Spontaneous nodulation in plants

Isolation of Spontaneous Nodule Formation (snf) Mutants of Lotus japonicus

Six genetically stable spontaneous root nodule formation (snf) mutant lines of Lotus japonicus, isolated from a mutagenised population, are described. Large scale screening for snf mutants was performed in the model legume Lotus japonicus ecotype Gifu, which has a small diploid genome, particularly suited for genetic studies. Mutant screening was carried out on plants derived from an EMS mutagenised population known to produce mutants with high frequency (Perry et al., 2003 Plant Physiol. 131: 866-871), according to the following procedure.

A. Six Genetically Stable snf Mutant Lines of Lotus japonicus Isolated

Seeds of 2400 M3 pools, composed of seeds from 12665 independent M2 plants, were surface sterilised, as described previously (Handberg and Stougaard, 1992 Plant J., 2: 487-496), pre-soaked in sterile water overnight and germinated in a double-tray arrangement containing clay granules in the upper tray. The l

Growth, Development and Nodulation of Spontaneous Nodule Formation (snf1) Mutants of Lotus japonicus

A. snf1 Mutants have a Normal Growth Phenotype

The snf1 mutants of Lotus, more specifically snf1-5g belonging to the snf1 category, show normal growth and development to maturity, indicating an absence of pleiotropic effects of the snf1 mutation. When snf1 mutant plants are grown under axenic nitrogen-deficient conditions, they show symptoms of nitrogen-starvation, including anthocyanin accumulation in the stem, light green leaves and poor growth of the aerial part of the plants (FIG. 1A). Their nodules are white, round shaped and distributed on a normal looking root system (FIGS. 1A, B, C, F).

B. Nodules of Axenically Grown snf1 Mutants are Devoid of Rhizobia

The bacterial content of nodules of snf mutants was examined in order to confirm that spontaneous nodule formation was a plant mutant phenotype and not, for example, due to delayed infection from accidentally contaminating Rhizobium

Loss of Function Alleles of the Snf Locus

When inoculated with M. loti the gain of function (snf1-5g) mutants develop normal nitrogen fixing and bacteria filled root nodules. In contrast, loss of function allelic mutants, snf1-2, are non-nodulating, while leaky snf1-1 mutants regularly develop up to two functional nodules. Loss of function snf1 mutants retain the ability to perceive Nod-factors applied to their roots, as indicated by root hair swelling and root deformation and Ca2+ spiking, comparable to the response of wild type plants. This indicates that ‘loss of function’ and ‘gain of function’ snf mutations affect a component of the nodulation signal transduction pathway that is down-stream of Ca2+ spiking.