Local auxin transport regulation in the nascent nodule - an overview in nodulating plants and an investigation into the cytokinin receptor, cre1 - mediated control of auxin transport in medicago truncatula

Abstract

Legumes form a symbiotic relationship with a group of bacteria, collectively known as rhizobia. The bacterial symbiont fixes atmospheric nitrogen within root nodules, thus providing the host with an assimilative nitrogen source. Nodule formation involves a complex signalling pathway within the legume host. The plant hormone auxin is involved in nodule organogenesis, but how auxin regulates nodulation is still poorly described. Several studies have found increased auxin signalling in nodule primordia, but so far auxin metabolites have never been quantified during the early stages of nodulation. Therefore, the first aim of this thesis was to establish methods for auxin quantification in legume roots. The presumed build-up of auxin in nodule primordia has been predicted to be due to inhibition of auxin export from cells at the nodule initiation site, but the regulation of auxin transport has not been tested systematically in different legumes. Therefore, the second aim was to compare auxin concentrations and auxin transport changes during nodulation in different legumes. Third, the regulation of auxin transport and auxin accumulation was placed into the known signalling pathway of nodulation in the model legume, Medicago truncatula. Auxins are naturally present in low quantities in the root. We developed an LC-MS/MS method for the accurate and sensitive quantification of auxins in root tissues. The method was validated and produced sensitive limits of detection / quantification and correlation coefficients. To compare the role of auxin between indeterminate and determinate nodule types, we measured auxin transport and auxin content in M. truncatula (forming indeterminate nodules) and Lotus japonicus (forming determinate nodules). In addition to acropetal auxin transport, basipetal auxin transport was regulated in response to rhizobia inoculation in both legumes. Different auxins with distinct levels of abundance were detected in separate legumes, with some unique to the nodule tissues. Auxin concentrations increased at the early stages of nodule formation in M. truncatula, but not Lotus japonicus. The inhibition of acropetal polar auxin transport by rhizobia occurred only in indeterminate nodule-forming legumes and correlated with the ability of synthetic auxin transport inhibitors to induce pseudonodules in those legumes. Finally, we investigated the role of the cytokinin receptor CRE1 in modulating auxin transport during nodulation in M. truncatula. We found that cytokinin signalling through CRE1 is necessary for inhibition of acropetal auxin transport, increased auxin concentration and auxin signalling in response to rhizobia. The CRE1 receptor was also required for the correct induction of several flavonoids, which could act as endogenous auxin transport inhibitors. External application of those flavonoids rescued nodulation in the cre1 nodulation-deficient mutant. In conclusion, we demonstrated that the auxin transport machinery is a crucial component in the host legume that is regulated in response to rhizobia. Auxin transport changes could explain measured changes in auxin concentrations during nodule initiation of M. truncatula, but not L. japonicus. Auxin transport control is mediated by endogenous flavonoids, and both flavonoid induction and auxin transport control are regulated by cytokinin signalling in M. truncatula. Show more