Structure and expression of the gene family encoding putrescine N-methyltransferase in Nicotiana tabacum: new clues to the evolutionary origin of cultivated tobacco.

The structure and nuclear genomic organization of the gene family encoding putrescine N-methyltransferase (PMT), the key enzyme in diverting polyamine metabolism towards the biosynthesis of nicotine and related alkaloids, was examined in Nicotiana tabacum. Five genes encoding PMT are present in the N. tabacum genome and all are expressed. The complete coding region and immediate 5'- and 3'- flanking regions were characterized for four members of the gene family and the Exon 1 region of the fifth member of the family was determined. Comparison of the nucleotide and deduced amino acid sequences of the N. tabacum PMT genes with those of presumed progenitor species, N. sylvestris, N. tomentosiformis and N. otophora, revealed that three members of the N. tabacum PMT gene family were most similar to the three genes present in N. sylvestris, whereas the two remaining PMT genes were similar to PMT genes present in N. tomentosiformis and N. otophora genomes, respectively. These data are consistent with an evolutionary origin of N. tabacum resulting from a cross involving N. sylvestris and an introgressed hybrid between N. tomentosiformis and N. otophora. The five PMT genes present in N. tabacum are expressed in the roots of wild-type plants, but not in other organs. The steady-state level of all five PMT transcripts is transiently increased in roots following topping (removal of the floral meristem), although the maximum level of induction for the individual transcripts varies considerably. In contrast to wild-type plants, no increase in PMT transcript levels was observed in a low-alkaloid (nic1nic2) mutant of Burley 21. These data support a role for nic1 and nic2 in the global regulation of alkaloid formation in tobacco and provide for the first time molecular confirmation of the presumed origin of cultivated tobacco.

Partial characterization of glutathione S-transferases from wheat (Triticum spp.) and purification of a safener-induced glutathione S-transferase from Triticum tauschii.

Hexaploid wheat (Triticum aestivum L.) has very low constitutive glutathione S-transferase (GST) activity when assayed with the chloroacetamide herbicide dimethenamid as a substrate, which may account for its low tolerance to dimethenamid in the field. Treatment of seeds with the herbicide safener fluxofenim increased the total GST activity extracted from T. aestivum shoots 9-fold when assayed with dimethenamid as a substrate, but had no effect on glutathione levels. Total GST activity in crude protein extracts from T. aestivum, Triticum durum, and Triticum tauschii was separated into several component GST activities by anion-exchange fast-protein liquid chromatography. These activities (isozymes) differed with respect to their activities toward dimethenamid or 1-chloro-2,4-dinitrobenzene as substrates and in their levels of induction by safener treatment. A safener-induced GST isozyme was subsequently purified by anion-exchange and affinity chromatography from etiolated shoots of the diploid wheat species T. tauschii (a progenitor of hexaploid wheat) treated with the herbicide safener cloquintocet-mexyl. The isozyme bound to a dimethenamid-affinity column and had a subunit molecular mass of 26 kD based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme (designated GST TSI-1) was recognized by an antiserum raised against a mixture of maize (Zea mays) GSTs. Amino acid sequences obtained from protease-digested GST TSI-1 had significant homology with the safener-inducible maize GST V and two auxin-regulated tobacco (Nicotiana tabacum) GST isozymes.

Surfactant-Increased Glyphosate Uptake into Plasma Membrane Vesicles Isolated from Common Lambsquarters Leaves.

Plasma membrane vesicles were isolated from mature leaves of lambsquarters (Chenopodium album L.) to investigate whether this membrane is a barrier to glyphosate uptake and whether surfactants possess differential abilities to enhance glyphosate permeability. Amino acids representing several structural classes showed [delta]pH-dependent transport, indicating that the proteins necessary for active, proton-coupled amino acid transport were present and functional. Glyphosate uptake was very low compared to the acidic amino acid glutamate, indicating that glyphosate is not utilizing an endogenous amino acid carrier to enter the leaf cells and that the plasma membrane appears to be a significant barrier to cellular uptake. In addition, glyphosate flux was much lower than that measured for either bentazon or atrazine, both lipid-permeable herbicides that diffuse through the bilayer. Glyphosate uptake was stimulated by 0.01% (v:v) MON 0818, the cationic surfactant used in the commercial formulation of this herbicide for foliar application. This concentration of surfactant did not disrupt the integrity of the plasma membrane vesicles, as evidenced by the stability of imposed pH gradients and active amino acid transport. Nonionic surfactants that disrupt the cuticle but that do not promote glyphosate toxicity in the field also increased glyphosate transport into the membrane vesicles. Thus, no correlation was observed between whole plant toxicity and surfactant-aided uptake. Current data suggest that surfactant efficacy may be the result of charged surfactants' ability to diffuse away from the cuticle into the subtending apoplastic space, where they act directly on the plasma membrane to increase glyphosate uptake.