Involvement of cytokinin in the stimulation of nodulation by low concentrations of ammonium in Pisum sativum

Nodulation in pea (Pisum sativum L.) grown in hydroponic and sand culture systems is stimulated by low concentrations (<1.0 mM ) of ammonium, but the physiological mechanisms underlying this stimulation are unknown. The current study involves a series of experiments, which investigate if the ammonium-induced stimulation of nodulation involves changes in endogenous hormone (auxin and cytokinin) levels. P. sativum L. cv. Express was grown in growth pouches for 1 week with mineral N (0.5 and 2.0 mM NH(4)(+) or NO(3)(-)) or for 3 weeks exposed to exogenous indole-3-acetic acid (IAA) or 6-benzylaminopurine (BAP) at a range of concentrations (10(-9) -10(-5) M ). Ammonium enhanced nodulation on the basis of both early whole plant (nodules plant(-1)) and specific nodulation (nodules g(-1) root DW), especially in 0.5 mM treatment in which nodulation was approximately 4-fold of the mineral-N-free control 1 week after inoculation. Correspondingly, the roots treated with ammonium contained much higher levels of t -zeatin (Z) and lower t -zeatin riboside (ZR) than that the control or nitrate-treated plants. There was no significant difference in IAA levels between the control and ammonium treatments. Exogenous application of BAP for 3 weeks at concentrations of 10(-7)-10(-5) M strongly inhibited nodulation. However, 10(-9) M BAP, but not IAA, significantly enhanced nodulation. These data support the theory that a relatively high ratio of cytokinin:auxin in roots is favourable for nodule initiation, but that an excessively high level of cytokinin inhibits nodulation. Based on these results we propose that stimulation of nodulation by low concentrations of ammonium may be mediated through increasing Z level in roots, which alters the balance of cytokinin and auxin, which in turn induces cortical cell divisions leading to nodule initiation.