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.

Single three-amino acid loop extension (TALE) homeodomain proteins, including the KNOTTED-like (KNOX) and BEL-like (BELL) families in plants, usually work as heterodimeric transcription factor complexes to regulate different developmental processes, often via effects on phytohormonal pathways. Nitrogen-fixing nodule formation in legumes is regulated by different families of homeodomain transcription factors. Whereas the role of KNOX transcription factors in the control of symbiosis was studied early, BELL transcription factors have received less attention. Here, we report the identification and expression analysis of BELL genes in the legume plants Medicago truncatula and Pisum sativum, which are involved in regulating symbiosis initiation and development. A more precise analysis was performed for the most significantly upregulated PsBELL1-2 gene in pea. We found that the PsBELL1-2 transcription factor could be a potential partner of PsKNOX9. In addition, we showed that PsBELL1-2 can interact with the PsDELLA1 (LA) protein-regulator of the gibberellin pathway, which has a previously demonstrated important role in symbiosis development.

The present work was designed to study the role of the phytohormones, cytokinin and auxin, in the symbiosis development between pea Pisum sativum L. and Rhizobium leguminosarum bv. viciae. To achieve this aim, the temporal expression patterns of the genes related to the cytokinin and auxin biosynthesis, perception, and response were studied. It was found that most of these genes showed specific transcriptional regulations upon nodulation at various stages following inoculation. Once they were purified using chromatographic methods, the endogenous cytokinins were detected by liquid chromatography-electrospray tandem mass spectrometry (LC[+]ES-MS). Our findings showed that cytokinin and auxin accumulation is tightly regulated during nodulation in order to control nodule organogenesis, infection events, and nodule function.

The IPD3/CYCLOPS transcription factor was shown to be involved in the regulation of nodule primordia development and subsequent stages of nodule differentiation. In contrast to early stages, the stages related to nodule differentiation remain less studied. Recently, we have shown that the accumulation of cytokinin at later stages may significantly impact nodule development. This conclusion was based on a comparative analysis of cytokinin localization between pea wild type and ipd3/cyclops mutants. However, the role of cytokinin at these later stages of nodulation is still far from understood. To determine a set of genes involved in the regulation of later stages of nodule development connected with infection progress, intracellular accommodation, as well as plant tissue and bacteroid differentiation, the RNA-seq analysis of pea mutant SGEFix--2 (sym33) nodules impaired in these processes compared to wild type SGE nodules was performed. To verify cytokinin’s influence on late nodule development stages, the comparative RNA-seq analysis of SGEFix--2 (sym33) mutant plants treated with cytokinin was also conducted. Findings suggest a significant role of cytokinin in the regulation of later stages of nodule development.

The key event that initiates nodule organogenesis is the perception of bacterial signal molecules, the Nod factors, triggering a complex of responses in epidermal and cortical cells of the root. The Nod factor signaling pathway interacts with plant hormones, including cytokinins and gibberellins. Activation of cytokinin signaling through the homeodomain-containing transcription factors KNOX is essential for nodule formation. The main regulators of gibberellin signaling, the DELLA proteins are also involved in regulation of nodule formation. However, the interaction between the cytokinin and gibberellin signaling pathways is not fully understood. Here, we show in Pisum sativum L. that the DELLA proteins can activate the expression of KNOX and BELL transcription factors involved in regulation of cytokinin metabolic and response genes. Consistently, pea la cry-s ( della1 della2 ) mutant showed reduced ability to upregulate expression of some cytokinin metabolic genes during nodulation. Our results suggest that DELLA proteins may regulate cytokinin metabolism upon nodulation.