A sucrose non-fermenting-1-related protein kinase 1 gene from wheat, TaSnRK1α regulates starch biosynthesis by modulating AGPase activity.

Major portion of wheat grain consist of carbohydrate, mainly starch. The proportion of amylose and amylopectin in starch greatly influence the end product quality. Advancement in understanding starch biosynthesis pathway and modulating key genes has enabled the genetic modification of crops resulting in enhanced starch quality. However, the regulation of starch biosynthesis genes still remains unexplored. So, to expand the limited knowledge, here, we characterized a Ser/Thr kinase, SnRK1α in wheat and determined its role in regulating starch biosynthesis. SnRK1 is an evolutionary conserved protein kinase and share homology to yeast SNF1. Yeast complementation assay suggests TaSnRK1α restores growth defect and promotes glycogen accumulation. Domain analysis and complementation assay with truncated domain proteins suggest the importance of ATP-binding and UBA domain in TaSnRK1α activity. Sub-cellular localization identified nuclear and cytoplasmic localization of TaSnRK1α in tobacco leaves. Further, heterologous over-expression (O/E) of TaSnRK1α in Arabidopsis not only led to increase in starch content but also enlarges the starch granules. TaSnRK1α was found to restore starch accumulation in Arabidopsis kin10. Remarkably, TaSnRK1α O/E increases the AGPase activity suggesting the direct regulation of rate limiting enzyme AGPase involved in starch biosynthesis. Furthermore, in vitro and in vivo interaction assay reveal that TaSnRK1α interacts with AGPase large sub-unit. Overall, our findings indicate that TaSnRK1α plays a role in starch biosynthesis by regulating AGPase activity.

Rethinking underutilized cereal crops: pan-omics integration and green system biology.

MAIN CONCLUSION: Due to harsh lifestyle changes, in the present era, nutritional security is needed along with food security so it is necessary to include underutilized cereal crops (UCCs) in our daily diet to counteract the rising danger of human metabolic illness. We can attain both the goal of zero hunger and nutritional security by developing improved UCCs using advanced pan-omics (genomics, transcriptomics, proteomics, metabolomics, nutrigenomics, phenomics and ionomics) practices. Plant sciences research progressed profoundly since the last few decades with the introduction of advanced technologies and approaches, addressing issues of food demand of the growing population, nutritional security challenges and climate change. However, throughout the expansion and popularization of commonly consumed major cereal crops such as wheat and rice, other cereal crops such as millet, rye, sorghum, and others were impeded, despite their potential medicinal and nutraceutical qualities. Undoubtedly neglected underutilized cereal crops (UCCs) also have the capability to withstand diverse climate change. To relieve the burden of major crops, it is necessary to introduce the new crops in our diet in the way of UCCs. Introgression of agronomically and nutritionally important traits by pan-omics approaches in UCCs could be a defining moment for the population's well-being on the globe. This review discusses the importance of underutilized cereal crops, as well as the application of contemporary omics techniques and advanced bioinformatics tools that could open up new avenues for future study and be valuable assets in the development and usage of UCCs in the perspective of green system biology. The increased and improved use of UCCs is dependent on number of factors that necessitate a concerted research effort in agricultural sciences. The emergence of functional genomics with molecular genetics might gear toward the reawakening of interest in underutilized cereals crops. The need of this era is to focus on potential UCCs in advanced agriculture and breeding programmes. Hence, targeting the UCCs, might provide a bright future for better health and scientific rationale for its use.

Interaction between long noncoding RNA (lnc663) and microRNA (miR1128) regulates PDAT-like gene activity in bread wheat (Triticum aestivum L.).

Amylose, a starch subcomponent, can bind lipids within its helical groove and form an amylose-lipid complex, known as resistant starch type 5 (RS-5). RS contributes to lower glycaemic index of grain with health benefits. Unfortunately, genes involved in lipid biosynthesis in wheat grain remain elusive. Our study aims to characterize the lipid biosynthesis gene and its post-transcriptional regulation using the parent bread wheat variety 'C 306' and its EMS-induced mutant line 'TAC 75' varying in amylose content. Quantitative analyses of starch-bound lipids showed that 'TAC 75' has significantly higher lipid content in grains than 'C 306' variety. Furthermore, expression analyses revealed the higher expression of wheat phospholipid: diacylglycerol acyltransferase-like (PDAT-like) in the 'TAC 75' compared to the 'C 306'. Overexpression and ectopic expression of TaPDAT in yeast and tobacco leaf confirmed its ability to accumulate lipids in vivo. Enzyme activity assay showed that TaPDAT catalyzes the triacylglycerol synthesis by acylating 1,2-diacylglycerol. Interestingly, the long non-coding RNA, lnc663, was upregulated with the TaPDAT gene, while the miRNA, miR1128, downregulated in the 'TAC 75', indicating a regulatory relationship. The GFP reporter assay confirmed that the lnc663 acts as a positive regulator, and the miR1128 as a negative regulator of the TaPDAT gene, which controls lipid accumulation in wheat grain. Our findings outline TaPDAT-mediated biosynthesis of lipid accumulation and reveal the molecular mechanism of the lnc663 and miR1128 mediated regulation of the TaPDAT gene in wheat grain.

Comparative transcriptome analyses revealed key genes involved in high amylopectin biosynthesis in wheat.

High amylopectin starch is an important modified starch for food processing industries. Despite a thorough understanding of starch biosynthesis pathway, the regulatory mechanism responsible for amylopectin biosynthesis is not well explored. The present study utilized transcriptome sequencing approach to understand the molecular basis of high amylopectin content in three high amylopectin mutant wheat lines ('TAC 6', 'TAC 358', and 'TAC 846') along with parent variety 'C 306'. Differential scanning calorimetry (DSC) of high amylopectin starch identified a high thermal transition temperature and scanning electron microscopy (SEM) revealed more spherical starch granules in mutant lines compared to parent variety. A set of 4455 differentially expressed genes (DEGs) were identified at two-fold compared to the parent variety in high amylopectin wheat mutants. At ten-fold, 279 genes, including two starch branching genes (SBEIIa and SBEIIb), were up-regulated and only 30 genes, including the starch debranching enzyme (DBE), were down-regulated. Among the genes, different isoforms of sucrose non-fermenting-1-related protein kinase-1 (TaSnRK1α2-3B and TaSnRK1α2-3D) and its regulatory subunit, sucrose non-fermenting-4 (SNF-4-2A, SNF-4-2B, and SNF-4-5D), were found to be highly up-regulated. Further, expression of the DEGs related to starch biosynthesis pathway and TaSnRK1α2 and SNF-4 was performed using qRT-PCR. High expression of TaSnRK1α2, SNF-4, and SBEII isoforms suggests their probable role in high amylopectin starch biosynthesis in grain endosperm. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03364-3.

Protein targeting to starch 1, a functional protein of starch biosynthesis in wheat (Triticum aestivum L.).

KEY MESSAGE: TaPTST1, a wheat homolog of AtPTST1 containing CBM can interact with GBSSI and regulate starch metabolism in wheat endosperm. In cereal endosperm, native starch comprising amylose and amylopectin is synthesized by the coordinated activities of several pathway enzymes. Amylose in starch influences its physio-chemical properties resulting in several human health benefits. The Granule-Bound Starch Synthase I (GBSSI) is the most abundant starch-associated protein. GBSSI lacks dedicated Carbohydrate-binding module (CBM). Previously, Protein Targeting To Starch 1 (PTST1) was identified as a crucial protein for the localization of GBSSI to the starch granules in Arabidopsis. The function of its homologous protein in the wheat endosperm is not known. In this study, TaPTST1, an AtPTST1 homolog, containing a CBM and a coiled-coil domain was identified in wheat. Protein-coding nucleotide sequence of TaPTST1 from Indian wheat variety 'C 306' was cloned and characterized. Homology modelling and molecular docking suggested the potential interaction of TaPTST1 with glucans and GBSSI. The TaPTST1 expression was higher in wheat grain than the other tissues, suggesting a grain-specific function. In vitro binding assays demonstrated different binding affinities of TaPTST1 for native starch, amylose, and amylopectin. Furthermore, the immunoaffinity pull-down assay revealed that TaPTST1 directly interacts with GBSSI, and the interaction is mediated by a coiled-coil domain. The direct protein-protein interaction was further confirmed by bimolecular fluorescence complementation assay (BiFC) in planta. Based on our findings we postulate a functional role for TaPTST1 in starch metabolism by targeting GBSSI to starch granules in wheat endosperm.

Genome-wide analysis of RING-type E3 ligase family identifies potential candidates regulating high amylose starch biosynthesis in wheat (Triticum aestivum L.).

In ubiquitin-mediated post-translational modifications, RING finger families are emerged as important E3 ligases in regulating biological processes. Amylose and amylopectin are two major constituents of starch in wheat seed endosperm. Studies have been found the beneficial effects of high amylose or resistant starch on health. The ubiquitin-mediated post-translational regulation of key enzymes for amylose/amylopectin biosynthesis (GBSSI and SBEII) is still unknown. In this study, the genome-wide analysis identified 1272 RING domains in 1255 proteins in wheat, which is not reported earlier. The identified RING domains classified into four groups-RING-H2, RING-HC, RING-v, RING-G, based on the amino acid residues (Cys, His) at metal ligand positions and the number of residues between them with the predominance of RING-H2 type. A total of 1238 RING protein genes were found to be distributed across all 21 wheat chromosomes. Among them, 1080 RING protein genes were identified to show whole genome/segmental duplication within the hexaploid wheat genome. In silico expression analysis using transcriptome data revealed 698 RING protein genes, having a possible role in seed development. Based on differential gene expression and correlation analysis of 36 RING protein genes in diverse (high and low) amylose mutants and parent, 10 potential RING protein genes found to be involved in high amylose biosynthesis and significantly associated with two starch biosynthesis genes; GBSSI and SBEIIa. Characterization of mutant lines using next-generation sequencing method identified unique mutations in 698 RING protein genes. This study signifies the putative role of RING-type E3 ligases in amylose biosynthesis and this information will be helpful for further functional validation and its role in other biological processes in wheat.

Understanding the regulatory relationship of abscisic acid and bZIP transcription factors towards amylose biosynthesis in wheat.

Starch is biosynthesized during seed development and this process is regulated by many bZIP proteins in bread wheat. Abscisic acid (ABA), an important phyto-hormone involved in various physiological processes mediated by bZIPs in plants including seed development. The 'Group A' TabZIP transcription factors play important roles in the ABA signaling pathway in Arabidopsis, rice and other cereal crops but their role in regulation of amylose biosynthesis in wheat is limited. In this study 83 'Group A' TabZIPs were characterized by gene expression analysis in wheat amylose mutants. A set of 17 TabZIPs was selected on the basis of differential expression (> 2 fold) in low and high amylose mutants from RNA-seq data and validated by qRT PCR. Based on qRT PCR and correlation analysis out of the 17 TabZIPs six differentially expressed candidate TabZIPs were identified, involving in high amylose biosynthesis. The TabZIP175.2, identified as upregulated in all high amylose lines and TabZIP90.2, TabZIP129.1, TabZIP132.2, TabZIP143 and TabZIP159.2 were found downregulated in all low amylose lines, after exogenous supply of ABA. Proximal promoter analysis of starch pathway genes revealed the presence of ABA-responsive elements (ABREs) that are putative binding sites for bZIPs. Collectively, these findings indicated the involvement of putative six candidate TabZIPs as transcriptional regulators of amylose related genes via an ABA-dependent pathway in wheat. This study could help the investigators to make an informed decision to edit wheat genome for high/low amylose content using gene-editing technologies.

Development and characterization of bZIP transcription factor based SSRs in wheat.

Assessment of existing diversity is the key for germplasm conservation and crop improvement. Wheat (Triticum aestivum L.) is among the most important cereal crop and consumed by two billion world's populations. DNA-based markers are predominantly used for diversity characterization because they are easy to develop and not influenced by environment. Among them microsatellites (simple sequence repeats, SSRs) are most suitable due to their genome-wide distribution, hypervariability and reproducibility for their applications in diversity, genetic improvement, and molecular breeding. bZIP transcription factors play major roles in plants in light and stress signalling, seed development, and defence. A total of 846 SSRs were identified from 370 wheat cDNA sequences and a sub-set of 35 polymorphic TabZIPMS (TriticumaestivumbZIP MicroSatellites) was used for diversity and genetic structure analysis of 92 Indian wheat varieties and related species. 114 SSR variants ranging from 2 to 5 per SSR locus were detected for 35 SSRs in the varieties. Average polymorphic information content (PIC) and observed heterozygosity was found to be 0.135 and 0.838, respectively. Thirty-four SSRs showed cross-transferability into different related species. Combined Bayesian model and Jaccard's similarity based genetic clustering analysis revealed two clusters of 80 bread wheat varieties and one separate cluster of related species. In this study, a total 35 novel bZIP-derived SSRs were identified in a set 370 bZIP genes and shown high polymorphism and cross-species transferability in wheat. The findings provide resources for future utilization in genetic resource conservation, trait introgression, breeding and varietal development.

Identification and characterization of long non-coding RNAs regulating resistant starch biosynthesis in bread wheat (Triticum aestivum L.).

Resistant starch (RS) also known as healthy starch has shown several health benefits. Enhancing the RS through genetic modification approaches has huge commercial importance. Regulatory RNA like long non-coding RNA (lncRNA) plays an important role in gene regulation. In this study, we mined 63 transcriptome datasets of wheat belonging to 35 genotypes representing two seed developmental stages. Contrasting expression of a subset of lncRNAs in RS mutant lines compared to parent wheat variety 'C 306' signifies their probable role in RS biosynthesis. Further, lncRNA- TCONS_00130663 showed strong positive correlation (r2 = 1) with LYPL gene and strong negative correlation with SBEIIb (r2 = -0.94). We found TCONS_00130663 as positive regulator of LYPL gene through interaction with miR1128. Based on relative expression, in silico interaction and DSC analysis we hypothesize the dual role of TCONS_00130663 in RS type 2 and type 5. The study provides a useful resource for functional mechanism of lncRNAs.

Discovery of 2-isoxazol-3-yl-acetamide analogues as heat shock protein 90 (HSP90) inhibitors with significant anti-HIV activity.

The recent burst of explorations on heat shock protein 90 (HSP90) in virus research supports its emergence as a promising target to overcome the drawbacks of current antiviral therapeutic regimen. In continuation of our efforts towards the discovery of novel anti-retroviral molecules, we designed, synthesized fifteen novels 2-isoxazol-3-yl-acetamide based compounds (2a-o) followed by analysis of their anti-HIV activity and cytotoxicity studies. 2a-b, 2e, 2j, and 2l-m were found to be active with inhibitory potentials >80% at their highest non-cytotoxic concentration (HNC). Further characterization of anti-HIV activity of these molecules suggests that 2l has ∼3.5 fold better therapeutic index than AUY922, the second generation HSP90 inhibitor. The anti-HIV activity of 2l is a cell type, virus isolate and viral load independent phenomena. Interestingly, 2l does not significantly modulate viral enzymes like Reverse Transcriptase (RT), Integrase (IN) and Protease (PR) as compared to their known inhibitors in a cell free in vitro assay system at its HNC. Further, 2l mediated inhibition of HSP90 attenuates HIV-1 LTR driven gene expression. Taken together, structural rationale, modeling studies and characterization of biological activities suggest that this novel scaffold can attenuate HIV-1 replication significantly within the host and thus opens a new horizon to develop novel anti-HIV therapeutic candidates.

Pivotal role of bZIPs in amylose biosynthesis by genome survey and transcriptome analysis in wheat (Triticum aestivum L.) mutants.

Starch makes up 70% of the wheat grain, and is an important source of calories for humans, however, the overconsumption of wheat starch may contribute to nutrition-associated health problems. The challenge is to develop resistant starch including high amylose wheat varieties with health benefits. Adapting advance genomic approaches in EMS-induced mutant lines differing in amylose content, basic leucine zipper (bZIP) regulatory factors that may play role in controlling amylose biosynthesis were identified in wheat. bZIP transcription factors are key regulators of starch biosynthesis genes in rice and maize, but their role in regulating these genes in wheat is poorly understood. A genome-wide survey identified 370 wheat bZIPs, clustered in 11 groups, showing variations in amino acids composition and predicted physicochemical properties. Three approaches namely, whole transcriptome sequencing, qRT-PCR, and correlation analysis in contrasting high and low amylose mutants and their parent line identified 24 candidate bZIP (positive and negative regulators), suggesting bZIPs role in high amylose biosynthesis. bZIPs positive role in high amylose biosynthesis is not known. In silico interactome studies of candidate wheat bZIP homologs in Arabidopsis and rice identified their putative functional role. The identified bZIPs are involved in stress-related pathways, flower and seed development, and starch biosynthesis. An in-depth analysis of molecular mechanism of novel candidate bZIPs may help in raising and improving high amylose wheat varieties.

Efficacy of Andrographis paniculata against extended spectrum ß-lactamase (ESBL) producing E. coli.

BACKGROUND: A. paniculata is widely known for its medicinal values and is traditionally used to treat a wide range of diseases such as cancer, diabetes, skin infections, influenza, diarrhoea, etc. The phytochemical constituents of this plant possess unique and interesting biological activities. The main focus of this study was to evaluate the antibacterial property of crude ethyl acetate (CEA) extract of A. paniculata against E. coli clinical isolates along with molecular docking of 10 different bioactive components from this plant with CTX-M-15. METHODS: CEA extract was subjected to phytochemical and FTIR analysis. The E. coli isolates were tested for antibiotic susceptibility through disk-diffusion method to observe their resistance pattern towards different antibiotics. Antibacterial activity and biofilm assay were performed through broth microdilution using a 96-well microplate. CEA extract was further utilized to observe its effect on the expression of a gene encoding CTX-M-15. Finally, in-silico studies were performed where 10 different bioactive compounds from A. paniculata were molecularly docked with CTX-M-15. RESULTS: Phytochemical and FTIR analysis detected the presence of various secondary metabolites and functional groups in CEA extract respectively. Molecular docking provided the number of residues and bond lengths together with a positive docking score. Antibiotic susceptibility showed the multi-drug resistance of all the clinical strains of E. coli. The antibacterial and antibiofilm efficiency of CEA extract (25, 50 and 100 µg/ml) was tested and 100 µg/ml of the extract was more effective in all the strains of E. coli. All 3 ESBL producing strains of E. coli were subjected to gene expression analysis through PCR. Strains treated with 100 µg/ml of the extract showed a downregulation of the gene encoding CTX-M-15 compared to untreated controls. CONCLUSIONS: The utilization of CEA extract of A. paniculata proved an economical way of controlling the growth and biofilm formation of ESBL strains of E. coli. CEA extract was also able to downregulate the expression of a gene encoding CTX-M-15. Molecular docking of 10 different bioactive compounds from A. paniculata with CTX-M-15 provided the residues and bond lengths with a positive docking score.

Trait Mapping Approaches Through Association Analysis in Plants.

Previously, association mapping (AM) methodology was used to unravel genetic complications in animal science by measuring the complex traits for candidate and non-candidate genes. Nowadays, this statistical approach is widely used to clarify the complexity in plant breeding program-based genome-wide breeding strategies, marker development, and diversity analysis. This chapter is particularly focused on methodologies with limitations and provides an overview of AM models and software used up to now. Association or linkage disequilibrium mapping has become a very popular method for discovering candidate and non-candidate genes and confirmation of quantitative trait loci (QTL) on various parts of the genome and in marker-assisted selection for breeding. Previously, various QTL investigations were carried out for different plants exclusively by linkage mapping. To help to understand the basics of modern molecular genetic techniques, in this chapter we summarize previous studies done on different crops. AM offers high-resolution power when there is large genotypic diversity and low linkage disequilibrium (LD) for the germplasm being investigated. The benefits of AM, compared with traditional QTL mapping, include a relatively detailed mapping resolution and a far less time-consuming approach since no mapping populations need to be generated. The advancements in genotyping and computational techniques have encouraged the use of AM. AM provides a fascinating approach for genetic investigation of QTLs, due to its resolution and the possibility to study the various genomic areas at the same time without construction of mapping populations. In this chapter we also discuss the advantages and disadvantages of AM, especially in the dicotyledonous crops Fabaceae and Solanaceae, with various genome-size reproductive strategies (clonal vs. sexual), and statistical models. The main objective of this chapter is to highlight the uses of association genetics in major and minor crop species that have trouble being analyzed for dissection of complex traits by identification of the factor responsible for controlling the effect of trait. Graphical Abstract.

Synthesis and anti-HCV determinant motif identification in pyranone carboxamide scaffold.

Hepatitis C Virus exhibits high genetic diversity. The current treatment for genotype-1 with ∼80% sustained virologic responses is a combination of pegylated interferon, ribavirin and boceprevir/telaprevir/simeprevir which is associated with several side effects and need close monitoring. Therefore, novel therapies are invited for safer and more efficient treatment. This study was designed for synthesis of new α-pyranone carboxamide analogs for evaluation of anti-HCV activity to delineate structure-activity relationship (SAR) and to identify anti-HCV determinant motif on this new scaffold. Forty four new α-pyranone carboxamide analogs were synthesized. Six potential anti-HCV candidates 11a (EC50=0.35 µM), 11e (EC50=0.48 µM), 12f (EC50=0.47 µM), 12g (EC50=0.39 µM), 12h (EC50=0.20 µM) and 12j (EC50=0.25 µM) with lower cytotoxicity (CC50>20 µM) were discovered through cell based HCV replicon system. The activity profile of forty four new α-pyranone carboxamide analogs suggests the role of an aromatic motif in the B region to add a synergistic effect to NHOH motif at 4-position and revels an anti-HCV activity determinants motif under this scaffold. The biochemical assay against most promising HCV target protein 'NS3 protease and NS5B polymerase' showed no activity and open a scope to explore new mechanism inhibitor.

Skeletal hybridization and PfRIO-2 kinase modeling for synthesis of α-pyrone analogs as anti-malarial agent.

The pharmacophoric hybridization and computational design approach were applied to generate a novel series of α-pyrone analogs as plausible anti-malarial lead candidate. A putative active site in flexible loop close to wing-helix domain of PfRIO2 kinase was explored computationally to understand the molecular basis of ligand binding. All the synthesized molecules (3a-g) exhibited in vitro antimalarial activity. Oxidative stress induced by 3a-d were calculated and found to be significantly higher in case of 3b. Therefore, 3b, which shown most significant result was identified as promising lead for further SAR study to develop potent anti-malarials.