Unusual branching in the seedlings of Lotus japonicus-gibberellins reveal the nitrogen-sensitive cell divisions within the pericycle on roots

Abstract

We studied the effects of several plant-growth regulators on the induction of nodule-like structures on roots of Lotus japonicus, which has been proposed as a candidate for a leguminous plant for molecular genetic analysis. Contrary to our expectations, the addition of gibberellin A3 (GA3) at concentrations of 10-7 M to 10-4 M resulted in the formation of nodule-like structures on roots when seedlings were plated on nitrogen- free Fahraeus agar medium. GA4 also induced such outgrowths but was less active than GA3. Application of an inhibitor of auxin transport, N-(1- naphthyl)-phthalamic acid (NPA) and of kinetin, which have been reported to induce pseudonodules in other legumes, had no effect on L. japonicus. Microscopic observations showed that GA3-induced nodule-like structures were caused by cell divisions within the pericycle on the roots. In addition, the outgrowths elicited by GA3 could be completely suppressed by the addition of 15 mM potassium nitrate or ammonium nitrate. These results show that the pericycle cells of the roots of L. japonicus are specifically sensitive to gibberellins and that potential for cell division might be modulated by nitrogen compounds. We also examined the effects of ancymidol and uniconazole [S-3307; (E)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4- triazol-1-yl)-1-penten-3-ol], two synthetic plant-growth retardants. Both compounds at 3 x 10-5 M significantly increased the number of stunted lateral roots. The unusual branching could not be counteracted by the exogenous addition of GA3 but by 10-6 M brassinolide. We discuss the physiological role of brassinolide in the initiation of lateral roots.