The sulfonylurea herbicide chlorsulfuron blocks the biosynthesis of the amino acids valine and isoleucine in plants. Addition of these two amino acids to excised pea root (Pisum sativum L. var Alaska) cultures incubated in the presence of chlorsulfuron completely alleviates herbicide-induced growth inhibition. The site of action of chlorsulfuron is the enzyme acetolactate synthase which catalyzes the fist step in the biosynthesis of valine and isoleucine. This enzyme is extremely sensitive to inhibition by chlorsulfuron having Iso values ranging from 18 to 36 nanomolar. In addition, acetolactate synthase from a wide variety of tolerant and sensitive plants species is highly sensitive to inhibition by chlorsulfuron.The selective sulfonylurea herbicide, chlorsulfuron (Fig. 1), represents a major advancement in weed control technology combining high herbicidal activity with outstanding tolerance to cereal crops and very low mammalian toxicity (3). Sensitive weeds are controlled at 10 to 20 g/ha which is equivalent to 1 to 2 tablespoons of active ingredient per acre. Chlorsulfuron acts to inhibit plant growth by blocking some process necessary for plant cell division (7-9). Recent studies have shown that another sulfonylurea herbicide, sulfometuron methyl, which is the active ingredient in Oust'( Weed Killer (Fig. 1 MATERIALS AND METHODS Pea Root Culture. Peas (Pisum sativum L. var Alaska) were surface sterilized by two 3-min soakings with gentle stirring in commercial bleach (5.25% NaOCI). After surface sterilization, the peas were sown in moist sterile vermiculite and incubated under sterile conditions for 72 h at 25 'C. Root sections, 10 mm long, including the root tip were excised from the resulting seedlings and grown in White's medium (12) supplemented with 20 g/l of sucrose as described by Van't Hof (10). All manipulations were carried out under sterile conditions. The pH of the culture medium was adjusted to 6.5 prior to autoclaving. Stock solutions of chlorsulfuron were prepared to a concentration of 500 mg/l in tetrahydrofuran. Aliquots of this stock were taken to dryness under nitrogen in small vials and the chlorsulfuron was then dissolved in 5 mM K2HPO4 (pH 7.5), diluted with phosphate buffer, and filter sterilized. These precautions were taken to avoid introducing organic solvents into the root cultures. Filter sterilized solutions of chlorsulfuron, casein hydrolysate (acid hydrolyzed), and L-amino acids were added to freshly inoculated sterile cultures of pea roots under sterile conditions. Culture inoculum consisted of 10 root sections which were added to 50 ml of White's medium in 250 ml Erlenmeyer flasks (12). The roots were grown in the dark at 25°C with gentle gyrotory shaking (60 rpm) and were collected after 96 h of incubation. Growth was determined by measuring root length. The net growth of the roots was determined by subtracting the initial 10 mm from the final length.Whole Plant Culture. Pea seeds were surface sterilized by two 3-min rinses in full strength commercial bleach (5.25% ...

Several mutants resistant to the herbicides chlorsulfuron and sulfometuron methyl were isolated form cultured cells of Nicotiana tabacum. Resistance was inherited as a single dominant or semidominant mutation in all cases. Linkage analysis of six mutants identified two unlinked genetic loci. Studies of plants homozygous for one mutation showed the mutant plants to be completely resistant to treatment with a concentration of chlorsulfuron 100 times higher than that which produces symptoms of phytotoxicity on normal plants.

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Photosynthesis in the AzoUa-Anabaena association was characterized with respect to photorespiration, early products of photosynthesis, and action spectra. Photorespiration as evidenced by an 02 inhibition of photosynthesis and an 02-dependent CO2 compensation concentration was found to occur in the association, and endophyte-free fronds, but not in the endophytic Anabaena. Analysis of the early products of photosynthesis indicated that both the fern and cyanobacterium fix CO2 via the Calvin cycle. The isolated endophytic Anabaena did not release significant amounts of amino acids synthesized from recently fixed carbon. The action spectra for photosynthesis in the Azoffa-Anabaena association indicated that the maximum quantum yield is between 650 and 670 nanometers, while in the endophyte the maximum is between 580 and 640 nanometers.Although the endophytic cyanobacterium is photosynthetically competent, any contribution it makes to photosynthesis in the intact association was not apparent in the action spectrum.Azolla is a genus of floating aquatic ferns in the Salviniaceae. An endophytic cyanobacterium, referred to as Anabaena azollae Strass., is associated with all stages of the fern's development. It colonizes cavities which are formed in the fern's dorsal aerial leaf lobes (21) and undergoes a pattern of development and differentiation which parallels that of the fern (10, 11). The endophyte can supply the association with its total N requirement by N2 fixation (18,19) and these associations have demonstrated potential as an N source for rice production (25,26).Previous studies showed that the association and filaments of Anabaena isolated from the leaf cavities could fix CO2 (16). In accord with studies on free-living N2-fixing cyanobacteria (1), these and related studies (19) have indicated the importance of photosynthesis in providing a source of reductant and ATP for nitrogenase activity. As an initial step toward understanding possible interactions of the two organisms' photosynthetic capabilities and their relationship to nitrogen fixation in the symbiotic state, this manuscript characterizes photosynthesis in the association and individual partners on the basis of products of "CO2 fixation, effects of 02 tension on CO2 fixation and CO2 compensation points, and action spectra. Preliminary accounts of some aspects of these investigations were presented previously (17,20 pooled, concentrated, and the products of CO2 fixation resolved by standard two-dimensional chromatographic methods and radioautography (2).Time course studies on "CO2 incorporation by the isolated endophyte were carried out at 29 C in small vials placed in a water-jacketed reaction vessel. A suspension of the endophyte in the N-free incubation medium (13) was maintained using a magnetic stir bar. After about 10 min of saturating illumination with white light, the vial containing the endophyte was sealed with a serum cap and 1 ml of a NaH 4CO3 solution containing 500 ,uCi of 14CO2 (58.5

The N2-fixing Azolla-Anabaena symbiotic association is characterized in regard to individual host and symbiont contributions to its total chlorophyll, protein, and levels of ammonia-assimilating enzymes. The phycocyanin content of the association and the isolated blue-green algal symbiont was used as a standard for this characterization. Phycocyanin was measured by absorption and fluorescence emission spectroscopy. The phycocyanin content and total phycobilin complement of the symbiotic algae were distinct from those of Anabaena cylindrica and a free-living isolate of the Azoila endophyte. The algal symbiont accounted for less than 20% of the association's chlorophyll and protein. Acetylene reduction rates in the association (based solely on the amount of algal chlorophyll) were 30 to 50% higher than those attained when the symbiont was isolated directly from the fern. More than 75% of the association's glutamate dehydrogenase and glutamine synthetase activities are contributed by the host plant. The specific activity of glutamate dehydrogenase is greater than that of glutamine synthetase in the association and individual partners. Both the host and symbiont have glutamate synthase activity. The net distribution of these enzymes is discussed in regard to the probable roles of the host and symbiont in the assimilation of ammonia resulting from N2 fixation by the symbiont.The Azolla-Anabaena azollae symbiosis is an N2-fixing association between a eukaryotic fern and a prokaryotic alga, both of which exhibit a higher plant type of photosynthesis (18,19).As an approach toward the characterization of the role of the host and symbiont4 in the association, it was considered important to determine the contribution of the partners to the association's Chl, protein, and ammonia-assimilating enzymes. While the alga could be isolated free of the fern, its removal from the host was never complete. Thus it was necessary to find an independent quantitative estimate for the amount of alga in the association. While nitrogenase is unique to the alga (21) its lability makes it unsuitable for quantitation. Chl a/b ratios provided another estimate, but this was based on the absence of Chl b from the alga (20) and was therefore not as sensitive. A third method, the quantitation of phycobilins which are unique to the alga in the association, was employed. A procedure involving fluorescence emission spectroscopy at 77 K permitted quantitative measurement of phycobilins in the association, where the fern's Chl b '