Project description:Phosphorus (P) deficiency is a major limitation for legume crop production. Although overall adaptations of plant roots to P deficiency have been extensively studied, fragmentary information is available in regards to root nodule responses to P deficiency. In this study, genome wide transcriptome analysis was conducted using RNA-seq analysis to investigate molecular mechanisms underlying soybean (Glycine max) nodule adaptation to phosphate (Pi) starvation. Phosphorus deficiency significantly decreased soybean nodule growth and nitrogenase activity. Nodule Pi concentrations declined by 49% in response to P deficiency, but this was well below the 87% and 88% decreases observed in shoots and roots, respectively. Nodule transcript profiling revealed that a total of 2,055 genes exhibited differential expression patterns between Pi sufficient and deficient conditions. A set of DEGs appeared to be involved in maintaining Pi homeostasis in soybean nodules, including 8 Pi transporters (PTs), 8 proteins containing the SYG1/PHO81/XPR1 domain (SPXs), and 16 purple acid phosphatases (PAPs). The results suggest that a complex transcriptional regulatory network participates in soybean nodule adaption to Pi starvation, most notable a Pi signaling pathway specifically involved in maintaining Pi homeostasis in nodules.
Project description:The soybean–Bradyrhizobium symbiosis enables symbiotic nitrogen fixation (SNF) within root nodules, reducing reliance on synthetic N fertilizers. However, nitrogen fixation is transient, peaking several weeks after Bradyrhizobium colonization and declining as nodules senesce in coordination with host development. To investigate the regulatory mechanisms governing SNF and senescence, we conducted a temporal transcriptomic analysis of soybean nodules colonized with Bradyrhizobium diazoefficiens USDA110. Weekly nodule samples (2 to 10 weeks postinoculation, wpi) were analyzed using RNA and small RNA sequencing, and acetylene reduction assays assessed nitrogenase activity from 4 to 7 wpi. We identified three major nodule developmental phases: early development (2 to 3 wpi), nitrogen fixation (3 to 8 wpi), and senescence (8 to 10 wpi). Soybean showed extensive transcriptional reprogramming during senescence, whereas Bradyrhizobium underwent major transcriptional shifts early in development before stabilizing during nitrogen fixation. We identified seven soybean genes and several microRNAs as candidate biomarkers of nitrogen fixation, including lipoxygenases (Lox), suggesting roles for oxylipin metabolism. Soy hemoglobin-2 (Hb2), previously classified as nonsymbiotic, was upregulated during senescence, implicating oxidative stress responses within aging nodules. Upregulation of the Bradyrhizobium paa operon and rpoH during senescence suggesting metabolic adaptation for survival beyond symbiosis. Additionally, Bradyrhizobium nif gene expression showed stage-specific regulation, with nifK peaking at 2 wpi, nifD and nifA at 2 and 10 wpi, and nifH, nifW, and nifS at 10 wpi. These findings provide insights into SNF regulation and nodule aging, revealing temporal gene expression patterns that could inform breeding or genetic engineering strategies to enhance nitrogen fixation in soybeans and other legume crops.
Project description:Metabolomics and transcriptomics of Bradyrhizobium diazoefficiens-induced root nodules Bradyrhizobium diazoefficiens is a nitrogen-fixing endosymbiont, which can grow inside root-nodule cells of the agriculturally important soybean and other host plants. Our previous studies described B. diazoefficiens host-specific global expression changes occurring during legume infection at the transcript and protein level. In order to further characterize nodule metabolism, we here determine by flow injection -time of flight mass spectrometry analysis the metabolome of i) nodules and roots from four different B. diazoefficiens host plants, ii) soybean nodules harvested at different time points during nodule development, and iii) soybean nodules infected by two strains mutated in key genes for nitrogen fixation, respectively. Ribose (soybean), tartaric acid (mungbean), hydroxybutanoyloxybutanoate (siratro) and catechol (cowpea) were among the metabolites found to be specifically elevated in one of the respective host plants. While the level of C4-dicarboxylic acids decreased during soybean nodule development, we observed an accumulation of trehalose-phosphate at 21 days post infection (dpi). Moreover, nodules from non-nitrogen-fixing bacteroids (nifA and nifH mutants) showed specific metabolic alterations; these were also supported by transcriptomics data that was generated for the two mutant strains and were helpful to separate for some examples the respective bacterial and plant contributions to the metabolic profile. The alterations included signs of nitrogen limitation in both mutants, and an increased level of a phytoalexin in nodules induced by the nifA mutant, suggesting that the tissue of these nodules exhibits defense and stress reactions.
Project description:The TRAP-seq process is dependent on the expression of a cell layer-specific His-FLAG-tagged ribosomal protein L18 (HF-GmRPL 18), which allows for the immunoprecipitation of ribosomes with their corresponding mRNA to produce tissue-specific translatomes (Zanetti et al., 2005; Castro‐Guerrero et al., 2016).To capture events occurring in the cortex during the early stages of infection and initial cortical cell divisions, we inoculated plants and performed a time-course collection of root samples at 72- and 96-hour post inoculation (hpi) followed by TRAP-seq. Immunoblot analysis indicated that an adequate amount of protein was present for immunoprecipitation. To study rhizobial-induced transcriptional changes in the cortex during early nodule development, we identified a soybean promoter (Glyma.18g53890, Figure 1B) expressed exclusively in the cortex cells using LCM (Kerk et al., 2003; Casson et al., 2008) followed by transcriptional analysis. The cortex-specific promoter was used to drive the expression of GmRPL18 in soybean hairy roots. To capture events occurring in the cortex during the early stages of infection and initial cortical cell divisions, we inoculated plants and performed a time-course collection of root samples at 72- and 96-hour post inoculation (hpi) followed by TRAP-seq. Immunoblot analysis indicated that an adequate amount of protein was present for immunoprecipitation (Figure 1C). Taken together, time-course cortex-specific TRAP-seq was established for studying of early nodule development in soybean.
Project description:To understand the responses of plants to environmental stresses will help mitigate the problems via creating stress-tolerant crop.Whole genome DNA microarrays were used to compare the nodule transcriptome profiling of soybean ‘Williams 82’ grown in vermiculite and subjected to low (0.1 mM; LP) or adequate (2.0 mM; HP) Pi levels. Plants were inoculated with two Bradyrhizobium diazoefficiens strains (USDA110 vs. CB1809), which exhibited contrasting growth and nodulation patterns under LP conditions, with the former model strain displaying a lower Pi-stress acclimatization in combination with the model Williams 82. Affymetrix’s whole Soybean Gene Expression Microarray (66K) was used. Microarrays were used to compare the nodule transcriptomes of the model soybean ‘Williams 82’ plants grown in vermiculite and subjected to low (0.1 mM) or sufficient (2.0 mM) inorganic phosphate levels