"Genome-wide identification of bZIP gene family in Pearl millet and transcriptional profiling under abiotic stress, phytohormonal treatments; and functional characterization of PgbZIP9".

Abiotic stresses are major constraints in crop production, and are accountable for more than half of the total crop loss. Plants overcome these environmental stresses using coordinated activities of transcription factors and phytohormones. Pearl millet an important C4 cereal plant having high nutritional value and climate resilient features is grown in marginal lands of Africa and South-East Asia including India. Among several transcription factors, the basic leucine zipper (bZIP) is an important TF family associated with diverse biological functions in plants. In this study, we have identified 98 bZIP family members (PgbZIP) in pearl millet. Phylogenetic analysis divided these PgbZIP genes into twelve groups (A-I, S, U and X). Motif analysis has shown that all the PgbZIP proteins possess conserved bZIP domains and the exon-intron organization revealed conserved structural features among the identified genes. Cis-element analysis, RNA-seq data analysis, and real-time expression analysis of PgbZIP genes suggested the potential role of selected PgbZIP genes in growth/development and abiotic stress responses in pearl millet. Expression profiling of selected PgbZIPs under various phytohormones (ABA, SA and MeJA) treatment showed differential expression patterns of PgbZIP genes. Further, PgbZIP9, a homolog of AtABI5 was found to localize in the nucleus and modulate gene expression in pearl millet under stresses. Our present findings provide a better understanding of bZIP genes in pearl millet and lay a good foundation for the further functional characterization of multi-stress tolerant PgbZIP genes, which could become efficient tools for crop improvement.

Unraveling the involvement of WRKY TFs in regulating plant disease defense signaling.

MAIN CONCLUSION: This review article explores the intricate role, regulation, and signaling mechanisms of WRKY TFs in response to biotic stress, particularly emphasizing their pivotal role in the trophism of plant-pathogen interactions. Transcription factors (TFs) play a vital role in governing both plant defense and development by controlling the expression of various downstream target genes. Early studies have shown the differential expression of certain WRKY transcription factors by microbial infections. Several transcriptome-wide studies later demonstrated that diverse sets of WRKYs are significantly activated in the early stages of viral, bacterial, and fungal infections. Furthermore, functional investigations indicated that overexpression or silencing of certain WRKY genes in plants can drastically alter disease symptoms as well as pathogen multiplication rates. Hence the new aspects of pathogen-triggered WRKY TFs mediated regulation of plant defense can be explored. The already recognized roles of WRKYs include transcriptional regulation of defense-related genes, modulation of hormonal signaling, and participation in signal transduction pathways. Some WRKYs have been shown to directly bind to pathogen effectors, acting as decoys or resistance proteins. Notably, the signaling molecules like salicylic acid, jasmonic acid, and ethylene which are associated with plant defense significantly increase the expression of several WRKYs. Moreover, induction of WRKY genes or heightened WRKY activities is also observed during ISR triggered by the beneficial microbes which protect the plants from subsequent pathogen infection. To understand the contribution of WRKY TFs towards disease resistance and their exact metabolic functions in infected plants, further studies are required. This review article explores the intrinsic transcriptional regulation, signaling mechanisms, and hormonal crosstalk governed by WRKY TFs in plant disease defense response, particularly emphasizing their specific role against different biotrophic, hemibiotrophic, and necrotrophic pathogen infections.

Synthetic sub-genomic transcript promoter from Horseradish Latent Virus (HRLV).

MAIN CONCLUSION: We characterized an efficient chimeric sub-genomic transcript promoter from Horseradish Latent Virus, FHS4, active in both dicot and monocot plants, and it could be a potential tool for plant biotechnology. Plant pararetroviruses are a rich source of novel plant promoters widely used for biotechnological applications. Here, we comprehensively characterized a unique sub-genomic transcript (Sgt) promoter of Horseradish Latent Virus (HRLV) and identified a fragment (HS4; - 340 to + 10; 351 bp) that showed the highest expression of reporter genes in both transient and transgenic assays as evidenced by biochemical, histochemical GUS reporter assay and transcript analysis of uidA gene by qRT-PCR. Phylogenetic analysis showed that the HSgt promoter was closely related to the sub-genomic promoter of the Cauliflower Mosaic Virus (CaMV19S). We found that the as-1 element and W-box played an important role in the transcriptional activity of the HS4 promoter. Furthermore, the HS4 promoter was also induced by salicylic acid. Alongside, we enhanced the activity of the HS4 promoter by coupling the enhancer region from Figwort Mosaic Virus (FMV) promoter to the upstream region of it. This hybrid promoter FHS4 was around 1.1 times stronger than the most commonly used promoter, 35S (Cauliflower Mosaic Virus full-length transcript promoter), and was efficient in driving reporter genes in both dicot and monocot plants. Subsequently, transgenic tobacco plants expressing an anti-microbial peptide BrLTP2.1 (Brassica rapa lipid transport protein 2.1), under the control of the FHS4 promoter, were developed. The in vitro anti-fungal assay revealed that the plant-derived BrLTP2.1 protein driven by an FHS4 promoter manifested increased resistance against an important plant fungal pathogen, Alternaria alternata. Finally, we concluded that the FHS4 promoter can be used as an alternative to the 35S promoter and has a high potential to become an efficient tool in plant biotechnology.

MYB Transcription Factor Family in Pearl Millet: Genome-Wide Identification, Evolutionary Progression and Expression Analysis under Abiotic Stress and Phytohormone Treatments.

Transcription factors (TFs) are the regulatory proteins that act as molecular switches in controlling stress-responsive gene expression. Among them, the MYB transcription factor family is one of the largest TF family in plants, playing a significant role in plant growth, development, phytohormone signaling and stress-responsive processes. Pearl millet (Pennisetum glaucum L.) is one of the most important C4 crop plants of the arid and semi-arid regions of Africa and Southeast Asia for sustaining food and fodder production. To explore the evolutionary mechanism and functional diversity of the MYB family in pearl millet, we conducted a comprehensive genome-wide survey and identified 279 MYB TFs (PgMYB) in pearl millet, distributed unevenly across seven chromosomes of pearl millet. A phylogenetic analysis of the identified PgMYBs classified them into 18 subgroups, and members of the same group showed a similar gene structure and conserved motif/s pattern. Further, duplication events were identified in pearl millet that indicated towards evolutionary progression and expansion of the MYB family. Transcriptome data and relative expression analysis by qRT-PCR identified differentially expressed candidate PgMYBs (PgMYB2, PgMYB9, PgMYB88 and PgMYB151) under dehydration, salinity, heat stress and phytohormone (ABA, SA and MeJA) treatment. Taken together, this study provides valuable information for a prospective functional characterization of the MYB family members of pearl millet and their application in the genetic improvement of crop plants.

Biodegradation of malathion by Micrococcus sp. strain MAGK3: kinetics and degradation fragments.

Malathion is widely used as an agricultural insecticide, but its toxic nature makes it a serious environmental contaminant. To screen indigenous bacteria for malathion degradation, a strain MAGK3 capable of utilizing malathion as its sole carbon and energy source was isolated from Pennisetum glaucum agricultural soil. Based on morphological and biochemical characteristics and 16S rDNA sequence analysis, strain MAGK3 was identified as Micrococcus aloeverae. The strain was cultured in the presence of malathion under aerobic and energy-restricting conditions, and it grew well in MSM containing malathion (1000 µl/L), showing the highest specific growth rate at 500 µl/L. Reverse-phase UHPLC-DAD analysis indicated that 100%, 90.48%, 84.27%, 75.46%, 66.65%, and 31.96% of malathion were degraded within 15 days in liquid culture augmented with 50, 100, 200, 300, 500, and 1000 µl/L concentrations of commercial malathion, respectively. Confirmation of malathion degradation to malathion mono, diacids, and phosphorus moiety was performed by Q-TOF-MS analysis, and a pathway of biodegradation was proposed. The influence of co-substrates was also examined to optimize biodegradation further. Kinetic studies based on different models were conducted, and the results demonstrated good conformity with the first-order model. Malathion degradation process by Micrococcus aloeverae was characterized by R2 of 0.95, and the initial concentration was reduced by 50% i.e. (DT50) in 8.11 d at an initial concentration of 500 µl/L. This establishes the Micrococcus sp. as a potent candidate for active bioremediation of malathion in liquid cultures as it can withstand high malathion load and can possibly impact the development strategies of bioremediation for its elimination.

Comprehensive identification and expression analysis of GRAS gene family under abiotic stress and phytohormone treatments in Pearl millet.

Pearl millet is an important C4 cereal plant that possesses enormous capacity to survive under extreme climatic conditions. It serves as a major food source for people in arid and semiarid regions of south-east Asia and Africa. GRAS is an important transcription factor gene family of plant that play a critical role in regulating developmental processes, stress responses and phytohormonal signalling. In the present study, we have identified a total number of 57 GRAS members in pearl millet. Phylogenetic analysis clustered all the PgGRAS genes into eight groups (GroupI-GroupVIII). Motif analysis has shown that all the PgGRAS proteins had conserved GRAS domains and gene structure analysis revealed a high structural diversity among PgGRAS genes. Expression patterns of PgGRAS genes in different tissues (leaf, stem and root) and under various abiotic stress (drought, heat and salinity) were determined. Further, expression analysis was also carried out in response to various hormones (SA, MeJA, GA and ABA). The results provide a clear understanding of GRAS transcription factor family in pearl millet, and lay a good foundation for the functional characterisation of GRAS genes in pearl millet.

Synthetic Promoters from Strawberry Vein Banding Virus (SVBV) and Dahlia Mosaic Virus (DaMV).

We have constructed two intra-molecularly shuffled promoters, namely S100 and D100. The S100 recombinant promoter (621 bp) was generated by ligation of 250 bp long upstream activation sequence (UAS) of Strawberry vein banding virus (SV10UAS; - 352 to - 102 relative to TSS) with its 371 bp long TATA containing core promoter domain (SV10CP; - 352 to + 19). Likewise, 726 bp long D100 promoter was constructed by fusion of 170 bp long UAS of Dahlia mosaic virus (DaMV14UAS; - 203 to - 33) with its 556 bp long core promoter domain (DaMV4CP; - 474 to + 82). S100 and D100 promoters showed 1.8 and 2.2 times stronger activities than that of the CaMV35S promoter. The activity of the promoters is comparable to that of the CaMV35S2 promoter. Transcript analysis employing qRT-PCR and histochemical assays supported the above findings. Abscisic acid and salicylic acid induce the activity of the D100 promoter. Leaf protein obtained from Nicotiana tabacum plant expressing NSD2 gene (Nigella sativa L. defensin 2) driven by the D100 promoter showed antifungal activity against Alternaria alternata and Phoma exigua var. exigua and antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus. Strong S100 and D100 promoters have potential to become efficient candidates for plant metabolic engineering and molecular pharming.

Genome-wide identification and expression analysis of WRKY transcription factors in pearl millet (Pennisetum glaucum) under dehydration and salinity stress.

BACKGROUND: Plants have developed various sophisticated mechanisms to cope up with climate extremes and different stress conditions, especially by involving specific transcription factors (TFs). The members of the WRKY TF family are well known for their role in plant development, phytohormone signaling and developing resistance against biotic or abiotic stresses. In this study, we performed a genome-wide screening to identify and analyze the WRKY TFs in pearl millet (Pennisetum glaucum; PgWRKY), which is one of the most widely grown cereal crops in the semi-arid regions. RESULTS: A total number of 97 putative PgWRKY proteins were identified and classified into three major Groups (I-III) based on the presence of WRKY DNA binding domain and zinc-finger motif structures. Members of Group II have been further subdivided into five subgroups (IIa-IIe) based on the phylogenetic analysis. In-silico analysis of PgWRKYs revealed the presence of various cis-regulatory elements in their promoter region like ABRE, DRE, ERE, EIRE, Dof, AUXRR, G-box, etc., suggesting their probable involvement in growth, development and stress responses of pearl millet. Chromosomal mapping evidenced uneven distribution of identified 97 PgWRKY genes across all the seven chromosomes of pearl millet. Synteny analysis of PgWRKYs established their orthologous and paralogous relationship among the WRKY gene family of Arabidopsis thaliana, Oryza sativa and Setaria italica. Gene ontology (GO) annotation functionally categorized these PgWRKYs under cellular components, molecular functions and biological processes. Further, the differential expression pattern of PgWRKYs was noticed in different tissues (leaf, stem, root) and under both drought and salt stress conditions. The expression pattern of PgWRKY33, PgWRKY62 and PgWRKY65 indicates their probable involvement in both dehydration and salinity stress responses in pearl millet. CONCLUSION: Functional characterization of identified PgWRKYs can be useful in delineating their role behind the natural stress tolerance of pearl millet against harsh environmental conditions. Further, these PgWRKYs can be employed in genome editing for millet crop improvement.