The feedback mechanism of nitrate inhibition of nitrogenase activity in soybean may involve asparagine and/or products of its metabolism

Abstract

A feedback mechanism which involves sensing of change in phloem N concentration has been proposed to control nodulation and dinitrogen fixation in the presence of external combined N. Whether this control is in response to a change in total N or in some specific signal compound(s) is not known. In the present study we reevaluated the hypothesis that control of nodulation and N2 fixation involves sensing of change in tissue N composition and attempted to identify potential signal molecule(s) involved. Two soybean (Glycine max [L.] Merr.) genotypes (Williams 82 and NOD1‐3) differing in nodule number and tolerance to nitrate were germinated in sand trays. Seven‐day‐old seedlings were inoculated with a solution of Bradyrhizobium japonicum and grown for 28 days in growth chambers, using a hydroponic system with limited N supply to promote nodulation. Half of 28‐day‐old plants were treated with 15 mM NO3−, then control and treated plants were sampled at the onset of nitrogenase inhibition (24 h following NO3−, treatment) for evaluation of nitrogenase activity and tissue concentration of total N and of each individual free amino acid. Phenylisothiocyanate‐(PITC) amino acid derivatives were separated and quantified using HPLC. The decline in nitrogenase activity following the short‐term nitrate treatment was associated with a dramatic asparagine concentration increase in the shoot and an increase in nodule aspartate and glutamate in both genotypes. Asparagine concentration in the shoot increased 35 times from a barely detectable level of 95 to 3 327 nmol g−1 fresh weight in Williams 82, and more than tripled from 509 to 1 753 nmol g−1 fresh weight in NOD1‐3. Increase in levels of free Asn and in total free amino acids in the shoot following the short‐term nitrate treatment was more pronounced in Williams 82 than in its partially nitrate‐tolerant mutant NOD1‐3. These results indicate that the feedback control of nodule activity may involve sensing changes in shoot asparagine levels and/or products of its metabolism (aspartate and glutamate) in the nodule. These results also indicate that partial‐nitrate tolerance of nodulation in the hypernodulated NOD1‐3 mutant is associated with a lesser change in tissue N following nitrate treatment.