Microbe-Responsive Proteomes During Plant-Microbe Interactions Between Rice Genotypes and the Multifunctional Methylobacterium oryzae CBMB20.

BACKGROUND: Rice is colonized by plant growth promoting bacteria such as Methylobacterium leading to mutually beneficial plant-microbe interactions. As modulators of the rice developmental process, Methylobacterium influences seed germination, growth, health, and development. However, little is known about the complex molecular responsive mechanisms modulating microbe-driven rice development. The application of proteomics to rice-microbe interactions helps us elucidate dynamic proteomic responses mediating this association. RESULTS: In this study, a total of 3908 proteins were detected across all treatments of which the non-inoculated IR29 and FL478 share up to 88% similar proteins. However, intrinsic differences appear in IR29 and FL478 as evident in the differentially abundant proteins (DAPs) and their associated gene ontology terms (GO). Successful colonization of M. oryzae CBMB20 in rice resulted to dynamic shifts in proteomes of both IR29 and FL478. The GO terms of DAPs for biological process in IR29 shifts in abundance from response to stimulus, cellular amino acid metabolic process, regulation of biological process and translation to cofactor metabolic process (6.31%), translation (5.41%) and photosynthesis (5.41%). FL478 showed a different shift from translation-related to response to stimulus (9%) and organic acid metabolic acid (8%). Both rice genotypes also showed a diversification of GO terms due to the inoculation of M. oryzae CBMB20. Specific proteins such as peptidyl-prolyl cis-trans isomerase (A2WJU9), thiamine thiazole synthase (A2YM28), and alanine-tRNA ligase (B8B4H5) upregulated in IR29 and FL478 indicate key mechanisms of M. oryzae CBMB20 mediated plant growth promotion in rice. CONCLUSIONS: Interaction of Methylobacterium oryzae CBMB20 to rice results in a dynamic, similar, and plant genotype-specific proteomic changes supporting associated growth and development. The multifaceted CBMB20 expands the gene ontology terms and increases the abundance of proteins associated with photosynthesis, diverse metabolic processes, protein synthesis and cell differentiation and fate potentially attributed to the growth and development of the host plant. The specific proteins and their functional relevance help us understand how CBMB20 mediate growth and development in their host under normal conditions and potentially link subsequent responses when the host plants are exposed to biotic and abiotic stresses.

Inoculation of ACC deaminase-producing endophytic bacteria down-regulates ethylene-induced pathogenesis-related signaling in red pepper (Capsicum annuum L.) under salt stress.

Pathogenesis-related (PR) signaling plays multiple roles in plant development under abiotic and biotic stress conditions and is regulated by a plethora of plant physiological as well as external factors. Here, our study was conducted to evaluate the role of an ACC deaminase-producing endophytic bacteria in regulating ethylene-induced PR signaling in red pepper plants under salt stress. We also evaluated the efficiency of the bacteria in down-regulating the PR signaling for efficient colonization and persistence in the plant endosphere. We used a characteristic endophyte, Methylobacterium oryzae CBMB20 and its ACC deaminase knockdown mutant (acdS- ). The wild-type M. oryzae CBMB20 was able to decrease ethylene emission by 23% compared to the noninoculated and acdS- M. oryzae CBMB20 inoculated plants under salt stress. The increase in ethylene emission resulted in enhanced hydrogen peroxide concentration, phenylalanine ammonia-lyase activity, ß-1,3 glucanase activity, and expression profiles of WRKY, CaPR1, and CaPTI1 genes that are typical salt stress and PR signaling factors. Furthermore, the inoculation of both the bacterial strains had shown induction of PR signaling under normal conditions during the initial inoculation period. However, wild-type M. oryzae CBMB20 was able to down-regulate the ethylene-induced PR signaling under salt stress and enhance plant growth and stress tolerance. Collectively, ACC deaminase-producing endophytic bacteria down-regulate the salt stress-mediated PR signaling in plants by regulating the stress ethylene emission levels and this suggests a new paradigm in efficient colonization and persistence of ACC deaminase-producing endophytic bacteria for better plant growth and productivity.

Label-free proteomics approach reveals candidate proteins in rice (Oryza sativa L.) important for ACC deaminase producing bacteria-mediated tolerance against salt stress.

The omics-based studies are important for identifying characteristic proteins in plants to elucidate the mechanism of ACC deaminase producing bacteria-mediated salt tolerance. This study evaluates the changes in the proteome of rice inoculated with ACC deaminase producing bacteria under salt-stress conditions. Salt stress resulted in a significant decrease in photosynthetic pigments, whereas inoculation of Methylobacterium oryzae CBMB20 had significantly increased pigment contents under normal and salt-stress conditions. A total of 76, 51 and 33 differentially abundant proteins (DAPs) were identified in non-inoculated salt-stressed plants, bacteria-inoculated plants under normal and salt stress conditions respectively. The abundances of proteins responsible for ethylene emission and programmed cell death were increased, and that of photosynthesis-related proteins were decreased in non-inoculated plants under salt stress. However, bacteria-inoculated plants had shown higher abundance of antioxidant proteins, RuBisCo and ribosomal proteins that are important for enhancing stress tolerance and improving plant physiological traits. Collectively, salt stress might affect plant physiological traits by impairing photosynthetic machinery and accelerating apoptosis leading to a decline in biomass. However, inoculation of plants with bacteria can assist in enhancing photosynthetic activity, antioxidant activities and ethylene regulation related proteins for attenuating salt-induced apoptosis and sustaining growth and development.

ACC deaminase-producing Brevibacterium linens RS16 enhances heat-stress tolerance of rice (Oryza sativa L.).

The rapid rise in global temperature has adverse effects on rice productivity. The lack of eminent resources for heat stress alleviation is threatening the agricultural sector. Heat stress alleviation by endophytic plant growth-promoting bacteria (PGPB) can be a sustainable and eco-friendly approach. The present study was conducted to check the colonization of Brevibacterium linens RS16 producing ACC (1-aminocyclopropane-1-carboxylate) deaminase in the rice endosphere and to characterize its efficiency in enhancing stress tolerance. The ethylene emission pathway, reactive oxygen species (ROS) concentrations, proline accumulation, expression of glutathione S-transferase (GST), and small heat shock proteins (sHSPs) were monitored at two different levels of heat stress (40°C and 45°C). Bacterial inoculation decreased ethylene emission levels by 26.9% and 24.4% in rice plants exposed to 40°C and 45°C, respectively, compared with the non-inoculated plants. B. linens RS16 also enhanced the expression profiles of glutathione S-transferase. The collective effect of GST expression profiles and decrease in ethylene emission due to bacterial ACC deaminase activity subsequently resulted in a decrease in ROS concentrations. Additionally, HSP16 and HSP26 increased expression in heat-stressed plants inoculated with B. linens RS16 resulted in enhanced stress tolerance (i.e., lesser proline accumulation) than non-inoculated plants. Hence, this study demonstrates the bacteria-mediated tolerance against heat stress by regulating the ethylene emission pathway and upregulating antioxidant enzymes and heat shock proteins.

Exposure to Oxy-Tetracycline Changes Gut Bacterial Community Composition in Rainbow Trout: A Preliminary Study.

The extensive use of antibiotics is evident in most of the livestock and aquaculture management for inhibiting pathogen infection. Korean aquaculture depends on the usage of oxy-tetracycline for growing rainbow trout. Hence, this study was conducted to evaluate the changes in gut bacterial community profiles of rainbow trout exposed to oxy-tetracycline and predict the metabolic functioning of the bacterial community. The gut bacterial community composition of oxy-tetracycline treated fish was assessed by amplicon sequencing targeting the 16S rRNA gene of bacteria and comparing with the control group that did not receive any antibiotic. The principle coordinate analysis and non-metric multidimensional scaling analysis had shown two distinct clusters that implies the changes in community composition. In phyla level, the relative abundances of Tenericutes and Firmicutes were observed to be significantly higher in oxy-tetracycline treated fish compared to the control. Furthermore, the prediction based metabolic profiling revealed the processes that are affected due to the shift in community profiles. For example, metabolic functioning of membrane efflux system, amino acid metabolism and glycolysis were significantly higher in oxy-tetracycline treated fish compared to the control. This study describes alteration in gut bacterial community composition and potential metabolic profiles of the community that might be responsible for surviving in antibiotic rich environment.

ACC deaminase and indole acetic acid producing endophytic bacterial co-inoculation improves physiological traits of red pepper (Capsicum annum L.) under salt stress.

Salinity induces myriad of physiological and biochemical perturbations in plants and its amelioration can be attained by the use of potential bacterial synthetic communities. The use of microbial consortia in contrast to single bacterial inoculation can additively enhance stress tolerance and productivity of agricultural crops. In this study, co-inoculation of Pseudomonas koreensis S2CB45 and Microbacterium hydrothermale IC37-36 isolated from arbuscular mycorrhizal fungi (AMF) spore and rice seed endosphere, respectively, were used to evaluate the physiological and biochemical effects on red pepper at two salt concentrations (75 mM and 150 mM). Plant growth promoting characteristics particularly 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, indole acetic acid (IAA) and cytokinin production were higher during co-culturing compared to the individual bacterial culture. The higher ACC deaminase activity had resulted in 20% and 22% decrease in stress ethylene emission compared to the non-inoculated plants at 75 mM and 150 mM salt stress, respectively. The decline in ethylene emission had eventually reduced ROS accumulation, and the co-inoculated plants had also harbored enhanced antioxidant enzyme activities and higher sugar accumulation compared to the other treatments suggesting enhanced tolerance to salinity. Collectively, these results put forward a novel consortium of bacterial strains that can be used for sustainable agricultural practices against salinity.

Proteome Changes Reveal the Protective Roles of Exogenous Citric Acid in Alleviating Cu Toxicity in Brassica napus L.

Citric acid (CA), as an organic chelator, plays a vital role in alleviating copper (Cu) stress-mediated oxidative damage, wherein a number of molecular mechanisms alter in plants. However, it remains largely unknown how CA regulates differentially abundant proteins (DAPs) in response to Cu stress in Brassica napus L. In the present study, we aimed to investigate the proteome changes in the leaves of B. L. seedlings in response to CA-mediated alleviation of Cu stress. Exposure of 21-day-old seedlings to Cu (25 and 50 µM) and CA (1.0 mM) for 7 days exhibited a dramatic inhibition of overall growth and considerable increase in the enzymatic activities (POD, SOD, CAT). Using a label-free proteome approach, a total of 6345 proteins were identified in differentially treated leaves, from which 426 proteins were differentially expressed among the treatment groups. Gene ontology (GO) and KEGG pathways analysis revealed that most of the differential abundance proteins were found to be involved in energy and carbohydrate metabolism, photosynthesis, protein metabolism, stress and defense, metal detoxification, and cell wall reorganization. Our results suggest that the downregulation of chlorophyll biosynthetic proteins involved in photosynthesis were consistent with reduced chlorophyll content. The increased abundance of proteins involved in stress and defense indicates that these DAPs might provide significant insights into the adaptation of Brassica seedlings to Cu stress. The abundances of key proteins were further verified by monitoring the mRNA expression level of the respective transcripts. Taken together, these findings provide a potential molecular mechanism towards Cu stress tolerance and open a new route in accelerating the phytoextraction of Cu through exogenous application of CA in B. napus.

Physiological response of tomato plant to chitosan-immobilized aggregated Methylobacterium oryzae CBMB20 inoculation under salinity stress.

The use of plant growth promoting bacteria as bioinoculant to alleviate salt stress is a sustainable and eco-friendly approach in agriculture. However, the maintenance of the bacterial population in the soil for longer period is a major concern. In the present study, chitosan-immobilized aggregated Methylobacterium oryzae CBMB20 was used as a bioinoculant to improve tomato plant (Solanum lycopersicum Mill.) growth under salt stress. The chitosan-immobilized aggregated M. oryzae CBMB20 was able to enhance plant dry weight, nutrient uptake (N, P, K and Mg2+), photosynthetic efficiency and decrease electrolyte leakage under salt stress conditions. The oxidative stress exerted by elevated levels of salt stress was also alleviated by the formulated bioinoculant, as it up-regulated the antioxidant enzyme activities and enhanced the accumulation of proline which acts as an osmolyte. The chitosan-immobilized aggregated M. oryzae CBMB20 was able to decrease the excess Na+ influx into the plant cells and subsequently decreasing the Na+/K+ ratio to improve tomato plant growth under salt stress conditions. Therefore, it is proposed that the chitosan-immobilized aggregated M. oryzae CBMB20 could be used as a bioinoculant to promote the plant growth under salt stress conditions.

Interactions between Pseudomonas spp. and their role in improving the red pepper plant growth under salinity stress.

Solitary inoculation of bacteria has been studied widely for plant growth development and amelioration of salinity stress but co-inoculation of bacteria for salt stress amelioration in red pepper plants has been less studied till date. Here, we investigated the co-inoculation effect of Pseudomonas frederiksbergensis OB139 and Pseudomonas vancouverensis OB155 in red pepper plant growth characteristics, plant photosynthesis pigments, ethylene emission, and antioxidant properties under 0, 50, 100 and 150 mM salt stress and compared them with non-inoculated control and single inoculation of each isolate. Results showed increasing concentrations of salinity stress arrested the normal plant growth, increased the stress ethylene levels, disrupted the photosynthetic parameters and also influenced the antioxidant enzymatic activities in non-inoculated control plants. Co-inoculation of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing Pseudomonas spp. significantly reduced the stress ethylene emission and contributed to a significant increase in plant growth compared to single inoculation and non-inoculated control. Catalase activity which was significantly increased in co-inoculated red pepper plants compared to other treatments imply its ability to efficiently neutralize the hydrogen peroxide ions formed as a result of oxidative stress in plants under salinity stress. Besides, significant reduction in malondialdehyde (MDA) content can be correlated to the increased salt tolerance in co-inoculated red pepper plants. Lastly, the increased content of photosynthetic pigments suggest the importance of co-inoculation in improving photosynthesis of red pepper plants. Together, the data demonstrated the functional compatibility of the ACC deaminase producing bacterial isolates and their role in improving the plant physical and biochemical characteristics under salinity stress.

Assessment of Methylobacterium oryzae CBMB20 aggregates for salt tolerance and plant growth promoting characteristics for bio-inoculant development.

Salinity is one of the major factors contributing to the loss of crop productivity and thereby impacting livelihood of people in more than 100 countries of the world and the area of land affected by salinity is increasing day by day. This will worsen due to various factors such as drought that might result in high soil salinity. Use of plant growth promoting rhizobacteria is one of the promising eco-friendly strategies for salinity stress management as part of sustainable agricultural practices. However, it requires selecting rhizobacteria with good survivability and adaptation to salt stress. In this study we report aggregation of Methylobacterium oryzae CBMB20 cells grown in media containing high C/N ratio (30:1) than in media containing low C/N ratio (7:1). Aggregated Methylobacterium oryzae CBMB20 cells exhibited enhanced tolerance to UV irradiation, heat, desiccation, different temperature regimes, oxidative stress, starvation and supported higher population in media. Poly-ß-hydroxybutyrate accumulation, exopolysaccharide production, proline accumulation and biofilm formation were good at 100 mM salt concentration with good microbial cell hydrophobicity at both 50 and 100 mM than other concentrations. Both the aggregated and non-aggregated cells grown under 0-200 mM salt concentrations produced IAA even at 200 mM salt concentration with a peak at 100 mM concentration with aggregated cells producing significantly higher quantities. ACC deaminase activity was observed in all NaCl concentrations studied with gradual and drastic reduction in aggregated and non-aggregated cells over increased salt concentrations.