Identification of miRNA and their target genes in Cestrum nocturnum L. and Cestrum diurnum L. in stress responses.

MicroRNAs (miRNAs) are small, highly conserved non-coding RNA molecules and products of primary miRNAs that regulate the target gene expression. Homology-based approaches were employed to identify miRNAs and their targets in Cestrum nocturnum L. and Cestrum diurnum L. A total of 32 and 12 miRNA candidates were identified in C. nocturnum and C. diurnum. These miRNAs belong to 26 and 10 miRNA families and regulate 1024 and 1007 target genes in C. nocturnum, and C. diurnum, respectively. The functional roles of these miRNAs have not been earlier elucidated in Cestrum. MiR815a, miR849, miR1089 and miR172 have a strong propensity to target genes controlling phytochrome-interacting factor 1 (PIF1), ubiquitin-specific protease 12 (UBP12), leucine-rich repeat (LRR) protein kinase and GAI, RGA, SCR (GRAS) family transcription factor in C. nocturnum. While miR5205a, miR1436 and miR530 regulate PATATIN-like protein 6 (PLP6), PHD finger transcription factor and myb domain protein 48 (MYB48) in C. diurnum. Overall, these miRNAs have regulatory responses in biotic and abiotic stresses in both plant species. Eight putative miRNAs and their target genes were selected for qRT-PCR validation. The validated results suggested the importance of miR815a, miR849, miR5205a, miR1089, miR172, miR1436, and miR530 in exerting control over stress responses in C. nocturnum and C. diurnum. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-022-01127-1.

Physicochemical analysis of Psophocarpus tetragonolobus (L.) DC seeds with fatty acids and total lipids compositions.

Psophocarpus tetragonolobus (L.) DC. is a tropical legume with potential nutritional properties. In present study, the physical properties and proximate composition of the seeds were evaluated. Besides, the physico-chemical properties of fatty oil from fully mature seeds were also studied. The fatty oil compositions of immature, mature and fully mature seeds were evaluated by GC-FID, GC/MS and (1)H-NMR. The study revealed that, fatty oil from fully mature seeds contained high proportion of unsaturated fatty acids (75.5 %), whereas immature seeds contained higher percentage of saturated fatty acid (61.3 %). In addition, unsaponification matter (0.25 %) of fatty oil was identified as stigmasterol (66.4 %) and ß-sitosterol (25.1 %). Total lipids of fully mature seeds were extracted and isolated as neutral, glyco- and phospholipids. Overall, the fatty oil of fully mature seeds was enriched with mono-unsaturated fatty acids (38.6 %) and poly-unsaturated fatty acids (36.9 %) without trans-fatty acids, thus meeting the edible oil standard.

Does the volatile hydrocarbon profile differ between the sexes: a case study on five aphidophagous ladybirds.

Insect hydrocarbons (HCs) primarily serve as a waterproofing cuticular layer and function extensively in chemical communication by facilitating species, sex, and colony recognition. In this study, headspace solid-phase microextraction is employed for investigating the sex-specific volatile HC profile of five ladybirds collected from Lucknow, India namely, Coccinella septempunctata (L.), Coccinella transversalis (Fabr.), Menochilus sexmaculatus (Fabr.), Propylea dissecta (Mulsant), and Anegleis cardoni (Weise) for the first time. Major compounds reported in C. septempunctata, C. transversalis, and A. cardoni are methyl-branched saturated HCs, whereas in M. sexmaculatus, and P. dissecta, they are unsaturated HCs. Other than A. cardoni, both the sexes of the other four ladybirds had similar compounds at highest peak but with statistically significant differences. However, in A. cardoni, which is a beetle with a narrow niche, the major compound in both male and female was different. The difference in volatile HC profile of the sexes of the five ladybirds indicates that gender-specific differences primarily exist due to quantitative differences in chemicals with only very few chemicals being unique to a gender. This variation in semiochemicals might have a role in behavioral or ecological aspects of the studied ladybirds.

Investigating the toxic mechanism of iron oxide nanoparticles-induced oxidative stress in tadpole (Duttaphrynus melanostictus): A combined biochemical and molecular study.

Metal oxide nanomaterials have toxicity towards aquatic organisms, especially microbes and invertebrates, but little is known about their impact on amphibians. We conducted a study on Duttaphrynus melanostictus (D. melanostictus) tadpoles to explore the chronic toxicity effects of iron oxide nanoparticles (IONPs) and the underlying mechanisms of IONPs-induced oxidative stress. IONPs exposure led to increased iron accumulation in the blood, liver, and kidneys of tadpoles, significantly affecting blood parameters and morphology. Higher IONPs concentrations (10 and 50 mg L-1) triggered reactive oxygen species generation, resulting in lipid peroxidation, oxidative stress, and pronounced toxicity in tadpoles. The activity levels of antioxidant enzymes/proteins (SOD, CAT, albumin, and lysozyme) decreased after IONPs exposure, and immunological measures in the blood serum were significantly reduced compared to the control group. Molecular docking analysis revealed that IONPs primarily attached to the surface of SOD/CAT/albumin/lysozyme through hydrogen bonding and hydrophobic forces. Overall, this study emphasizes the ability of IONPs to induce oxidative damage by decreasing immunological profiles such as ACH50 (34.58 ± 2.74 U mL-1), lysozyme (6.94 ± 0.82 U mL-1), total Ig (5.00 ± 0.35 g dL-1), total protein (1.20 ± 0.17 g dL-1), albumin (0.52 ± 0.01 g dL-1) and globulin (0.96 ± 0.01 g dL-1) and sheds light on their potential toxic effects on tadpoles.

Ecotoxicity risk assessment of copper oxide nanoparticles in Duttaphrynus melanostictus tadpoles.

In recent years, copper oxide nanoparticles (CONPs) have gained considerable importance in ecotoxicology studies. CONP ecotoxicity studies on amphibians are limited, particularly on Duttaphrynus melanostictus (D. melanostictus) tadpoles, and most CONP ecotoxicity studies have shown developmental effects on amphibians. Therefore, the present study aimed to determine the ecotoxicity of CONPs in D. melanostictus tadpoles by assessing multi-biomarkers including bioaccumulation, antioxidants, biochemical, haematological, immunological and oxidative stress biomarkers. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to characterize the morphology and physicochemical properties of CONPs. After 30 d of the experiment, blood and organs were collected to measure the levels of multiple biomarkers. The dissolution rate of copper ions in exposed media was observed in all studied groups. According to the results, significant (p < 0.05) increase in copper ion bioaccumulation (blood, liver and kidney), oxidative stress and biochemical biomarkers in the blood serum of CONPs exposed tadpoles compared to control tadpoles, which was accompanied by significant variations in morphological and haematological parameters. In contrast to the untreated tadpoles, the CONPs-exposed tadpoles showed statistically significant (p < 0.05) decreases in antioxidants and immunological indices of blood serum. Based on our results, we concluded that the ecotoxicity of CONPs is due to the production of reactive oxygen species (ROS), which can cause oxidative stress in tadpoles, resulting in impairments. According to our knowledge, the present study was the first to use a multi-biomarker ecotoxicity approach on D. melanostictus tadpoles that could be used as an ecological bioindicator to assess aquatic toxicity.

Reducing the biosynthesis of condensed tannin in winged bean (Psophocarpus tetragonolobus (L.) DC.) by virus-induced gene silencing of anthocyanidin synthase (ANS) gene.

The Underutilized legume-winged bean (Psophocarpus tetragonolobus (L.) DC.) and its various parts are infested with condensed tannin (CT) or proanthocyanidin (PA). CT has anti-nutritional effect as it adversely affects the digestion of proteins, minerals and vitamin among ruminants and humans. It is also responsible for low protein digestibility and decreased amino acid availability. One of the probable reasons of underutilization of P. tetragonolobus is due to its infestation with CT. Histochemical staining of various tissues of P. tetragonolobus with dimethylcinnmaldehyde (DMACA) developed a deep-blue colour indicating the presence of polyphenolic condensed tannin. Structural monomeric unit catechin and epi-catechin were reported to be responsible for biosynthesis of CT in P. tetragonolobus. The enzyme anthocyanidin synthase (ANS) and its corresponding transcripts were identified and phylogenetically mapped. The transcript was subjected to virus-induced gene silencing (VIGS) through agro-infiltration in P. tetragonolobus for reducing the CT-content. The WbANS-VIGS induced P. tetragonolobus resulted in four-fold decrease of CT as compared to the control P. tetragonolobus. A decrease of 73% of CT level was reported in VIGS silenced Wb-ANS line of P. tetragonolobus. This study resulted and confirmed that, the silencing of (ANS) gene in P. tetragonolobus has a regulatory effect on the condensed tannin biosynthesis. This study will pave way for further manipulation of ANS enzyme for reducing the biosynthesis of the anti-nutrient CT. Reducing the CT content will make this underutilized legume more acceptable. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03435-5.

Autophagy Analysis: A Step-by-Step Simple Practical Guide for Immunofluorescence and Western Blotting.

Autophagy is a vital cellular process responsible for breaking down faulty cellular components and organelles, ultimately routed through lysosomes for degradation. This intricate mechanism involves the translocation of LC3, a cytoplasmic protein, onto the autophagosome membranes. As a result, it becomes feasible to discern cells engaged in autophagy by employing fluorescent markers designed for LC3 or other indicative autophagy markers. Although a variety of techniques such as immunofluorescence and western blotting serve as indispensable tools for assessing autophagy, the definitive confirmation comes from the visualization of autophagosomes using transmission electron microscopy. While numerous protocols for antibody staining can be found in scientific literature and on antibody suppliers' websites, these procedures often demand significant time and financial resources for setup. This chapter endeavors to provide a user-friendly and cost-effective guide for practitioners seeking proficiency in immunofluorescence staining and western blotting techniques.

Genome-wide identification and expression analysis of the HD2 protein family and its response to drought and salt stress in Gossypium species.

Histone deacetylase 2 (HD2) proteins play an important role in the regulation of gene expression. This helps with the growth and development of plants and also plays a crucial role in responses to biotic and abiotic stress es. HD2s comprise a C2H2-type Zn2+ finger at their C-terminal and an HD2 label, deacetylation and phosphorylation sites, and NLS motifs at their N-terminal. In this study, a total of 27 HD2 members were identified, using Hidden Markov model profiles, in two diploid cotton genomes (Gossypium raimondii and Gossypium arboretum) and two tetraploid cotton genomes (Gossypium hirsutum and Gossypium barbadense). These cotton HD2 members were classified into 10 major phylogenetic groups (I-X), of which group III was found to be the largest with 13 cotton HD2 members. An evolutionary investigation showed that the expansion of HD2 members primarily occurred as a result of segmental duplication in paralogous gene pairs. Further qRT-PCR validation of nine putative genes using RNA-Seq data suggested that GhHDT3D.2 exhibits significantly higher levels of expression at 12h, 24h, 48h, and 72h of exposure to both drought and salt stress conditions compared to a control measure at 0h. Furthermore, gene ontology, pathways, and co-expression network study of GhHDT3D.2 gene affirmed their significance in drought and salt stress responses.

Transcriptome Meta-Analysis Associated Targeting Hub Genes and Pathways of Drought and Salt Stress Responses in Cotton (Gossypium hirsutum): A Network Biology Approach.

Abiotic stress tolerance is an intricate feature controlled through several genes and networks in the plant system. In abiotic stress, salt, and drought are well known to limit cotton productivity. Transcriptomics meta-analysis has arisen as a robust method to unravel the stress-responsive molecular network in crops. In order to understand drought and salt stress tolerance mechanisms, a meta-analysis of transcriptome studies is crucial. To confront these issues, here, we have given details of genes and networks associated with significant differential expression in response to salt and drought stress. The key regulatory hub genes of drought and salt stress conditions have notable associations with functional drought and salt stress-responsive (DSSR) genes. In the network study, nodulation signaling pathways 2 (NSP2), Dehydration-responsive element1 D (DRE1D), ethylene response factor (ERF61), cycling DOF factor 1 (CDF1), and tubby like protein 3 (TLP3) genes in drought and tubby like protein 1 (TLP1), thaumatin-like proteins (TLP), ethylene-responsive transcription factor ERF109 (EF109), ETS-Related transcription Factor (ELF4), and Arabidopsis thaliana homeodomain leucine-zipper gene (ATHB7) genes in salt showed the significant putative functions and pathways related to providing tolerance against drought and salt stress conditions along with the significant expression values. These outcomes provide potential candidate genes for further in-depth functional studies in cotton, which could be useful for the selection of an improved genotype of Gossypium hirsutum against drought and salt stress conditions.

Assessment of toxicity in the freshwater tadpole Polypedates maculatus exposed to silver and zinc oxide nanoparticles: A multi-biomarker approach.

Nanoparticles (NPs), especially silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs), are widely used in various industrial applications and are released into the surrounding environment through industrial and household wastewater. They have enormous toxic effects on aquatic animals and amphibians. In the current study, a multi-biomarker approach was used to assess toxicity on Polypedates maculatus (P. maculatus) tadpoles collected from a freshwater pond and exposed to sub-lethal concentrations of Ag-NPs (1, 5 and 10 mg L-1) and ZnO-NPs (1, 10 and 50 mg L-1). A significant bioaccumulation of silver (Ag) and Zinc (Zn) was observed in the blood, liver, kidney and bones in comparison to control tadpoles. Blood parameters (Red blood cells (RBC), Hematocrit (Htc), White blood cells (WBC), monocytes, lymphocytes and neutrophils), immunological markers (ACH50, lysozyme, total Ig, total protein, albumin, and globulin), biochemical markers (glucose, cortisol, cholesterol, triglycerides, alanine transaminase (ALT), asparatate transaminase (AST), alkaline phosphatase (ALP), urea and creatinine) and the oxidative stress marker (LPO) of serum were increased significantly (p < 0.05) in Ag/ZnO-NPs exposed groups when compared to the control groups. The levels of mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV) and haemoglobin (Hb) in the ZnO NP-exposed groups were significantly different from those in the control group. Antioxidant (SOD and CAT) levels were significantly declined in the treatment groups. Based on the results, Ag/ZnO-NPs are toxic to aquatic organisms and amphibians at sub-lethal concentrations. The species P. maculatus can be used as a bioindicator for the nanomaterial (NM) contamination of freshwater systems.

A review on arsenic pollution, toxicity, health risks, and management strategies using nanoremediation approaches.

OBJECTIVES: Over 50 countries are affected by arsenic contamination. The problem is becoming worse as the number of affected people increases and new sites are reported globally. CONTENT: Various human activities have increased arsenic pollution, notably in both terrestrial and aquatic environments. Contamination of our water and soil by arsenic poses a threat to our environment and natural resources. Arsenic poisoning harms several physiological systems and may cause cancer and death. Excessive exposure may cause toxic build-up in human and animal tissues. Arsenic-exposed people had different skin lesion shapes and were vulnerable to extra arsenic-induced illness risks. So far, research shows that varying susceptibility plays a role in arsenic-induced diseases. Several studies have revealed that arsenic is a toxin that reduces metabolic activities. Diverse remediation approaches are being developed to control arsenic in surrounding environments. SUMMARY AND OUTLOOK: A sustainable clean-up technique (nanoremediation) is required to restore natural equilibrium. More research is therefore required to better understand the biogeochemical processes involved in removing arsenic from soils and waters.

Genome-Wide Identification and Evolutionary Analysis of Gossypium Tubby-Like Protein (TLP) Gene Family and Expression Analyses During Salt and Drought Stress.

Tubby-like proteins (TLPs) possess a highly conserved closed ß barrel tubby domain at C-terminal and N-terminal F-box. The role of TLP gene family members has been widely discussed in numerous organisms; however, the detailed genome-wide study of this gene family in Gossypium species has not been reported till date. Here, we systematically identified 105 TLP gene family members in cotton (Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense) genomes and classified them into eight phylogenetic groups. Cotton TLP12 gene family members clustered into two groups, 4 and 8. They experienced higher evolutionary pressure in comparison to others, indicating the faster evolution in both diploid as well as in tetraploid cotton. Cotton TLP gene family members expanded mainly due to segmental duplication, while only one pair of tandem duplication was found in cotton TLPs paralogous gene pairs. Subsequent qRT-PCR validation of seven putative key candidate genes of GhTLPs indicated that GhTLP11A and GhTLP12A.1 genes were highly sensitive to salt and drought stress. The co-expression network, pathways, and cis-regulatory elements of GhTLP11A and GhTLP12A.1 genes confirmed their functional importance in salt and drought stress responses. This study proposes the significance of GhTLP11A and GhTLP12A.1 genes in exerting control over salt and drought stress responses in G. hirsutum and also provides a reference for future research, elaborating the biological roles of G. hirsutum TLPs in both stress responses.

De novo sequencing and comparative analysis of leaf transcriptomes of diverse condensed tannin-containing lines of underutilized Psophocarpus tetragonolobus (L.) DC.

Condensed tannin (CT) or proanthocyanidin (PA) is a unique group of phenolic metabolite with high molecular weight with specific structure. It is reported that, the presence of high-CT in the legumes adversely affect the nutrients in the plant and impairs the digestibility upon consumption by animals. Winged bean (Psophocarpus tetragonolobus (L.) DC.) is one of the promising underutilized legume with high protein and oil-content. One of the reasons for its underutilization is due to the presence of CT. Transcriptome sequencing of leaves of two diverse CT-containing lines of P. tetragonolobus was carried out on Illumina Nextseq 500 sequencer to identify the underlying genes and contigs responsible for CT-biosynthesis. RNA-Seq data generated 102586 and 88433 contigs for high (HCTW) and low CT (LCTW) lines of P. tetragonolobus, respectively. Based on the similarity searches against gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) database revealed 5210 contigs involved in 229 different pathways. A total of 1235 contigs were detected to differentially express between HCTW and LCTW lines. This study along with its findings will be helpful in providing information for functional and comparative genomic analysis of condensed tannin biosynthesis in this plant in specific and legumes in general.

Establishment of an efficient and rapid method of multiple shoot regeneration and a comparative phenolics profile in in vitro and greenhouse-grown plants of Psophocarpus tetragonolobus (L.) DC.

An in vitro method of multiple shoot induction and plant regeneration in Psophocarpus tetragonolobus (L.) DC was developed. Cotyledons, hypocotyls, epicotyls, internodal and young seedling leaves were used as explants. MS media supplemented with various concentrations of either thidiazuron (TDZ) or N6-benzylaminopurine (BAP) along with NAA or IAA combinations were used to determine their influence on multiple shoot induction. MS media supplemented with TDZ induced direct shoot regeneration when epicotyls and internodal segments were used as explants. TDZ at 3 mg L(-1) induced highest rate (89.2 ± 3.28%) of regeneration with (13.4 ± 2.04) shoots per explant. MS media supplemented with BAP in combination with NAA or IAA induced callus mediated regeneration when cotyledons and hypocotyls were used as explants. BAP (2.5 mg L(-1)) and IAA (0.2 mg L(-1)) induced highest rate (100 ± 2.66%) of regeneration with (23.2 ± 2.66) shoots per explant. Mature plants produced from regenerated shoots were transferred successfully to the greenhouse. In a comparative study, the phenolics contents of various parts of greenhouse-grown plants with that of in vitro-raised plants showed significant variations.