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Nitrogen and Ethylene Control the Action of enod40. (A) Number of nodules on control and Se40 roots 18 DPI. Plants were grown on nitrogen-free medium or on medium supplemented with potassium nitrate at a final concentration of 0, 1, 2, or 5 mM. Twenty to 40 plants were used in at least two independent experiments. (B) Plants harboring developing nodules were treated at 10 DPI with a 20 mM potassium nitrate solution. Nodule numbers in independent plant pools (20 plants per time point) were counted 0, 4, 8, or 16 days after treatment (T). (C) Mean root length (in centimeters) of plants grown for 5 days on agar plates in a medium containing 0.25 mM potassium nitrate supplemented with different concentrations of ACC. Root length is expressed as the mean SD. The asterisk indicates the only slight albeit statistically significant difference (P 0.01). (D) Number of nodules on plant roots infected with Sm41 or supplemented with ACC and AVG at 15 and 23 DPI. In (A), (B), and (D), nodule number is expressed as the mean SE.
Source publication
Molecular mechanisms involved in the control of root nodule organogenesis in the plant host are poorly understood. One of the nodulin genes associated with the earliest phases of this developmental program is enod40. We show here that transgenic Medicago truncatula plants overexpressing enod40 exhibit accelerated nodulation induced by Sinorhizobium...
Contexts in source publication
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... days after inoculation, the nod- ules on these plants were counted. Figure 5A shows that enod40-mediated acceleration of nodulation was repressed significantly by potassium nitrate concentrations as low as 1 mM (50% reduction in nodule number). To test whether enod40 action could be regulated by nitrogen once nodules were formed, at 10 DPI, potassium nitrate was applied at a concentration of 20 mM to transgenic plants harboring de- veloping nodules. ...
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... test whether enod40 action could be regulated by nitrogen once nodules were formed, at 10 DPI, potassium nitrate was applied at a concentration of 20 mM to transgenic plants harboring de- veloping nodules. Counting nodules at 0, 4, 8, and 16 days after nitrate treatment indicated that the supply of potas- sium nitrate blocked nodulation both in control and enod40 transformants ( Figure 5B; data not shown). Plants that were maintained 8 or 16 days after the nitrogen treatment showed an increase in nodule number (Figure 5B), probably because the plant metabolized the added nitrogen. ...
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... nodules at 0, 4, 8, and 16 days after nitrate treatment indicated that the supply of potas- sium nitrate blocked nodulation both in control and enod40 transformants ( Figure 5B; data not shown). Plants that were maintained 8 or 16 days after the nitrogen treatment showed an increase in nodule number (Figure 5B), probably because the plant metabolized the added nitrogen. ...
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... control and Se40 seedlings grown on low-nitrogen agar medium were treated with ACC for 5 days. No consid- erable differences in primary root length (or in the other re- sponses) were observed in the Se40 plants compared with control plants ( Figure 5C; data not shown), except for a slight but statistically significant (P 0.01) increase in root length at 0.01 mM ACC. However, the degree of inhibition of root elongation at different ACC concentrations indicated that both sets of plants were sensitive to ethylene. ...
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... addi- tion, plants were treated with ACC or aminoethoxyvinylgly- cine (AVG; an inhibitor of ACC synthase) with or without concomitant S. meliloti inoculation. A marked reduction in nodule number at 15 and 23 DPI was found after treatment with either 10 5 M ( Figure 5D) or 10 7 M ACC (data not shown), confirming that in M. truncatula plants, ethylene negatively regulates nodulation (Penmetsa and Cook, 1997). Despite the repression of nodule formation by ACC in Se40 plants, their differential nodulation kinetics were main- tained at both concentrations. ...
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... the repression of nodule formation by ACC in Se40 plants, their differential nodulation kinetics were main- tained at both concentrations. When the ACC synthase in- hibitor AVG (10 7 M) was applied to roots of bacterially infected plants, the control plants treated with AVG showed enhanced nodulation similar to that of enod40 transformants ( Figure 5D). However, microscopic analyses of these control roots revealed only a few cases of extensive cortical cell di- visions in the infected region, unlike our observations in the Se40 plants (data not shown). ...
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... Recent experimental evidences have demonstrated that lncRNAs regulate the expression of proximal protein-coding genes (Engreitz et al. 2016;Wang et al. 2018;Zhao et al. 2018). LncRNA COLDAIR governs flowering time by regulating the chromatin dynamics at proximal FLC gene locus, while ENOD40 acts as a scaffold to localize the proteins in specific loci to regulate root nodule formation in legumes (Charon and Crespi 1999;Heo and Sung 2011). We identified a large number of mRNAs proximally located to the differentially expressed lncRNAs. ...
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Abstract
Long non-coding RNAs (lncRNAs) are important regulators of various biological processes. Here, we identified lncRNAs at seven successive stages of seed development in small-seeded and large-seeded chickpea cultivars. In total, 4751 lncRNAs implicated in diverse biological processes were identified. Most of lncRNAs were conserved between the two cultivars, whereas only a few of them were conserved in other plants, suggesting their species-specificity. A large number of lncRNAs differentially expressed between the two chickpea cultivars associated with seed development-related processes were identified. The lncRNAs acting as precursors of miRNAs and those mimicking target protein-coding genes of miRNAs involved in seed size/weight determination, including HAIKU1, BIG SEEDS1, and SHB1, were also revealed. Further, lncRNAs located within seed size/weight associated quantitative trait loci were also detected. Overall, we present a comprehensive resource and identified candidate lncRNAs that may play important roles during seed development and seed size/weight determination in chickpea.
... SymREM1 and VPY are important for rhizobial infection (Lefebvre et al., 2010;Murray et al., 2011). Early nodulin 40 (ENOD40) is required for the induction of cortical cell divisions (Charon et al., 1999). The two remaining nodulin genes, AeSBT (subtilase) and AeCRK, were selected based on transcriptomic and genetic studies on A. evenia symbiosis Gully et al., 2018). ...
Intensive research on nitrogen-fixing symbiosis in two model legumes has uncovered the molecular mechanisms whereby rhizobial Nod factors activate a plant symbiotic signaling pathway that controls infection and nodule organogenesis. By constrast, the so-called Nod-independent symbiosis found between Aeschynomene evenia and photosynthetic bradyrhizobia, which does not involve Nod factor recognition nor infection thread formation, is less well known. To gain knowledge on how Nod-independent symbiosis is established, we conducted a phenotypic and molecular characterization of A. evenia lines carrying mutations in different nodulation genes. Besides investigating the effect of the mutations on rhizobial symbiosis, we examined their consequences on mycorrhizal symbiosis and in non-symbiotic conditions. Analysing allelic mutant series for AePOLLUX, AeCCaMK (Ca2+/calmodulin dependent kinase), AeCYCLOPS, AeNSP2 (nodulation signaling pathway 2), and AeNIN (nodule inception) demonstrated that these genes intervene at several stages of intercellular infection and during bacterial accommodation. We provide evidence that AeNSP2 has an additional nitrogen-dependent regulatory function in the formation of axillary root hairs at lateral root bases, which are rhizobia-colonized infection sites. Our investigation of the recently discovered symbiotic actor AeCRK (cysteine-rich receptor-like kinase) specified that it is not involved in mycorrhization; however, it is essential for both symbiotic signaling and early infection during nodulation. These findings provide important insights on the modus operandi of Nod-independent symbiosis and contribute to the general understanding of how rhizobial-legume symbioses are established by complementing the information acquired in model legumes.
... When soybean roots are inoculated with rhizobia or treated with NFs, the expression of ENOD40 is rapidly upregulated (Kouchi & Hata, 1993;Yang et al., 1993;Minami et al., 1996), and alteration of ENOD40 expression significantly influences nodulation (Charon et al., 1999;Kumagai et al., 2006;Wan et al., 2007). ENOD40 can act as a regulatory long noncoding RNA (Campalans et al., 2004) that also encodes two small peptides of 12 or 13 amino acids (Sousa et al., 2001), and these ENOD40 peptides can bind to and enhance the stability of sucrose synthase (Hardin et al., 2003). ...
Symbiotic nodulation is initiated in the roots of legumes in response to low nitrogen and rhizobial signal molecules and is dynamically regulated by a complex regulatory network that coordinates rhizobial infection and nodule organogenesis.
It has been shown that the miR156‐SPL module mediates nodulation in legumes; however, conclusive evidence of how this module exerts its function during nodulation remains elusive.
Here, we report that the miR156b‐GmSPL9d module regulates symbiotic nodulation by targeting multiple key regulatory genes in the nodulation signalling pathway of soybean. miR156 family members are differentially expressed during nodulation, and miR156b negatively regulates nodulation by mainly targeting soybean SQUAMOSA promoter‐binding protein‐like 9d (GmSPL9d), a positive regulator of soybean nodulation. GmSPL9d directly binds to the miR172c promoter and activates its expression, suggesting a conserved role of GmSPL9d. Furthermore, GmSPL9d was coexpressed with the soybean nodulation marker genes nodule inception a (GmNINa) and GmENOD40‐1 during nodule formation and development. Intriguingly, GmSPL9d can bind to the promoters of GmNINa and GmENOD40‐1 and regulate their expression.
Our data demonstrate that the miR156b‐GmSPL9d module acts as an upstream master regulator of soybean nodulation, which coordinates multiple marker genes involved in soybean nodulation.
... GmENOD40 is a downstream component of NFs perception (Ferguson et al., 2010), which is expressed in peripheral cells, cortical cells, nodule primordia and developing nodules of root vascular bundles (Ferguson & Mathesius, 2014). Charon et al. (1999) reported that the change of ENOD40 expression abundance could affect nodulation, suggesting that it plays an important role in the organogenesis of nodules. In the present study, the expression abundance of GmENOD40 was up-regulated by H 2 S and rhizobia in soybean roots under whatever water condition (Fig. 10A). ...
Hydrogen sulphide (H2S), as a new gas signal molecule, participates in the regulation of a variety of abiotic stresses in plants. However, it was unclear how H2S and rhizobia can together to affect the adaptation of soybean to water deficiency. Here, the adaptation mechanism of H2S and rhizobia in soybean to water deficiency was studied. Our results showed that H2S and rhizobia jointly enhanced leaf chlorophyll content, the relative water content (RWC) and caused an increase biomass in soybean under water deficiency. Besides, under water deficiency, H2S enhanced biomass by affecting nodule numbers and nitrogenase activity during the growth of soybean. The expression of soybean nodulation marker genes including early nodulin 40 (GmENOD40), ERF required for nodulation (GmERN), and nodulation inception genes were up-regulated by H2S and rhizobia in nodules. Moreover, the combined effect of H2S and rhizobia were proved to affect the enzyme activities and gene expression level of antioxidant, as well as osmotic protective substance under water deficiency. In addition, the metabolomics results provided that the changes of lipids and lipid-like molecules were remarkably promoted by the combined effect of H2S and rhizobia. Thus, H2S and rhizobia synergistically subsided the oxidative damage by increasing the accumulation of metabolites and strengthening the antioxidant capacity under water deficiency.
... The soybean gene early nodulin 40 (ENOD40) played a pivotal role in nodule organogenesis (Charon et al., 1999;Kumagai et al., 2006;Wan et al., 2007). NNC1 regulated the expression of ENOD40 by binding to the AP2 cis-elements of ENOD40 promoter, which consequently represses ENOD40 expression and negatively regulated nodulation (Wang et al., 2014c). ...
Soybean is one of the most important oilseed and fodder crops. Benefiting from the efforts of soybean breeders and the development of breeding technology, large number of germplasm have been generated over the last 100 years. Nevertheless, soybean breeding needs to be accelerated to meet the needs of a growing world population, to promote sustainable agriculture, and to address future environmental changes. The acceleration is highly reliant on the discoveries in gene functional studies. The release of the reference soybean genome in 2010 has significantly facilitated the advance in soybean functional genomics. Here, we review the research progress in soybean omics (genomics, transcriptomics, epigenomics, and proteomics), germplasm development (germplasm resources and databases), gene discovery (genes that are responsible for important soybean traits including yield, flowering and maturity, seed quality, stress resistance, nodulation, and domestication), and transformation technology during the past decade. At the end, we also briefly discuss current challenges and future directions.
... It is expressed in the root pericycle and in the differentiating cells of the nodule primordia (Crespi et al., 1994;Compaan et al., 2001). The overexpression of ENOD40 leads to accelerated nodulation, mainly caused by increased initiation of primordia and an enhanced sensitivity to nodulation signals (Charon et al., 1999). ...
In recent years, the extensive use of high-throughput technologies led to the discovery of thousands of long non-coding RNAs (lncRNAs) in eukaryotes, questioning their roles. Many of them have already emerged as major regulators of a plethora of key cellular and molecular processes. In particular, lncRNAs were shown to impact the transcriptome content via different mechanisms, including alternative splicing (AS). In order to modulate AS, lncRNAs can either act as chromatin remodelers, form duplexes with DNA or RNA sequences, or interact with specific splicing factors to alter their activity. The plant lncRNA named ALTERNATIVE SPLICING COMPETITOR (ASCO) interacts in vivo with the NUCLEAR SPECKLE RNA-BINDING PROTEINS (NSRs) splicing factors, leading to AS modulation during root development in Arabidopsis thaliana. In this study, we aimed to characterize the role of the ASCO-NSR complex within plant development. We demonstrated that NSRs bind a large subset of pre-messenger RNAs involved in auxin pathway and defense responses. Consistently, NSRs appear to be crucial for the establishment of proper immune responses. Furthermore, the impact of deregulation of ASCO expression was studied at both phenotypical and molecular levels, revealing its involvement in the plant response to flagellin and in the regulation of AS for key defense genes, respectively. Additionally, ASCO was shown to bind to the core spliceosomal proteins PRP8a and SmD1b. As for NSRs, ASCO can hijack PRP8a by altering its binding to pre-mRNA targets. Finally, an unsuspected link between ASCO and temperature acclimation was uncovered, suggesting that this lncRNA can act as a potential integrator of multiple stresses in the plant. Therefore, this work sheds light over new biological roles for lncRNA-splicing factor complexes in plants, describing non-coding RNA-mediated splicing regulatory mechanisms and their integration in plant development and stress responses.
... The possible functions of legume ENOD40 have been argued mainly in favor of the induction of cortical cell divisions that lead to the initiation of nodule primordia and appropriate differentiation and development of nodules (Charon et al. 1997(Charon et al. , 1999Mylona et al. 1995). Expression of infection-related genes is independent of ENOD40 activation, indicating rhizobial invasion to be independent of ENOD40 (Kumagai et al. 2006). ...
The lncRNA ENOD40 is required for cortical cell division during root nodule symbiosis (RNS) of legumes, though it is not essential for actinorhizal RNS. Our objective was to understand whether ENOD40 was required for aeschynomenoid nodule formation in Arachis hypogaea. AhENOD40 express from chr5 (AhENOD40-1) and chr15 (AhENOD40-2) during symbiosis, and RNA interference of these transcripts drastically affected nodulation indicating the importance of ENOD40 in A.hypogaea. Furthermore, we demonstrated several distinct characteristics of ENOD40: (i) Natural antisense transcript of ENOD40 was detected from the AhENOD40-1 locus (designated as NAT-AhDONE40). (ii) Both AhENOD40-1 and AhENOD40-2 had two exons, whereas NAT-AhDONE40 was monoexonic. RT-qPCR analysis indicated both sense and antisense transcripts to be present in both cytoplasm and nucleus, and their expression increased with the progress of symbiosis. (iii) RNA Pulldown from whole cell extracts of infected roots at 4DPI indicated NAT-AhDONE40 to interact with the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain containing Ash (Absent Small homeotic disc) family protein AhASHR3 and this interaction was further validated using RNA immunoprecipitation and EMSA. (iv) ChIP assays indicate deposition of ASHR3 specific histone marks H3K36me3 and H3K4me3 in both the ENOD40 loci during the progress of symbiosis. ASHR3 is known for its role in optimizing cell proliferation and reprogramming. Since both ASHR3 and ENOD40 from legumes cluster away from those in actinorhizal plants and other nonlegumes in phylogenetic distance trees, we hypothesize that the interaction of DONE40 with ASHR3 could have evolved for adapting the nodule organogenesis program for legumes.
... The possible functions of legume ENOD40 have been argued mainly in favor of the induction of cortical cell divisions that lead to the initiation of nodule primordia and appropriate differentiation and development of nodules (Charon et al. 1997(Charon et al. , 1999Mylona et al. 1995). Expression of infection-related genes is independent of ENOD40 activation, indicating rhizobial invasion to be independent of ENOD40 (Kumagai et al. 2006). ...
... In the preinfection phase, the infection of roots by rhizobia is triggered by the perception by the plant of specific bacterial signals, called Nod factors (NFs) [12,13]. Among the early nodulin genes, ENOD40 is considered to be a key gene, and its expression strongly affects the number of nodules [14,15]. The perception of NFs is mediated by nodule factor receptor (NFR) which encoded by the nodule factor receptor gene [16]. ...
Soybean (Glycine max [L.] Merr.) is a food and oil crop whose growth and yield are influenced by root and nodule development. In the present study, GmNMHC5 was found to promote the formation of nodules in overexpressing mutants. In contrast, the number of nodules in Gmnmhc5 edited with CRISPR/Cas9 decreased sharply. In 35S:GmNMHC5 mutants, expression levels of genes involved in nodulation were significantly up-regulated. Both in vitro and in vivo biochemical analyses showed that GmNMHC5 directly interacted with GmGAI (a DELLA protein), and the content of gibberellin 3 (GA3) in overexpressing mutants was lower than that in the wild type. These results revealed that GmNMHC5 participates in the classical GA signaling pathway, and may regulate the content of GA3 to match the optimal concentration required for nodule formation, thereby promoting nodulation by directly interacting with GmGAI. A model illustrating the mechanism by which GmNMHC5 promotes soybean nodulation is presented.
... Plant genes play a central role in regulating and controlling nodulation processes (Roy et al. 2019). Therefore, we examined 10 genes that are required in the rhizobial NF signaling pathway (DMI2, DMI3, NIN, Enod40, DELLA and NPL1) and the plant defense response (PR1, PR10, RhobA and ERF1) to explore the similarities and differences in the responses induced by the NF-and T3SS-mediated signaling pathways ( Figure 6) (Charon et al. 1999, Endre et al. 2002, Laporte et al. 2014, Fonouni-Farde et al. 2016. ...
Under nitrogen-limiting conditions, symbiotic nodulation promotes the growth of legume plants via the fixation of atmospheric nitrogen to ammonia by rhizobia in root nodules. The rhizobial Nod factor (NF) and type III secretion system (T3SS) are two key signaling pathways for establishing the legume–rhizobium symbiosis. However, whether NF signaling is involved in the nodulation of Robinia pseudoacacia and Mesorhizobium amorphae CCNWGS0123, and its symbiotic differences compared to T3SS signaling remain unclear. Therefore, to elucidate the function of NF signaling in nodulation, we mutated nodC in M. amorphae CCNWGS0123, which aborted NF synthesis. Compared to the plants inoculated with the wild type strain, the plants inoculated with the NF-deficient strain exhibited shorter shoots with etiolated leaves. These phenotypic characteristics were similar to those of the plants inoculated with the T3SS-deficient strain, which served as a nod− (non-effective nodulation) control. Both the plants inoculated with the NF- and T3SS-deficient strains formed massive root hair swellings, but no normal infection threads were detected. Sections of the nodules showed that inoculation with the NF- and T3SS-deficient strains induced small, white bumps without any rhizobia inside. Analyzing the accumulation of six plant hormones and the expression of ten plant genes indicated that the NF- and T3SS-deficient strains activated plant defense reactions while suppressing plant symbiotic signaling during the perception and nodulation processes. The requirement for NF signaling appeared to be conserved in two other leguminous trees that can establish symbiosis with M. amorphae CCNWGS0123. In contrast, the function of the T3SS might differ among species, even within the same subfamily (Faboideae). Overall, this work demonstrated that nodulation of R. pseudoacacia and M. amorphae CCNWGS0123 was both NF and T3SS dependent.
