MicroRNA 4407 modulates nodulation in soybean by repressing a root-specific ISOPENTENYLTRANSFERASE (GmIPT3).

MicroRNAs (miRNAs) are important regulators of plant biological processes, including soybean nodulation. One miRNA, miR4407, was identified in soybean roots and nodules. However, the function of miR4407 in soybean is still unknown. MiR4407, unique to soybean, positively regulates lateral root emergence and root structures and represses a root-specific ISOPENTENYLTRANSFERASE (GmIPT3). By altering the expression of miR4407 and GmIPT3, we investigated the role of miR4407 in lateral root and nodule development. Both miR4407 and GmIPT3 are expressed in the inner root cortex and nodule primordia. Upon rhizobial inoculation, miR4407 was downregulated while GmIPT3 was upregulated. Overexpressing miR4407 reduced the number of nodules in transgenic soybean hairy roots while overexpressing the wild-type GmIPT3 or a miR4407-resistant GmIPT3 mutant (mGmIPT3) significantly increased the nodule number. The mechanism of miR4407 and GmIPT3 functions was also linked to autoregulation of nodulation (AON), where miR4407 overexpression repressed miR172c and activated its target, GmNNC1, turning on AON. Exogenous CK mimicked the effects of GmIPT3 overexpression on miR172c, supporting the notion that GmIPT3 regulates nodulation by enhancing root-derived CK. Overall, our data revealed a new miRNA-mediated regulatory mechanism of nodulation in soybean. MiR4407 showed a dual role in lateral root and nodule development.

GmWRI1c Increases Palmitic Acid Content to Regulate Seed Oil Content and Nodulation in Soybean (Glycine max).

Soybean (Glycine max) is an important oil crop, but the regulatory mechanisms underlying seed oil accumulation remain unclear. We identified a member of the GmWRI1s transcription factor family, GmWRI1c, that is involved in regulating soybean oil content and nodulation. Overexpression of GmWRI1c in soybean hairy roots increased the expression of genes involved in glycolysis and de novo lipogenesis, the proportion of palmitic acid (16:0), and the number of root nodules. The effect of GmWRI1c in increasing the number of root nodules via regulating the proportion of palmitic acid was confirmed in a recombinant inbred line (RIL) population. GmWRI1c shows abundant sequence diversity and has likely undergone artificial selection during domestication. An association analysis revealed a correlation between seed oil content and five linked natural variations (Hap1/Hap2) in the GmWRI1c promoter region. Natural variations in the GmWRI1c promoter were strongly associated with the GmWRI1c transcript level, with higher GmWRI1c transcript levels in lines carrying GmWRI1cHap1 than in those carrying GmWRI1cHap2. The effects of GmWRI1c alleles on seed oil content were confirmed in natural and RIL populations. We identified a favourable GmWRI1c allele that can be used to breed new varieties with increased seed oil content and nodulation.

Shoot-derived miR2111 controls legume root and nodule development.

Legumes control their nodule numbers through the autoregulation of nodulation (AON). Rhizobia infection stimulates the production of root-derived CLE peptide hormones that are translocated to the shoot where they regulate a new signal. We used soybean to demonstrate that this shoot-derived signal is miR2111, which is transported via phloem to the root where it targets transcripts of Too Much Love (TML), a negative regulator of nodulation. Shoot perception of rhizobia-induced CLE peptides suppresses miR2111 expression, resulting in TML accumulation in roots and subsequent inhibition of nodule organogenesis. Feeding synthetic mature miR2111 via the petiole increased nodule numbers per plant. Likewise, elevating miR2111 availability by over-expression promoted nodulation, while target mimicry of TML induced the opposite effect on nodule development in wild-type plants and alleviated the supernodulating and stunted root growth phenotypes of AON-defective mutants. Additionally, in non-nodulating wild-type plants, ectopic expression of miR2111 significantly enhanced lateral root emergence with a decrease in lateral root length and average root diameter. In contrast, hairy roots constitutively expressing the target mimic construct exhibited reduced lateral root density. Overall, these findings demonstrate that miR2111 is both the critical shoot-to-root factor that positively regulates root nodule development and also acts to shape root system architecture.

Insights into nitrogen fixing traits and population structure analyses in cowpea (Vigna unguiculata L. Walp) accessions grown in Ghana.

With legumes, symbiotic N2 fixation can meet the species N demand and reduce the over-reliance on chemical fertilizers in tropical regions where N deficiency is a major factor limiting crop yields and increased agricultural sustainability. Therefore, to optimize the use of cowpea (Vigna unguiculata L. Walp) germplasm in effective breeding, evaluation of genetic diversity and quantification of N2 fixation are essential prerequisites. The aim of this study was to explore the level of diversity using SSR markers and N2-fixing traits in a set of cowpea germplasm grown in Ghana. We analysed 49 cowpea accessions collected from Northern Ghana using qualitative vegetative and N2 fixation traits, and simple sequence repeat (SSR) markers. Experimental field results revealed considerable morpho-physiological variation for plant growth habits, grain yield and symbiotic performance between and among the cowpea accessions. Results from both the 15N natural abundance and ureides in the xylem sap were able to descriminate between high and low levels of N2 fixation in cowpea accessions. Five subpopulations were identified within accessions inferred from STRUCTURE 2.3.4. A general linear model was used to assess the association of SSR markers with N2-fixing traits. There were significant (p ≤ 0.05) links between SSR markers and symbiosis-related traits such as nodule number, nodule dry weight, shoot dry weight, N-fixed, N derived from air (Ndfa), and relative uried-N (RU-N).

QTL analysis of nodule traits and the identification of loci interacting with the type III secretion system in soybean.

Symbiotic nitrogen fixation is the main source of nitrogen for soybean growth. Since the genotypes of rhizobia and soybean germplasms vary, the nitrogen-fixing ability of soybean after inoculation also varies. A few studies have reported that quantitative trait loci (QTLs) control biological nitrogen fixation traits, even soybean which is an important crop. The present study reported that the Sinorhizobium fredii HH103 gene rhcJ belongs to the tts (type III secretion) cluster and that the mutant HH103ΩrhcJ can clearly decrease the number of nodules in American soybeans. However, few QTLs of nodule traits have been identified. This study used a soybean (Glycine max (L.) Merr.) 'Charleston' × 'Dongnong 594' (C × D, n = 150) recombinant inbred line (RIL). Nodule traits were analysed in the RIL population after inoculation with S. fredii HH103 and the mutant HH103ΩrhcJ. Plants were grown in a greenhouse with a 16-h light cycle at 26 °C and an 8-h dark cycle at 18 °C. Then, 4 weeks after inoculation, plants were harvested for evaluation of nodule traits. Through QTL mapping, 16 QTLs were detected on 8 chromosomes. Quantitative PCR (qRT-PCR) and RNA-seq analysis determined that the genes Glyma.04g060600, Glyma.18g159800 and Glyma.13g252600 might interact with rhcJ.

GmTIR1/GmAFB3-based auxin perception regulated by miR393 modulates soybean nodulation.

Auxins play important roles in the nodulation of legumes. However, the mechanism by which auxin signaling regulates root nodulation is largely unknown. In particular, the role of auxin receptors and their regulation in determinate nodule development remains elusive. We checked the expression pattern of the auxin receptor GmTIR1/GmAFB3 genes in soybean. We analyzed the functions of GmTIR1/AFB3 in the regulation of rhizobial infection and nodule number, and also tested the functions of miR393 during nodulation and its relationship with GmTIR1/AFB3. The results showed that GmTIR1 and GmAFB3 genes exhibit diverse expression patterns during nodulation and overexpression of GmTIR1 genes significantly increased inflection foci and eventual nodule number. GmTIR1/AFB3 genes were post-transcriptionally cleaved by miR393 family and knock-down of the miR393 family members significantly increased rhizobial infection and the nodule number. Overexpression of the mutated form of GmTIR1C at the miR393 cleavage site that is resistant to miR393 cleavage led to a further increase in the number of infection foci and nodules, suggesting that miR393s modulate nodulation by directly targeting GmTIR1C. This study demonstrated that GmTIR1- and GmAFB3-mediated auxin signaling, that is spatio-temporally regulated by miR393, plays a crucial role in determinate nodule development in soybean.

Symbiotic Functioning and Photosynthetic Rates Induced by Rhizobia Associated with Jack Bean (Canavalia ensiformis L.) Nodulation in Eswatini.

Improving the efficiency of the legume-rhizobia symbiosis in African soils for increased grain yield would require the use of highly effective strains capable of nodulating a wide range of legume plants. This study assessed the photosynthetic functioning, N2 fixation, relative symbiotic effectiveness (%RSE) and C assimilation of 22 jack bean (Canavalia ensiformis L.) microsymbionts in Eswatini soils as a first step to identifying superior isolates for inoculant production. The results showed variable nodule number, nodule dry matter, shoot biomass and photosynthetic rates among the strains tested under glasshouse conditions. Both symbiotic parameters and C accumulation differed among the test isolates at the shoot, root and whole-plant levels. Although 7 of the 22 jack bean isolates showed much greater relative symbiotic efficiency than the commercial Bradyrhizobium strain XS21, only one isolate (TUTCEeS2) was statistically superior to the inoculant strain, which indicates its potential for use in inoculant formulation after field testing. Furthermore, the isolates that recorded high %RSE elicited greater amounts of fixed N.

Total nodule number as an independent prognostic factor in resected stage III non-small cell lung cancer: a deep learning-powered study.

Background: Almost every patient with lung cancer has multiple pulmonary nodules; however, the significance of nodule multiplicity in locally advanced non-small cell lung cancer (NSCLC) remains unclear. Methods: We identified patients who had undergone surgical resection for stage I-III NSCLC at the Peking University People's Hospital from 2005 to 2018 for whom preoperative chest computed tomography (CT) scans were available. Deep learning-based artificial intelligence (AI) algorithms using convolutional neural networks (CNN) were applied to detect and classify pulmonary nodules (PNs). Maximally selected log-rank statistics were used to determine the optimal cutoff value of the total nodule number (TNN) for predicting survival. Results: A total of 33,410 PNs were detected by AI among the 2,126 participants. The median TNN detected per person was 12 [interquartile range (IQR) 7-20]. It was revealed that AI-detected TNN (analyzed as a continuous variable) was an independent prognostic factor for both recurrence-free survival (RFS) [hazard ratio (HR) 1.012, 95% confidence interval (CI): 1.002 to 1.022, P=0.021] and overall survival (OS) (HR 1.013, 95% CI: 1.002 to 1.025, P=0.021) in multivariate analyses of the stage III cohort. In contrast, AI-detected TNN was not significantly associated with survival in the stage I and II cohorts. In a survival tree analysis, rather than using traditional IIIA and IIIB classifications, the model grouped cases according to AI-detected TNN (lower vs. higher: log-rank P<0.001), which led to a more effective determination of survival rates in the stage III cohort. Conclusions: The AI-detected TNN is significantly associated with survival rates in patients with surgically resected stage III NSCLC. A lower TNN detected on preoperative CT scans indicates a better prognosis for patients who have undergone complete surgical resection.

A GmNINa-miR172c-NNC1 Regulatory Network Coordinates the Nodulation and Autoregulation of Nodulation Pathways in Soybean.

Symbiotic root nodules are root lateral organs of plants in which nitrogen-fixing bacteria (rhizobia) convert atmospheric nitrogen to ammonia. The formation and number of nodules in legumes are precisely controlled by a rhizobia-induced signal cascade and host-controlled autoregulation of nodulation (AON). However, how these pathways are integrated and their underlying mechanisms are unclear. Here, we report that microRNA172c (miR172c) activates soybean (Glycine max) Rhizobia-Induced CLE1 (GmRIC1) and GmRIC2 by removing the transcriptional repression of these genes by Nodule Number Control 1 (NNC1), leading to the activation of the AON pathway. NNC1 interacts with GmNINa, the soybean ortholog of Lotus NODULE INCEPTION (NIN), and hampers its transcriptional activation of GmRIC1 and GmRIC2. Importantly, GmNINa acts as a transcriptional activator of miR172c. Intriguingly, NNC1 can transcriptionally repress miR172c expression, adding a negative feedback loop into the NNC1 regulatory network. Moreover, GmNINa interacts with NNC1 and can relieve the NNC1-mediated repression of miR172c transcription. Thus, the GmNINa-miR172c-NNC1 network is a master switch that coordinately regulates and optimizes NF and AON signaling, supporting the balance between nodulation and AON in soybean.

Genetic Analysis and Mapping of QTLs for Soybean Biological Nitrogen Fixation Traits Under Varied Field Conditions.

Soybean is an important economic and green manure crop that is widely used in intercropping and rotation systems due to its high biological nitrogen fixation (BNF) capacity and the resulting reduction in N fertilization. However, the genetic mechanisms underlying soybean BNF are largely unknown. Here, two soybean parent genotypes contrasting in BNF traits and 168 F9:11 recombinant inbred lines (RILs) were evaluated under four conditions in the field. The parent FC1 always produced more big nodules, yet fewer nodules in total than the parent FC2 in the field. Furthermore, nodulation in FC1 was more responsive to environmental changes than that in FC2. Broad-sense heritability (h2 b ) for all BNF traits varied from 0.48 to 0.87, which suggests that variation in the observed BNF traits was primarily determined by genotype. Moreover, two new QTLs for BNF traits, qBNF-16 and qBNF-17, were identified in this study. The qBNF-16 locus was detected under all of the four tested conditions, where it explained 15.9-59.0% of phenotypic variation with LOD values of 6.31-32.5. Meanwhile qBNF-17 explained 12.6-18.6% of observed variation with LOD values of 4.93-7.51. Genotype group analysis indicated that the FC1 genotype of qBNF-16 primarily affected nodule size (NS), while the FC2 genotype of qBNF-16 promoted nodule number (NN). On the other hand, the FC1 genotype of qBNF-17 influenced NN and the FC2 genotype of qBNF-17 impacted NS. The results on the whole suggest that these two QTLs might be valuable markers for breeding elite soybean varieties with high BNF capacities.