N2-Acetylornithine deacetylase functions as a Cys-Gly dipeptidase in the cytosolic glutathione degradation pathway in Arabidopsis thaliana.

Glutathione (GSH) is required for various physiological processes in plants, including redox regulation and detoxification of harmful compounds. GSH also functions as a repository for assimilated sulfur and is actively catabolized in plants. In Arabidopsis, GSH is mainly degraded initially by cytosolic enzymes, γ-glutamyl cyclotransferase, and γ-glutamyl peptidase, which release cysteinylglycine (Cys-Gly). However, the subsequent enzyme responsible for catabolizing this dipeptide has not been identified to date. In the present study, we identified At4g17830 as a Cys-Gly dipeptidase, namely cysteinylglycine peptidase 1 (CGP1). CGP1 complemented the phenotype of the yeast mutant that cannot degrade Cys-Gly. The Arabidopsis cgp1 mutant had lower Cys-Gly degradation activity than the wild type and showed perturbed concentrations of thiol compounds. Recombinant CGP1 showed reasonable Cys-Gly degradation activity in vitro. Metabolomic analysis revealed that cgp1 exhibited signs of severe sulfur deficiency, such as elevated accumulation of O-acetylserine (OAS) and the decrease in sulfur-containing metabolites. Morphological changes observed in cgp1, including longer primary roots of germinating seeds, were also likely associated with sulfur starvation. Notably, At4g17830 has previously been reported to encode an N2-acetylornithine deacetylase (NAOD) that functions in the ornithine biosynthesis. The cgp1 mutant did not show a decrease in ornithine content, whereas the analysis of CGP1 structure did not rule out the possibility that CGP1 has Cys-Gly dipeptidase and NAOD activities. Therefore, we propose that CGP1 is a Cys-Gly dipeptidase that functions in the cytosolic GSH degradation pathway and may play dual roles in GSH and ornithine metabolism.

Involvement of Peptidoglycan Receptor Proteins in Mediating the Growth-Promoting Effects of Bacillus pumilus TUAT1 in Arabidopsis thaliana.

Bacillus pumilus TUAT1 acts as plant growth-promoting rhizobacteria for various plants like rice and Arabidopsis. Under stress conditions, B. pumilus TUAT1 forms spores with a thick peptidoglycan (PGN) cell wall. Previous research showed that spores were significantly more effective than vegetative cells in enhancing plant growth. In Arabidopsis, lysin motif proteins, LYM1, LYM3 and CERK1, are required for recognizing bacterial PGNs to mediate immunity. Here, we examined the involvement of PGN receptor proteins in the plant growth promotion (PGP) effects of B. pumilus TUAT1 using Arabidopsis mutants defective in PGN receptors. Root growth of wild-type (WT), cerk1-1, lym1-1 and lym1-2 mutant plants was significantly increased by TUAT1 inoculation, but this was not the case for lym3-1 and lym3-2 mutant plants. RNA-seq analysis revealed that the expression of a number of defense-related genes was upregulated in lym3 mutant plants. These results suggested that B. pumilus TUAT1 may act to reduce the defense response, which is dependent on a functional LYM3. The expression of the defense-responsive gene, WRKY29, was significantly induced by the elicitor flg-22, in both WT and lym3 mutant plants, while this induction was significantly reduced by treatment with B. pumilus TUAT1 and PGNs in WT, but not in lym3 mutant plants. These findings suggest that the PGNs of B. pumilus TUAT1 may be recognized by the LYM3 receptor protein, suppressing the defense response, which results in plant growth promotion in a trade-off between defense and growth.

Retrograde sulfur flow from glucosinolates to cysteine in Arabidopsis thaliana.

Specialized (secondary) metabolic pathways in plants have long been considered one-way routes of leading primary metabolite precursors to bioactive end products. Conversely, endogenous degradation of such "end" products in plant tissues has been observed following environmental stimuli, including nutrition stress. Therefore, it is of general interest whether specialized metabolites can be reintegrated into primary metabolism to recover the invested resources, especially in the case of nitrogen- or sulfur-rich compounds. Here, we demonstrate that endogenous glucosinolates (GLs), a class of sulfur-rich plant metabolites, are exploited as a sulfur source by the reallocation of sulfur atoms to primary metabolites such as cysteine in Arabidopsis thaliana Tracer experiments using 34S- or deuterium-labeled GLs depicted the catabolic processing of GL breakdown products in which sulfur is mobilized from the thioglucoside group in GL molecules, potentially accompanied by the release of the sulfate group. Moreover, we reveal that beta-glucosidases BGLU28 and BGLU30 are the major myrosinases that initiate sulfur reallocation by hydrolyzing particular GL species, conferring sulfur deficiency tolerance in A. thaliana, especially during early development. The results delineate the physiological function of GL as a sulfur reservoir, in addition to their well-known functions as defense chemicals. Overall, our findings demonstrate the bidirectional interaction between primary and specialized metabolism, which enhances our understanding of the underlying metabolic mechanisms via which plants adapt to their environments.

Glutathione degradation activity of γ-glutamyl peptidase 1 manifests its dual roles in primary and secondary sulfur metabolism in Arabidopsis.

Glutathione (GSH) functions as a major sulfur repository and hence occupies an important position in primary sulfur metabolism. GSH degradation results in sulfur reallocation and is believed to be carried out mainly by γ-glutamyl cyclotransferases (GGCT2;1, GGCT2;2, and GGCT2;3), which, however, do not fully explain the rapid GSH turnover. Here, we discovered that γ-glutamyl peptidase 1 (GGP1) contributes to GSH degradation through a yeast complementation assay. Recombinant proteins of GGP1, as well as GGP3, showed high degradation activity of GSH, but not of oxidized glutathione (GSSG), in vitro. Notably, the GGP1 transcripts were highly abundant in rosette leaves, in agreement with the ggp1 mutants constantly accumulating more GSH regardless of nutritional conditions. Given the lower energy requirements of the GGP- than the GGCT-mediated pathway, the GGP-mediated pathway could be a more efficient route for GSH degradation than the GGCT-mediated pathway. Therefore, we propose a model wherein cytosolic GSH is degraded chiefly by GGP1 and likely also by GGP3. Another noteworthy fact is that GGPs are known to process GSH conjugates in glucosinolate and camalexin synthesis; indeed, we confirmed that the ggp1 mutant contained higher levels of O-acetyl-l-Ser, a signaling molecule for sulfur starvation, and lower levels of glucosinolates and their degradation products. The predicted structure of GGP1 further provided a rationale for this hypothesis. In conclusion, we suggest that GGP1 and possibly GGP3 play vital roles in both primary and secondary sulfur metabolism.

Degradation of glutathione and glutathione conjugates in plants.

Glutathione (GSH) is a ubiquitous, abundant, and indispensable thiol for plants that participates in various biological processes, such as scavenging reactive oxygen species, redox signaling, storage and transport of sulfur, detoxification of harmful substances, and metabolism of several compounds. Therefore knowledge of GSH metabolism is essential for plant science. Nevertheless, GSH degradation has been insufficiently elucidated, and this has hampered our understanding of plant life. Over the last five decades, the γ-glutamyl cycle has been dominant in GSH studies, and the exoenzyme γ-glutamyl transpeptidase has been regarded as the major GSH degradation enzyme. However, recent studies have shown that GSH is degraded in cells by cytosolic enzymes such as γ-glutamyl cyclotransferase or γ-glutamyl peptidase. Meanwhile, a portion of GSH is degraded after conjugation with other molecules, which has also been found to be carried out by vacuolar γ-glutamyl transpeptidase, γ-glutamyl peptidase, or phytochelatin synthase. These findings highlight the need to re-assess previous assumptions concerning the γ-glutamyl cycle, and a novel overview of the plant GSH degradation pathway is essential. This review aims to build a foundation for future studies by summarizing current understanding of GSH/glutathione conjugate degradation.

Phytochelatin-mediated metal detoxification pathway is crucial for an organomercurial phenylmercury tolerance in Arabidopsis.

KEY MESSAGE: An organomercurial phenylmercury activates AtPCS1, an enzyme known for detoxification of inorganic metal(loid) ions in Arabidopsis and the induced metal-chelating peptides phytochelatins are essential for detoxification of phenylmercury. Small thiol-rich peptides phytochelatins (PCs) and their synthases (PCSs) are crucial for plants to mitigate the stress derived from various metal(loid) ions in their inorganic form including inorganic mercury [Hg(II)]. However, the possible roles of the PC/PCS system in organic mercury detoxification in plants remain elusive. We found that an organomercury phenylmercury (PheHg) induced PC synthesis in Arabidopsis thaliana plants as Hg(II), whereas methylmercury did not. The analyses of AtPCS1 mutant plants and in vitro assays using the AtPCS1-recombinant protein demonstrated that AtPCS1, the major PCS in A. thaliana, was responsible for the PheHg-responsive PC synthesis. AtPCS1 mutants cad1-3 and cad1-6, and the double mutant of PC-metal(loid) complex transporters AtABCC1 and AtABCC2 showed enhanced sensitivity to PheHg as well as to Hg(II). The hypersensitivity of cad1-3 to PheHg stress was complemented by the own-promoter-driven expression of AtPCS1-GFP. The confocal microscopy of the complementation lines showed that the AtPCS1-GFP was preferentially expressed in epidermal cells of the mature and elongation zones, and the outer-most layer of the lateral root cap cells in the meristematic zone. Moreover, in vitro PC-metal binding assay demonstrated that binding affinity between PC and PheHg was comparable to Hg(II). However, plant ionomic profiles, as well as root morphology under PheHg and Hg(II) stress, were divergent. These results suggest that PheHg phytotoxicity is different from Hg(II), but AtPCS1-mediated PC synthesis, complex formation, and vacuolar sequestration by AtABCC1 and AtABCC2 are similarly functional for both PheHg and Hg(II) detoxification in root surficial cell types.

Effects of Thiosulfate as a Sulfur Source on Plant Growth, Metabolites Accumulation and Gene Expression in Arabidopsis and Rice.

Plants are considered to absorb sulfur from their roots in the form of sulfate. In bacteria like Escherichia coli, thiosulfate is a preferred sulfur source. It is converted into cysteine (Cys). This transformation consumes less NADPH and ATP than sulfate assimilation into Cys. In Saccharomyces cerevisiae, thiosulfate promoted growth more than sulfate. In the present study, the availability of thiosulfate, the metabolite transformations and gene expressions it induces were investigated in Arabidopsis and rice as model dicots and monocots, respectively. In Arabidopsis, the thiosulfate-amended plants had lower biomass than those receiving sulfate when sulfur concentrations in the hydroponic medium were above 300 µM. In contrast, rice biomass was similar for plants raised on thiosulfate and sulfate at 300 µM sulfur. Therefore, both plants can use thiosulfate but it is a better sulfur source for rice. In both plants, thiosulfate levels significantly increased in roots following thiosulfate application, indicating that the plants absorbed thiosulfate into their root cells. Thiosulfate is metabolized in plants by a different pathway from that used for sulfate metabolism. Thiosulfate increases plant sulfide and cysteine persulfide levels which means that plants are in a more reduced state with thiosulfate than with sulfate. The microarray analysis of Arabidopsis roots revealed that 13 genes encoding Cys-rich proteins were upregulated more with thiosulfate than with sulfate. These results together with those of the widely targeted metabolomics analysis were used to proposes a thiosulfate assimilation pathway in plants.

AtOPT6 Protein Functions in Long-Distance Transport of Glutathione in Arabidopsis thaliana.

The involvement of the Arabidopsis oligopeptide transporter AtOPT6, which was previously shown to take up glutathione (GSH) when expressed in yeast cells or in Xenopus laevis oocytes, in GSH transport was analyzed using opt6 knockout mutant lines. The concentration of GSH in flowers or siliques was lower in opt6 mutants relative to wild-type plants, suggesting involvement of AtOPT6 in long-distance transport of GSH. The GSH concentration in phloem sap was similar between opt6 mutants and wild-type plants. These results, combined with earlier reports showing expression of AtOPT6 in the vascular bundle, especially in the cambial zone, suggest that AtOPT6 functions to transport GSH into cells surrounding the phloem in sink organs. The opt6 mutant plants showed delayed bolting, implying the importance of AtOPT6 for regulation of the transition from vegetative to reproductive growth. After cadmium (Cd) treatment, the concentration of the major phytochelatin PC2 was lower in flowers in the opt6 mutants and Cd was accumulated in roots of opt6 mutant plants compared with wild-type plants. These results suggest that AtOPT6 is likely to be involved in transporting GSH, PCs and Cd complexed with these thiols into sink organs.

Identification of C-terminal Regions in Arabidopsis thaliana Phytochelatin Synthase 1 Specifically Involved in Activation by Arsenite.

Phytochelatins (PCs) are major chelators of toxic elements including inorganic arsenic (As) in plant cells. Their synthesis confers tolerance and influences within-plant mobility. Previous studies had shown that various metal/metalloid ions differentially activate PC synthesis. Here we identified C-terminal parts involved in arsenite- [As(III)] dependent activation of AtPCS1, the primary Arabidopsis PC synthase. The T-DNA insertion in the AtPCS1 mutant cad1-6 causes a truncation in the C-terminal regulatory domain that differentially affects activation by cadmium (Cd) and zinc (Zn). Comparisons of cad1-6 with the AtPCS1 null mutant cad1-3 and the double mutant of tonoplast PC transporters abcc1/2 revealed As(III) hypersensitivity of cad1-6 equal to that of cad1-3. Both cad1-6 and cad1-3 showed increased As distribution to shoots compared with Col-0, whereas Zn accumulation in shoots was equally lower in cad1-6 and cad1-3. Supporting these phenotypes of cad1-6, PC accumulation in the As(III)-exposed plants were at trace level in both cad1-6 and cad1-3, suggesting that the truncated AtPCS1 of cad1-6 is defective in PCS activity in response to As(III). Analysis of a C-terminal deletion series of AtPCS1 using the PCS-deficient mutant of fission yeast suggested important regions within the C-terminal domain for As(III)-dependent PC synthesis, which were different from the regions previously suggested for Cd- or Zn-dependent activation. Interestingly, we identified a truncated variant more strongly activated than the wild-type protein. This variant could potentially be used as a tool to better restrict As mobility in plants.

In vitro rhizobia response and symbiosis process under aluminum stress.

Aluminum (Al) toxicity is a major problem affecting soil fertility, microbial diversity, and nutrient uptake of plants. Rhizobia response and legume interaction under Al conditions are still unknown; it is important to understand how to develop and improve legume cultivation under Al stress. In this study, rhizobia response was recorded under different Al concentrations. Al effect on rhizobial cells was characterized by combination with different two pH conditions. Symbiosis process was compared between α- and ß-rhizobia inoculated onto soybean varieties. Rhizobial cell numbers was decreased as Al concentration increased. However, induced Al tolerance considerably depended on rhizobia types and their origins. Accordingly, organic acid results were in correlation with growth rate and cell density which suggested that citric acid might be a positive selective force for Al tolerance and plant interaction on rhizobia. Al toxicity delayed and interrupted the plant-rhizobia interaction and the effect was more pronounced under acidic conditions. Burkholderia fungorum VTr35 significantly improved plant growth under acid-Al stress in combination with all soybean varieties. Moreover, plant genotype was an important factor to establish an effective nodulation and nitrogen fixation under Al stress. Additionally, tolerant rhizobia could be applied as an inoculant on stressful agroecosystems. Furthermore, metabolic pathways have still been unknown under Al stress.

Phytochelatin Synthase has Contrasting Effects on Cadmium and Arsenic Accumulation in Rice Grains.

Phytochelatin (PC) synthesis has been well demonstrated as a major metal tolerance mechanism in Arabidopsis thaliana, whereas its contribution to long-distance element transport especially in monocots remains elusive. Using rice as a cereal model, we examined physiological roles of Oryza sativa phytochelatin synthase 1 (OsPCS1) in the distribution and detoxification of arsenic (As) and cadmium (Cd), two toxic elements associated with major food safety concerns. First, we isolated four different transcript variants of OsPCS1 as well as one from OsPCS2. Quantitative real-time reverse transcription-PCR (RT-PCR) of each OsPCS transcript in rice seedlings suggested that expression of OsPCS1full, the longest OsPCS1 variant, was most abundant, followed by OsPCS2. Heterologous expression of OsPCS variants in PCS-deficient mutants of Schizosaccharomyces pombe and A. thaliana suggested that OsPCS1full possessed PCS activity in response to As(III) and Cd while the activity of other PCS variants was very low. To address physiological functions in toxic element tolerance and accumulation, two independent OsPCS1 mutant rice lines (a T-DNA and a Tos17 insertion line) were identified. The OsPCS1 mutants exhibited increased sensitivity to As(III) and Cd in hydroponic experiments, showing the importance of OsPCS1-dependent PC synthesis for rice As(III) and Cd tolerance. Elemental analyses of rice plants grown in soil with environmentally relevant As and Cd concentrations showed increased As accumulation and decreased Cd accumulation in grains of the T-DNA line. The Tos17 mutant also exhibited the reduced Cd accumulation phenotype. These contrasting effects on As and Cd distribution to grains suggest the existence of at least partially distinct PC-dependent pathways for As and Cd.

Post-Translational Regulation of the Dicing Activities of Arabidopsis DICER-LIKE 3 and 4 by Inorganic Phosphate and the Redox State.

In Arabidopsis thaliana, small interfering RNAs (siRNAs) generated by two Dicer isoforms, DCL3 and DCL4, function in distinct epigenetic processes, i.e. RNA-directed DNA methylation and post-transcriptional gene silencing, respectively. Plants often respond to their environment by producing a distinct set of small RNAs; however, the mechanism for controlling the production of different siRNAs from the same dsRNA substrate remains unclear. We established a simple biochemical method to visualize the dsRNA-cleaving activities of DCL3 and DCL4 in cell-free extracts prepared from Arabidopsis seedlings. Here, we demonstrate that different nutrient statuses of a host plant affect the post-translational regulation of the dicing activity of DCL3 and DCL4. Phosphate deficiency inhibited DCL3, and the activity of DCL3 was directly activated by inorganic phosphate. Sulfur deficiency inhibited DCL4 but not DCL3, and the activity of DCL4 was recovered by supplementation of the cell-free extracts with reductants containing a thiol group. Immunopurified DCL4 was activated by recombinant Arabidopsis thioredoxin-h1 with dithiothreitol. Therefore, DCL4 is subject to redox regulation. These results demonstrate that post-translational regulation of DCL activities fine-tunes the balance between branches of the gene silencing pathway according to the growth environment.

Effects of Cadmium Treatment on the Uptake and Translocation of Sulfate in Arabidopsis thaliana.

Cadmium (Cd) is a highly toxic and non-essential element for plants, whereas phytochelatins and glutathione are low-molecular-weight sulfur compounds that function as chelators and play important roles in detoxification. Cadmium exposure is known to induce the expression of sulfur-assimilating enzymes and sulfate uptake by roots. However, the molecular mechanism underlying Cd-induced changes remains largely unknown. Accordingly, we analyzed the effects of Cd treatment on the uptake and translocation of sulfate and accumulation of thiols in Arabidopsis thaliana Both wild type (WT) and null mutant (sel1-10 and sel1-18) plants of the sulfate transporter SULTR1;2 exhibited growth inhibition when treated with CdCl2 However, the mutant plants exhibited a lower growth rate and lower Cd accumulation. Cadmium treatment also upregulated the transcription of SULTR1;2 and sulfate uptake activity in WT plants, but not in mutant plants. In addition, the sulfate, phytochelatin and total sulfur contents were preferentially accumulated in the shoots of both WT and mutant plants treated with CdCl2, and sulfur K-edge XANES spectra suggested that sulfate was the main compound responsible for the increased sulfur content in the shoots of CdCl2-treated plants. Our results demonstrate that Cd-induced sulfate uptake depends on SULTR1;2 activity, and that CdCl2 treatment greatly shifts the distribution of sulfate to shoots, increases the sulfate concentration of xylem sap and upregulates the expression of SULTRs involved in root-to-shoot sulfate transport. Therefore, we conclude that root-to-shoot sulfate transport is stimulated by Cd and suggest that the uptake and translocation of sulfate in CdCl2-treated plants are enhanced by demand-driven regulatory networks.

Evaluation of the possibility to use the plant-microbe interaction to stimulate radioactive 137Cs accumulation by plants in a contaminated farm field in Fukushima, Japan.

Field experiments in a contaminated farmland in Nihonmatsu city, Fukushima were conducted to assess the effectiveness of the plant-microbe interaction on removal of radiocesium. Before plowing, 93.3% of radiocesium was found in the top 5 cm layer (5,718 Bq kg DW(-1)). After plowing, Cs radioactivity in the 0-15 cm layer ranged from 2,037 to 3,277 Bq kg DW(-1). Based on sequential extraction, the percentage of available radiocesium (water soluble + exchangeable) was fewer than 10% of the total radioactive Cs. The transfer of (137)Cs was investigated in three agricultural crops; komatsuna (four cultivars), Indian mustard and buckwheat, inoculated with a Bacillus or an Azospirillum strains. Except for komatsuna Nikko and Indian mustard, inoculation with both strains resulted in an increase of biomass production by the tested plants. The highest (137)Cs radioactivity concentration in above-ground parts was found in Bacillus-inoculated komatsuna Nikko (121 Bq kg DW(-1)), accompanied with the highest (137)Cs TF (0.092). Furthermore, komatsuna Nikko-Bacillus and Indian mustard-Azospirillum associations gave the highest (137)Cs removal, 131.5 and 113.8 Bq m(-2), respectively. Despite the beneficial effect of inoculation, concentrations of (137)Cs and its transfer to the tested plants were not very high; consequently, removal of (137)Cs from soil would be very slow.

Stable cesium uptake and accumulation capacities of five plant species as influenced by bacterial inoculation and cesium distribution in the soil.

The effects of inoculation with Bacillus and Azospirillum strains on growth and cesium accumulation of five plant species, Komatsuna, Amaranth, sorghum, common millet and buckwheat, grown on cesium-spiked soil were assessed for potential use in cesium remediation. Pot experiments were performed using "artificially" Cs-contaminated soil. Three treatments were applied based on Cs location in the soil. For a soil height of 15 cm in the pots, Cs was added as follows: in the top five cm to imitate no ploughing condition; in the bottom five cm simulating inverted ploughing; and uniformly distributed Cs reproducing normal plowing. Generally, inoculation of Cs-exposed plants significantly enhanced growth and tolerance to this element. Transfer factor (ratio of Cs concentration in the plant tissues to that in surrounding soil) was strongly influenced by Cs distribution, with higher values in the top-Cs treatment. Within this treatment, inoculation of Komatsuna with Bacillus and Azospirillum strains resulted in the greatest transfer factors of 6.55 and 6.68, respectively. Cesium content in the shoots was high in the Azospirillum-inoculated Komatsuna, Amaranth, and buckwheat, i.e., 1,830, 1,220, and 1,030 µg per pot, respectively (five plants were grown in each pot). Therefore, inoculation of Komatsuna and Amaranth with the strains tested here could be effective in enhancing Cs accumulation. The decrease of Cs transfer under uniform- and bottom-Cs treatments would suggest that countermeasures aiming at decreasing the transfer of Cs could rely on ploughing practices.

Effect of Bacterial Extracellular Polymeric Substances from Enterobacter spp. on Rice Growth under Abiotic Stress and Transcriptomic Analysis.

Plant biostimulants have received attention as sustainable alternatives to chemical fertilizers. Extracellular polymeric substances (EPSs), among the compounds secreted by plant growth-promoting rhizobacteria (PGPRs), are assumed to alleviate abiotic stress. This study aims to investigate the effect of purified EPSs on rice under abiotic stress and analyze their mechanisms. A pot experiment was conducted to elucidate the effects of inoculating EPSs purified from PGPRs that increase biofilm production in the presence of sugar on rice growth in heat-stress conditions. Since all EPSs showed improvement in SPAD after the stress, Enterobacter ludwigii, which was not characterized as showing higher PGP bioactivities such as phytohormone production, nitrogen fixation, and phosphorus solubilization, was selected for further analysis. RNA extracted from the embryos of germinating seeds at 24 h post-treatment with EPSs or water was used for transcriptome analysis. The RNA-seq analysis revealed 215 differentially expressed genes (DEGs) identified in rice seeds, including 139 up-regulated and 76 down-regulated genes. A gene ontology (GO) enrichment analysis showed that the enriched GO terms are mainly associated with the ROS scavenging processes, detoxification pathways, and response to oxidative stress. For example, the expression of the gene encoding OsAAO5, which is known to function in detoxifying oxidative stress, was two times increased by EPS treatment. Moreover, EPS application improved SPAD and dry weights of shoot and root by 90%, 14%, and 27%, respectively, under drought stress and increased SPAD by 59% under salt stress. It indicates that bacterial EPSs improved plant growth under abiotic stresses. Based on our results, we consider that EPSs purified from Enterobacter ludwigii can be used to develop biostimulants for rice.

Diversity of Fast-Growth Spore-Forming Microbes and Their Activity as Plant Partners.

Biofertilizers are agricultural materials capable of reducing the usage amounts of chemical fertilizers. Spore-forming microorganisms (SFM) could be used for plant growth promotion or to improve plant health. Until now, biofertilizers based on SFM have been applied for rice and other crops. In this study, we isolated and characterized SFM, which were colonized on the Oryza sativa L. roots. SFM were analyzed regarding the short-term effects of biofertilization on the nursery growths. Analysis was performed without nitrogen or any inorganic fertilizer and was divided into two groups, including bacteria and fungi. SF-bacteria were dominated by the Firmicutes group, including species from Viridibacillus, Lysinibacillus, Solibacillus, Paenibacillus, Priestia, and mainly Bacillus (50%). The fungi group was classified as Mucoromycota, Basidiomycota, and mainly Ascomycota (80%), with a predominance of Penicillium and Trichoderma species. In plant performance in comparison with B. pumilus TUAT1, some bacteria and fungus isolates significantly improved the early growth of rice, based on 48 h inoculum with 107 CFU mL-1. Furthermore, several SFM showed positive physiological responses under abiotic stress or with limited nutrients such as phosphorous (P). Moreover, the metabolic fingerprint was obtained. The biofertilizer based on SFM could significantly reduce the application of the inorganic fertilizer and improve the lodging resistances of rice, interactively enhancing better plant health and crop production.

The Application of Sulfur Influences Microbiome of Soybean Rhizosphere and Nutrient-Mobilizing Bacteria in Andosol.

This study aimed to determine the effect of sulfur (S) application on a root-associated microbial community resulting in a rhizosphere microbiome with better nutrient mobilizing capacity. Soybean plants were cultivated with or without S application, the organic acids secreted from the roots were compared. High-throughput sequencing of 16S rRNA was used to analyze the effect of S on microbial community structure of the soybean rhizosphere. Several plant growth-promoting bacteria (PGPB) isolated from the rhizosphere were identified that can be harnessed for crop productivity. The amount of malic acid secreted from the soybean roots was significantly induced by S application. According to the microbiota analysis, the relative abundance of Polaromonas, identified to have positive association with malic acid, and arylsulfatase-producing Pseudomonas, were increased in S-applied soil. Burkholderia sp. JSA5, obtained from S-applied soil, showed multiple nutrient-mobilizing traits among the isolates. In this study, S application affected the soybean rhizosphere bacterial community structure, suggesting the contribution of changing plant conditions such as in the increase in organic acid secretion. Not only the shift of the microbiota but also isolated strains from S-fertilized soil showed PGPB activity, as well as isolated bacteria that have the potential to be harnessed for crop productivity.

Plant Growth-promoting Effects of Viable and Dead Spores of Bacillus pumilus TUAT1 on Setaria viridis.

Spores are a stress-resistant form of Bacillus spp., which include species that are plant growth-promoting rhizobacteria (PGPR). Previous studies showed that the inoculation of plants with vegetative cells or spores exerted different plant growth-promoting effects. To elucidate the spore-specific mechanism, we compared the effects of viable vegetative cells, autoclaved dead spores, and viable spores of Bacillus pumilus TUAT1 inoculated at 107 CFU plant-1 on the growth of the C4 model plant, Setaria viridis A10.1. B. pumilus TUAT1 spores exerted stronger growth-promoting effects on Setaria than on control plants 14 days after the inoculation. Viable spores increased shoot weight, root weight, shoot length, root length, and nitrogen uptake efficiency 21 days after the inoculation. These increases involved primary and crown root formation. Additionally, autoclaved dead spores inoculated at 108 or 109 CFU plant-1 had a positive impact on crown root differentiation, which increased total lateral root length, resulting in a greater biomass and more efficient nitrogen uptake. The present results indicate that an inoculation with viable spores of B. pumilus TUAT1 is more effective at enhancing the growth of Setaria than that with vegetative cells. The plant response to dead spores suggests that the spore-specific plant growth-promoting mechanism is at least partly independent of symbiotic colonization.

Genetic and Physiological Characterization of Soybean-Nodule-Derived Isolates from Bangladeshi Soils Revealed Diverse Array of Bacteria with Potential Bradyrhizobia for Biofertilizers.

Genetic and physiological characterization of bacteria derived from nodules of leguminous plants in the exploration of biofertilizer is of paramount importance from agricultural and environmental perspectives. Phylogenetic analysis of the 16S rRNA gene of 84 isolates derived from Bangladeshi soils revealed an unpredictably diverse array of nodule-forming and endosymbiotic bacteria-mostly belonging to the genus Bradyrhizobium. A sequence analysis of the symbiotic genes (nifH and nodD1) revealed similarities with the 16S rRNA gene tree, with few discrepancies. A phylogenetic analysis of the partial rrn operon (16S-ITS-23S) and multi-locus sequence analysis of atpD, glnII, and gyrB identified that the Bradyrhizobium isolates belonged to Bradyrhizobium diazoefficiens, Bradyrhizobium elkanii, Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense species. In the pot experiment, several isolates showed better activity than B. diazoefficiens USDA110, and the Bho-P2-B2-S1-51 isolate of B. liaoningense showed significantly higher acetylene reduction activity in both Glycine max cv. Enrei and Binasoybean-3 varieties and biomass production increased by 9% in the Binasoybean-3 variety. Tha-P2-B1-S1-68 isolate of B. diazoefficiens significantly enhanced shoot length and induced 10% biomass production in Binasoybean-3. These isolates grew at 1-4% NaCl concentration and pH 4.5-10 and survived at 45 °C, making the isolates potential candidates for eco-friendly soybean biofertilizers in salty and tropical regions.