The identification of auxin transport inhibiting flavonoids that are active during nodule formation

Abstract

Soil bacteria called rhizobia induce the formation of root nodules on legume roots, in which they fix atmospheric nitrogen that the plant can use as a nitrogen source. The mechanism of nodule development involves the spatiotemporal control of the action of two plant hormones, auxin and cytokinin, but it is not well understood how rhizobia manipulate the changes in plant hormones. This thesis examined the function and identity of secondary metabolites called flavonoids in coordinating auxin and cytokinin action during nodule development. There is evidence that rhizobia induce certain flavonoids which inhibit polar auxin transport during nodule formation, one way of directing auxin into developing nodules. However, the identity of these flavonoids remained unknown for the past 20 years, and their identification was one focus of this thesis. It was argued that the putative auxin transport inhibitor should (1) inhibit auxin transport similarity to rhizobia: (2) be induced in the root shaft by rhizobia at the timepoint of auxin transport inhibition and (3) complement the loss of nodulation in roots lacking flavonoids. Using these criteria, the quercetin glycoside, isoquercetrin (quercetin-3-O-glucofuranoside), was identified and characterised as the best candidate for a flavonoid which acts as the regulator of auxin transport during nodule formation in the model legume Medicago truncatula. Cytokinin, a second hormone necessary for nodule development, acts upstream of flavonoid induction and auxin transport control. A mutation in the cytokinin receptor CRE1 inhibits nodule development and prevents auxin transport control by rhizobia. Flavonoids have been shown to complement nodulation and auxin response in the cre1 mutant but the active flavonoid had not been identified. In this thesis, several flavonoids, including isoquercetrin were examined for their ability to complement nodulation in the cre1 mutant. Isoquercetrin was able to partially restore nodulation in the cre1 mutant. Surprisingly, another flavonoid, 4,4'-dihydroxy-2'-methoxychalcone, a flavonoid likely required for the infection of rhizobia was a better complementor of nodulation in the cre1 mutant. These results suggest a link between the role of cytokinin, the process of infection and nodule development. Finally, it was previously shown that the application of the synthetic cytokinin, 6-benzylaminopurine (BAP) was able to induce the formation of nodule-like structures without 3 rhizobia infection, known as pseudonodules. It was found here that flavonoids are also necessary for the formation of pseudonodules. Both isoquercetrin and 4,4'-dihydroxy-2'-methoxychalcone were able to fully complement pseudonodule formation in roots deficient in the flavonoid pathway. This validated the hypothesis that cytokinin works upstream of the flavonoid pathway and both pseudonodule and nodule development include an overlappiny signalling pathway during organogenesis. Show more