conversion of Ser 264 to Gly.…”

“…Transgenic plants that express a nuclear gene encoding a modified Q B protein with a single peptide for chloroplast targeting of the protein also exhibited increased atrazine tolerance (Cheung et al, 1988 will provide growers new options for designing weed management strategies (Duke et al, 1991). This will facilitate the development of cultural practices that reduce the continuous use of specific herbicides, thereby reducing the selection pressure for herbicide-resistant weeds (Gressel and Segel, 1982).…”

Ectopic Expression of Rubisco Subunits in Maize Mesophyll Cells Does Not Overcome Barriers to Cell Type-Specific Accumulation

“…This led to the accumulation of approximately 10% of the wild-type level of Rubisco. Similarly, a psbA gene expressed in the nucleus yielded low amounts of protein relative to its endogenous counterpart (Cheung et al, 1988). We reasoned that codon optimization might increase the production of nucleus-encoded LS.…”

Herbicide-Resistant Field Crops

“…These transgenic products rely on both target site and metabolic detoxification resistance mechanisms. A s-triazineresistant gene construct composed of the mutant resistance coding sequence, expression level control sequences, and a transit-peptide encoding sequence resulted in a resistant transgenic tobacco plant line (Cheung et al, 1988 Metabolic detoxification resistance has been transferred from microbial species to crop plants. The bromoxynil-specific nitrilase gene, encoded by the bxn gene, has been transfered into cotton, potato, tomato, and rapeseed (Dyer el a]., 1993b).…”