Mutational analysis indicates that abnormalities in rhizobial infection and subsequent plant cell and bacteroid differentiation in pea (Pisum sativum) nodules coincide with abnormal cytokinin responses and localization

Abstract

Background and Aims Recent findings indicate that Nod factor signalling is tightly interconnected with phytohormonal regulation that affects the development of nodules. Since the mechanisms of this interaction are still far from understood, here the distribution of cytokinin and auxin in pea (Pisum sativum) nodules was investigated. In addition, the effect of certain mutations blocking rhizobial infection and subsequent plant cell and bacteroid differentiation on cytokinin distribution in nodules was analysed. Methods Patterns of cytokinin and auxin in pea nodules were profiled using both responsive genetic constructs and antibodies. Key Results In wild-type nodules, cytokinins were found in the meristem, infection zone and apical part of the nitrogen fixation zone, whereas auxin localization was restricted to the meristem and peripheral tissues. We found significantly altered cytokinin distribution in sym33 and sym40 pea mutants defective in IPD3/CYCLOPS and EFD transcription factors, respectively. In the sym33 mutants impaired in bacterial accommodation and subsequent nodule differentiation, cytokinin localization was mostly limited to the meristem. In addition, we found significantly decreased expression of LOG1 and A-type RR11 as well as KNOX3 and NIN genes in the sym33 mutants, which correlated with low cellular cytokinin levels. In the sym40 mutant, cytokinins were detected in the nodule infection zone but, in contrast to the wild type, they were absent in infection droplets. Conclusions In conclusion, our findings suggest that enhanced cytokinin accumulation during the late stages of symbiosis development may be associated with bacterial penetration into the plant cells and subsequent plant cell and bacteroid differentiation.

General Patterns and Species-Specific Differences in the Organization of the Tubulin Cytoskeleton in Indeterminate Nodules of Three Legumes

“…Recently, it was demonstrated that cytokinins control the rearrangement of cortical microtubules from transverse orientation in the transition and elongation zones to oblique orientation in the differentiation zone in the epidermal cells of A. thaliana root [ 47 ]. Intriguingly, the localization of auxin in the symbiotic nodules of L. japonicus [ 48 ] and P. sativum [ 49 ] is limited to the meristem and vascular tissues. Active accumulation of cytokinins was observed in P. sativum nodules not only in the meristem, but also in the infection zone and the distal part of the nitrogen fixation zone [ 49 ].…”

Identification of BELL Transcription Factors Involved in Nodule Initiation and Development in the Legumes Pisum sativum and Medicago truncatula

“…Approximately 50–100 mg of ground plant tissue was used for RNA extraction, as previously described [ 39 ]. Around 1–2.5 μg of total RNA was used to synthesize cDNA using 20 μL RevertAid Reverse Transcriptase (Thermo Fisher Scientific, USA), and then the cDNA samples were diluted to 1:10.…”

Structure and Development of the Legume-Rhizobial Symbiotic Interface in Infection Threads

“…These include enzymes such as the E3 ubiquitin ligase in L. japonicus [ 87 ], the putative E3 ubiquitin ligase with the Nodule specific RING finger domain (LjnsRING) in L. japonicus [ 163 ], the E3 ubiquitin ligase with ‘zinc finger’ type domain, Seven in absentia (MtSINA) in M. truncatula [ 164 ] and Cystathionine-β-synthase-like1 (MtCBS1) in M. truncatula [ 165 ]. Recently, using pea gibberellin-deficient and della -deficient mutants, it was shown that the phytohormone gibberellin suppresses the formation of infection threads [ 166 ], and its amount in infection threads is much lower than in bacteroids [ 167 ], on the contrary, phytohormones cytokinins and auxins play an important role in the development and propagation of infection threads, as well as in the release of bacteria from infection droplets [ 132 , 168 , 169 , 170 ]. A putative role of ethylene in infection thread maturation was also suggested [ 171 ].…”