Regulation of the Later Stages of Nodulation Stimulated by IPD3/CYCLOPS Transcription Factor and Cytokinin in Pea Pisum sativum L.

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.

Laser Microdissection of Pisum sativum L. Nodules Followed by RNA-Seq Analysis Revealed Crucial Transcriptomic Changes During Infected Cell Differentiation

Garden pea (Pisum sativum L.) is a globally important legume crop. Like other legumes, it forms beneficial symbiotic interactions with the soil bacteria rhizobia, gaining the ability to fix atmospheric nitrogen. In pea nodules, the meristem is long-lasting and results in the formation of several histological zones that implicate a notable differentiation of infected host cells. However, the fine transcriptional changes that accompany differentiation are still unknown. In this study, using laser microdissection followed by RNA-seq analysis, we performed transcriptomic profiling in the early infection zone, late infection zone, and nitrogen fixation zone of 11-day-old nodules of pea wild-type line SGE. As a result, a list of functional groups of differentially expressed genes (DEGs) in different nodule histological zones and a list of genes with the most prominent expression changes during nodule development were obtained. Their analyses demonstrated that the highest amount of DEGs was associated with the nitrogen fixation zone. Among well-known genes controlling nodule development, we revealed genes that can be novel players throughout nodule formation. The characterized genes in pea were compared with those previously described in other legumes and their possible functions in nodule development are discussed.

At the Root of Nodule Organogenesis: Conserved Regulatory Pathways Recruited by Rhizobia

The interaction between legume plants and soil bacteria rhizobia results in the formation of new organs on the plant roots, symbiotic nodules, where rhizobia fix atmospheric nitrogen. Symbiotic nodules represent a perfect model to trace how the pre-existing regulatory pathways have been recruited and modified to control the development of evolutionary “new” organs. In particular, genes involved in the early stages of lateral root development have been co-opted to regulate nodule development. Other regulatory pathways, including the players of the KNOX-cytokinin module, the homologues of the miR172-AP2 module, and the players of the systemic response to nutrient availability, have also been recruited to a unique regulatory program effectively governing symbiotic nodule development. The role of the NIN transcription factor in the recruitment of such regulatory modules to nodulation is discussed in more details.

Transcriptomic analysis of sym28 and sym29 supernodulating mutants of pea (Pisum sativum L.) under complex inoculation with beneficial microorganisms

The garden pea (Pisum sativum L.), like most members of Fabaceae family, is capable of forming symbioses with beneficial soil microorganisms such as nodule bacteria (rhizobia), arbuscular mycorrhizal (AM) fungi and plant growth promoting bacteria (PGPB). The autoregulation of nodulation (AON) system is known to play an important role in controlling both the number of nodules and the level of root colonization by AM via root-to-shoot signaling mediated by CLAVATA/ESR-related (CLE) peptides and their receptors. In the pea, mutations in genes Sym28 (CLV2-like) and Sym29 (CLV1-like), which encode receptors for CLE peptides, lead to the supernodulation phenotype, i.e., excessive nodule formation. The aim of the present study was to analyze the response of pea cv. ‘Frisson’ (wild type) and mutants P64 (sym28) and P88 (sym29) to complex inoculation with rhizobia, AM fungi and PGPB, with regard to biomass accumulation, yield and transcriptomic alterations. The plants were grown in quartz sand for 2 and 4 weeks after inoculation with either rhizobia (Rh) or complex inoculation with Rh + AM, Rh + PGPB, and Rh+AM+PGPB, and the biomass and yield were assessed. Transcriptome sequencing of whole shoots and roots was performed using a modified RNAseq protocol named MACE (Massive Analysis of cDNA Ends). In the experimental conditions, P88 (sym29) plants demonstrated the best biomass accumulation and yield, as compared to the wild type and P64 (sym28) plants, whereas P64 (sym28) had the lowest rate of biomass and seed yield. The transcriptome analysis showed that both supernodulating mutants more actively responded to biotic and abiotic factors than the wild-type plants and demonstrated increased expression of genes characteristic to late stages of nodule development. The roots of P64 (sym28) plants responded to AM+Rh treatment with upregulation of genes encoding plastid proteins, which can be connected with the activation of carotenoid biosynthesis (namely, the non-mevalonate pathway that takes place in root plastids). The more active response to symbionts in P88 (sym29) plants, as compared to cv. ‘Frisson’, was associated with counterregulation of transcripts involved in chloroplast functioning and development in leaves, which accompanies successful plant development in symbiotic conditions. Finally, the effect of retardation of plant aging upon mycorrhization on a transcriptomic level was recorded for cv. ‘Frisson’ but not for P64 (sym28) and P88 (sym29) mutants, which points towards its possible connection with the AON system. The results of this work link the plant’s autoregulation with the responsiveness to inoculation with beneficial soil microorganisms.

Nitrate restricts nodule organogenesis through inhibition of cytokinin biosynthesis in Lotus japonicus

Legumes balance nitrogen acquisition from soil nitrate with symbiotic nitrogen fixation. Nitrogen fixation requires establishment of a new organ, which is a cytokinin dependent developmental process in the root. We found cytokinin biosynthesis is a central integrator, balancing nitrate signalling with symbiotic acquired nitrogen. Low nitrate conditions provide a permissive state for induction of cytokinin by symbiotic signalling and thus nodule development. In contrast, high nitrate is inhibitory to cytokinin accumulation and nodule establishment in the root zone susceptible to nodule formation. This reduction of symbiotic cytokinin accumulation was further exacerbated in cytokinin biosynthesis mutants, which display hypersensitivity to nitrate inhibition of nodule development, maturation and nitrogen fixation. Consistent with this, cytokinin application rescues nodulation and nitrogen fixation of biosynthesis mutants in a concentration dependent manner. These inhibitory impacts of nitrate on symbiosis occur in a Nlp1 and Nlp4 dependent manner and contrast with the positive influence of nitrate on cytokinin biosynthesis that occurs in species that do not form symbiotic root nodules. Altogether this shows that legumes, as exemplified by Lotus japonicus, have evolved a different cytokinin response to nitrate compared to non-legumes.

Identification of the Important Genes of Bradyrhizobium diazoefficiens 113-2 Involved in Soybean Nodule Development and Senescence

Legume nodule development and senescence directly affect nitrogen fixation efficiency and involve a programmed series of molecular events. These molecular events are carried out synchronously by legumes and rhizobia. The characteristics and molecular mechanisms of nitrogen fixation at soybean important developmental stages play critical roles in soybean cultivation and fertilizer application. Although the gene expression of soybean were analyzed in nodules at five important soybean developmental stages, information on the expression of rhizobial genes in these nodule samples is limited. In the present study, we investigated the expression of Bradyrhizobium diazoefficiens 113-2 genes in the nodule samples from five developmental stages of soybean (Branching stage, flowering stage, fruiting stage, pod stage and harvest stage). Similar gene expression patterns of B. diazoefficiens 113-2 were existed during optimal symbiotic functioning, while different expression patterns were found among early nodule development, nitrogen fixation progress and nodule senescence. Besides, we identified 164 important different expression genes (DEGs) associated with nodule development and senescence. These DEGs included those encoding nod, nif, fix proteins and T3SS secretion system-related proteins, as well as proteins involved in nitrogen metabolism, ABC transporters and two-component system pathways. Gene Ontology, KEGG pathway and homology analysis of the identified DEGs revealed that most of these DEGs are uncharacterized genes associated with nodule development and senescence, and they are not core genes among the rhizobia genomes. Our results provide new clues for the understanding of the genetic determinants of soil rhizobia in nodule development and senescence, and supply theoretical basis for the creation of high efficiency soybean cultivation technology.

Microscopic and transcriptomic analyses of Dalbergoid legume peanut reveal a divergent evolution leading to Nod Factor dependent epidermal crack-entry and terminal bacteroid differentiation.

Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS come from model legumes that exhibit a root-hair mode of bacterial infection in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step towards understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated a comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen scavenging mechanisms in the Papilionoid family. Finally, absence of cysteine-rich motif-1 containing-NCRs, but the recruitment of defensin like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobial symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.

Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.