Bacterial exopolysaccharide amendment improves the shelf life and functional efficacy of bioinoculant under salinity stress.

AIM: Bacterial exopolysaccharide (EPS) possesses numerous properties beneficial for the growth of microbes and plants under hostile conditions. The study aimed to develop a bioformulation with bacterial EPS to enhance the bioinoculant's shelf-life and functional efficacy under salinity stress. METHODS AND RESULTS: High EPS-producing and salt-tolerant bacterial strain (SD2) exhibiting auxin-production, phosphate-solubilization, and biofilm-forming ability was selected. EPS-based bioformulation of SD2 improved the growth of three legumes under salt stress, from which pigeonpea was selected for further experiments. SD2 improved the growth and lowered the accumulation of stress markers in plants under salt stress. Bioformulations with varying EPS concentrations (1% and 2%) were stored for 6 months at 4°C, 30°C, and 37°C to assess their shelf-life and functional efficacy. The shelf life and efficacy of EPS-based bioformulation was sustained at higher temperature, enhancing pigeonpea growth under stress after six months of storage in both control and natural conditions. However, the efficacy of non-EPS-based bioformulation declined following four months of storage. The bioformulation modulated bacterial abundance in the plant's rhizosphere under stress conditions. CONCLUSIONS AND IMPACT STATEMENT: The study brings forth a new strategy for developing next-generation bioformulations with higher shelf-life and efficacy for salinity stress management in pigeonpea under saline conditions.

Transplantation of soil from organic field confers disease suppressive ability to conducive soil.

Organic agriculture is a sustainable method of farming, and confers disease-suppressing abilities to disease-conducive soils via specialized soil microbiomes. This study aimed at transforming a disease-conducive soil from a conventional field into disease-suppressive soil by inoculating soil from an organic field previously established as "disease-suppressive". The effectiveness of the transformed soil was established with the model plant wheat (Triticum aestivum) grown under natural conditions, with regard to its potential in inhibiting fungal phytopathogens, Rhizoctonia solani and Fusarium oxysporum. The conducive soil inoculated with the disease-suppressive soil performed better than the control conducive soil in terms of reduced disease severity in plants, improved soil nutrient content, increased activity of hydrolytic enzymes, and increased abundance of structural and functional microbial markers. The study demonstrates the efficacy of the soil microbiome under long-term organic agriculture in transforming disease-conducive soil into disease-suppressive soils. Such practises are simple and easy to implement, and could greatly improve the sustainability and crop yield in developing countries.

Composition of fungal communities upon multiple passaging of rhizosphere microbiome for salinity stress mitigation in Vigna radiata.

The top-down approach of microbiome-mediated rhizosphere engineering has emerged as an eco-friendly approach for mitigating stress and enhancing crop productivity. It has been established to mitigate salinity stress in Vigna radiata using multi-passaging approach. During the process of acclimatization under increasing levels of salinity stress, the structure of rhizospheric microbial community undergoes dynamic changes, while facilitating stress mitigation in plants. In this study, using ITS-based amplicon sequencing, the dynamics of rhizosphere fungal community was unravelled over successive passages under salinity stress in V. radiata. Clear shifts were evident among the fungal community members under stress and non-stress conditions, upon application of acclimatized rhizosphere microbiome in V. radiata across successive passages. These shifts correlated with enhanced plant biometrics and reduced stress marker levels in plant. Significant changes in the fungal community structure were witnessed in the rhizosphere across specific passaging cycles under salinity stress, which possibly facilitated stress mitigation in V. radiata.

Employing mutants to study the role of a three-membered bacterial consortium in growth promotion of Cajanus cajan.

Bacterial consortia, comprising plant growth promoting (PGP) rhizobacteria, are known to outcompete their impacts on plant attributes compared to their individual application. However, tracking of individual bacterial strains post application as consortium, remains challenging. The primary goal of this study was to develop an efficient method of tracking bioinoculants by generating spontaneous mutants of three different bacterial strains in an established consortium, using antibiotic-based screening, followed by their enumeration after application in Cajanus cajan. Mutants were generated for consortium members, viz. Azotobacter chroococcum (A), Priestia megaterium (formerly Bacillus megaterium) (B), and Pseudomonas sp. (P), against streptomycin, kanamycin and rifampicin, respectively. Those mutants having similar growth rates and PGP properties as compared to wild type bacterial strains were selected to test their efficacy in plant growth promotion. Selected mutant strains were applied as mono, dual and triple cultures to C. cajan grown hydroponically. Enumeration of mutant bacterial strains was carried out to check their viability. Bacterial colonization on roots was also analyzed. The application of triple (mutant) inoculants improved plant growth attributes significantly in comparison to mono and dual culture treatments and control. Cell enumeration revealed that the abundance of each bacterial strain increased till the 5th day of treatment. No significant change was observed later in their abundance for any treatment. The triple culture treatment showed greater abundance of bacterial mutant strains in comparison to mono- or dual cultures. To the best of our knowledge, this is the first mutant-based study to have reported the successful tracking and enumeration of bacterial consortium members, post their application in C. cajan.

Management of abiotic stresses by microbiome-based engineering of the rhizosphere.

Abiotic stresses detrimentally affect both plant and soil health, threatening food security in an ever-increasing world population. Sustainable agriculture is necessary to augment crop yield with simultaneous management of stresses. Limitations of conventional bioinoculants have shifted the focus to more effective alternatives. With the realization of the potential of rhizospheric microbiome engineering in enhancing plant's fitness under stress, efforts have accelerated in this direction. Though still in its infancy, microbiome-based engineering has gained popularity because of its advantages over the microbe-based approach. This review briefly presents major abiotic stresses afflicting arable land, followed by an introduction to the conventional approach of microbe-based enhancement of plant attributes and stress mitigation with its inherent limitations. It then focuses on the significance of the rhizospheric microbiome and possibilities of harnessing its potential by its strategic engineering for stress management. Further, success stories related to two major approaches of microbiome engineering (generation of synthetic microbial community/consortium, and host-mediated artificial selection) pertaining to stress management have been critically presented. Together with bringing forth the challenges associated with the wide application of rhizospheric microbiome engineering in agriculture, the review proposes the adoption of a combinational scheme for the same, bringing together ecological and reductionist approaches for improvised sustainable agricultural practices.

Storage of soil microbiome for application in sustainable agriculture: prospects and challenges.

Soil microbiome is a dynamic micro-ecosystem driving and fine-tuning several biological processes in the global macro-ecosystems. Its tremendous potential towards mediating sustainability in the ecosystem necessitates the urgent need to store it optimally and efficiently as "next-generation biologicals" for future applications via soil transplantation. The challenge, therefore, is to devise a strategy for the storage of soil microbiome such that its "functionality" is preserved for later application. This review discusses the current endeavours made towards storage of the soil microbiome. The methods for assessing the integrity of soil microbiome by targeting the structural diversity and functional potential of the preserved microbiomes have also been discussed. Further, the success stories related to the storage of fecal microbiome for application in transplants have also been highlighted. This is done primarily with the objective of learning lessons, and parallel application of the knowledge gained, in bringing about improvement in the research domain of soil microbiome storage. Subsequently, the limitations of current techniques of preservation have also been delineated. Further, the open questions in the area have been critically discussed. In conclusion, possible alternatives for storage, comprehensive analyses of the composition of the stored microbiome and their potential have been presented.

Serotonin and Melatonin Biosynthesis in Plants: Genome-Wide Identification of the Genes and Their Expression Reveal a Conserved Role in Stress and Development.

Serotonin (Ser) and melatonin (Mel) serve as master regulators of plant growth and development by influencing diverse cellular processes. The enzymes namely, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H) catalyse the formation of Ser from tryptophan. Subsequently, serotonin N-acetyl transferase (SNAT) and acetyl-serotonin methyltransferase (ASMT) form Mel from Ser. Plant genomes harbour multiple genes for each of these four enzymes, all of which have not been identified. Therefore, to delineate information regarding these four gene families, we carried out a genome-wide analysis of the genes involved in Ser and Mel biosynthesis in Arabidopsis, tomato, rice and sorghum. Phylogenetic analysis unravelled distinct evolutionary relationships among these genes from different plants. Interestingly, no gene family except ASMTs showed monocot- or dicot-specific clustering of respective proteins. Further, we observed tissue-specific, developmental and stress/hormone-mediated variations in the expression of the four gene families. The light/dark cycle also affected their expression in agreement with our quantitative reverse transcriptase-PCR (qRT-PCR) analysis. Importantly, we found that miRNAs (miR6249a and miR-1846e) regulated the expression of Ser and Mel biosynthesis under light and stress by influencing the expression of OsTDC5 and OsASMT18, respectively. Thus, this study may provide opportunities for functional characterization of suitable target genes of the Ser and Mel pathway to decipher their exact roles in plant physiology.

ACC deaminase positive Enterobacter-mediated mitigation of salinity stress, and plant growth promotion of Cajanus cajan: a lab to field study.

Salinity is a major abiotic stress that negatively impacts plant health and soil microbiota. ACC (1-aminocyclopropane carboxylic acid) deaminase producing microorganisms act as natural stress busters that protect plants from different kinds of stresses. The study focused on the isolation of potent, indigenous, multi-trait ACC deaminase producers. The shortlisted ACC deaminase producers were checked for their ability to promote growth of Cajanus cajan, and mitigate stress under laboratory conditions followed by validation of their potency in naturally saline field conditions. Physiological stress markers were assessed to evaluate the impact of salinity in plants treated with ACC deaminase producer, compared to controls. Further, the contribution of ACC deaminase in stress mitigation was demonstrated by using a chemical inhibitor for ethylene biosynthesis. This study presents a polyphasic approach, transitioning from the rhizospheric soil to the laboratory to validation in the field, and puts forth a promising eco-friendly alternative for sustainable agriculture. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01031-0.

Genome-wide discovery of OsHOX24-binding sites and regulation of desiccation stress response in rice.

KEY MESSAGE: OsHOX24 mediates regulation of desiccation stress response via complex regulatory network as indicated by its binding to several target genes including transcription factors in rice. HD-ZIP I subfamily of homeobox transcription factors (TFs) are involved in abiotic stress responses and plant development. Previously, we demonstrated the role of OsHOX24, a member of HD-ZIP I subfamily, in abiotic stress responses. In this study, we identified downstream targets of OsHOX24 under control and desiccation stress conditions via chromatin immunoprecipitation-sequencing (ChIP-seq) approach in wild-type and OsHOX24 over-expression transgenic in rice. OsHOX24-binding sites in each sample and differential binding sites between the samples were detected at various genomic locations, including genic and intergenic regions. Gene ontology enrichment analysis revealed that OsHOX24 direct target genes were involved in several biological processes, including plant development, ABA-mediated signalling pathway, ubiquitin-dependent protein catabolic process, ion transport, abiotic and biotic stress responses besides transcriptional and translational regulation. The enrichment of several cis-regulatory motifs representing binding sites of other TFs, such as ABFs, ERF1, MYB1, LTREs and SORLIP2, suggested the involvement of OsHOX24 in a complex regulatory network. These findings indicate that OsHOX24-mediated desiccation stress regulation involves modulation of a plethora of target genes, which participate in diverse pathways in rice.

Over-Expression of OsHOX24 Confers Enhanced Susceptibility to Abiotic Stresses in Transgenic Rice via Modulating Stress-Responsive Gene Expression.

Homeobox transcription factors play critical roles in plant development and abiotic stress responses. In the present study, we raised rice transgenics over-expressing stress-responsive OsHOX24 gene (rice homeodomain-leucine zipper I sub-family member) and analyzed their response to various abiotic stresses at different stages of development. At the seed germination stage, rice transgenics over-expressing OsHOX24 exhibited enhanced sensitivity to abiotic stress conditions and abscisic acid as compared to wild-type (WT). OsHOX24 over-expression rice seedlings showed reduced root and shoot growth under salinity and desiccation stress (DS) conditions. Various physiological and phenotypic assays confirmed higher susceptibility of rice transgenics toward abiotic stresses as compared to WT at mature and reproductive stages of rice development too. Global gene expression profiling revealed differential regulation of several genes in the transgenic plants under control and DS conditions. Many of these differentially expressed genes were found to be involved in transcriptional regulatory activities, besides carbohydrate, nucleic acid and lipid metabolic processes and response to abiotic stress and hormones. Taken together, our findings highlighted the role of OsHOX24 in regulation of abiotic stress responses via modulating the expression of stress-responsive genes in rice.

Characterization of Rice Homeobox Genes, OsHOX22 and OsHOX24, and Over-expression of OsHOX24 in Transgenic Arabidopsis Suggest Their Role in Abiotic Stress Response.

Homeobox transcription factors are well known regulators of plant growth and development. In this study, we carried out functional analysis of two candidate stress-responsive HD-ZIP I class homeobox genes from rice, OsHOX22, and OsHOX24. These genes were highly up-regulated under various abiotic stress conditions at different stages of rice development, including seedling, mature and reproductive stages. The transcript levels of these genes were enhanced significantly in the presence of plant hormones, including abscisic acid (ABA), auxin, salicylic acid, and gibberellic acid. The recombinant full-length and truncated homeobox proteins were found to be localized in the nucleus. Electrophoretic mobility shift assay established the binding of these homeobox proteins with specific DNA sequences, AH1 (CAAT(A/T)ATTG) and AH2 (CAAT(C/G)ATTG). Transactivation assays in yeast revealed the transcriptional activation potential of full-length OsHOX22 and OsHOX24 proteins. Homo- and hetero-dimerization capabilities of these proteins have also been demonstrated. Further, we identified putative novel interacting proteins of OsHOX22 and OsHOX24 via yeast-two hybrid analysis. Over-expression of OsHOX24 imparted higher sensitivity to stress hormone, ABA, and abiotic stresses in the transgenic Arabidopsis plants as revealed by various physiological and phenotypic assays. Microarray analysis revealed differential expression of several stress-responsive genes in transgenic lines as compared to wild-type. Many of these genes were found to be involved in transcriptional regulation and various metabolic pathways. Altogether, our results suggest the possible role of OsHOX22/OsHOX24 homeobox proteins as negative regulators in abiotic stress responses.

Transcriptome analysis in different rice cultivars provides novel insights into desiccation and salinity stress responses.

Drought and salinity are the major environmental factors that affect rice productivity. Comparative transcriptome analysis between tolerant and sensitive rice cultivars can provide insights into the regulatory mechanisms involved in these stress responses. In this study, the comparison of transcriptomes of a drought-tolerant [Nagina 22 (N22)] and a salinity-tolerant (Pokkali) rice cultivar with IR64 (susceptible cultivar) revealed variable transcriptional responses under control and stress conditions. A total of 801 and 507 transcripts were exclusively differentially expressed in N22 and Pokkali rice cultivars, respectively, under stress conditions. Gene ontology analysis suggested the enrichment of transcripts involved in response to abiotic stress and regulation of gene expression in stress-tolerant rice cultivars. A larger number of transcripts encoding for members of NAC and DBP transcription factor (TF) families in N22 and members of bHLH and C2H2 TF families in Pokkali exhibited differential regulation under desiccation and salinity stresses, respectively. Transcripts encoding for thioredoxin and involved in phenylpropanoid metabolism were up-regulated in N22, whereas transcripts involved in wax and terpenoid metabolism were up-regulated in Pokkali. Overall, common and cultivar-specific stress-responsive transcripts identified in this study can serve as a helpful resource to explore novel candidate genes for abiotic stress tolerance in rice.

Genome-wide analysis of homeobox gene family in legumes: identification, gene duplication and expression profiling.

Homeobox genes encode transcription factors that are known to play a major role in different aspects of plant growth and development. In the present study, we identified homeobox genes belonging to 14 different classes in five legume species, including chickpea, soybean, Medicago, Lotus and pigeonpea. The characteristic differences within homeodomain sequences among various classes of homeobox gene family were quite evident. Genome-wide expression analysis using publicly available datasets (RNA-seq and microarray) indicated that homeobox genes are differentially expressed in various tissues/developmental stages and under stress conditions in different legumes. We validated the differential expression of selected chickpea homeobox genes via quantitative reverse transcription polymerase chain reaction. Genome duplication analysis in soybean indicated that segmental duplication has significantly contributed in the expansion of homeobox gene family. The Ka/Ks ratio of duplicated homeobox genes in soybean showed that several members of this family have undergone purifying selection. Moreover, expression profiling indicated that duplicated genes might have been retained due to sub-functionalization. The genome-wide identification and comprehensive gene expression profiling of homeobox gene family members in legumes will provide opportunities for functional analysis to unravel their exact role in plant growth and development.

Gene discovery and tissue-specific transcriptome analysis in chickpea with massively parallel pyrosequencing and web resource development.

Chickpea (Cicer arietinum) is an important food legume crop but lags in the availability of genomic resources. In this study, we have generated about 2 million high-quality sequences of average length of 372 bp using pyrosequencing technology. The optimization of de novo assembly clearly indicated that hybrid assembly of long-read and short-read primary assemblies gave better results. The hybrid assembly generated a set of 34,760 transcripts with an average length of 1,020 bp representing about 4.8% (35.5 Mb) of the total chickpea genome. We identified more than 4,000 simple sequence repeats, which can be developed as functional molecular markers in chickpea. Putative function and Gene Ontology terms were assigned to at least 73.2% and 71.0% of chickpea transcripts, respectively. We have also identified several chickpea transcripts that showed tissue-specific expression and validated the results using real-time polymerase chain reaction analysis. Based on sequence comparison with other species within the plant kingdom, we identified two sets of lineage-specific genes, including those conserved in the Fabaceae family (legume specific) and those lacking significant similarity with any non chickpea species (chickpea specific). Finally, we have developed a Web resource, Chickpea Transcriptome Database, which provides public access to the data and results reported in this study. The strategy for optimization of de novo assembly presented here may further facilitate the transcriptome sequencing and characterization in other organisms. Most importantly, the data and results reported in this study will help to accelerate research in various areas of genomics and implementing breeding programs in chickpea.

Comprehensive expression analysis suggests overlapping and specific roles of rice glutathione S-transferase genes during development and stress responses.

BACKGROUND: Glutathione S-transferases (GSTs) are the ubiquitous enzymes that play a key role in cellular detoxification. Although several GSTs have been identified and characterized in various plant species, the knowledge about their role in developmental processes and response to various stimuli is still very limited. In this study, we report genome-wide identification, characterization and comprehensive expression analysis of members of GST gene family in crop plant rice, to reveal their function(s). RESULTS: A systematic analysis revealed the presence of at least 79 GST genes in the rice genome. Phylogenetic analysis grouped GST proteins into seven classes. Sequence analysis together with the organization of putative motifs indicated the potential diverse functions of GST gene family members in rice. The tandem gene duplications have contributed a major role in expansion of this gene family. Microarray data analysis revealed tissue-/organ- and developmental stage-specific expression patterns of several rice GST genes. At least 31 GST genes showed response to plant hormones auxin and cytokinin. Furthermore, expression analysis showed the differential expression of quite a large number of GST genes during various abiotic stress (20), arsenate stress (32) and biotic stress (48) conditions. Many of the GST genes were commonly regulated by developmental processes, hormones, abiotic and biotic stresses. CONCLUSION: The transcript profiling suggests overlapping and specific role(s) of GSTs during various stages of development in rice. Further, the study provides evidence for the role of GSTs in mediating crosstalk between various stress and hormone response pathways and represents a very useful resource for functional analysis of selected members of this family in rice.