Mechanistic elucidation of germination potential and growth of Sesbania sesban seedlings with Bacillus anthracis PM21 under heavy metals stress: An in vitro study.

Soils contaminated with heavy metals such as Chromium (Cr) and Cadmium (Cd) severely impede plant growth. Several rhizospheric microorganisms support plant growth under heavy metal stress. In this study, Cr and Cd stress was applied to in vitro germinating seedlings of a Legume plant species, Sesbania sesban, and investigated the plant growth potential in presence and absence of Bacillus anthracis PM21 bacterial strain under heavy metal stress. The seedlings were exposed to different concentrations of Cr (25-75 mg/L) and Cd (100-200 mg/L) in Petri plates. Growth curve analysis of B. anthracis PM21 revealed its potential to adapt Cr and Cd stress. The bacteria supported plant growth by exhibiting ACC-deaminase activity (1.57-1.75 µM of α-ketobutyrate/h/mg protein), producing Indole-3-acetic acid (99-119 µM/mL) and exopolysaccharides (2.74-2.98 mg/mL), under heavy metal stress condition. Analysis of variance revealed significant differences in growth parameters between the seedlings with and without bacterial inoculation in metal stress condition. The combined Cr+Cd stress (75 + 200 mg/L) significantly reduced root length (70%), shoot length (24%), dry weight (54%) and fresh weight (57%) as compared to control. Conversely, B. anthracis PM21 inoculation to seedlings significantly increased (p ≤ 0.05) seed germination percentage (5%), root length (31%), shoot length (23%) and photosynthetic pigments (Chlorophyll a: 20%; Chlorophyll b: 16% and total chlorophyll: 18%), as compared to control seedlings without B. anthracis PM21 inoculation. The B. anthracis PM21 inoculation also enhanced activities of antioxidant enzymes, including superoxide dismutase (52%), peroxidase (66%), and catalase (21%), and decreased proline content (56%), electrolyte leakage (50%), and malondialdehyde concentration (46%) in seedlings. The B. anthracis PM21 inoculated seedlings of S. sesban exhibited significantly high (p ≤ 0.05) tissue deposition of Cr (17%) and Cd (16%) as compared to their control counterparts. Findings of the study suggested that B. anthracis PM21 endured metal stress through homeostasis of antioxidant activities, and positively impacted S. sesban growth and biomass. Further experiments in controlled conditions are necessary for investigating phytoremediation potential of S. sesban in metal-contaminated soils in presence of B. anthracis PM21 bacterial strain.

CRISPR/Cas9 genome editing shows the important role of AZC_2928 gene in nitrogen-fixing bacteria of plants.

AZC_2928 gene (GenBank accession no. BAF88926.1) of Azorhizobium caulinodans ORS571 has sequence homology to 2,3-aminomutases. However, its function is unknown. In this study, we are for the first time to knock out the gene completely in A. caulinodans ORS571 using the current advanced genome editing tool, CRISPR/Cas9. Our results show that the editing efficiency is 34% and AZC_2928 plays an extremely important role in regulating the formation of chemotaxis and biofilm. CRISPR/Cas9 knockout of AZC_2928 (△AZC_2928) significantly enhanced chemotaxis and biofilm formation. Both chemotaxis and biofilm formation play an important role in nitrogen-fixing bacteria and their interaction with their host plants. Interestingly, AZC_2928 did not affect the motility of A. caulinodans ORS571 and the nodulation formation in their natural host plant, Sesbania rostrata. Due to rhizobia needing to form bacteroids for symbiotic nitrogen fixation in mature nodules, AZC_2928 might have a direct influence on nitrogen fixation efficiency rather than the number of nodulations.

Assessment of chromium phytotoxicity, phytoremediation and tolerance potential of Sesbania sesban and Brachiaria mutica grown on chromite mine overburden dumps and garden soil.

The addition of toxic chromium in agrarian soils from mine overburden dump is of serious concern. To combat the toxicity of chromium an experiment was carried out at the Department of Botany, Utkal University, India by taking the chromium rich overburden dump (OBD) from Sukinda chromite mine, India. Different proportions of chromium rich OBD (10%, 20%, 30%, 40%, 50%, 70% and 100% OBD) were supplied to garden soil and plants of legume and non-legume categories (Sesbania sesban L. and Brachiaria mutica L.) were grown for assessment of phytoaccumulation ability of chromium, tolerance index, chlorophyll, protein and proline and the activity of oxidative inhibitors enzymes. After 60 days of experimentation, Cr phytotoxicity on physiological and biochemical parameters were assessed. The outcome of the results showed that the activity of antioxidant enzymes enhanced considerably in roots as compared to shoots with enhancing concentration of chromium. To compare the phytoaccumulation ability, the tolerance index (TI), bio-concentration factor (BCF) and transportation index (Ti) were measured in two different species. The results showed that at 100% OBD the TI, BCF and Ti of S. sesban have 22.30, 0.45 and 71.06 and B. mutica have 20.83, 0.43 and 42.45 respectively. The result showed that S. sesban not only had the highest bioaccumulation capacity of chromium but also have high tolerance index and transportation index as compared to B. mutica. Hence S. sesban can be recommended for phytostabilization programme to alleviate toxic chromium from chromite overburden mining sites.

Role of abscisic acid in strigolactone-induced salt stress tolerance in arbuscular mycorrhizal Sesbania cannabina seedlings.

BACKGROUND: Strigolactones (SLs) are considered to be a novel class of phytohormone involved in plant defense responses. Currently, their relationships with other plant hormones, such as abscisic acid (ABA), during responses to salinity stress are largely unknown. RESULTS: In this study, the relationship between SL and ABA during the induction of H2O2 - mediated tolerance to salt stress were studied in arbuscular mycorrhizal (AM) Sesbania cannabina seedlings. The SL levels increased after ABA treatments and decreased when ABA biosynthesis was inhibited in AM plants. Additionally, the expression levels of SL-biosynthesis genes in AM plants increased following treatments with exogenous ABA and H2O2. Furthermore, ABA-induced SL production was blocked by a pre-treatment with dimethylthiourea, which scavenges H2O2. In contrast, ABA production was unaffected by dimethylthiourea. Abscisic acid induced only partial and transient increases in the salt tolerance of TIS108 (a SL synthesis inhibitor) treated AM plants, whereas SL induced considerable and prolonged increases in salt tolerance after a pre-treatment with tungstate. CONCLUSIONS: These results strongly suggest that ABA is regulating the induction of salt tolerance by SL in AM S. cannabina seedlings.

A Chemotaxis Receptor Modulates Nodulation during the Azorhizobium caulinodans-Sesbania rostrata Symbiosis.

UNLABELLED: Azorhizobium caulinodans ORS571 is a free-living nitrogen-fixing bacterium which can induce nitrogen-fixing nodules both on the root and the stem of its legume host Sesbania rostrata This bacterium, which is an obligate aerobe that moves by means of a polar flagellum, possesses a single chemotaxis signal transduction pathway. The objective of this work was to examine the role that chemotaxis and aerotaxis play in the lifestyle of the bacterium in free-living and symbiotic conditions. In bacterial chemotaxis, chemoreceptors sense environmental changes and transmit this information to the chemotactic machinery to guide motile bacteria to preferred niches. Here, we characterized a chemoreceptor of A. caulinodans containing an N-terminal PAS domain, named IcpB. IcpB is a soluble heme-binding protein that localized at the cell poles. An icpB mutant strain was impaired in sensing oxygen gradients and in chemotaxis response to organic acids. Compared to the wild-type strain, the icpB mutant strain was also affected in the production of extracellular polysaccharides and impaired in flocculation. When inoculated alone, the icpB mutant induced nodules on S. rostrata, but the nodules formed were smaller and had reduced N2-fixing activity. The icpB mutant failed to nodulate its host when inoculated competitively with the wild-type strain. Together, the results identify chemotaxis and sensing of oxygen by IcpB as key regulators of the A. caulinodans-S. rostrata symbiosis. IMPORTANCE: Bacterial chemotaxis has been implicated in the establishment of various plant-microbe associations, including that of rhizobial symbionts with their legume host. The exact signal(s) detected by the motile bacteria that guide them to their plant hosts remain poorly characterized. Azorhizobium caulinodans ORS571 is a diazotroph that is a motile and chemotactic rhizobial symbiont of Sesbania rostrata, where it forms nitrogen-fixing nodules on both the roots and the stems of the legume host. We identify here a chemotaxis receptor sensing oxygen in A. caulinodans that is critical for nodulation and nitrogen fixation on the stems and roots of S. rostrata These results identify oxygen sensing and chemotaxis as key regulators of the A. caulinodans-S. rostrata symbiosis.

EDTA-enhanced phytoremediation of contaminated calcareous soils: heavy metal bioavailability, extractability, and uptake by maize and sesbania.

Natural and chemically enhanced phytoextraction potentials of maize (Zea mays L.) and sesbania (Sesbania aculeata Willd.) were explored by growing them on two soils contaminated with heavy metals. The soils, Gujranwala (fine, loamy, mixed, hyperthermic Udic Haplustalf) and Pacca (fine, mixed, hyperthermic Ustollic Camborthid), were amended with varying amounts of ethylenediaminetetraacetic acid (EDTA) chelating agent, at 0, 1.25, 2.5, and 5.0 mM kg(-1) soil to enhance metal solubility. The EDTA was applied in two split applications at 46 and 60 days after sowing (DAS). The plants were harvested at 75 DAS. Addition of EDTA significantly increased the lead (Pb) and cadmium (Cd) concentrations in roots and shoots, uptake, bioconcentration factor, and phytoextraction rate over the control. Furthermore, addition of EDTA also significantly increased the soluble fractions of Pb and Cd in soil over the controls; the maximum increase of Pb and Cd was 13.1-fold and 3.1-fold, respectively, with addition of 5.0 mM EDTA kg(-1)soil. Similarly, the maximum Pb and Cd root and shoot concentrations, translocation, bioconcentration, and phytoextraction efficiency were observed at 5.0 mM EDTA kg(-1) soil. The results suggest that both crops can successfully be used for phytoremediation of metal-contaminated calcareous soils.

The effects of triclosan on spore germination and hyphal growth of the arbuscular mycorrhizal fungus Glomus intraradices.

The effect of triclosan (5-chloro-2-[2,4-dichlorophenoxy]phenol; TCS), on spore germination, hyphal growth, and hyphal branching of the arbuscular mycorrhizal (AM) fungus, Glomus intraradices spores was evaluated at exposure concentrations of 0.4 and 4.0 µg/L in a static renewal exposure system. To determine if potential effects were mycotoxic or a consequence of impaired signaling between a host plant and the fungal symbiont, spores were incubated with and without the addition of a root exudate. Exposed spores were harvested at days 7, 14, and 21. AM spore germination, hyphal growth, and hyphal branching were significantly lower in both TCS concentrations compared to controls in non-root exudate treatments suggesting direct mycotoxic effects of TCS on AM development. Greater hyphal growth and hyphal branching in controls and 0.4µg/L TCS treatments with root exudate compared to non-root exudate treatments demonstrated growth stimulation by signaling chemicals present in the root exudate. This stimulatory effect was absent in the 4.0 µg/L TCS treatments indicating a direct effect on plant signaling compounds or plant signal response.

Nodule development on the tropical legume Sesbania virgata under flooded and non-flooded conditions.

The interaction between the Brazilian pioneer legume Sesbania virgata and its microsymbiont Azorhizobium doebereinerae leads to the formation of nitrogen-fixing nodules on roots that grow either in well-aerated soils or in wetlands. We studied the initiation and development of nodules under these alternative conditions. To this end, light and fluorescence microscopy were used to follow the bacterial colonisation and invasion into the host and, by means of transmission electron microscopy, we could observe the intracellular entry. Under hydroponic conditions, intercellular invasion took place at lateral root bases and mature nodules were round and determinate. However, on roots grown in vermiculite that allows aerated growth, bacteria also entered via root hair invasion and nodules were both of the determinate and indeterminate type. Such versatility in entry and developmental plasticity, as previously described in Sesbania rostrata, enables efficient nodulation in both dry and wet environments and are an important adaptive feature of this group of semi-tropical plants that grow in temporarily flooded habitats.

Lon protease of Azorhizobium caulinodans ORS571 is required for suppression of reb gene expression.

Bacterial Lon proteases play important roles in a variety of biological processes in addition to housekeeping functions. In this study, we focused on the Lon protease of Azorhizobium caulinodans, which can fix nitrogen both during free-living growth and in stem nodules of the legume Sesbania rostrata. The nitrogen fixation activity of an A. caulinodans lon mutant in the free-living state was not significantly different from that of the wild-type strain. However, the stem nodules formed by the lon mutant showed little or no nitrogen fixation activity. By microscopic analyses, two kinds of host cells were observed in the stem nodules formed by the lon mutant. One type has shrunken host cells containing a high density of bacteria, and the other type has oval or elongated host cells containing a low density or no bacteria. This phenotype is similar to a praR mutant highly expressing the reb genes. Quantitative reverse transcription-PCR analyses revealed that reb genes were also highly expressed in the lon mutant. Furthermore, a lon reb double mutant formed stem nodules showing higher nitrogen fixation activity than the lon mutant, and shrunken host cells were not observed in these stem nodules. These results suggest that Lon protease is required to suppress the expression of the reb genes and that high expression of reb genes in part causes aberrance in the A. caulinodans-S. rostrata symbiosis. In addition to the suppression of reb genes, it was found that Lon protease was involved in the regulation of exopolysaccharide production and autoagglutination of bacterial cells.

NAD(P)+-malic enzyme mutants of Sinorhizobium sp. strain NGR234, but not Azorhizobium caulinodans ORS571, maintain symbiotic N2 fixation capabilities.

C(4)-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N(2)-fixing bacteria (bacteroids) within legume nodules. In Sinorhizobium meliloti bacteroids from alfalfa, NAD(+)-malic enzyme (DME) is required for N(2) fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont Rhizobium leguminosarum, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report that dme mutants of the broad-host-range Sinorhizobium sp. strain NGR234 formed nodules whose level of N(2) fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while single pckA and single dme mutants fixed N(2) at reduced rates, a pckA dme double mutant had no N(2)-fixing activity (Fix(-)). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix(-) phenotype of S. meliloti dme mutants may be specific to the alfalfa-S. meliloti symbiosis. We therefore examined the ME-like genes azc3656 and azc0119 from Azorhizobium caulinodans, as azc3656 mutants were previously shown to form Fix(-) nodules on the tropical legume Sesbania rostrata. We found that purified AZC3656 protein is an NAD(P)(+)-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N(2) fixation in A. caulinodans and S. meliloti, in other rhizobia this activity can be bypassed via another pathway(s).

Involvement of the azorhizobial chromosome partition gene (parA) in the onset of bacteroid differentiation during Sesbania rostrata stem nodule development.

A parA gene in-frame deletion mutant of Azorhizobium caulinodans ORS571 (ORS571-ΔparA) was constructed to evaluate the roles of the chromosome-partitioning gene on various bacterial traits and on the development of stem-positioned nodules. The ΔparA mutant showed a pleiomorphic cell shape phenotype and was polyploid, with differences in nucleoid sizes due to dramatic defects in chromosome partitioning. Upon inoculation of the ΔparA mutant onto the stem of Sesbania rostrata, three types of immature nodule-like structures with impaired nitrogen-fixing activity were generated. Most showed signs of bacteroid early senescence. Moreover, the ΔparA cells within the nodule-like structures exhibited multiple developmental-stage phenotypes. Since the bacA gene has been considered an indicator for bacteroid formation, we applied the expression pattern of bacA as a nodule maturity index in this study. Our data indicate that the bacA gene expression is parA dependent in symbiosis. The presence of the parA gene transcript was inversely correlated with the maturity of nodule; the transcript was switched off in fully mature bacteroids. In summary, our experimental evidence demonstrates that the parA gene not only plays crucial roles in cellular development when the microbe is free-living but also negatively regulates bacteroid formation in S. rostrata stem nodules.

Water-tolerant legume nodulation.

Water-tolerant nodulation is an adaptation of legumes that grow in wet or temporarily flooded habitats. This nodulation mode takes place at lateral root bases via intercellular bacterial invasion in cortical infection pockets. The tropical legume Sesbania rostrata has become a model for the study of the molecular basis of crack entry nodulation compared with root hair curl nodulation. For intercellular invasion, Nodulation Factor (NF) signalling recruits an ethylene-dependent, common Sym gene-independent pathway, leading to local cell death. The NF structure requirements are less stringent than for intracellular invasion in root hairs, which is correlated with a very specific NF-induced calcium spiking signature, presumably necessary for correct gene expression to assemble a functional entry complex in the epidermis.

Sesbania rostrata: a case study of natural variation in legume nodulation.

Legumes acquired the ability to engage in a symbiotic interaction with soil-borne bacteria and establish a nitrogen-fixing symbiosis in a novel root organ, the nodule. Most legume crops and the model legumes Medicago truncatula and Lotus japonicus are infected intracellularly in root hairs via infection threads that lead the bacteria towards a nodule primordium in the root cortex. This infection process, however, does not reflect the great diversity of infection strategies that are used by leguminous plants. An alternative, intercellular invasion occurs in the semiaquatic legume Sesbania rostrata. Bacteria colonize epidermal fissures at lateral root bases and trigger cortical cell death for infection pocket formation and subsequent intercellular and intracellular infection thread progression towards the primordium. This infection mode evolved as an adaptation to waterlogged conditions that inhibit intracellular invasion. In this review, we discuss the molecular basis for this adaptation and how insights into this process contribute to general knowledge of the rhizobial infection process.

Comparative transcriptome analysis reveals common and specific tags for root hair and crack-entry invasion in Sesbania rostrata.

The tropical legume Sesbania rostrata provides its microsymbiont Azorhizobium caulinodans with versatile invasion strategies to allow nodule formation in temporarily flooded habitats. In aerated soils, the bacteria enter via the root hair curling mechanism. Submergence prevents this epidermal invasion by accumulation of inhibiting concentrations of ethylene and, under these conditions, the bacterial colonization occurs via intercellular cortical infection at lateral root bases. The transcriptome of both invasion ways was compared by cDNA-amplified fragment length polymorphism analysis. Clusters of gene tags were identified that were specific for either epidermal or cortical invasion or were shared by both. The data provide insight into mechanisms that control infection and illustrate that entry via the epidermis adds a layer of complexity to rhizobial invasion.

Bioaccumulation and physiological effects of mercury in Sesbania drummondii.

The accumulation of mercury and its effect on growth, photosynthesis and antioxidative responses were studied in Sesbania drummondii seedlings. Mercury concentration in shoots as well as in the roots increased with increasing Hg concentrations in the growth solution. The accumulation of Hg was more in roots than shoots. At 100 mg l-1 Hg concentration, shoots accumulated 998 mg Hg kg -1 dry weight (dw) while roots accumulated 41,403 mg Hg kg-1 dw. Seedlings growth was not significantly affected at lower concentrations of Hg. A concentration of 100 mg l-1 Hg inhibited growth by 36.8%, with respect to control. Photosynthetic activity was assessed by measuring chlorophyll a fluorescence by determination of Fv/Fm and Fv/Fo values. Photosynthetic integrity was not affected up to 50 mg l-1 Hg concentration, however, concentrations higher than 50 mg l-1 affected photosynthetic integrity. Sesbania responded to Hg induced oxidative stress by modulating non-enzymatic antioxidants [glutathione (GSH) and non-protein thiols (NPSH)] and enzymatic antioxidants: superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR). Glutathione content and GSH/GSSG ratio increased up to a concentration of 50 mg l-1 while slight down at 100 mg l-1 Hg. The content of NPSH significantly increased with increasing Hg concentrations in the growth medium. The activities of antioxidative enzymes, SOD, APX and GR followed the same trends as antioxidants first increased up to a concentration of 50 mg l-1 Hg and then slight decreased. The results of present study suggest that Sesbania plants were able to accumulate and tolerate Hg induced stress using an effective antioxidative defense mechanisms.

Gibberellins are involved in nodulation of Sesbania rostrata.

Upon submergence, Azorhizobium caulinodans infects the semiaquatic legume Sesbania rostrata via the intercellular crack entry process, resulting in lateral root-based nodules. A gene encoding a gibberellin (GA) 20-oxidase, SrGA20ox1, involved in GA biosynthesis, was transiently up-regulated during lateral root base nodulation. Two SrGA20ox1 expression patterns were identified, one related to intercellular infection and a second observed in nodule meristem descendants. The infection-related expression pattern depended on bacterially produced nodulation (Nod) factors. Pharmacological studies demonstrated that GAs were involved in infection pocket and infection thread formation, two Nod factor-dependent events that initiate lateral root base nodulation, and that they were also needed for nodule primordium development. Moreover, GAs inhibited the root hair curling process. These results show that GAs are Nod factor downstream signals for nodulation in hydroponic growth.