Rhizobium Lipo-chitooligosaccharide Signaling Triggers Accumulation of Cytokinins in Medicago truncatula Roots

Abstract

Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides (LCOs). Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accumulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizobium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and isopentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase (CCaMK), a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein2/Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

“…Typically in temperate legumes, including the model legume Medicago truncatula, alfalfa (Medicago sativa), or clover (Trifolium sp), indeterminate nodules are initiated from pericycle and inner cortical cell divisions, and nodules maintain a persistent meristem. In many tropical legumes, however, including common bean (Phaseolus vulgaris) and soybean (Glycine max), determinate nodules form from outer cortical cell divisions, and the resulting nodules contain a temporary meristem that later differentiates (Hirsch, 1992;van Spronsen et al, 2001) The mechanism of nodule initiation is only partially elucidated. Nod factors produced by rhizobia are in most cases necessary and in some legumes sufficient to induce nodules (Truchet et al, 1991).…”

“…Nod factors produced by rhizobia are in most cases necessary and in some legumes sufficient to induce nodules (Truchet et al, 1991). The signaling cascade mediating Nod factor action leads to the activation of cytokinin signaling in the cortex of the root Oldroyd et al, 2011), which is accompanied by activation of the gene encoding the cytokinin biosynthesis enzyme LOG1, a cytokinin riboside 5-monophosphate phosphoribohydrolase (Mortier et al, 2014), and an increase in cytokinin concentration at the nodule initiation site in M. truncatula (van Zeijl et al, 2015). Several studies have implicated cytokinin as a central regulator in nodule development .…”

“…In response to rhizobia and cytokinins, the cre1 mutant fails to induce several cytokinin primary response genes, such as RESPONSE REGULATOR4 and NSP2 (NODULATION SIGNALING PATHWAY2) (Ariel et al, 2012). A recent transcriptome comparison of gene expression changes in wild-type and cre1 mutant roots in response to a 3-h Nod factor treatment in M. truncatula demonstrated that cytokinin signaling is necessary for the majority (;75% or ;600 transcripts) of Nod factor-induced transcriptional changes (van Zeijl et al, 2015). However, the mechanism of how cytokinin controls nodule development is still not defined.…”

“…Early studies showed that application of synthetic auxin transport inhibitors can induce pseudonodules in some legumes that are characterized by a peripheral vasculature, which does not extend into the distal part of the nodule, an uninfected central zone, and a diffuse meristem (Allen et al, 1953;Hirsch et al, 1989). During nodulation, acropetal root auxin transport is temporarily inhibited by rhizobia-inducing indeterminate nodules, and this is followed by a local increase in auxin response in the pericycle and inner cortex at the site of nodule initiation (Mathesius et al, 1998;Boot et al, 1999;van Noorden et al, 2007). In the cre1 mutant, rhizobia are unable to inhibit acropetal auxin transport and this is associated with a misregulation of some PIN auxin carrier-encoding gene expression and protein accumulation (Plet et al, 2011).…”

Different Pathways Act Downstream of the CEP Peptide Receptor CRA2 to Regulate Lateral Root and Nodule Development

“…NODULE INCEPTION and NODULATION SIGNALING PATHWAY1 and 2 [Kaló et al, 2005;Smit et al, 2005] and MtCLV3/ESR-RELATED12 and 13 [MtCLE12 and 13; Mortier et al, 2010;Saur et al, 2011]). Nodulation is also positively and negatively controlled by complex, context-dependent interactions with various hormones and peptides (Penmetsa and Cook, 1997;Gonzalez-Rizzo et al, 2006;Penmetsa et al, 2008;Mortier et al, 2010;Plet et al, 2011;Saur et al, 2011;Mortier et al, 2012;Larrainzar et al, 2015;van Zeijl et al, 2015). Collectively, these signals, along with the NF/SYM pathway, determine how, when, and where nodules form, as well as the overall nodule number on the root system (Penmetsa and Cook, 1997;Oldroyd et al, 2011).…”