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.
Highlights
Root nodule symbiosis (RNS) is a key innovation allowing certain plants to associate with bacteria capable of converting atmospheric nitrogen into ammonia, a process known as nitrogen fixation (Sprent and Sprent, 1990)
Datisca glomerata was supplied by Katharina Pawlowski (Stockholm University), Sesbania rostrata by Barry Rolfe, Cicer arietinum by Angela Pattison (University of Sydney), Lotus japonicus by Brett Ferguson (University of Queensland), N. tabacum Wis. #381 by Spencer Whitney (Australian National University), the cre1-1 mutant by Florian Frugier (Institute of Plant Sciences Paris-Saclay, France), and A. thaliana TCSn::GFP seeds by Bruno Müller (University of Zürich)
No pseudonodules or cortical cell divisions were seen after application of 1 μM BAP to roots of the Medicago cre-1 mutant (0/20 roots), which is defective in the cytokinin receptor gene CRE-1 (Supplementary Figure 1e), compared to successful pseudonodules found in wildtype roots (9/20 roots)
Summary
Root nodule symbiosis (RNS) is a key innovation allowing certain plants to associate with bacteria capable of converting atmospheric nitrogen into ammonia, a process known as nitrogen fixation (Sprent and Sprent, 1990). Exogenous application of cytokinin has been shown to lead to the formation of nodule-like, but uninfected, cortical cell-derived structures known as “pseudonodules” in M. sativa, L. japonicus, Trifolium repens, and Macroptilium atropurpureum (Joshi et al, 1991; Relicet al., 1993; Hirsch et al, 1997; Mathesius et al, 2000a; Heckmann et al, 2011) In some cases, these pseudonodules expressed early nodulation genes and developed peripheral vasculature, suggesting that they closely resemble rhizobiainduced nodules (Hirsch et al, 1997; Fang and Hirsch, 1998; Mathesius et al, 2000a; Heckmann et al, 2011). We investigated the actions of cytokinin across a broad phylogenetic and functional range of nodulating and non-nodulating legumes as well as non-legumes to find out whether the ability of plants to form pseudonodules in response to cytokinin correlates with their ability to nodulate with rhizobia or actinorhizal bacteria
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