Cytokinin Biosynthesis Promotes Cortical Cell Responses during Nodule Development

Abstract

Legume mutants have shown the requirement for receptor-mediated cytokinin signaling in symbiotic nodule organogenesis. While the receptors are central regulators, cytokinin also is accumulated during early phases of symbiotic interaction, but the pathways involved have not yet been fully resolved. To identify the source, timing, and effect of this accumulation, we followed transcript levels of the cytokinin biosynthetic pathway genes in a sliding developmental zone of Lotus japonicus roots. LjIpt2 and LjLog4 were identified as the major contributors to the first cytokinin burst. The genetic dependence and Nod factor responsiveness of these genes confirm that cytokinin biosynthesis is a key target of the common symbiosis pathway. The accumulation of LjIpt2 and LjLog4 transcripts occurs independent of the LjLhk1 receptor during nodulation. Together with the rapid repression of both genes by cytokinin, this indicates that LjIpt2 and LjLog4 contribute to, rather than respond to, the initial cytokinin buildup. Analysis of the cytokinin response using the synthetic cytokinin sensor, TCSn, showed that this response occurs in cortical cells before spreading to the epidermis in L. japonicus. While mutant analysis identified redundancy in several biosynthesis families, we found that mutation of LjIpt4 limits nodule numbers. Overexpression of LjIpt3 or LjLog4 alone was insufficient to produce the robust formation of spontaneous nodules. In contrast, overexpressing a complete cytokinin biosynthesis pathway leads to large, often fused spontaneous nodules. These results show the importance of cytokinin biosynthesis in initiating and balancing the requirement for cortical cell activation without uncontrolled cell proliferation.

Dynamics of Ethylene Production in Response to Compatible Nod Factor

“…Our finding that the snf2 gain-of-function mutation in the L. japonicus Lhk1 cytokinin receptor increases ethylene is consistent with this well-established cytokinin-ethylene crosstalk. The elevated ethylene in snf1 may suggest that elevated cytokinin levels exist in this mutant, further highlighting the role of the nodulation signaling pathway in the induction of cytokinin production (Reid et al, 2017).…”

“…Additionally, NIN and the AP2/ERF transcription factor ERN1 (Cerri et al, 2012(Cerri et al, , 2016(Cerri et al, , 2017Kawaharada et al, 2017;Yano et al, 2017) are transcriptionally activated by the GRAS-type transcription factors NSP1 and NSP2 (Kaló et al, 2005;Smit et al, 2005;Heckmann et al, 2006;Murakami et al, 2006;Hirsch et al, 2009). Nodulation signaling also induces cytokinin biosynthesis (Reid et al, 2017), and activation of the cytokinin receptor LHK1 is sufficient to induce nodule organogenesis (Tirichine et al, 2007;Murray et al, 2007).…”

Legume nodulation: The host controls the party

“…Under favourable growing conditions, ethylene has a role in infection thread formation, calcium spiking initiation, and nodule primordium maintenance (Goodlass & Smith, ; Lee & LaRue, ; Sun et al, ; Penmetsa et al, ; Lohar, Stiller, Kam, Stacey, & Gresshoff, ; reviewed in Ferguson & Mathesius, ). It is also thought to act in positioning the nodule around the root (Heidstra et al, ) and interacts with other phytohormones, such as gibberellin (Ferguson, Foo, Ross, & Reid, ), abscisic acid (Ding et al, ), auxin (Prayitno, Rolfe, & Mathesius, ), and cytokinin (Heckmann et al, ; Lorteau, Ferguson, & Guinel, ; Reid et al, ) to regulate nodule organogenesis. Interestingly, ethylene also has a role in determining the nodule type in Sesbania rostrata , a semiaquatic tropical legume that can form both determinate and indeterminate nodules (Fernández‐López et al, ).…”

Inside out: root cortex‐localized LHK1 cytokinin receptor limits epidermal infection of Lotus japonicus roots by Mesorhizobium loti

“…Two of the most potent endogenous plant cytokinins, trans‐zeatin and isopentenyl adenine, were shown to accumulate rapidly upon rhizobial inoculation and in roots exposed to minute amounts of lipo‐chitooligosaccharide signalling molecules, known as nodulation factors (NFs) (Van Zeijl et al ., ; Reid et al ., , ). Synthesized by rhizobia in the presence of a compatible plant host (Lerouge et al ., ), NFs initiate symbiotic root development by selectively activating specific plant lysine motif (LysM) receptor‐like kinase molecules, known as NF receptors (Limpens et al ., ; Madsen et al ., ; Radutoiu et al ., , ; Arrighi et al ., ; Den Camp et al ., ; Broghammer et al ., ; Liang et al ., ; Murakami et al ., ).…”

“…The LHK1 promoter is active in both the root epidermis and the root cortex and is further upregulated by M. loti infection, initially within the root cortex and subsequently also in the root epidermis (Held et al ., ). The cytokinin activity, induced in L. japonicus roots by M. loti inoculation and measured as an output of a TCS cytokinin sensor, follows a similar pattern (Held et al ., ; Reid et al ., ), suggesting that presence of LHK1 in both cellular locations may be relevant to symbiotic infection. By performing cell‐specific complementation experiments, we demonstrate here that, as well as being essential for nodule formation, presence of LHK1 in the root cortex is also required and sufficient to limit M. loti infection at the root epidermis, which underscores the existence of an LHK1‐dependent root cortex‐to‐epidermis feedback mechanism.…”