Molecular Basis for Differential Metabolic Responses to Monosulfuron in Three Nitrogen-Fixing Cyanobacteria

Nitrogen-fixing cyanobacteria are vital photosynthetic microorganisms that contribute to soil fertility by fixing atmospheric nitrogen and are also important for maintaining ecosystem stability. These microorganisms can be very sensitive to herbicides because they possess many characteristics of higher plants. Six days after the application of monosulfuron at 0.03 to 0.3 nmol L 21 under laboratory conditions, growth of the nitrogen-fixing cyanobacteria Anabaena flos-aquae, Anabaena azollae, and Anabaena azotica was stimulated, but at higher concentrations (30 to 300 nmol L 21 ) protein synthesis was inhibited. The production of 16 amino acids in A. flos-aquae was reduced from 7 to 69% with increasing monosulfuron concentration. Application of monosulfuron at 3 to 300 nmol L 21 substantially inhibited in vitro acetolactate synthase (ALS) activity as indicated by 50% inhibition index values of 3.3, 65.2, and 101.3 nmol L 21 for A. flos-aquae, A. azollae, and A. azotica, respectively. In contrast, extractable ALS activity was not affected in these algal species with monosulfuron treatments ranging from 0.03 to 300 nmol L 1 except in A. flos-aquae at higher concentrations (30 to 300 nmol L 21 ). The most sensitive species to monosulfuron was A. flos-aquae, followed by A. azollae and A. azotica. Molecular analyses showed that the genomic DNA of A. azollae and A. azotica differed in only one amino acid. Results from photogenetic analyses revealed a high degree of homology between these algae. In contrast, the genomic DNA of A. flos-aquae differed from that of A. azollae and A. azotica in 44 and 45 amino acids, respectively. Our findings support the view that monosulfuron toxicity in these three nitrogen-fixing cyanobacteria is due to interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly ALS activity. Nomenclature: Monosulfuron; Anabaena flos-aquae (Lyngb) Breb; Anabaena azollae Strasb; Anabaena azotica Ley. Nitrogen-fixing cyanobacteria are vital photosynthetic microorganisms that contribute to soil fertility by fixing atmospheric nitrogen (Habte and Alexander 1980; Sinha and Kumar 1992) and maintaining ecosystem stability (Irisarri et al. 2001; Shen and Lu 2005). The agronomic importance of free-living symbiotic cyanobacteria as biofertilizers has been recognized (Habte and Alexander 1980). Some cyanobacterial strains that thrive in fields release small quantities of ammonia, as well as other small nitrogenous polypeptides during active growth. Recent research by Shen and Lu (2005) demonstrated that the application of a mixture of 12 species of nitrogen-fixing cyanobacteria (cultured for 90 d; 1 g dry algae weight per liter) after the sowing of a rice (Oryza sativa L.) crop was more beneficial to rice yield and soil fertility than the application (0.78 g L 21 ) of a urea fertilizer [CO(NH2)2] or a nonfertilizer control treatment. After the addition of the algal mixture to the rice paddies, total nitrogen content of rice stems and leaves increased by 27 and 39%, and rice yields increased by 9 and 12% relative to the urea fertilizer and control treatments, respectively. Three of the most common cyanobacteria in agricultural fields in China are the filamentous nitrogen-fixing species Anabaena azotica Ley (a free-living soil cyanobacterium with a high nitrogen-fixing capability), Anabaena flos-aquae (Lyngb) Breb. (a free-living soil cyanobacterium that may be toxic to some organisms), and Anabaena azollae Strasb (a cyanobacterium that forms a symbiotic relationship with the water fern, Azolla spp.) (Li 1962; Shen and Li 1993; Wu and Zhou 2004).