Nodulating Legumes Are Distinguished by a Sensitivity to Cytokinin in the Root Cortex Leading to Pseudonodule Development

Abstract

Root nodule symbiosis (RNS) is a feature confined to a single clade of plants, the Fabids. Among Fabids capable of RNS, legumes form root cortex-based nodules in symbioses with rhizobia, while actinorhizal species form lateral root-based nodules with actinomycetes. Cytokinin has previously been shown to be sufficient for “pseudonodule” initiation in model legumes. Here, we tested whether this response correlates with the ability to nodulate across a range of plant species. We analyzed the formation of pseudonodules in 17 nodulating and non-nodulating legume species, and 11 non-legumes, including nodulating actinorhizal species, using light and fluorescence microscopy. Cytokinin-induced pseudonodules arising from cortical cell divisions occurred in all nodulating legume species, but not in any of the other species, including non-nodulating legumes. Pseudonodule formation was dependent on the CRE1 cytokinin receptor in Medicago truncatula. Inhibition of root growth by cytokinin occurred across plant groups, indicating that pseudonodule development is the result of a specific cortical cytokinin response unique to nodulating legumes. Lack of a cortical cytokinin response from the Arabidopsis thaliana cytokinin reporter TCSn::GFP supported this hypothesis. Our results suggest that the ability to form cortical cell-derived nodules was gained in nodulating legumes, and likely lost in non-nodulating legumes, due to a specific root cortical response to cytokinin.

Evolution of NIN and NIN-like Genes in Relation to Nodule Symbiosis

“…As the CE region does not occur in the Parasponia NIN promoter, the mobile signal most likely does not regulate NIN expression through cytokinin signaling. This conclusion is in line with a recent study in which cytokinin is applied to different plant species [ 53 ]. It was shown that exogenous application of cytokinin induces nodule-like structures on nodulating legume species, and in Lotus, for example, this depends on NIN [ 54 ].…”

“…It was shown that exogenous application of cytokinin induces nodule-like structures on nodulating legume species, and in Lotus, for example, this depends on NIN [ 54 ]. However, nodule-like structures were not induced by cytokinin on either non-nodulating legumes or on actinorhizal species [ 53 ]. The non-nodulating legumes most likely have lost the ability to form cytokinin-induced nodule-like structures due to the loss of NIN , whereas in actinorhizal plants this might be due to the absence of a CE region in their NIN promoter.…”

NODULE INCEPTION Recruits the Lateral Root Developmental Program for Symbiotic Nodule Organogenesis in Medicago truncatula

“…While nodulating and non-nodulating species of plants respond to auxin treatment with emergence of nodule-like structures [47], only nodulating legumes do so in response to cytokinin [48]. Our work suggests that cytokinin promotion of NIN activates LBD16 that then promotes local auxin biosynthesis through the induction of STY and YUC .…”

Accumulation of and Response to Auxins in Roots and Nodules of the Actinorhizal Plant Datisca glomerata Compared to the Model Legume Medicago truncatula

“…Furthermore, cytokinin applied exogenously or constitutively secreted by Nod − rhizobial strains was sufficient to activate cortical cell divisions and initiate some of the morphological and molecular events typical for nodulation (Cooper and Long, 1994; Mathesius et al, 2000). This response is only found in nodulating legumes but not in non-nodulating species nor in actinorhizal species (Gauthier-Coles et al, 2019). Cytokinin synthesis genes are activated during nodulation in various legumes (see, e.g., Mortier et al, 2014; Dolgikh et al, 2017; Reid et al, 2017), and increased cytokinin concentrations were measured in the root zone susceptible to rhizobia within 3 h after application of Nod factors (van Zeijl et al, 2015).…”