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.

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.

Development of efficient synthetic promoters derived from pararetrovirus suitable for translational research.

MAIN CONCLUSION: In this study, useful hybrid promoters were developed for efficient ectopic gene expression in monocot and dicot plants, and they hold strong prominence in both transgenic research and biotech industries. This study deals with developing novel synthetic promoters derived from Rice Tungro Bacilliform Virus (RTBV) and Mirabilis Mosaic Virus (MMV). Despite numerous availability, there is a severe scarcity of promoters universally suitable for monocot and dicot plants. Here, eight chimeric promoter constructs were synthesized as gBlocks gene fragments through domain swapping and hybridization by incorporating important domains of previously characterized RTBV and MMV promoters. The developed promoter constructs were assessed for transient GUS expression in tobacco protoplast (Xanthi Brad) and agro-infiltrated tobacco, petunia, rice and pearl millet. Protoplast expression analysis showed that two promoter constructs, namely pUPMA-RP1-MP1GUS and pUPMA-RP4-MP1GUS exhibited 3.56 and 2.5 times higher activities than that of the CaMV35S promoter. We had observed the similar type of expression patterns of these promoters in agroinfiltration-based transient studies. RP1-MP1 and RP4-MP1 promoters exhibited 1.87- and 1.68-fold increase expression in transgenic tobacco plants; while, a 1.95-fold increase was found in RP1-MP1 transgenic rice plants when compared their activities with CaMV35S promoter. Furthermore, on evaluating these promoter constructs for their expression in the bacterial system, pUPMA-RP1-MP1GFP was found to have the highest GFP expression. Moreover, the promoter construct was also evaluated for its capacity to express the HMP3 gene. Biobeads of encapsulated bacterial cells expressing HMP3 gene under control of the pUPMA-RP4-MP1 promoter were found to reduce 72.9% copper and 29.2% zinc concentration from wastewater. Our results had demonstrated that the developed promoter constructs could be used for translational research in dicot, monocot plants and bacterial systems for efficient gene expression.

Synthetic promoters from blueberry red ringspot virus (BRRV).

MAIN CONCLUSION: We analyzed the synthetic full-length transcript promoter of Blueberry red ringspot virus (BRRV) and developed two chimeric promoters (MBR3 and FBR3). Transcriptional activities of these chimeric promoters were found equivalent to that of the CaMV35S2 promoter. Chimeric promoters driven plant-derived PaDef protein showed high antimicrobial activities against several pathogens. Blueberry red ringspot virus (BRRV) is a pararetrovirus under the genus, Soymovirus belongs to the Caulimoviridae family. We have made a synthetic version of the BRRV-Flt promoter and analyzed its activity in detail. A 372 bp promoter fragment BR3 (- 212 to + 160) showed the strongest transcriptional activity compared with other fragments in both transient and transgenic assays; its activity was found near equivalent to that of the CaMV35S promoter. We constructed two chimeric promoters; MBR3 and FBR3 by fusing the UASs (Upstream activation sequences) of Mirabilis mosaic virus (MUAS; - 297 to - 38; 335 bp) and Figwort mosaic virus (FUAS; - 249 to - 54; 303 bp) respectively to the core promoter domain of BR3 (BR3; - 212 to + 160; 372 bp). The activities of MBR3 and FBR3 promoters were found equivalent to that of the activity of the CaMV35S2 promoter and approximately 4.0 (four) times stronger than that of the CaMV35S promoter. Histochemical and fluorometric GUS assays confirmed the above observation. The transcriptional efficacies of these recombinant promoters were tested by evaluating the antibacterial and antifungal activities of recombinant plant-derived antimicrobial peptide Persea americana var. drymifolia defensin (PaDef) driven under these promoters. Bioassays showed promising antifungal activities of the plant made PaDef against Alternaria alternata and antibacterial property against Gram-positive (S. aureus and R. fascians) and Gram-negative bacteria (E. coli and P. aeruginosa). Based upon the above results, MBR3 and FBR3 could be useful promoters for plant genetic engineering and can become useful substitutes for the widely used CaMV35S2 promoter in plant biology.

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.

Enhancement of ABA Sensitivity Through Conditional Expression of the ARF10 Gene in Brassica juncea Reveals Fertile Plants with Tolerance Against Alternaria brassicicola.

Concomitant increase of auxin-responsive factors ARF16 and ARF17, along with enhanced expression of ARF10 in resistant Sinapis alba compared with that in susceptible Brassica juncea upon challenge with Alternaria brassicicola, revealed that abscisic acid (ABA)-auxin crosstalk is a critical factor for resistance response. Here, we induced the ABA response through conditional expression of ARF10 in B. juncea using the A. brassicicola-inducible GH3.3 promoter. Induced ABA sensitivity caused by conditional expression of ARF10 in transgenic B. juncea resulted in tolerance against A. brassicicola and led to enhanced expression of several ABA-responsive genes without affecting the auxin biosynthetic gene expression. Compared with ABI3 and ABI4, ABI5 showed maximum upregulation in the most tolerant transgenic lines upon pathogen challenge. Moreover, elevated expression of ARF10 by different means revealed a direct correlation between ARF10 expression and the induction of ABI5 protein in B. juncea. Through in vitro DNA-protein experiments and chromosome immunoprecipitation using the ARF10 antibody, we demonstrated that ARF10 interacts with the auxin-responsive elements of the ABI5 promoter. This suggests that ARF10 may function as a modulator of ABI5 to induce ABA sensitivity and mediate the resistance response against A. brassicicola.

Structural characterization of a novel full-length transcript promoter from Horseradish Latent Virus (HRLV) and its transcriptional regulation by multiple stress responsive transcription factors.

KEY MESSAGE: The promoter fragment described in this study can be employed for strong transgene expression under both biotic and abiotic stress conditions. Plant-infecting Caulimoviruses have evolved multiple regulatory mechanisms to address various environmental stimuli during the course of evolution. One such mechanism involves the retention of discrete stress responsive cis-elements which are required for their survival and host-specificity. Here we describe the characterization of a novel Caulimoviral promoter isolated from Horseradish Latent Virus (HRLV) and its regulation by multiple stress responsive Transcription factors (TFs) namely DREB1, AREB1 and TGA1a. The activity of full length transcript (Flt-) promoter from HRLV (- 677 to + 283) was investigated in both transient and transgenic assays where we identified H12 (- 427 to + 73) as the highest expressing fragment having ~ 2.5-fold stronger activity than the CaMV35S promoter. The H12 promoter was highly active and near-constitutive in the vegetative and reproductive parts of both Tobacco and Arabidopsis transgenic plants. Interestingly, H12 contains a distinct cluster of cis-elements like dehydration-responsive element (DRE-core; GCCGAC), an ABA-responsive element (ABRE; ACGTGTC) and as-1 element (TGACG) which are known to be induced by cold, drought and pathogen/SA respectively. The specific binding of DREB1, AREB1 and TGA1a to DRE, ABRE and as-1 elements respectively were confirmed by the gel-binding assays using H12 promoter-specific probes. Detailed mutational analysis of the H12 promoter suggested that the presence of DRE-core and as-1 element was indispensable for its activity which was further confirmed by the transactivation assays. Our studies imply that H12 could be a valuable genetic tool for regulated transgene expression under diverse environmental conditions.

Interaction of Arabidopsis TGA3 and WRKY53 transcription factors on Cestrum yellow leaf curling virus (CmYLCV) promoter mediates salicylic acid-dependent gene expression in planta.

MAIN CONCLUSION: This paper highlighted a salicylic acid-inducible Caulimoviral promoter fragment from Cestrum yellow leaf curling virus (CmYLCV). Interaction of Arabidopsis transcription factors TGA3 and WRKY53 on CmYLCV promoter resulted in the enhancement of the promoter activity via NPR1-dependent salicylic acid signaling. Several transcriptional promoters isolated from plant-infecting Caulimoviruses are being presently used worldwide as efficient tools for plant gene expression. The CmYLCV promoter has been isolated from the Cestrum yellow leaf curling virus (Caulimoviruses) and characterized more than 12 years ago; also we have earlier reported a near-constitutive, pathogen-inducible CmYLCV promoter fragment (-329 to +137 from transcription start site; TSS) that enhances stronger (3×) expression than the previously reported fragments; all these fragments are highly efficient in monocot and dicot plants (Sahoo et al. Planta 240: 855-875, 2014). Here, we have shown that the full-length CmYLCV promoter fragment (-729 to +137 from TSS) is salicylic acid (SA) inducible. In this context, we have performed an in-depth study to elucidate the factors responsible for SA-inducibility of the CmYLCV promoter. We found that the as-1 1 and W-box1 elements (located at -649 and -640 from the TSS) of the CmYLCV promoter are required for SA-induced activation by recruiting Arabidopsis TGA3 and WRKY53 transcription factors. Consequently, as a nascent observation, we established the physical interaction between TGA3 and WYKY53; also demonstrated that the N-terminal domain of TGA3 is sufficient for the interaction with the full-length WRKY53. Such interaction synergistically activates the CmYLCV promoter activity in planta. Further, we found that activation of the CmYLCV promoter by SA through TGA3 and WRKY53 interaction depends on NPR1. Finally, the findings presented here provide strong support for the direct regulatory roles of TGA3 and WRKY53 in the SA and NPR1-dependent activation of a Caulimoviral promoter (CmYLCV).

Synthetic promoters in planta.

MAIN CONCLUSION: This paper reviews the importance, prospective and development of synthetic promoters reported in planta. A review of the synthetic promoters developed in planta would help researchers utilize the available resources and design new promoters to benefit fundamental research and agricultural applications. The demand for promoters for the improvement and application of transgenic techniques in research and agricultural production is increasing. Native/naturally occurring promoters have some limitations in terms of their induction conditions, transcription efficiency and size. The strength and specificity of native promoter can be tailored by manipulating its 'cis-architecture' by the use of several recombinant DNA technologies. Newly derived chimeric promoters with specific attributes are emerging as an efficient tool for plant molecular biology. In the last three decades, synthetic promoters have been used to regulate plant gene expression. To better understand synthetic promoters, in this article, we reviewed promoter structure, the scope of cis-engineering, strategies for their development, their importance in plant biology and the total number of such promoters (188) developed in planta to date; we then categorized them under different functional regimes as biotic stress-inducible, abiotic stress-inducible, light-responsive, chemical-inducible, hormone-inducible, constitutive and tissue-specific. Furthermore, we identified a set of 36 synthetic promoters that control multiple types of expression in planta. Additionally, we illustrated the differences between native and synthetic promoters and among different synthetic promoter in each group, especially in terms of efficiency and induction conditions. As a prospective of this review, the use of ideal synthetic promoters is one of the prime requirements for generating transgenic plants suitable for promoting sustainable agriculture and plant molecular farming.

Utilization of plant-derived recombinant human ß-defensins (hBD-1 and hBD-2) for averting salmonellosis.

We describe the use of plant-made ß-defensins as effective antimicrobial substances for controlling salmonellosis, a deadly infection caused by Salmonella typhimurium (referred to further as S. typhi). Human ß-defensin-1 (hBD-1) and -2 (hBD-2) were expressed under the control of strong constitutive promoters in tobacco plants, and bio-active ß-defensins were successfully extracted. In the in vitro studies, enriched recombinant plant-derived human ß-defensin-1 (phBD-1) and -2 (phBD-2) obtained from both T1 and T2 transgenic plants showed significant antimicrobial activity against Escherichia coli and S. typhi when used individually and in various combinations. The 2:1 peptide combination of phBD-1:phBD-2 with peptides isolated from T1-and T2-generation plants reduced the growth of S. typhi by 96 and 85 %, respectively. In vivo studies employing the mouse model (Balb/c) of Salmonella infection clearly demonstrated that the administration of plant-derived defensins individually and in different combinations enhanced the mean survival time of Salmonella-infected animals. When treatment consisted of the 2:1 phBD-1:phBD-2 combination, approximately 50 % of the infected mice were still alive at 206 h post-inoculation; the lowest number of viable S. typhi was observed in the liver and spleen of infected animals. We conclude that plant-made recombinant ß-defensins (phBD-1 and phBD-2) are promising antimicrobial substances and have the potential to become additional tools against salmonellosis, particularly when used in combination.

Effective control of Salmonella infections by employing combinations of recombinant antimicrobial human ß-defensins hBD-1 and hBD-2.

We successfully produced two human ß-defensins (hBD-1 and hBD-2) in bacteria as functional peptides and tested their antibacterial activities against Salmonella enterica serovar Typhi, Escherichia coli, and Staphylococcus aureus employing both spectroscopic and viable CFU count methods. Purified peptides showed approximately 50% inhibition of the bacterial population when used individually and up to 90% when used in combination. The 50% lethal doses (LD50) of hBD-1 against S. Typhi, E. coli, and S. aureus were 0.36, 0.40, and 0.69 µg/µl, respectively, while those for hBD-2 against the same bacteria were 0.38, 0.36, and 0.66 µg/µl, respectively. Moreover, we observed that bacterium-derived antimicrobial peptides were also effective in increasing survival time and decreasing bacterial loads in the peritoneal fluid, liver, and spleen of a mouse intraperitoneally infected with S. Typhi. The 1:1 hBD-1/hBD-2 combination showed maximum effectiveness in challenging the Salmonella infection in vitro and in vivo. We also observed less tissue damage and sepsis formation in the livers of infected mice after treatment with hBD-1 and hBD-2 peptides individually or in combination. Based on these findings, we conclude that bacterium-derived recombinant ß-defensins (hBD-1 and hBD-2) are promising antimicrobial peptide (AMP)-based substances for the development of new therapeutics against typhoid fever.

Comparative analysis of synthetic DNA promoters for high-level gene expression in plants.

MAIN CONCLUSION: We have designed two near- constitutive and stress-inducible promoters (CmYLCV9.11 and CmYLCV4); those are highly efficient in both dicot and monocot plants and have prospective to substitute the CaMV 35S promoter. We performed structural and functional studies of the full-length transcript promoter from Cestrum yellow leaf curling virus (CmYLCV) employing promoter/leader deletion and activating cis-sequence analysis. We designed a 465-bp long CmYLCV9.11 promoter fragment (-329 to +137 from transcription start site) that showed enhanced promoter activity and was highly responsive to both biotic and abiotic stresses. The CmYLCV9.11 promoter was about 28-fold stronger than the CaMV35S promoter in transient and stable transgenic assays using ß-glucuronidase (GUS) reporter gene. The CmYLCV9.11 promoter also demonstrated stronger activity than the previously reported CmYLCV promoter fragments, CmpC (-341 to +5) and CmpS (-349 to +59) in transient systems like maize protoplasts and onion epidermal cells as well as transgenic systems. A good correlation between CmYLCV9.11 promoter-driven GUS-accumulation/enzymatic activities with corresponding uidA-mRNA level in transgenic tobacco plants was shown. Histochemical (X-Gluc) staining of transgenic seedlings, root and floral parts expressing the GUS under the control of CmYLCV9.11, CaMV35S, CmpC and CmpS promoters also support the above findings. The CmYLCV9.11 promoter is a constitutive promoter and the expression level in tissues of transgenic tobacco plants was in the following order: root > leaf > stem. The tobacco transcription factor TGA1a was found to bind strongly to the CmYLCV9.11 promoter region, as shown by Gel-shift assay and South-Western blot analysis. In addition, the CmYLCV9.11 promoter was regulated by a number of abiotic and biotic stresses as studied in transgenic Arabidopsis and tobacco plants. The newly derived CmYLCV9.11 promoter is an efficient tool for biotechnological applications.

Efficient chimeric plant promoters derived from plant infecting viral promoter sequences.

In the present study, we developed a set of three chimeric/hybrid promoters namely FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt incorporating different important domains of Figwort Mosaic Virus sub-genomic transcript promoter (FSgt, -270 to -60), Mirabilis Mosaic Virus sub-genomic transcript promoter (MSgt, -306 to -125) and Peanut Chlorotic Streak Caulimovirus full-length transcript promoter (PFlt-, -353 to +24 and PFlt-UAS, -353 to -49). We demonstrated that these chimeric/hybrid promoters can drive the expression of reporter genes in different plant species including tobacco, Arabidopsis, petunia, tomato and spinach. FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt promoters showed 4.2, 1.5 and 1.2 times stronger GUS activities compared to the activity of the CaMV35S promoter, respectively, in tobacco protoplasts. Protoplast-derived recombinant promoter driven GFP showed enhanced accumulation compared to that obtained under the CaMV35S promoter. FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt promoters showed 3.0, 1.3 and 1.0 times stronger activities than the activity of the CaMV35S² (a modified version of the CaMV35S promoter with double enhancer domain) promoter, respectively, in tobacco (Nicotiana tabacum, var. Samsun NN). Alongside, we observed a fair correlation between recombinant promoter-driven GUS accumulation with the corresponding uidA-mRNA level in transgenic tobacco. Histochemical (X-gluc) staining of whole transgenic seedlings and fluorescence images of ImaGene Green™ treated floral parts expressing the GUS under the control of recombinant promoters also support above findings. Furthermore, we confirmed that these chimeric promoters are inducible in the presence of 150 µM salicylic acid (SA) and abscisic acid (ABA). Taken altogether, we propose that SA/ABA inducible chimeric/recombinant promoters could be used for strong expression of gene(s) of interest in crop plants.

Protocol for imbibed seed piercing for Agrobacterium-mediated transformation of jute.

Here, we present a streamlined Agrobacterium-mediated transformation protocol for jute (Corchorus sp.). We describe steps to pierce and vacuum infiltrate imbibed jute seeds with Agrobacterium suspension. We then detail procedures for selecting transformed seeds by using a hygromycin-B-supplemented medium. This approach can achieve transformation efficiencies of 20.44% ± 1.17% and 15.55% ± 0.58% for tossa (C. olitorius) and white (C. capsularis) jute, respectively. Demanding minimal resources and time, this protocol can elevate genetic engineering research in jute fiber crops. For complete details on the use and execution of this protocol, please refer to Majumder et al. (2020).1.

A review on ubiquitin ligases: Orchestrators of plant resilience in adversity.

Ubiquitin- proteasome system (UPS) is universally present in plants and animals, mediating many cellular processes needed for growth and development. Plants constantly defend themselves against endogenous and exogenous stimuli such as hormonal signaling, biotic stresses such as viruses, fungi, nematodes, and abiotic stresses like drought, heat, and salinity by developing complex regulatory mechanisms. Ubiquitination is a regulatory mechanism involving selective elimination and stabilization of regulatory proteins through the UPS system where E3 ligases play a central role; they can bind to the targets in a substrate-specific manner, followed by poly-ubiquitylation, and subsequent protein degradation by 26 S proteasome. Increasing evidence suggests different types of E3 ligases play important roles in plant development and stress adaptation. Herein, we summarize recent advances in understanding the regulatory roles of different E3 ligases and primarily focus on protein ubiquitination in plant-environment interactions. It also highlights the diversity and complexity of these metabolic pathways that enable plant to survive under challenging conditions. This reader-friendly review provides a comprehensive overview of E3 ligases and their substrates associated with abiotic and biotic stresses that could be utilized for future crop improvement.

"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.

Functional characterization of a serine-threonine protein kinase from Bambusa balcooa that implicates in cellulose overproduction and superior quality fiber formation.

BACKGROUND: Molecular markers allow rapid identification of biologically important germplasm/s having desired character. Previously we have reported a genotype specific molecular marker, Balco1128 [GenBank ID EU258678] of Bambusa balcooa containing an ORF (375 bp) having high similarity with receptor like cytoplasmic kinase of Arabidopsis and Oryza. Balco1128 was found to be associated only with bamboo genotypes endowed with high cellulose and low lignin contents of fibers. Under the above backdrop, it was necessitated to characterize this genetic marker for better understanding of its biological significance in context of superior quality fiber development. RESULTS: The full length cDNA (3342 bp) of BbKst, a serine-threonine protein kinase was isolated from B. balcooa comprising of six LRR domains at the N-terminal end and a kinase domain at the C-terminal end. Bacteria-expressed BbKst-kinase domain (3339 bp long) showed Mg(2+) dependent kinase activity at pH 7.0, 28°C. Bioinformatics study followed by phospho-amino analysis further confirmed that BbKst-kinase belongs to the serine/threonine protein kinase family. Transcript analysis of the BbKst gene following RNA slot blot hybridization and qPCR revealed higher expression of BbKst during initiation and elongation stages of fiber development. Tissue specific expression studies showed much higher expression of BbKst transcript in stems and internodes of B. balcooa than in leaves and rhizomes. Southern analysis revealed single copy insertion of BbKst in most of the Agrobacterium mediated transgenic tobacco plants. Real-time PCR detected 150-200 fold enhanced expression of BbKst in different T1 tobacco lines than that of the vector transformed plants. Heterologous expression of BbKst under control of 35S promoter in transgenic tobacco showed high cellulose deposition in the xylem fibers. Number of xylary fibers was higher in transgenic T0 and T1 plants than that of empty-vector transformed tobacco plants offering enhanced mechanical strength to the transgenic plants, which was also substantiated by their strong upright phenotypes, significantly higher cellulose contents, flexibility coefficient, slenderness ratio, and lower Runkel ratio of the fibers. CONCLUSIONS: This finding clearly demonstrated that BbKst gene (GenBank ID JQ432560) encodes a serine/threonine protein kinase. BbKst induced higher cellulose deposition/synthesis in transgenic tobacco plants, an important attribute of fiber quality bestowing additional strength to the plant.

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.

CRISPR-mediated iron and folate biofortification in crops: advances and perspectives.

Micronutrient deficiency conditions, such as anemia, are the most prevalent global health problem due to inadequate iron and folate in dietary sources. Biofortification advancements can propel the rapid amelioration of nutritionally beneficial components in crops that are required to combat the adverse effects of micronutrient deficiencies on human health. To date, several strategies have been proposed to increase micronutrients in plants to improve food quality, but very few approaches have intrigued `clustered regularly interspaced short palindromic repeats' (CRISPR) modules for the enhancement of iron and folate concentration in the edible parts of plants. In this review, we discuss two important approaches to simultaneously enhance the bioavailability of iron and folate concentrations in rice endosperms by utilizing advanced CRISPR-Cas9-based technology. This includes the 'tuning of cis-elements' and 'enhancer re-shuffling' in the regulatory components of genes that play a vital role in iron and folate biosynthesis/transportation pathways. In particular, base-editing and enhancer re-installation in native promoters of selected genes can lead to enhanced accumulation of iron and folate levels in the rice endosperm. The re-distribution of micronutrients in specific plant organs can be made possible using the above-mentioned contemporary approaches. Overall, the present review discusses the possible approaches for synchronized iron and folate biofortification through modification in regulatory gene circuits employing CRISPR-Cas9 technology.

Further Characterization of MUAS35SCP and FUAS35SCP Recombinant Promoters and Their Implication in Translational Research.

Recombinant promoters are of high value in translational research. Earlier, we developed two recombinant promoters, namely MUAS35SCP and FUAS35SCP, and their transcriptional activities were found to be stronger than that of the most widely used CaMV35S promoter in dicot plants. Presently, we are reporting constitutive expression of both GUS and GFP reporters under the control of these promoters in several monocots, including rice, wheat, and pearl millet. We observed that these promoters could express the reporter genes constitutively, and their expression abilities were almost equal to that of the CaMV35S2 promoter. Plant-derived enriched PaDef (Persea americana var. drymifolia defensin) and NsDef2 (Nigella sativa L. defensin 2) antimicrobial peptides expressed under the control of these promoters arrest the growth of devastating phytopathogens like Pseudomonas syringae, Rhodococcus fascians, and Alternaria alternata. We observed that plant-derived NsDef2 and PaDef under control of these promoters showed approximately 80-90% inhibitory activity against Pseudomonas syringae. Hence, these promoters were constitutive and universal, as they can drive the expression of transgenes in both dicot and monocot plants. Alongside, these promoters could become a valuable tool for raising genetically modified plants with in-built resistance toward phytopathogens.