Legumes are unique in their ability to form a symbiotic relationship with soil
bacteria called rhizobia which reside in root nodules and fix atmospheric nitrogen. This
is beneficial to the environment as the need for nitrogen fertiliser is eliminated or at
least reduced. However, biological, like industrial nitrogen fixation is energetically
expensive and the plant has developed several mechanisms, both local and systemic
(autoregulation of nodulation), to control the number of nodules. Both processes, the
formation of the nodules (and nitrogen fixation) as well as the control of nodule
number, have been extensively studied.
Plant hormones are involved in all known processes of plant development
including nodulation. Roles of auxins and cytokinins in particular are well
documented. Involvement of the plant hormone abscisic acid (ABA) in nodulation is
less known. ABA plays a critical role in physiological processes such as germination,
seed dormancy, growth, lateral root formation, desiccation and environmental stress
responses. ABA may also be also involved in various stages of nodulation such as
growth of nodules, autoregulation of nodule number as well as desiccation of nodules.
This project was undertaken to further understand the role of ABA in
nodulation by working on the model legume Lotus japonicus and its bacterial partner
Mesorhizobium loti. The study was done using three approaches; (i) isolation and
characterisation of an ABA insensitive mutant, (ii) creation of a transgenic line with
ABA insensitivity conferring gene abi1 from Arabidopsis, and (iii) physiological
studies, including the effect of external application of ABA.
The ABA insensitive mutant Beyma was isolated in Lotus japonicus MG-20
from an EMS mutagenesis population using root growth inhibition to applied ABA as
the screening criterion. This stable mutant that segregates as a dominant Mendelian
mutation, is insensitive to ABA induced inhibition of germination, vegetative growth,
stomatal opening as well as nodulation. Tissue ABA levels were normal, suggesting a
‘sensitivity’ rather than ‘biosynthesis’ mutation. It is slow growing (50-70 % of wild
type MG-20) and has a near-constitutive wilty phenotype associated with its inability
to regulate stomatal opening. Whilst showing a wide range of ABA insensitivephenotypes, Beyma did not show alteration of nodule number control, as in the absence
of added ABA, the number and patterning (but not size) of nodules formed in the
mutant was similar to MG-20.
Transgenic lines containing the abi1, ABA insensitive mutant gene from
Arabidopsis was created in Lotus japonicus Gifu B-129. The presence of the transgene
was confirmed by PCR. The lines generated showed ABA insensitive phenotypes, such
as defective stomatal opening leading to wiltyness and frequent drying of branches and
leaves. However, like Beyma the transgenic lines did not show alteration in nodule
number control but did show an alteration in nodule size.
Through physiological studies it was found that for germination, ABA sensing
differed in different ecotypes of Lotus japonicus. Gifu was more sensitive to ABA than
MG-20 and this reduced sensitivity in MG-20 was clearly due to a single dominant
locus as confirmed by the segregation ratio in an F1 population. Variations in
germination sensitivity to ABA were also observed between various mutants of both
Gifu and MG-20. There was lesser effect of ABA in shoot growth in MG-20 and its
mutants, though Gifu and its mutants were somewhat more sensitive, again
emphasising the difference in sensitivity between the two ecotypes. This same trend
was also observed with root growth. In Lotus japonicus, stimulation of lateral root
growth was only observed in MG-20 and MG-20 mutants, and not in Gifu and its
mutants. A change in the orientation of growth was observed in both Gifu and MG-20
possibly due to gravitropic response to ABA. Gifu was again seen to be less sensitive
when a nodulation study was done in closed jars, leading to the possibility that sensing
of ABA for germination maybe through a different pathway to nodulation.
Split root experiments demonstrated that application of ABA to one side of the
root, inhibited nodulation locally but not systemically. Stomata of such plants were
completely closed, yet the root portion not exposed to the inhibitory level of ABA
nodulated normally. This result along with the fact that both the ABA insensitive
mutant and the Atabi1 transgenics do not show nodule number defects lead to the
proposal that ABA is not involved directly in systemic autoregulation of nodulation
Coloured pages - 40-45, 58, 61, 62, 64, 66, 67, 77, 82, 83, 85.