Heterologous expression of nattokinase in E. coli: Biochemical characterization and functional analysis of fibrin binding residues.

Heterologous expression of nattokinase, a potent fibrinolytic enzyme, has been successfully carried out in various microorganisms. However, the successful expression of this enzyme as a soluble protein was not achieved in E. coli. This study delves into the expression of nattokinase in E. coli as a soluble protein followed by its biochemical characterization and functional analysis for fibrinolytic activity. E. coli BL21C41 and pET32a vector host strain with pGro7 protein chaperone induced with IPTG at 16 °C 180 rpm for 16 h enabled the production of recombinant nattokinase in soluble fraction. Enzymatic assays demonstrated its protease activity, while characterization revealed optimal catalytic conditions at 37 °C and pH 8.0, with remarkable stability over a broad pH range (6.0-10.0) and up to 50 °C. The kinetic constants were determined as follows: Km = 25.83 ± 3.43 µM, Vmax = 62.91 ± 1.68 µM/s, kcat = 38.45 ± 1.06 s-1, and kcat/Km = 1.49 × 106 M-1 s-1. In addition, the fibrinolytic activity of NK, quantified by the fibrin plate hydrolysis assay was 1038 ± 156 U/ml, with a corresponding specific activity of 1730 ± 260 U/mg and the assessment of clot lysis time on an artificial clot (1 mg) was found to be 51.5 ± 2.5 min unveiling nattokinase's fibrinolytic potential. Through molecular docking, a substantial binding energy of -6.46 kcal/mol was observed between nattokinase and fibrin, indicative of a high binding affinity. Key fibrin binding residues, including Ser300, Leu302, and Asp303, were identified and confirmed. These mutants affected specifically the fibrin binding and not the proteolytic activity of NK. This comprehensive study provides crucial conditions for the expression of protein in soluble form in E. coli and biochemical properties paving the way for future research and potential applications in medicine and biotechnology.

Investigation of two-pore K+ (TPK) channels in Triticum aestivum L. suggests their role in stress response.

Two-pore K+ (TPK) channels are voltage-independent and involved in stress response in plants. Herein, we identified 12 TaTPK genes located on nine chromosomes in the Triticum aestivum genome. The majority of TaTPK genes comprised two exons. Each TaTPK channel comprised four transmembrane (TM) helices, N- and C-terminal ion-channel domains, two EF-hand domains and one 14-3-3 binding site. Additionally, highly conserved 'GYGD' motif responsible for K+ ion specificity, was found in between the TMs in both the ion-channel domains. Nine TaTPK channels were predicted to be localised at the plasma membrane, while three were vacuolar. The protein-protein and protein-chemical interactions indicated the coordinated functioning of the TaTPK channels with the other K+ transporters and their possible interaction with the Ca2+-signaling pathway. Expression studies suggested their importance in both vegetative and reproductive tissues development. Significantly modulated expression of various TaTPK genes during heat, drought, combined heat and drought and salt stresses, and after fungal infestation, depicted their function in stress responses. The miRNAs and transcription factors interaction analyses suggested their role in the hormone, light, growth and development-related, and stress-responsive signaling cascades. The current study suggested vital functions of various TaTPK genes, especially in stress response, and would provide an opportunity for their detailed characterization in future studies.

Modulation in gene expression and enzyme activity suggested the roles of monodehydroascorbate reductase in development and stress response in bread wheat.

Monodehydroascorbate reductase (MDHAR) is a crucial enzymatic antioxidant of the ascorbate-glutathione pathway involved in reactive oxygen species scavenging. Herein, we identified 15 TaMDHAR genes in bread wheat. Phylogenetic analysis revealed their clustering into three groups, which are also related to the subcellular localization in the peroxisome matrix, peroxisome membrane, and chloroplast. Each TaMDHAR protein consisted of two conserved domains; Pyr_redox and Pyr_redox_2 of the pyridine nucleotide disulfide oxidoreductase family. The occurrence of diverse groups of cis-regulatory elements in the promoter region and their interaction with numerous transcription factors suggest assorted functions of TaMDHARs in growth and development and in light, phytohormones, and stress responses. Expression analysis in various tissues further revealed their importance in vegetative and reproductive development. In addition, the differential gene expression and enhanced enzyme activity during drought, heat, and salt treatments exposed their role in abiotic stress response. Interaction of MDHARs with various antioxidant enzymes and biochemicals related to the ascorbate-glutathione cycle exposed their synchronized functioning. Interaction with auxin indicated the probability of cross-talk between antioxidants and auxin signaling. The miR168a, miR169, miR172 and others interaction with various TaMDHARs further directed their association with developmental processes and stress responses. The current study provides extensive information about the importance of TaMDHARs, moreover, the precise role of each gene needs to be established in future studies.

Genome-wide identification of 2-oxoglutarate and Fe (II)-dependent dioxygenase family genes and their expression profiling under drought and salt stress in potato.

The 2-Oxoglutatrate-dependent dioxygenases (2OGDs) comprise the 2-Oxoglutatrate and Fe(II)-dependent dioxygenases (2ODD) enzyme families that facilitate the biosynthesis of various compounds like gibberellin, ethylene, etc. The 2OGDs are also involved in various catabolism pathways, such as auxin and salicylic acid catabolism. Despite their important roles, 2ODDs have not been studied in potato, which is the third most important crop globally. In this study, a comprehensive genome wide analysis was done to identify all 2ODDs in potatoes, and the putative genes were analysed for the presence of the signature 2OG-FeII_Oxy (PF03171) domain and the conserved DIOX_N (PF14226) domain. A total of 205 St2ODDs were identified and classified into eight groups based on their function. The physiochemical properties, gene structures, and motifs were analysed, and gene duplication events were also searched for St2ODDs. The active amino acid residues responsible for binding with 2-oxoglutarate and Fe (II) were conserved throughout the St2ODDs. The three-dimensional (3D) structures of the representative members of flavanol synthase (FNS), 1-aminocyclopropane-1-carboxylic acid oxidases (ACOs), and gibberellin oxidases (GAOXs) were made and docked with their respective substrates, and the potential interactions were visualised. The expression patterns of the St2ODDs under abiotic stressors such as heat, salt, and drought were also analysed. We found altered expression levels of St2ODDs under abiotic stress conditions, which was further confirmed for drought and salt stress using qRT-PCR. The expression levels of St2ODD115, St2ODD34, and St2ODD99 were found to be upregulated in drought stress with 2.2, 1.8, and 2.6 fold changes, respectively. After rewatering, the expression levels were normal. In salt stress, the expression levels of St2ODD151, St2ODD76, St2ODD91, and St2ODD34 were found to be upregulated after 24 hours (h), 48 hours (h), 72 hours (h), and 96 hours (h). Altogether, the elevated expression levels suggest the importance of St2ODDs under abiotic stresses, i.e., drought and salt. Overall, our study provided a knowledge base for the 2ODD gene family in potato, which can be used further to study the important roles of 2ODDs in potato plants.

TaGPX1-D overexpression provides salinity and osmotic stress tolerance in Arabidopsis.

Glutathione peroxidases (GPXs) are known to play an essential role in guarding cells against oxidative stress by catalyzing the reduction of hydrogen peroxide and organic hydroperoxides. The current study aims functional characterization of the TaGPX1-D gene of bread wheat (Triticum aestivum) for salinity and osmotic stress tolerance. To achieve this, we initially performed the spot assays of TaGPX1-D expressing yeast cells. The growth of recombinant TaGPX1-D expressing yeast cells was notably higher than the control cells under stress conditions. Later, we generated transgenic Arabidopsis plants expressing the TaGPX1-D gene and investigated their tolerance to various stress conditions. The transgenic plants exhibited improved tolerance to both salinity and osmotic stresses compared to the wild-type plants. The higher germination rates, increased antioxidant enzymes activities, improved chlorophyll, carotenoid, proline and relative water contents, and reduced hydrogen peroxide and MDA levels in the transgenic lines supported the stress tolerance mechanism. Overall, this study demonstrated the role of TaGPX1-D in abiotic stress tolerance, and it can be used for improving the tolerance of crops to environmental stressors, such as salinity and osmotic stress in future research.

MSMEG_5850, a stress-induced TetR protein, involved in global transcription regulation in Mycobacterium smegmatis.

Aim: To decipher the role of MSMEG_5850 in the physiology of mycobacteria. Methods: MSMEG_5850 was knocked out and RNA sequencing was performed. MSMEG_5850 protein was purified from the Escherichia coli pET28a system. Electrophoretic mobility shift assay and size exclusion chromatography were used to determine the binding of MSMEG_5850 to its motif and binding stoichiometry. The effect of nutritional stress was monitored. Results: Transcriptome analysis revealed the differential expression of 148 genes in an MSMEG_5850 knockout strain. MSMEG_5850 had control over 50 genes because those genes had a binding motif upstream of their sequence. The electrophoretic mobility shift assay showed MSMEG_5850 bound to its motif as a monomer. MSMEG_5850 was upregulated under nutritional stress and promoted the survival of mycobacteria. Conclusion: The study confirms the role of MSMEG_5850 in global transcriptional regulation.

CRISPR/Cas9-mediated editing of phytoene desaturase (PDS) gene in an important staple crop, potato.

The gene editing using the CRISPR/Cas9 system has become an important biotechnological tool for studying gene function and improving crops. In this study, we have used CRISPR/Cas9 system for editing the phytoene desaturase gene (PDS) in popular Indian potato cultivar Kufri Chipsona-I. A construct (pHSE401) carrying two target gRNAs with glycine tRNA processing system under the control of Arabidopsis U6 promoter and the Cas9 protein was constructed and transformed in potato plants using Agrobacterium-mediated genetic transformations. The regeneration efficiency of 45% was observed in regenerated plants, out of which 81% of the putative transformants shoot lines exhibited mutant or bleached phenotype (albinism). The deletion mutations were detected within the StPDS gene in the genotyped plants and a mutation efficiency of 72% for gRNA1 and gRNA2 has been detected using Sanger sequencing. Hence, we set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and generates mutations (deletions) within StPDS gene in potato. The bleached phenotype is easily detectable after only few weeks after Agrobacterium-mediated transformation. This is the first report as a proof of concept for CRISPR/Cas9-based editing of PDS gene in Indian potato cv. Kufri Chipsona-I. This study demonstrates that CRISPR/Cas9 can be used to edit genes at high frequency within the genome of the potato for various traits. Therefore, this study will aid in creating important mutants for modifying molecular mechanisms controlling traits of agronomic importance.

Expression of TaNCL2-A ameliorates cadmium toxicity by increasing calcium and enzymatic antioxidants activities in arabidopsis.

Cadmium (Cd) is a heavy metal that occurs naturally in the environment and is toxic to both animals and plants. The impact of Cd toxicity is shown to be reduced by the exogenous application of calcium (Ca) in crop plants. The sodium/calcium exchanger-like (NCL) protein is involved in Ca enrichment in the cytoplasm by transporting it from the vacuole in the exchange of cytosolic sodium (Na). However, it has not been utilized to ameliorate the Cd toxicity, to date. An elevated expression of TaNCL2-A gene in the root and shoot tissues of bread wheat seedlings, and a higher growth rate of recombinant yeast cells, suggested its role in Cd stress response. The TaNCL2-A expressing transgenic Arabidopsis lines exhibited significant Cd tolerance with increased Ca (∼10-fold) accumulation. The proline content and antioxidant enzymes activities were increased while oxidative stress-related molecules such as H2O2 and MDA were reduced in the transgenic lines. In addition, the growth and yield parameters of transgenic lines such as seed germination rate, root length, leaf biomass, leaf area index, rosette diameter, leaf length and width, and silique count, along with various physiological indicators like chlorophyll, carotenoid, and relative water contents were also improved in comparison to the control plants. Further, the transgenic lines exhibited significant salinity and osmotic stress tolerance, as well. Taken together, these results suggested that the TaNCL2-A could mitigate Cd toxicity along with salinity and osmotic stress. This gene may also be utilized for phytoremediation and Cd sequestration in future studies.

Heterologous expression of Chlorophytum borivilianum Squalene epoxidase in tobacco modulates stigmasterol production and alters vegetative and reproductive growth.

KEYMESSAGE: CbSE overexpression increased stigmasterol levels and altered plant morphology. The genes upstream and downstream of CbSE were found to be upregulated, which confirms its regulatory role in the saponin biosynthetic pathway. Chlorophytum borivilianum is a high-value medicinal plant with many promising preclinical applications that include saponins as a major active ingredient. Squalene epoxidase (SE) is one of the major rate-limiting enzymes of the saponin biosynthetic pathway. Here, we functionally characterized C. borivilianum SE (CbSE) by over-expressing heterologously in Nicotiana tabacum. The heterologous expression of CbSE resulted in stunted pant growth with altered leaf and flower morphology. Next, RT-qPCR analysis of transgenic plants overexpressing CbSE revealed increased expression levels of Cycloartenol synthase (CAS), Beta amyrin synthase (ßAS), and cytochrome P450 monooxygenase 51 (CYP51) (Cytochrome P450), which encode key enzymes for triterpenoid and phytosterol biosynthesis in C. borivilianum. Further, Methyl Jasmonate (MeJa) treatment upregulated Squalene synthase (SQS), SE, and Oxidosqualene cyclases (OSCs) to a significant level. GC-MS analysis of the leaf and hairy roots of the transformants showed an increased stigmasterol content (0.5-1.0 fold) compared to wild type (WT) plants. These results indicate that CbSE is a rate-limiting gene, which encodes an efficient enzyme responsible for phytosterol and triterpenoid production in C. borivilianum.

Integrated analysis of smRNAome, transcriptome, and degradome data to decipher microRNAs regulating costunolide biosynthesis in Saussurea lappa.

Saussurea lappa (S. lappa) has been known to synthesize medicinally important, costunolide. Due to its immense therapeutic importance, understanding of regulatory mechanism associated with its biosynthesis is crucial. The identification of genes and transcription factors (TFs) in S. lappa, created a clear picture of costunolide biosynthesis pathways. Further to understand the regulation of costunolide biosynthesis by miRNAs, an integrated study of transcriptome, miRNAs, and degradome was performed. Identified candidate miRNAs and associated feed-forward loops (FFLs) illustrates their regulatory role in secondary metabolite biosynthesis. Small RNA and degradome sequencing were performed for leaf and root tissues to determine miRNAs-targets pairs. A total of 711 and 525 such targets were obtained for novel and known miRNAs respectively. This data was used to generate costunolide-specific miRNA-TF-gene interactome to perform systematic analyses through graph theoretical approach. Interestingly, miR171c.1 and sla-miR121 were identified as key regulators to connect and co-regulate both mevalonate and sesquiterpenoid pathways to bio-synthesize costunolide. Tissue-specific FFLs were identified to be involved in costunolide biosynthesis which further suggests the evolutionary co-relation of root-specific networks in synthesis of secondary metabolites in addition to leaf-specific networks. This integrative approach allowed us to determine candidate miRNAs and associated tissue-specific motifs involved in the diversification of secondary metabolites. MiRNAs identified in present study can provide alternatives for bioengineering tool to enhance the synthesis of costunolide and other secondary metabolites in S. lappa.

Genome-wide analysis of Catalase gene family reveal insights into abiotic stress response mechanism in Brassica juncea and B. rapa.

Environmental stresses affect the yield and productivity of Brassica crops. Catalases are important antioxidant enzymes involved in reducing excess hydrogen peroxide produced by environmental stresses. In the present study, nine and seven CAT family members in two oilseed Brassica species (B. juncea and B. rapa) were identified with complete characterization based on gene and protein structure. Phylogenetic classification categorized CAT proteins into three classes and differentiated the monocot and dicot-specific CAT proteins. Further, the gene and protein characterizations revealed a high degree of conservation across the CAT family members. Differences were observed in the CAT-HEME binding affinity in CAT1, CAT2, and CAT3 isozymes, which could suggest their differential enzyme activities in different conditions. Furthermore, protein-protein interaction with other antioxidant proteins suggested their coordinated role in ROS scavenging mechanisms. Notably, the differential gene expression of BjuCATs and BraCATs and CAT enzyme activities suggested their crucial roles in major abiotic stresses faced by Brassica species. Promoter analysis in BjuCATs and BraCATs suggested the presence of abiotic-stress responsive cis-regulatory elements. Gene regulatory network analysis suggested miRNA and TF mediated stress response in BjuCATs and BraCATs. CAT family screening and characterization in Brassica sp. has established a basic ground for further functional validation in abiotic and heavy-metal stresses which can help in developing stress tolerant crops.

OSCA Genes in Bread Wheat: Molecular Characterization, Expression Profiling, and Interaction Analyses Indicated Their Diverse Roles during Development and Stress Response.

The hyperosmolality-gated calcium-permeable channels (OSCA) are pore-forming transmembrane proteins that function as osmosensors during various plant developmental processes and stress responses. In our analysis, through in silico approaches, a total of 42 OSCA genes are identified in the Triticum aestivum genome. A phylogenetic analysis reveals the close clustering of the OSCA proteins of Arabidopsis thaliana, Oryza sativa, and T. aestivum in all the clades, suggesting their origin before the divergence of dicots and monocots. Furthermore, evolutionary analyses suggest the role of segmental and tandem duplication events (Des) and purifying selection pressure in the expansion of the OSCA gene family in T. aestivum. Expression profiling in various tissue developmental stages and under abiotic and biotic stress treatments reveals the probable functioning of OSCA genes in plant development and the stress response in T. aestivum. In addition, protein-protein and protein-chemical interactions reveal that OSCA proteins might play a putative role in Ca2+-mediated developmental processes and adaptive responses. The miRNA interaction analysis strengthens the evidence for their functioning in various biological processes and stress-induced signaling cascades. The current study could provide a foundation for the functional characterization of TaOSCA genes in future studies.

Excess nitrogen responsive HvMADS27 transcription factor controls barley root architecture by regulating abscisic acid level.

Nitrogen (N) is an important element for plant growth and development. Although several studies have examined plants' response to N deficiency, studies on plants' response to excess N, which is common in fertilizer-based agrosystems, are limited. Therefore, the aim of this study was to examine the response of barley to excess N conditions, specifically the root response. Additionally, genomic mechanism of excess N response in barley was elucidated using transcriptomic technologies. The results of the study showed that barley MADS27 transcription factor was mainly expressed in the roots and its gene contained N-responsive cis-regulatory elements in the promoter region. Additionally, there was a significant decrease in HvMADS27 expression under excess N condition; however, its expression was not significantly affected under low N condition. Phenotypic analysis of the root system of HvMADS27 knockdown and overexpressing barley plants revealed that HvMADS27 regulates barley root architecture under excess N stress. Further analysis of wild-type (WT) and transgenic barley plants (hvmads27 kd and hvmads27 c-Myc OE) revealed that HvMADS27 regulates the expression of HvBG1 ß-glucosidase, which in turn regulates abscisic acid (ABA) level in roots. Overall, the findings of this study showed that HvMADS27 expression is downregulated in barley roots under excess N stress, which induces HvBG1 expression, leading to the release of ABA from ABA-glucose conjugate, and consequent shortening of the roots.

A grobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC.

A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg L-1 and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg L-1. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), ß-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understanding of the metabolic pathways of this medicinally important plant and its genetic improvement. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03251-x.

Identification of Pseudo-R genes in Vitis vinifera and characterization of their role as immunomodulators in host-pathogen interactions.

INTRODUCTION: Duplication events are fundamental to co-evolution in host-pathogen interactions. Pseudogenes (Ψs) are dysfunctional paralogs of functional genes and resistance genes (Rs) in plants are the key to disarming pathogenic invasions. Thus, deciphering the roles of pseudo-R genes in plant defense is momentous. OBJECTIVES: This study aimed to functionally characterize diverse roles of the resistance Ψs as novel gene footprints and as significant gene regulators in the grapevine genome. METHODS: PlantPseudo pipeline and HMM-profiling identified whole-genome duplication-derived (WGD) Ψs associated with resistance genes (Ψ-Rs). Further, novel antifungal and antimicrobial peptides were characterized for fungal associations using protein-protein docking with Erysiphe necator proteins. miRNA and tasiRNA target sites and transcription factor (TF) binding sites were predicted in Ψ-Rs. Finally, differential co-expression patterns in Ψ-Rs-lncRNAs-coding genes were identified using the UPGMA method. RESULTS: 2,746 Ψ-Rs were identified from 31,032 WGD Ψs in the genome of grapevine. 69-antimicrobial and 81-antifungal novel peptides were generated from Ψ-Rs. The putative genic potential was predicted for five novel antifungal peptides which were further characterized by docking against E. necator proteins. 395 out of 527 resistance loci-specific Ψ-Rs were acting as parental gene mimics. Further, to explore the diverse roles of Ψ-Rs in plant-defense, we identified 37,026 TF-binding sites, 208 miRNA, and 99 tasiRNA targeting sites on these Ψ-Rs. 194 Ψ-Rs were exhibiting tissue-specific expression patterns. The co-expression network analysis between Ψs-lncRNA-genes revealed six out of 79 pathogen-responsive Ψ-Rs as significant during pathogen invasion. CONCLUSIONS: Our study provides pathogen responsive Ψ-Rs integral for pathogen invasion, which will offer a useful resource for future experimental validations. In addition, our findings on novel peptide generations from Ψ-Rs offer valuable insights which can serve as a useful resource for predicting novel genes with the futuristic potential of being investigated for their bioactivities in the plant system.

Stress responsive OsHyPRP16 promoter driven early expression of resistance gene Pi54 potentiate the resistance against Magnaporthe oryzae in transgenic rice.

The rice Hybrid Proline Rich Protein (HyPRP) encoding gene, OsHyPRP16 expression exhibit early upregulation in response to Magnaporthe oryzae inoculation. Here, we functionally characterized the OsHyPRP16 promoter through deletion analysis in transgenic Arabidopsis using GUS (ß-glucuronidase) reporter assay. The promoter fragments, sequentially deleted from the 5' end could induce differential GUS activity in response to stresses induced by different hormones and abiotic stress conditions. In addition, a strong GUS induction was observed in M. oryzae inoculated transgenic Arabidopsis. Based on the insilico and stress-inducibility of D1 promoter fragment against various phytohormones and rice blast fungus, and with no basal activity under control conditions, we rationally selected D1 promoter fragment to drive the expression of a major rice blast resistance gene; Pi54 in the genetic background of blast susceptible TP309 rice line. The D1 promoter fragment was able to induce the expression of Pi54 at immediate-early stages of M. oryzae infection in transgenic rice. The transgenic plants with Pi54 under the control of D1 promoter fragment displayed complete resistance against M. oryzae infection as compared to control plants. The present study suggests that the D1 fragment of OsHyPRP16 promoter is a valuable tool for breeding and development of rice lines with early-inducible and pathogen-responsive enhanced disease resistance.

Insight into the Roles of Proline-Rich Extensin-like Receptor Protein Kinases of Bread Wheat (Triticum aestivum L.).

Proline-rich extensin-like receptor protein kinases (PERKs) are known for their roles in the developmental processes and stress responses of many plants. We have identified 30 TaPERK genes in the genome of T. aestivum, exploring their evolutionary and syntenic relationship and analyzing their gene and protein structures, various cis-regulatory elements, expression profiling, and interacting miRNAs. The TaPERK genes formed 12 homeologous groups and clustered into four phylogenetic clades. All the proteins exhibited a typical domain organization of PERK and consisted of conserved proline residue repeats and serine-proline and proline-serine repeats. Further, the tyrosine-x-tyrosine (YXY) motif was also found conserved in thirteen TaPERKs. The cis-regulatory elements and expression profiling under tissue developmental stages suggested their role in plant growth processes. Further, the differential expression of certain TaPERK genes under biotic and abiotic stress conditions suggested their involvement in defense responses as well. The interaction of TaPERK genes with different miRNAs further strengthened evidence for their diverse biological roles. In this study, a comprehensive analysis of obtained TaPERK genes was performed, enriching our knowledge of TaPERK genes and providing a foundation for further possible functional analyses in future studies.

Long Non-Coding RNAs as Emerging Regulators of Pathogen Response in Plants.

Long non-coding RNAs (lncRNAs) are transcripts without protein-coding potential that contain more than 200 nucleotides that play important roles in plant survival in response to different stresses. They interact with molecules such as DNA, RNA, and protein, and play roles in the regulation of chromatin remodeling, RNA metabolism, and protein modification activities. These lncRNAs regulate the expression of their downstream targets through epigenetic changes, at the level of transcription and post-transcription. Emerging information from computational biology and functional characterization of some of them has revealed their diverse mechanisms of action and possible roles in biological processes such as flowering time, reproductive organ development, as well as biotic and abiotic stress responses. In this review, we have mainly focused on the role of lncRNAs in biotic stress response due to the limited availability of knowledge in this domain. We have discussed the available molecular mechanisms of certain known lncRNAs against specific pathogens. Further, considering that fungal, viral, and bacterial diseases are major factors in the global food crisis, we have highlighted the importance of lncRNAs against pathogen responses and the progress in plant research to develop a better understanding of their functions and molecular mechanisms.

Characterization of APX and APX-R gene family in Brassica juncea and B. rapa for tolerance against abiotic stresses.

KEY MESSAGE: APX and APX-R gene families were identified and characterized in two important oilseed species of Brassica. Gene expression under abiotic stress conditions, recombinant protein expression, and analysis further divulged their drought, heat, and salt-responsive behavior. Ascorbate peroxidases (APX) are heme-dependent enzymes that rid the cells of H2O2 and regulate diverse biological processes. In the present study, we performed APX gene family characterization in two Brassica sp. (B. juncea and B. rapa) as these are commercially important oilseed crops and affected severely by abiotic stresses. We identified 16 BjuAPX and 9 BraAPX genes and 2 APX-R genes each in B. juncea and B. rapa genomes, respectively. Phylogenetic analysis divided the APX genes into five distinct clades, which exhibited conservation in the gene structure, motif organization, and sub-cellular location within the clade. Structural analysis of APX and APX-R proteins revealed the amino acid substitutions in conserved domains of APX-R proteins. The expression profiling of BjuAPX and BraAPX genes showed that 3 BjuAPX, 7BraAPX, and 2 BraAPX-R genes were drought and heat responsive. Notably, BjuAAPX1a, BjuAPX1d, BjuAAPX6, BraAAPX1a, BraAAPX2, and BraAAPX3b showed high expression levels in RT-qPCR. Cis-regulatory elements in APX and APX-R gene promoters supported the differential behavior of these genes. Further, two stress-responsive genes BjuAPX1d and BraAAPX2 were cloned, characterized, and their roles were validated under heat, drought, salt, and cold stress in bacterial expression system. This study for the first time reports the presence of APX activity in dimeric and LMW form of purified BraAAPX2 protein. The study may help pave way for developing abiotic stress-tolerant Brassica crops.