CYTOKININ OXIDASE/DEHYDROGENASE3 Maintains Cytokinin Homeostasis during Root and Nodule Development in Lotus japonicus

Abstract

Cytokinins are required for symbiotic nodule development in legumes, and cytokinin signaling responses occur locally in nodule primordia and in developing nodules. Here, we show that the Lotus japonicus Ckx3 cytokinin oxidase/dehydrogenase gene is induced by Nod factor during the early phase of nodule initiation. At the cellular level, pCkx3::YFP reporter-gene studies revealed that the Ckx3 promoter is active during the first cortical cell divisions of the nodule primordium and in growing nodules. Cytokinin measurements in ckx3 mutants confirmed that CKX3 activity negatively regulates root cytokinin levels. Particularly, tZ and DHZ type cytokinins in both inoculated and uninoculated roots were elevated in ckx3 mutants, suggesting that these are targets for degradation by the CKX3 cytokinin oxidase/dehydrogenase. The effect of CKX3 on the positive and negative roles of cytokinin in nodule development, infection and regulation was further clarified using ckx3 insertion mutants. Phenotypic analysis indicated that ckx3 mutants have reduced nodulation, infection thread formation and root growth. We also identify a role for cytokinin in regulating nodulation and nitrogen fixation in response to nitrate as ckx3 phenotypes are exaggerated at increased nitrate levels. Together, these findings show that cytokinin accumulation is tightly regulated during nodulation in order to balance the requirement for cell divisions with negative regulatory effects of cytokinin on infection events and root development.

Cytokinin Biosynthesis Promotes Cortical Cell Responses during Nodule Development

“…Overexpression of a complete biosynthetic pathway produced apparently uncontrolled cell divisions in a broad zone. This is consistent with the negative effects on root growth and infections observed in cytokinin overaccumulation or gain-of-function mutants (Tirichine et al, 2007;Reid et al, 2016). The absence of spontaneous nodules (Ipt overexpression) or primordia proliferation without full nodule development (Log overexpression) indicates that increases in IPT or LOG activity alone are insufficient to trigger the sufficient cytokinin increase or cell divisions required for normal nodule organogenesis.…”

“…It is likely that both cytokinin accumulation and receptor availability modulate these cytokinin response domains. Changes in root cytokinin levels have been detected in response to rhizobia and purified NF (van Zeijl et al, 2015;Reid et al, 2016). The early nodulation signaling pathway is required for this response, as it was not detected in the Mtdmi3 (ccamk) background (van Zeijl et al, 2015).…”

“…Legumes forming indeterminate nodules share the requirement for cytokinin perception, which in Medicago truncatula is controlled primarily by the MtCRE1/CHK1 receptor together with some redundant activity of MtCHK2, MtCHK3, and MtCHK4 (Gonzalez-Rizzo et al, 2006;Plet et al, 2011;Boivin et al, 2016). In contrast to organogenesis, cytokinin signaling plays a negative regulatory role in rhizobia infection, as the Ljlhk1-1 mutant exhibits hyperinfection while mutations in LjCkx3, which cause increased root cytokinin, reduce infection (Murray et al, 2007;Reid et al, 2016). Therefore, cytokinin signaling is central to both pathways (Miri et al, 2016).…”

A Laser Dissection-RNAseq Analysis Highlights the Activation of Cytokinin Pathways by Nod Factors in the Medicago truncatula Root Epidermis

“…speed of CK translocation to the cortex versus degradation by AtCKX3 in the epidermis). In L. japonicus, the mutation of a CKX3 gene expressed in nodule primordia but not in the epidermis leads to a reduction of both nodulation and infection thread formation (Reid et al, 2016), suggesting that CK overproduction in the cortex induces a negative feedback on infection and that CK may have temporally and spatially restricted antagonistic functions. In M. truncatula, the opposite impact on nodulation observed for the epidermal pEPI:AtCKX3 and cortical pCO:AtCKX3 constructs suggests distinct predominant roles for CK in outer and inner root tissues during NF signaling and nodulation, with a stronger negative component in the epidermis.…”

“…Our study generated evidence for the NF regulation of classic hormone pathways such as those for auxins, GAs, strigolactones, and CK (consistent with Breakspear et al, 2014), as well as peptide hormones such as CLE and CEP (Table III). We were particularly interested in CKs, known for their critical role in the initiation of nodule organogenesis and shown to accumulate rapidly in response to NF Reid et al, 2016) but not considered as early epidermal signals. Indeed, in Rhizobium spp.-inoculated L. japonicus roots, TCS:GUS expression is detected first in the cortex (Held et al, 2014).…”