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With the advancement of plant synthetic biology, complex genetic engineering circuits are being developed, which require more diverse genetic regulatory elements (promoters) to operate. Constitutive promoters are widely used for such gene engineering projects, but the list of strong, constitutive plant promoters with strength surpassing the widely used promoter, the CaMV35S, is limited. In this work, we attempted to increase the constitutive promoter library by developing efficient synthetic promoters suitable for high-level gene expression. To do that, we selected three strong pararetroviral-based promoters from Mirabilis mosaic virus (MMV), Figwort mosaic virus (FMV), and Horseradish latent virus (HRLV) and rationally designed and combined their promoter elements. We then tested the newly developed promoters in Nicotiana benthamiana and found a highly active tri-hybrid promoter, MuasFuasH17 (MFH17). We further used these promoter elements in generating random mutant promoters by DNA shuffling techniques in an attempt to change/improve the MFH17 promoter. We further evaluated the activity of the MFH17 promoter in Oryza sativa seedlings and studied the effect of as-1 elements present in it. Finally, we tested the efficacy and tissue specificity of the MFH17 promoter in planta by developing transgenic Nicotiana tabacum and Arabidopsis thaliana plants and found it highly constitutive and efficient in driving the gene throughout the plant tissues. Overall, we conclude that this tripartite synthetic promoter MFH17 is a strong, highly constitutive, and dual-species (dicot and monocot) expressing promoter, which can be a valuable addition to the constitutive plant promoter library for plant synthetic biology. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Promoters are one of the most important components for many gene-based research as they can fine-tune precise gene expression. Many unique plant promoters have been characterized, but strong promoters with dual expression in both monocot and dicot systems are still lacking. In this study, we attempted to make such a promoter by combining specific domains from monocot-infecting pararetroviral-based promoters sugarcane bacilliform virus (SCBV) and banana streak virus (BSV) to a strong dicot-infecting pararetroviral-based promoter mirabilis mosaic virus (MMV). The generated chimeric promoters, MS, SM, MB, and BM, were tested in monocot and dicot systems and further validated in transgenic tobacco plants. We found that the developed chimeric promoters were species-specific (monocot or dicot), which depended on their respective core promoter (CP) region. Furthermore, with this knowledge, deletion-hybrid promoters were developed and evaluated, which led to the development of a unique dual-expressing promoter, MSD3, with high gene expression efficiency (GUS and GFP reporter genes) in rice, pearl millet, and tobacco plants. We conclude that the MSD3 promoter can be an important genetic tool and will be valuable in plant biology research and application. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The genus Vigna comprises fast-growing, diploid legumes, cultivated in tropical and subtropical parts of the world. It comprises more than 200 species among which Vigna angularis, Vigna radiata, Vigna mungo, Vigna aconitifolia, Vigna umbellata, Vigna unguiculata, and Vigna vexillata are of enormous agronomic importance. Human selection along with natural variability within these species encompasses a vital source for developing new varieties. The present review convokes the early domestication history of Vigna species based on archeological pieces of evidence and domestication-related traits (DRTs) together with genetics of domestication. Traces of early domestication of Vigna have been evidenced to spread across several temperate and tropical regions of Africa, Eastern Asia, and few parts of Europe. Several DRTs of Vigna species, such as pod shattering, pod and seed size, dormancy, seed coat, seed color, maturity, and pod dehiscence, can clearly differentiate wild species from their domesticates. With the advancement in next-generation highthroughput sequencing techniques, exploration of genetic variability using recently released reference genomes along with de novo sequencing of Vigna species have provided a framework to perform genome-wide association and functional studies to figure out different genes related to DRTs. In this review, genes and quantitative trait loci (QTLs) related to DRTs of different Vigna species have also been summarized. Information provided in this review will enhance the in-depth understanding of the selective pressures that causes crop domestication along with nature of evolutionary selection made in unexplored Vigna species. Furthermore, correlated archeological and domestication-related genetic evidence will facilitate Vigna species to be considered as suitable model plants. [ABSTRACT FROM AUTHOR]