Further investigation of the roles of auxin and cytokinin in the NH4+-induced stimulation of nodulation using white clover transformed with the auxin-sensitive reporter GH3:gusA

Abstract

Although mineral N (nitrate and ammonium) is believed to have generally negative effects on nodulation in legume-rhizobia symbioses, previous studies have shown that low, static concentrations of ammonium stimulate nodulation in pea, and that this enhancement may be due to an elevation in cytokinin to auxin levels in roots. Here, the effects of ammonium (0.0, 0.1, 0.5 and 2.5mM) on nodulation and auxin levels were investigated in wild-type (WT) white clover (Trifolium repens cv. Haifa) and its transformants (lines 38 and 41) which contain the auxin-sensitive reporter gene (GH3:gusA). The effects of exogenous application (10 -10 , 10 -9 and 10 -8 M) of the cytokinin 6-benzylaminopurine (BAP) were also assessed. Whole-plant nodulation (nodules plant -1 ) and dry weight (DW)-specific nodulation (nodules g -1 root DW) were stimulated (up to 49%) in all white clover lines by 0.1mM NH 4 +. This represents the first confirmation of an NH 4 +-induced stimulation of DW-specific nodulation in a species other than pea. At 2.5mM NH 4 +, the effect was lost on whole-plant nodulation and was inhibitory on DW-specific nodulation. Rhizobial inoculation resulted in a decline in the expression of GH3:gusA in root tips as expected; however, ammonium treatment did not affect GH3 expression in any root zones. Exogenous application of BAP at 10 -9 and 10 -8 M stimulated whole-plant and DW-specific nodulation in wild-type white clover to a similar degree as treatment with 0.1 mM NH 4 +. These results support our previous hypothesis that the stimulation of nodulation by low concentrations of ammonium involves the alteration of the ratio of cytokinin to auxin, specifically by increasing cytokinin.