Soybean Root Nodules Research Articles

Polyphasic analysis reveals correlation between phenotypic and genotypic analysis in soybean bradyrhizobia (Bradyrhizobium spp.)

Soybean bradyrhizobia (Bradyrhizobium spp.) are bacteria that fix atmospheric nitrogen within the root nodules of soybean, a crop critical for meeting global nutritional protein demand. Members of this group differ in symbiotic effectiveness, and historically both phenotypic and genotypic approaches have been used to assess bradyrhizobial diversity. However, agreement between various approaches of assessment is poorly known. A collection (n=382) of soybean bradyrhizobia (Bradyrhizobium japonicum, B. diazoefficiens, and B. elkanii) were characterized by Internal Transcribed Spacer – Restriction Fragment Length Polymorphism (ITS-RFLP), cellular fatty acid composition (fatty acid methyl esters, FAME), and serological reactions to assess agreement between phenotypic and genotypic methods. Overall, 76% of the accessions demonstrated identical clustering with each of these techniques. FAME was able to identify all 382 accessions, whereas 14% were non-reactive serologically. One ITS-RFLP group, containing 36 Delaware isolates, produced multiple ITS amplicons indicating they possess multiple ribosomal RNA (rrn) operons. Cloning and sequencing revealed that these strains contained as many as three heterogenous rrn operons, a trait previously unknown in bradyrhizobia. A representative subset of 96 isolates was further characterized using 16S rRNA and Internal Transcribed Spacer (ITS) amplicon sequencing. ITS sequences showed better inter- and intra-species discrimination (65–99% identity) than 16S sequences (96–99% identity). This study shows that phenotypic and genotypic approaches are strongly correlated at the species level but should be approached with caution. We also suggest using combined 16S and ITS genotyping data to obtain better inter- and intra-species resolution in bradyrhizobia classification.

Gene regulatory networks associated with lateral root and nodule development in soybean

AbstractLegume plants such as soybean produce two major types of root lateral organs, lateral roots and root nodules. A robust computational framework was developed to predict potential gene regulatory networks (GRNs) associated with root lateral organ development in soybean. A genome-scale expression data set was obtained from soybean root nodules and lateral roots and subjected to biclustering using QUBIC (QUalitative BIClustering algorithm). Biclusters and transcription factor (TF) genes with enriched expression in lateral root tissues were converged using different network inference algorithms to predict high-confidence regulatory modules that were repeatedly retrieved in different methods. The ranked combination of results from all different network inference algorithms into one ensemble solution identified 21 GRN modules of 182 co-regulated genes networks, potentially involved in root lateral organ development stages in soybean. The workflow correctly predicted previously known nodule- and lateral root-associated TFs including the expected hierarchical relationships. The results revealed distinct high-confidence GRN modules associated with early nodule development involving AP2, GRF5 and C3H family TFs, and those associated with nodule maturation involving GRAS, LBD41 and ARR18 family TFs. Knowledge from this work supported by experimental validation in the future is expected to help determine key gene targets for biotechnological strategies to optimize nodule formation and enhance nitrogen fixation.

SOIL PROTECTION STUDY THROUGH THE APPLICATION OF COMPLEX METHODS FOR SUSTAINABLE MANAGEMENT OF THE SOYBEAN AGROSYSTEM

"The degradation of natural soil fertility is caused by practicing the classic tillage system (ploughing with the furrow return) and using high doses of chemical fertilizers. It is necessary to adopt soil tillage techniques that aim to preserve and improve soil fertility without decrease production. In Romania, soybean crop, could be an economic boost for the agriculture sector because it reduces the costs required for the procurement of vegetable protein needed for the livestock and food sector, soybeans being a plant that fixes nitrogen from various sources (precipitation, activity of Bradyrhizobium japonicum bacteria, humus mineralization and residual nitrogen). From the research conducted between 2018-2020 at ARDS Turda, regarding the soybean root nodules cultivated in four tillage systems (CS - plough, MC - chisel, MD - disk and NT- direct sowing) with the technology specific to each system, the results indicate that in a conservative system the soybeans yield correlates positively with the number and weight of nodules formed. Organo-mineral fertilization (green fertilizer - autumn rape + gulle manure + N40P40) contributes significantly positively to the number and the weight of nodules and increases the yield. As an alternative to the CS, soybean can be cultivated in a minimum tillage system (MC), the yield difference compared to the classical system (plough) being insignificant, just 38 kg. The pedo-climatic conditions of the experimental area it is favourable for this crop and for the fixation of atmospheric nitrogen, without requiring high doses of mineral fertilizers."

GmMAX2-D14 and -KAI interaction-mediated SL and KAR signaling play essential roles in soybean root nodulation.

Despite of important functions of strigolactones (SLs) and karrikins (KARs) in plant development, plant-parasite and plant-fungi interactions, their roles in soybean-rhizobia interaction remain elusive. SL/KAR signaling genes GmMAX2a, GmD14s, and GmKAIs are activated by rhizobia infection. GmMAX2a restored atmax2 root hair defects and soybean root hairs were changed in GmMAX2a overexpression (GmMAX2a-OE) or knockdown (GmMAX2a-KD) mutants. GmMAX2a-KD gave fewer, whereas GmMAX2a-OE produced more nodules than GUS hairy roots. Mutation of GmMAX2a in its KD or OE transgenic hairy roots affected the rhizobia infection-induced increases in early nodulation gene expression. Both mutant hairy roots also displayed the altered auxin, jasmonate and abscisic acid levels, as further verified by transcriptomic analyses of their synthetic genes. Overexpression of an auxin synthetic gene GmYUC2a also affected SL and KAR signaling genes. GmMAX2a physically interacted with SL/KAR receptors GmD14s, GmKAIs, and GmD14Ls with different binding affinities, depending on variations in the critical amino acids, forming active D14/KAI-SCFMAX2 complexes. The knockdown mutant roots of the nodule-specifically expressing GmKAIs and GmD14Ls gave fewer nodules, with altered expression of several early nodulation genes. The expression levels of GmKAIs, and GmD14Ls were markedly changed in GmMAX2a mutant roots, so did their target repressor genes GmD53s and GmSMAX1s. Thus, SL and KAR signaling were involved in soybean-rhizobia interaction and nodulation partly through interactions with hormones, and this may explain the different effects of MXA2 orthologs on legume determinate and indeterminate nodulation. The study provides fresh insights into the roles of GmMAX2-mediated SL/KAR signaling in soybean root hair and nodule formation.

High-Throughput Mass Spectrometric Analysis of the Whole Proteome and Secretome From Sinorhizobium fredii Strains CCBAU25509 and CCBAU45436.

Sinorhizobium fredii is a dominant rhizobium on alkaline-saline land that can induce nitrogen-fixing symbiotic root nodules in soybean. Two S. fredii strains, CCBAU25509 and CCBAU45436, were used in this study to facilitate in-depth analyses of this species and its interactions with soybean. We have previously completed the full assembly of the genomes and detailed transcriptomic analyses for these two S. fredii strains, CCBAU25509 and CCBAU45436, that exhibit differential compatibility toward some soybean hosts. In this work, we performed high-throughput Orbitrap analyses of the whole proteomes and secretomes of CCBAU25509 and CCBAU45436 at different growth stages. Our proteomic data cover coding sequences in the chromosome, chromid, symbiotic plasmid, and other accessory plasmids. In general, we found higher levels of protein expression by genes in the chromosomal genome, whereas proteins encoded by the symbiotic plasmid were differentially accumulated in bacteroids. We identified secreted proteins from the extracellular medium, including seven and eight Nodulation Outer Proteins (Nops) encoded by the symbiotic plasmid of CCBAU25509 and CCBAU45436, respectively. Differential host restriction of CCBAU25509 and CCBAU45436 is regulated by the allelic type of the soybean Rj2(Rfg1) protein. Using sequencing data from this work and available in public databases, our analysis confirmed that the soybean Rj2(Rfg1) protein has three major allelic types (Rj2/rfg1, rj2/Rfg1, rj2/rfg1) that determine the host restriction of some Bradyrhizobium diazoefficiens and S. fredii strains. A mutant defective in the type 3 protein secretion system (T3SS) in CCBAU25509 allowed this strain to nodulate otherwise-incompatible soybeans carrying the rj2/Rfg1 allelic type, probably by disrupting Nops secretion. The allelic forms of NopP and NopI in S. fredii might be associated with the restriction imposed by Rfg1. By swapping the NopP between CCBAU25509 and CCBAU45436, we found that only the strains carrying NopP from CCBAU45436 could nodulate soybeans carrying the rj2/Rfg1 allelic type. However, no direct interaction between either forms of NopP and Rfg1 could be observed.

Compatibility of MO PLUS biofertilizer and Paenybacillus polymyxa to stimulate rice germination

The level of productivity of rice plantations can be increased through technical improvements in culture, namely spur plant growth. Increasing the germination rate is done by using growth regulators. Plants have limited synthesizing growth hormones in support of optimal growth. Therefore, it is necessary to add additional growth hormone from outside which can be given through fertilizers and symbiosis of microorganisms, including through the help of growth-regulating bacteria. MO Plus which is a combination of biological fertilizers and microorganisms Bradyrhizobium japonicum which is forming soybean root nodules to take nitrogen directly from the air and Streptomycetes as biological control of plant diseases. Efforts to merge MO Plus with P. polymyxa isolates in a consortium of biological product formulations need to be initiated to stimulate rice germination. The research stage consisted of synergistic tests by growing P. polymyxa bacteria and microbial consortium and calculating the number of bacterial colonies 48 hours in liquid media Nutrient Broth. The ability test produced IAA using L-Triptofan as a precursor, and the ability to produce GA3. The results showed that the levels of IAA and GA3 in Mo plus were 0.095 and 2.225 µg.l−1 respectively, while P. polymyxa: 0.087 and 2.251 µg.l−1. Synergism test results show that P. polymyxa and MO Plus bacteria can be grown on the same media simultaneously with the number of colonies of 2.3.108 CFU/ml. The treatment of MO Plus + Paenybacillus has the best influence on germination, wet weight, and dry weight of rice seeds.

Nitrogen Cycling in Soybean Rhizosphere: Sources and Sinks of Nitrous Oxide (N2O).

Nitrous oxide (N2O) is the third most important greenhouse gas after carbon dioxide and methane, and a prominent ozone-depleting substance. Agricultural soils are the primary anthropogenic source of N2O because of the constant increase in the use of industrial nitrogen (N) fertilizers. The soybean crop is grown on 6% of the world’s arable land, and its production is expected to increase rapidly in the future. In this review, we summarize the current knowledge on N-cycle in the rhizosphere of soybean plants, particularly sources and sinks of N2O. Soybean root nodules are the host of dinitrogen (N2)-fixing bacteria from the genus Bradyrhizobium. Nodule decomposition is the main source of N2O in soybean rhizosphere, where soil organisms mediate the nitrogen transformations that produce N2O. This N2O is either emitted into the atmosphere or further reduced to N2 by the bradyrhizobial N2O reductase (N2OR), encoded by the nos gene cluster. The dominance of nos– indigenous populations of soybean bradyrhizobia results in the emission of N2O into the atmosphere. Hence, inoculation with nos+ or nos++ (mutants with enhanced N2OR activity) bradyrhizobia has proved to be promising strategies to reduce N2O emission in the field. We discussed these strategies, the molecular mechanisms underlying them, and the future perspectives to develop better options for global mitigation of N2O emission from soils.

Analysis of the denitrification pathway and greenhouse gases emissions in Bradyrhizobium sp. strains used as biofertilizers in South America.

Greenhouse gases are considered as potential atmospheric pollutants, with agriculture being one of the main emission sources. The practice of inoculating soybean seeds with Bradyrhizobium sp. might contribute to nitrous oxide (N2 O) emissions. We analysed this capacity in five of the most used strains of Bradyrhizobium sp. in South America. We analysed the denitrification pathway and N2 O production by Bradyrhizobium japonicum E109 and CPAC15, Bradyrhizobium diazoefficiens CPAC7 and B. elkanii SEMIA 587 and SEMIA 5019, both in free-living conditions and in symbiosis with soybean. The in silico analysis indicated the absence of nosZ genes in B. japonicum and the presence of all denitrification genes in B. diazoefficiens strains, as well as the absence of nirK, norC and nosZ genes in B. elkanii. The in planta analysis confirmed N2 O production under saprophytic conditions or symbiosis with soybean root nodules. In the case of symbiosis, up to 26.1 and 18.4 times higher in plants inoculated with SEMIA5019 and E109, respectively, than in those inoculated with USDA110. The strains E109, SEMIA 5019, CPAC15 and SEMIA 587 showed the highest N2 O production both as free-living cells and in symbiotic conditions in comparison with USDA110 and CPAC7, which do have the nosZ gene. Although norC and nosZ could not be identified in silico or invitro in SEMIA 587 and SEMIA 5019, these strains showed the capacity to produce N2 O in our experimental conditions. This is the first report to analyse and confirm the incomplete denitrification capacity and N2 O production in four of the five most used strains of Bradyrhizobium sp. for soybean inoculation in South America.

Nitrogen application after plant growth regulator herbicide drift reduces soybean growth and yield

AbstractThe success of dicamba-tolerant soybean [Glycine max(L.) Merr.] has revived concerns about plant growth regulator (PGR) herbicide exposure to conventional soybean. In laboratory studies, soybean root nodulation is inhibited by excess auxin, which is the mechanism of action of PGR herbicides. Soybean exposed to PGRs in a field environment may have a similar response, and if nodulation is compromised, nitrogen (N) fixation may be reduced, with subsequent seed yield or protein content decreases. Many soybean–N studies report minimal impact to soybean yield. However, if soybeans show foliar PGR injury symptoms, could N application compensate for a potential nodulation inhibition response? This study examined the response of non–PGR tolerant soybean to N after exposure to low doses of 2,4-D and dicamba applied once (at soybean growth stages V1, V3, and early reproduction [R1 or R2]) or twice (V1 + V3 or V3 + R). N was either foliar or soil applied at early (∼5 d after PGR application) or late (10 d after PGR application) timings. Nodulation and plant growth were evaluated at R3, and grain yield and seed protein and oil content were quantified at maturity. Plant biomass and nodulation were reduced by 10% with some PGR treatments, and early foliar N application after PGR injury resulted in reduction up to 25%. N applications to non–PGR treated soybean did not increase yield. Soybean treated with PGR at V1 or V3, with or without N, had yields similar to control treatments. However, yield reductions of up to 20% were observed when PGRs were applied at V5 or R stages or when double PGR applications were followed by early foliar N application. Seed protein and oil content were not affected by PGR or N treatment.

A convenient, soil-free method for the production of root nodules in soybean to study the effects of exogenous additives.

Legumes develop root nodules that harbor endosymbiotic bacteria, rhizobia. These rhizobia convert nitrogen to ammonia by biological nitrogen fixation. A thorough understanding of the biological nitrogen fixation in legumes and its regulation is key to develop sustainable agriculture. It is well known that plant hormones affect nodule formation; however, most studies are limited to model legumes due to their suitability for in vitro, plate‐based assays. Specifically, it is almost impossible to measure the effects of exogenous hormones or other additives during nodule development in crop legumes such as soybean as they have huge root system in soil. To circumvent this issue, the present research develops suitable media and growth conditions for efficient nodule development under in vitro, soil‐free conditions in an important legume crop, soybean. Moreover, we also evaluate the effects of all major phytohormones on soybean nodule development under identical growing conditions. Phytohormones such as abscisic acid (ABA) and jasmonic acid (JA) had an overall inhibitory effect and those such as gibberellic acid (GA) or brassinosteroids (BRs) had an overall positive effect on nodule formation. This versatile, inexpensive, scalable, and simple protocol provides several advantages over previously established methods. It is extremely time‐ and resource‐efficient, does not require special training or equipment, and produces highly reproducible results. The approach is expandable to other large legumes as well as for other exogenous additives.

Estimation of stability of soybean root nodule bacterial strains to the recommended chemical fungicides

The work determines the resistance of various strains of soybean nodule bacteria to the recommended chemical fungicides in the tank solution applied to the seed surface. As experimental rhizobia there were studied such soybean bacteria of the Bradyrhizobium japonicum of the following strains as 634b, 640, BR-648, BR-733. The fungicides “Maksim”, “Protekt”, “Protekt Forte” were used as disinfectants. In the tank solution there was determined compatibility by mixing the biopreparation and the disinfectant, keeping the mixture at room temperature in the dark, with further determining percentage of the survived rhizobia depending on the disinfectant type and the keeping time of the solution. It has been established that different soya rhizobia strains do not practically differ in resistance to disinfectants in the tank solution. However, the disinfectants possess different degrees of toxicity to the studied strains, which made it possible to arrange the disinfectants due to their increasing toxicity for rhizobia “Maksim”, “Protekt”, “Protekt Forte”. Since the strains taken for the study practically did not differ in their resistance to disinfectants in the tank solution, there was investigated only the strain B. japonicum 634b to determine the dynamics of rhizobia death on the treated seeds. Determination of the disinfectants' toxicity was carried out by placing the washes from treated seeds on the plates with nutrient medium. The treated seeds were kept at room temperature in the dark. The selection of the treated seeds was carried out after 2, 4 and 8 hours after treatment. The nature of the curves for rhizobia reduction on seeds showed that “Protekt Forte” was the most toxic to bacteria on the seed surface, and “Maksim” was the most “soft”. This study will allow optimizing the process of soybean seeds inoculation with their simultaneous treatment with chemical disinfectants.

GmNMH7, a MADS-box transcription factor, inhibits root development and nodulation of soybean (Glycine max [L.] Merr.)

As an important food crop and oil crop, soybean (Glycine max [L.] Merr.) is capable of nitrogen-fixing by root nodule. Previous studies showed that GmNMH7, a transcription factor of MADS-box family, is associated with nodule development, but its specific function remained unknown. In this study, we found that GmNMH7 was specifically expressed in root and nodule and the expression pattern of GmNMH7 was similar to several genes involved in early development of nodule (GmENOD40-1, GmENOD40-2, GmNFR1a, GmNFR5a, and GmNIN) after rhizobia inoculation. The earlier expression peak of GmNMH7 compared to the other genes (GmENOD40-1, GmENOD40-2, GmNFR1a, GmNFR5a, and GmNIN) indicated that the gene is related to the nod factor (NF) signaling pathway and functions at the early development of nodule. Over-expression of GmNMH7 in hairy roots significantly reduced the nodule number and the root length. In the transgenic hairy roots, over-expression of GmNMH7 significantly down-regulated the expression levels of GmENOD40-1, GmENOD40-2, and GmNFR5α. Moreover, the expression of GmNMH7 could respond to abscisic acid (ABA) and gibberellin (GA3) treatment in the root of Zigongdongdou seedlings. Over-expressing GmNMH7 gene reduced the content of ABA, and increased the content of GA3 in the positive transgenic hairy roots. Therefore, we concluded that GmNMH7 might participate in the NF signaling pathway and negatively regulate nodulation probably through regulating the content of GA3.

Isolation, characterization and symbiotic performance evaluation of soybean (Glycine max) nodulating rhizobia from different districts of Bangladesh

Rhizobia can meet up nitrogen requirement of legumes by biological nitrogen fixation through symbiosis. The main objective of this study was to find out the morpho-physiological diversity of indigenous soybean nodulating rhizobia and to find effective rhizobial strains for enhancing better soybean production at different environmental conditions. Fourteen rhizobial strains were isolated and studied their morph-physiological characteristics; evaluated their nodulation and symbiotic efficiency at potted soil. Among the strains, seven were slow growing, four were intermediate to slow growing and three were fast growing. The strains exposed creamy, milky-white and transparent single colonies between 1.17 and 3.67 mm in diameter after 3 to 7 days on growth medium. Twelve strains were alkali producers and only two strains were acid producers. Most of them tolerated both acidic pH (4.0 and 5.0) and alkaline pH (9.0 and 10.0) conditions. All strains tolerated to 1.0% NaCl but none of them survived at 5.0% salt stress. They retained their normal growth up to 37oC but most of them showed growth susceptibility at 45oC and growth was inhibited at 50oC. The strains were inoculated as treatments on soybean plants to compare their performance on growth of soybean along with urea and control treatment. Overall, rhizobial treatments significantly increased nodulation and growth of soybean plants over recommended dose of urea and negative control. Diverse rhizobial strains were associated with soybean root nodules in Bangladesh. The strains SB-27, SB-28 and SB-212 were very effective and produced almost double plant dry matter weight over some other strains, dose of urea and negative control. The physical stress tolerant and highly symbiotic strains deserve to be effective as bio-fertilizer for soybean crop production.

Antitumor activity and ability to prevent acrylamide formation in fried foods of asparaginase from soybean root nodules.

A novel asparaginase (designated srnASNase) has been purified from soybean root nodules and identified by MALDI-TOF/TOF-MS. And the enzymatic properties, antitumor activity and the ability to prevent acrylamide formation in fried foods of srnASNase were evaluated. SrnASNase had high specific activity (531.37 U/mg) toward L-asparagine under optimum conditions (pH 8.0 and 40°C), no activity toward L-glutamine and D-glutamine, but trace activity toward D-asparagine. It was stable in the pH range of 7.0-9.0 and up to 40°C. The Km and Vmax of srnASNase were 0.36mM and 51.64mM/min, respectively. Further, in vitro anti-proliferative activity on human cancer cells assay showed that srnASNase was superior to commercial asparaginase in solution by controlling the tumor cell growth with time. In addition, srnASNase showed more effective acrylamide mitigation than commercial asparaginase in fried foods. These results indicate that srnASNase is a potential candidate for applications in the food processing and pharmaceutical industry. PRACTICAL APPLICATIONS: L-asparaginase (L-asparagine amidohydrolase; EC 3.5.1.1) is an enzyme that catalyzes the hydrolysis of the amide group of the side-chain of L-asparagine into aspartic acid and ammonia. It has long been used as a primary component in the treatment of acute lymphoblastic leukemia (All) and other related blood cancers. Apart from its clinical usage, L-asparaginase has attracted more attention in the food processing industries as a promising acrylamide-mitigating agent in recent years. This research revealed that soybean root nodules might be good sources of novel asparaginase.

Burkholderia and Paraburkholderia are Predominant Soybean Rhizobial Genera in Venezuelan Soils in Different Climatic and Topographical Regions.

The climate, topography, fauna, and flora of Venezuela are highly diverse. However, limited information is currently available on the characterization of soybean rhizobia in Venezuela. To clarify the physiological and genetic diversities of soybean rhizobia in Venezuela, soybean root nodules were collected from 11 soil types located in different topographical regions. A total of 395 root nodules were collected and 120 isolates were obtained. All isolates were classified in terms of stress tolerance under different concentrations of NaCl and Al3+. The tolerance levels of isolates to NaCl and Al3+ varied. Based on sampling origins and stress tolerance levels, 44 isolates were selected for further characterization. An inoculation test indicated that all isolates showed the capacity for root nodulation on soybean. Based on multilocus sequence typing (MLST), 20 isolates were classified into the genera Rhizobium and Bradyrhizobium. The remaining 24 isolates were classified into the genus Burkholderia or Paraburkholderia. There is currently no evidence to demonstrate that the genera Burkholderia and Paraburkholderia are the predominant soybean rhizobia in agricultural fields. Of the 24 isolates classified in (Para) Burkholderia, the nodD–nodB intergenic spacer regions of 10 isolates and the nifH gene sequences of 17 isolates were closely related to the genera Rhizobium and Bradyrhizobium, respectively. The root nodulation numbers of five (Para) Burkholderia isolates were higher than those of the 20 α-rhizobia. Furthermore, among the 44 isolates tested, one Paraburkholderia isolate exhibited the highest nitrogen-fixation activity in root nodules.

Backscattered electron imaging of high pressure frozen soybean root nodules visualizes formation of symbiosome membranes.

High-pressure frozen soybean root nodules were fractured and backscattered electron images were obtained from uncoated samples in a low vacuum scanning electron microscope equipped with a cryo-transfer system. Structures of infected cells were well preserved: numerous symbiosomes, as well as nuclei, plastids and mitochondria were observed without ice crystal damage. After appropriate sublimation of water, bacteria included in symbiosomes were visualized. Membrane accumulation near nuclei, and vesicles and tubular membranes, which possibly contribute to symbiosome membrane formation, could be observed in a near native state. The method promises to be widely applicable to visualize interaction between membranes in various biological systems.

Complete Genome Sequence of Bradyrhizobium ottawaense OO99T, an Efficient Nitrogen-Fixing Symbiont of Soybean.

We present the complete genome sequence of Bradyrhizobium ottawaense strain OO99T, a nitrogen-fixing bacterium from root nodules of soybean. The genome consists of a single 8.6-Mb chromosome and includes a symbiosis island. Genes involved in symbiotic nitrogen fixation, stress response, resistance to antibiotics, and toxic compounds were detected.

Metabolic Noise and Distinct Subpopulations Observed by Single Cell LAESI Mass Spectrometry of Plant Cells in situ.

Phenotypic variations and stochastic expression of transcripts, proteins, and metabolites in biological tissues lead to cellular heterogeneity. As a result, distinct cellular subpopulations emerge. They are characterized by different metabolite expression levels and by associated metabolic noise distributions. To capture these biological variations unperturbed, highly sensitive in situ analytical techniques are needed that can sample tissue embedded single cells with minimum sample preparation. Optical fiber-based laser ablation electrospray ionization mass spectrometry (f-LAESI-MS) is a promising tool for metabolic profiling of single cells under ambient conditions. Integration of this MS-based platform with fluorescence and brightfield microscopy provides the ability to target single cells of specific type and allows for the selection of rare cells, e.g., excretory idioblasts. Analysis of individual Egeria densa leaf blade cells (n = 103) by f-LAESI-MS revealed significant differences between the prespecified subpopulations of epidermal cells (n = 97) and excretory idioblasts (n = 6) that otherwise would have been masked by the population average. Primary metabolites, e.g., malate, aspartate, and ascorbate, as well as several glucosides were detected in higher abundance in the epidermal cells. The idioblasts contained lipids, e.g., PG(16:0/18:2), and triterpene saponins, e.g., medicoside I and azukisaponin I, and their isomers. Metabolic noise for the epidermal cells were compared to results for soybean (Glycine max) root nodule cells (n = 60) infected by rhizobia (Bradyrhizobium japonicum). Whereas some primary metabolites showed lower noise in the latter, both cell types exhibited higher noise for secondary metabolites. Post hoc grouping of epidermal and root nodule cells, based on the abundance distributions for certain metabolites (e.g., malate), enabled the discovery of cellular subpopulations characterized by different mean abundance values, and the magnitudes of the corresponding metabolic noise. Comparison of prespecified populations from epidermal cells of the closely related E. densa (n = 20) and Elodea canadensis (n = 20) revealed significant differences, e.g., higher sugar content in the former and higher levels of ascorbate in the latter, and the presence of species-specific metabolites. These results demonstrate that the f-LAESI-MS single cell analysis platform has the potential to explore cellular heterogeneity and metabolic noise for hundreds of tissue-embedded cells.

Proteomic and ecophysiological responses of soybean (Glycine max L.) root nodules to Pb and hg stress

BackgroundLead (Pb) and mercury (Hg) are persistent hazardous metals in industrially polluted soils which can be toxic in low quantities. Metal toxicity can cause changes at cellular and molecular level which should be studied for better understanding of tolerance mechanism in plants. Soybean (Glycine max L.) is an important oilseed crop of the world including India. Indian soils growing soybean are often contaminated by Pb and Hg. The aim of this study was to explore how soybean root nodule responds to Pb and Hg through proteomic and ecophysiological alterations in order to enhance tolerance to metal stress.ResultsSoybean plants were exposed to Pb (30 ppm PbCl2) and Hg (0.5 ppm HgCl2) to study histological, histochemical, biochemical and molecular response of N2-fixing symbiotic nodules. Both Pb and Hg treatment increased the level of oxidative stress in leaves and nodules. Chlorosis in leaves and morphological/anatomical changes in nodules were observed. Activities of ascorbate peroxidase, glutathione reductase and catalase were also modulated. Significant changes were observed in abundance of 76 proteins by Pb and Hg. Pb and Hg influenced abundance of 33 proteins (17 up and 16 down) and 43 proteins (33 up and 10 down), respectively. MS/MS ion search identified 55 proteins which were functionally associated with numerous cellular functions. Six crucial proteins namely catalase (CAT), allene oxide synthase (AOS), glutathione S-transferase (GST), calcineurin B like (CBL), calmodulin like (CML) and rapid alkalinisation factor (RAF) were selected for transcript abundance estimation. The qRT-PCR based real time expression exhibited a positive correlation with proteomics expression except for GST and RAF.ConclusionSoybean root nodule responds to metal stress by increased abundance of defence, development and repair related proteins. An efficient proteomic modulation might lead to metal-induced stress tolerance in N2-fixing nodules. Although concentrations of Pb and Hg used in the study cannot be considered equimolar, yet Hg seems to induce more changes in nodule proteomic profile, and higher damage to both bacteroides and root anatomy.

Wound-induced polypeptides improve resistance against Pseudomonas syringae pv. tomato DC3000 in Arabidopsis

Wound-induced polypeptides (WIPs) are a novel class of polypeptides with the length less than 100 amino acids. Our previous research has identified a number of WIP genes in soybean (Glycine max) root nodules. However, functions of WIPs in planta remains largely unknown. Here, we identified five WIP-encoding genes, AtWIP1-5, in Arabidopsis. Among them, AtWIP1 and -2 are ubiquitously expressed in a partially overlapping pattern as revealed by both qRT-PCR and promoter:GUS assays. Subcellular localization analyses reveal that both AtWIP1 and -2 are localized at the plasma membrane while AtWIP1 shows a punctate distribution pattern. AtWIP1, -2 are transcriptionally induced by flg22 treatment, but repressed by effector(s) of Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Heterologous overexpression of GmWIP genes enhances resistance of Arabidopsis to Pst DC3000 at the cost of growth inhibition. Moreover, overexpression of GmWIP genes promotes pattern-triggered immunity (PTI) evidenced by increased expressions of flg22-inducible genes and enhanced seedling growth inhibition under flg22 treatment. Taken together, our results indicate that WIPs positively regulate plant resistance against Pst DC3000 by enhancing PTI responses.