Physiological alterations and heavy metal accumulation in the transplanted lichen Pyxine cocoes (Sw.) Nyl. in Lucknow city, Uttar Pradesh.

Air pollution has become a major concern due to its detrimental effects on living beings. The present study is aimed at assessing the current status of air pollution in Lucknow city using lichen transplantation technique and assesing its effect on physiology of Pyxine cocoes. The samples of P. cocoes were collected from relatively pollution-free area Malihabad and transplanted in 10 designated sites in five regions for 30 days. Various parameters such as heavy metals, chlorophyll pigments, carotenoid, chlorophyll degradation, and electrolyte conductivity were estimated in transplanted lichens. The study revealed that the concentration of all 10 heavy metals was higher in all transplanted samples than in the control sample, which was found in order of Al > Fe > Mn > Zn > Cu > Cr > Pb > Ni > Co > Cd. Among all 10 transplanted sites, the significantly increased accumulation of aluminum (5.11 to 5.47 µg L-1), iron (4.73 to 5.46 µg L-1), manganese (110.99 to 144.58 µg g-1), and zinc (87.96 to 97.40 µg g-1) was found in Charbagh, Qaisarbagh, and Alambagh sites. Further, in all samples, chlorophyll a (3.98 µg L-1), chlorophyll b (1.22 µg L-1), total chlorophyll (5.20 µg L-1), and chlorophyll degradation (0.55 µg g-1) were significantly decreased, whereas elevated levels of carotenoid (0.71 µg g-1), and electrolyte conductivity (64.99 µS cm-1), were observed. The scanning electron microscope (SEM) investigated the morphological changes in transplanted lichen samples, and significant damage to the anatomy of mycelium was found in most of the polluted site's samples, which correlated with the pollution levels. The present study clearly demonstrated that the transplanted lichen P. cocoes is an efficient bioaccumulator and bioindicator of air quality in urban environments.

Comparative transcriptomic analysis and antioxidant defense mechanisms in clusterbean (Cyamopsis tetragonoloba (L.) Taub.) genotypes with contrasting drought tolerance.

To understand drought tolerance mechanism(s) in clusterbean (Cyamopsis tetragonoloba), we conducted physiological, biochemical, and de novo comparative transcriptome analysis of drought-tolerant (RGC-1002) and drought-sensitive (RGC-1066) genotypes subjected to 30 days of drought stress. Relative water content (RWC) was maintained in tolerant genotype but was reduced in sensitive genotype. Leaf pigment concentrations were higher in tolerant genotype. Net photosynthesis was significantly decreased in sensitive genotype but insignificant reduction was found in tolerant genotype. Enzymatic antioxidant (GR, APX, DHAR) activities were enhanced in tolerant genotype, while there were insignificant changes in these enzymes in sensitive genotype. The ratios of antioxidant molecules (ASC/DHA and GSH/GSSG) were higher in tolerant genotype as compared to sensitive genotype. In sensitive genotype, 6625 differentially expressed genes (DEGs) were upregulated and 5365 genes were downregulated. In tolerant genotype, 5206 genes were upregulated and 2793 genes were downregulated. In tolerant genotype, transketolase family protein, phosphoenolpyruvate carboxylase 3, temperature-induced lipocalin, and cytochrome oxidase were highly upregulated. Moreover, according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the drought tolerance may be attributed to upregulated starch and sucrose metabolism-related genes in tolerant genotype. Finally, quantitative real-time PCR confirmed the reproducibility of the RNA-seq data.

Genome-wide profiling of drought-tolerant Arabidopsis plants over-expressing chickpea MT1 gene reveals transcription factors implicated in stress modulation.

Drought, a major abiotic limiting factor, could be modulated with in-built reprogramming of plants at molecular level by regulating the activity of plant developmental processes, stress endurance and adaptation. The transgenic Arabidopsis thaliana over-expressing metallothionein 1 (MT1) gene of desi chickpea (Cicer arietinum L.) was subjected to transcriptome analysis. We evaluated drought tolerance of 7 days old plants of Arabidopsis thaliana in both wild-type (WT) as well as transgenic plants and performed transcriptome analysis. Our analysis revealed 24,737 transcripts representing 24,594 genes out of which 5,816 were differentially expressed genes (DEGs) under drought conditions and 841 genes were common in both genotypes. A total of 1251 DEGs in WT and 2099 in MT1 were identified in comparison with control. Out of the significant DEGs, 432 and 944 were upregulated, whereas 819 and 1155 were downregulated in WT and MT1 plants, respectively. The physiological and molecular parameters involving germination assay, root length measurements under different stress treatments and quantitative expression analysis of transgenic plants in comparison to wild-type were found to be enhanced. CarMT1 plants also demonstrated modulation of various other stress-responsive genes that reprogrammed themselves for stress adaptation. Amongst various drought-responsive genes, 24 DEGs showed similar quantitative expression as obtained through RNA sequencing data. Hence, these modulatory genes could be used as a genetic tool for understanding and delineating the mechanisms for fine-tuning of stress responses in crop plants.

An overview on miRNA-encoded peptides in plant biology research.

MicroRNAs (miRNAs) are short (21-23 nt) regulatory RNA molecules present in plants and animals which are known for regulating the mRNA target gene expression either by cleavage or translational repression. With the advancements in miRNAs research in plants towards their biogenesis and applications has directed the recent discovery of pri-miRNAs encoding functional peptides or microRNA peptides (miPEPs). These miPEPs are encoded by 5' of pri-miRs containing short ORFs (miORFs). miPEPs are known to enhance the activity of their associated miRNAs by increasing their accumulation and hence downregulating the target genes. Since miPEPs are very specific for each miRNA, they are considered as novel and effective tools for improving traits of interest for plant growth promotion and plant-microbe interaction. Entire peptidome research is the need of the hour. This review thus summarizes recent advancements in miPEP research and its applications as a technology with important agronomical implications with miRNAs augmentation.

microRNA 166: an evolutionarily conserved stress biomarker in land plants targeting HD-ZIP family.

MicroRNAs (miRNAs) are significant class of noncoding RNAs having analytical investigating and modulatory roles in various signaling mechanisms in plants related to growth, development and environmental stress. Conserved miRNAs are an affirmation of land plants evolution and adaptation. They are a proof of indispensable roles of endogenous gene modulators that mediate plant survival on land. Out of such conserved miRNA families, is one core miRNA known as miR166 that is highly conserved among land plants. This particular miRNA is known to primarily target HD ZIP-III transcription factors. miR166 has roles in various developmental processes, as well as regulatory roles against biotic and abiotic stresses in major crop plants. Major developmental roles indirectly modulated by miR166 include shoot apical meristem and vascular differentiation, leaf and root development. In terms of abiotic stress, it has decisive regulatory roles under drought, salinity, and temperature along with biotic stress management. miR166 and its target genes are also known for their beneficial synergy with microorganisms in leguminous crops in relation to lateral roots and nodule development. Hence it is important to study the roles of miR166 in different crop plants to understand its defensive roles against environmental stresses and improve plant productivity by reprogramming several gene functions at molecular levels. This review is hence a summary of different regulatory roles of miR166 with its target HD-ZIP III and its modulatory and fine tuning against different environmental stresses in various plants.

Chickpea glutaredoxin (CaGrx) gene mitigates drought and salinity stress by modulating the physiological performance and antioxidant defense mechanisms.

Glutaredoxins (Grxs) are short, cysteine-rich glutathione (GSH)-mediated oxidoreductases. In this study, a chickpea (Cicer arietinum L.) glutaredoxin [LOC101493651 (CaGrx)] gene has been selected based on screening experiments with two contrasting varieties of chickpea, PUSA-362 (drought-tolerant) and ICC-1882 (drought-sensitive) under drought and salinity. The tolerant variety showed higher CaGrx gene expression, as compared to less in the sensitive variety, under both the stresses. The CaGrx gene was then over-expressed in Arabidopsis thaliana and were exposed to drought and salinity. The over-expression of CaGrx elevated the activity of glutaredoxin, which induced antioxidant enzymes (glutathione reductase; GR, glutathione peroxidase; GPX, catalase; CAT, ascorbate peroxidase; APX, glutathione-S-transferase; GST, superoxide dismutase; SOD, monodehydroascorbate reductase; MDHAR, and dehydroascorbate reductase; DHAR), antioxidants (GSH and ascorbate) and stress-responsive amino acids (cysteine and proline). Enhancement in the antioxidant defense system possibly administered tolerance in transgenics against both stresses. CaGrx reduced stress markers (H2O2, TBARS, and electrolyte leakage) and enhanced root growth, seed germination, and survival against both stresses. The physiological parameters (net photosynthesis; P N, water use efficiency; WUE, stomatal conductance; g s, transpiration; E, electron transport rate; ETR, and photochemical quenching; qP), chlorophylls and carotenoids, were improved in the transgenics during both stresses, that maintained the photosynthetic apparatus and protected the plants from damage. The enhanced activity of the cysteine biosynthesis enzyme, o-acetylserine (thiol) lyase (OAS-TL), increased the cysteine level in the transgenics, which elevated glutathione biosynthesis to maintain the ascorbate-glutathione cycle under both stresses. This investigation verified that the CaGrx gene provides tolerance against salinity and drought, maintaining physiological and morphological performances, and could be exploited for genetic engineering approaches to overcome both the stresses in various crops. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00999-z.

Over-expression of chickpea metallothionein 1 gene confers tolerance against major toxic heavy metal stress in Arabidopsis.

Heavy metals are ubiquitously present in nature, including soil, water, and thus in plants, thereby causing a potential health risk. This study has investigated the role and efficiency of the chickpea metallothionein 1 (MT1) gene against the major toxic heavy metals, i.e., As [As(III) and As(V)], Cr(VI), and Cd toxicity. MT1 over-expressing transgenic lines had reduced As(V) and Cr(VI) accumulation, whereas Cd accumulation was enhanced in the L3 line. The physiological responses (WUE, A, Gs, E, ETR, and qP) were noted to be enhanced in transgenic plants, whereas qN was decreased. Similarly, the antioxidant molecules and enzymatic activities (GSH/GSSG, Asc/DHA, APX, GPX, and GRX) were higher in the transgenic plants. The activity of antioxidant enzymes, i.e., SOD, APX, GPX, and POD, were highest in the Cd-treated lines, whereas higher CAT activity was observed in As(V)-L1 and GRX in Cr-L3 line. The stress markers TBARS, H2O2, and electrolyte leakage were lower in transgenic lines in comparison to WT, while RWC was enhanced in the transgenic lines, and the transcript of MT1 gene was accumulated in the transgenic lines. Similarly, the level of stress-responsive amino acid cysteine was higher in transgenic plants as compared to WT plants. Among all the heavy metals, MT1 over-expressing lines showed a highly increased accumulation of Cd, whereas a non-significant effect was observed with As(III) treatment. Overall, the results demonstrate that Arabidopsis thaliana transformed with the MT1 gene mitigates heavy metal stress by regulating the defense mechanisms in plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01103-1.

Over-expression of chickpea glutaredoxin (CaGrx) provides tolerance to heavy metals by reducing metal accumulation and improved physiological and antioxidant defence system.

Glutaredoxins (Grxs) are small multifunctional redox proteins. Grxs have glutathione-dependent oxidoreductase activity in the presence of glutathione reductase and NADPH. The role of Grxs is well studied in heavy metal tolerance in prokaryotic and mammalian systems but not in plant genera. In the present study, a chickpea glutaredoxin (CaGrx) gene (LOC101493651) has been investigated against metal stress based on its primary screening in chickpea which revealed higher up-regulation of CaGrx gene under various heavy metals (AsIII-25 µM, AsV-250 µM, Cr(VI)-300 µM, and Cd-500 µM) stress. This CaGrx gene was overexpressed in Arabidopsis thaliana and investigated various biochemical and physiological performances under each metal stress. Transgenic plants showed significant up-regulation of the CaGrx gene during qRT-PCR analysis as well as longer roots, higher seed germination, and survival efficiency during each metal stress. The levels of stress markers, TBARS, H2O2, and electrolyte leakage were found to be less in transgenic lines as compared to WT revealed less toxicity in transgenics. The total accumulation of AsIII, AsV, and Cr(VI) were significantly reduced in all transgenic lines except Cd, which was slightly reduced. The physiological parameters such as net photosynthetic rate (PN), stomatal conductance (gs), transpiration (E), water use efficiency (WUE), photochemical quenching (qP), and electron transport rate (ETR), were maintained in transgenic lines during metal stress. Various antioxidant enzymes such as glutaredoxin (GRX), glutathione reductase (GR), glutathione peroxidase (GPX), glutathione-S-transferase (GST), ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), antioxidant molecules (ascorbate, GSH) and stress-responsive amino acids (proline and cysteine) levels were significantly increased in transgenic lines which provide metal tolerance. The outcome of this study strongly indicates that the CaGrx gene participates in the moderation of metal stress in Arabidopsis, which can be utilized in biotechnological interventions to overcome heavy metal stress conditions in different crops.

Overexpression of rice glutaredoxin genes LOC_Os02g40500 and LOC_Os01g27140 regulate plant responses to drought stress.

Glutaredoxins (Grxs) are small (10-15 kDa) glutathione (GSH) - dependent redox proteins. The role of Grxs are well documented in tolerance to heavy metal stress in prokaryotic and mammalian systems and a few plant genera, but is poorly understood in plants against drought. In the present study, two rice glutaredoxin (Osgrx) genes (LOC_Os02g40500 and LOC_Os01g27140) responsible for tolerance against heavy metal stress have been studied for investigating their role against drought. Each glutaredoxin gene was over-expressed in Arabidopsis thaliana to reveal their role in drought stress. The relative expression of both Osgrx genes was higher in the transgenic lines. Transgenic lines of both Osgrxs showed longer roots, higher seed germination, and survival efficiency during drought stress. The physiological parameters (PN, gs, E, WUE, qP, NPQ and ETR), antioxidant enzymes (GRX, GR, GPX, GST, APX, POD, SOD, CAT, DHAR, and MDHAR), antioxidant molecules (ascorbate and GSH) and stress-responsive amino acids (cysteine and proline) levels were additionally increased in transgenic lines of both Osgrxs to provide drought tolerance. The outcomes from this study strongly determined that each Osgrx gene participated in the moderation of drought and might be utilized in biological engineering strategies to overcome drought conditions in different crops.

Over-expression of CarMT gene modulates the physiological performance and antioxidant defense system to provide tolerance against drought stress in Arabidopsis thaliana L.

Drought is one of the major abiotic stresses which negatively affect plant growth and crop yield. Metallothionein (MTs) is a low molecular weight protein, mainly involved in metal homeostasis, while, its role in drought stress is still to be largely explored. The present study was aimed to investigate the role of MT gene against drought stress. The chickpea MT based on its up-regulation under drought stress was overexpressed in Arabidopsis thaliana to explore its role in mitigation of drought stress. The total transcript of MT gene was up to 30 fold higher in transgenic lines. Arabidopsis plants transformed with MT gene showed longer roots, better efficiency of survival and germination, larger siliques and higher biomass compared to WT. The physiological variables (A, WUE, G, E, qP and ETR) of WT plants were reduced during drought stress which recovered in transgenic Arabidopsis lines. The enzymatic and non-enzymatic antioxidant (APX, GPX, POD, GR, GRX, GST, CAT, MDHAR, ASc and GSH) levels were also enhanced in transgenic lines to provide tolerance. Simultaneously, drought responsive amino acids, i.e. proline and cysteine contents were higher in transgenic lines. Overall, the results suggest that MT gene is actively involved in the mitigation of drought stress and could be the choice for genetic engineering strategy to overcome drought stress.

Application of glycine reduces arsenic accumulation and toxicity in Oryza sativa L. by reducing the expression of silicon transporter genes.

The present study was intended to investigate the role of amino acid glycine in detoxification of As in Oryza sativa L. The growth parameters such as, shoot length and fresh weight were decreased during As(III) and As(V) toxicity. However, the application of glycine recovered the growth parameters against As stress. The application of glycine reduced the As accumulation in all the treatments, and it was more effective against As(III) treatment and reduced the accumulation by 68% in root and 71% in shoot. Similarly, the translocation of As from root to shoot, was higher against As(III) and As(V) treatments, whereas, reduced upon glycine application. The translocation of Fe and Na was also affected by As, which was lower under As(III) and As(V) treatments. However, the application of glycine significantly enhanced the translocation of Fe and Na in the shoot. Besides, the expression of lower silicon transporters i.e. Lsi-1 and Lsi-2 was observed to be significantly suppressed in the root with the application of glycine against As treatment. Similarly, the expression of three GRX and two GST gene isoforms were found to be significantly increased with glycine application. Simultaneously, the activities of antioxidant enzymes i.e. l-arginine dependent NOS, SOD, NTR and GRX were found to be significantly enhanced in the presence of glycine. Increased activities of antioxidant enzymes coincided with the decreased level of TBARS and H2O2 in rice seedlings. Overall, the results suggested that the application of glycine reduces As accumulation through suppressing the gene expression of lower silicon transporters and ameliorates As toxicity by enhancing antioxidants defense mechanism in rice seedlings.

Single amino acid substitutions in recombinant plant-derived human α1-proteinase inhibitor confer enhanced stability and functional efficacy.

BACKGROUND: Human α1-proteinase inhibitor (α1-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α1-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern. METHODS: Five variants of plant-expressed recombinant α1-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α1-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy. RESULTS: Urea-induced unfolding of recombinant α1-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4M to 4.3M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~5-fold at 54°C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α1-PI but provided enhanced resistance to oxidative inactivation. CONCLUSIONS: Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α1-PI protein, without inflicting the inhibitory activity of the protein. GENERAL SIGNIFICANCE: Our results demonstrate the significance of engineered modifications in plant-derived recombinant α1-PI protein molecule for further therapeutic development.

Thermodynamics of Ga2O3 Heteroepitaxy and Material Growth Via Metal Organic Chemical Vapor Deposition.

Heteroepitaxy of gallium oxide (Ga2O3) is gaining popularity to address the absence of p-type doping, limited thermal conductivity of Ga2O3 epilayers, and toward realizing high-quality p-n heterojunction. During the growth of ß-Ga2O3 on 4H-SiC (0001) substrates using metal-organic chemical vapor deposition, we observed formation of incomplete, misoriented particles when the layer was grown at a temperature between 650 °C and 750 °C. We propose a thermodynamic model for Ga2O3 heteroepitaxy on foreign substrates which shows that the energy cost of growing ß-Ga2O3 on 4H-SiC is slightly lower as compared to sapphire substrates, suggesting similar high-temperature growth as sapphire, typically in the range of 850 °C-950 °C, that can be used for the growth of ß-Ga2O3 on SiC. A two-step modified growth method was developed where the nucleation layer was grown at 750 °C followed by a buffer layer grown at various temperatures from 920 °C to 950 °C. 2θ-ω scan of X-ray diffraction (XRD) and transmission electron microscope images confirm the ß-polymorph of Ga2O3 with dominant peaks in the (-201) direction. The buffer layer grown at 950 °C using a "ramp-growth" technique exhibits root-mean-square surface roughness of 3 nm and full width of half maxima of XRD rocking curve as low as 0.79°, comparable to the most mature ß-Ga2O3 heteroepitaxy on sapphire, as predicted by the thermodynamic model. Finally, the interface energy of an average Ga2O3 island grown on 4H-SiC is calculated to be 0.2 J/m2 from the cross-section scanning transmission electron microscope image, following the Wulff-Kaishew theorem of the equilibrium island shape.

An Overview of Targeted Genome Editing Strategies for Reducing the Biosynthesis of Phytic Acid: an Anti-nutrient in Crop Plants.

Anti-nutrients are substances either found naturally or are of synthetic origin, which leads to the inactivation of nutrients and limits their utilization in metabolic processes. Phytic acid is classified as an anti-nutrient, as it has a strong binding affinity with most minerals like Fe, Zn, Mg, Ca, Mn, and Cd and impairs their proper metabolism. Removing anti-nutrients from cereal grains may enable the bioavailability of both macro- and micronutrients which is the desired goal of genetic engineering tools for the betterment of agronomic traits. Several strategies have been adopted to minimize phytic acid content in plants. Pursuing the molecular strategies, there are several studies, which result in the decrement of the total phytic acid content in grains of major as well as minor crops. Biosynthesis of phytic acid mainly takes place in the seed comprising lipid-dependent and lipid-independent pathways, involving various enzymes. Furthermore, some studies show that interruption of these enzymes may involve the pleiotropic effect. However, using modern biotechnological approaches, undesirable agronomic traits can be removed. This review presents an overview of different genes encoding the various enzymes involved in the biosynthetic pathway of phytic acid which is being targeted for its reduction. It also, highlights and enumerates the variety of potential applications of genome editing tools such as TALEN, ZFN, and CRISPR/Cas9 to knock out the desired genes, and RNAi for their silencing.

Epitaxial Growth of (-201) ß-Ga2O3 on (001) Diamond Substrates.

Heteroepitaxial growth of ß-Ga2O3 on (001) diamond by metal-organic chemical vapor deposition (MOCVD) is reported. A detailed study was performed with Transmission Electron Microscopy (TEM) elucidating the epitaxial relation of (-201) ß-Ga2O3||(001) diamond and [010]/[-13-2] ß-Ga2O3 ||[110]/[1-10] diamond, with the presence of different crystallographically related epitaxial variants apparent from selected area diffraction patterns. A model explaining the arrangement of atoms along ⟨110⟩ diamond is demonstrated with a lattice mismatch of 1.03-3.66% in the perpendicular direction. Dark field imaging showed evidence of arrays of discrete defects at the boundaries between different grains. Strategies to reduce the density of defects are discussed.

Alterations in plant anatomy and higher lignin synthesis provides drought tolerance in cluster bean [Cyamopsis tetragonoloba (L.) Taub.].

Four contrasting varieties of guar, RGC-1002 and RGC-1038, drought tolerant, while, Sarada and RGC-936, drought sensitive, were monitored in watered and drought. The water status, phenolics, plant anatomy and transcript level of genes related to anatomical traits were assessed. The study aimed to decipher the anatomical adaptations of guar plants in response to water stress. The physiological determinants, relative water content (RWC), water potential (ψ), and leaf membrane damage, declined under drought in all four varieties although, the decrement was lesser in the tolerant varieties. Furthermore, the tolerant cultivars subjected to water stress recorded higher accumulation of total phenolic content, anthocyanin and lignin, which efficiently scavenge the reactive oxygen species. The results suggest that the cultivars RGC-1002 and RGC-1038 are better able to resist drought-induced oxidative stress than Sarada and RGC-936. Moreover, leaf, petiole, stem and root anatomical traits viz. size of epidermal cell, parenchyma, width of cortex layer, and diameter of xylem vessels were narrowed in all the varieties although, the decrement was lesser in the tolerant varieties under drought. The expression analysis of genes revealed that drought-tolerant varieties showed enhanced mechanical support for water conduction by up-regulation of genes, Phenylalanine ammonia-lyase1 (PAL1), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), cinnamoyl-CoA reductase (CCR), caffeoyl-CoA O-methyltransferase (CCOMT), and cinnamyl-alcohol dehydrogenase (CAD6) in water stress conditions. The alterations in physio-anatomical, biochemical and gene expression traits in tolerant guar varieties enabled them to maintain steady nutrient transport while reducing the risk of embolisms and increasing water-flow resistance for better survival in water stressed conditions.

Evaluating the pesticidal impact of plant protease inhibitors: lethal weaponry in the co-evolutionary battle.

In the arsenal of plant defense, protease inhibitors (PIs) are well-designed defensive products to counter field pests. PIs are produced in plant tissues by means of 'stable defense metabolite' and triggered on demand as the perception of the signal and well established as a part of plant active defense. PIs have been utilized for approximately four decades, initially as a gene-alone approach that was later replaced by multiple gene pyramiding/gene stacking due to insect adaptability towards the PI alone. By considering the adaptive responses of the pest to the single insecticidal gene, the concept of gene pyramiding gained continuous appreciation for the development of transgenic crops to deal with co-evolving pests. Gene pyramiding approaches are executed to bypass the insect's adaptive responses against PIs. Stacking PIs with additional insecticidal proteins, plastid engineering, recombinant proteinase inhibitors, RNAi-based methods and CRISPR/Cas9-mediated genome editing are the advanced tools and methods for next-generation pest management. Undoubtedly, the domain associated with the mechanism of PIs in the course of plant-pest interactions will occupy a central role for the advancement of more efficient and sustainable pest control strategies. © 2021 Society of Chemical Industry.

High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants.

To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding ß-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD(600) 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 µM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T(0) transgenic plants were generated, representing 3.6% transformation frequency. T(0) transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T(1) progeny. PCR, RT-PCR, and Southern hybridization analysis of T(0) and T(1) transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T(0) and T(1) transgenic chickpea plants was achieved maximum up to 116 ng mg(-1) of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.

In silico designing of vaccine candidate against Clostridium difficile.

Clostridium difficile is a spore-forming gram-positive bacterium, recognized as the primary cause of antibiotic-associated nosocomial diarrhoea. Clostridium difficile infection (CDI) has emerged as a major health-associated infection with increased incidence and hospitalization over the years with high mortality rates. Contamination and infection occur after ingestion of vegetative spores, which germinate in the gastro-intestinal tract. The surface layer protein and flagellar proteins are responsible for the bacterial colonization while the spore coat protein, is associated with spore colonization. Both these factors are the main concern of the recurrence of CDI in hospitalized patients. In this study, the CotE, SlpA and FliC proteins are chosen to form a multivalent, multi-epitopic, chimeric vaccine candidate using the immunoinformatics approach. The overall reliability of the candidate vaccine was validated in silico and the molecular dynamics simulation verified the stability of the vaccine designed. Docking studies showed stable vaccine interactions with Toll-Like Receptors of innate immune cells and MHC receptors. In silico codon optimization of the vaccine and its insertion in the cloning vector indicates a competent expression of the modelled vaccine in E. coli expression system. An in silico immune simulation system evaluated the effectiveness of the candidate vaccine to trigger a protective immune response.

Effect of point mutations in translation initiation context on the expression of recombinant human alpha(1)-proteinase inhibitor in transgenic tomato plants.

The functional and biological significance of translation initiation context sequence in determining high-level expression of modified synthetic human alpha(1)-proteinase inhibitor (alpha(1)-PI) gene was documented in stable transgenic tomato plants. Context sequence of initiator ATG codon derived from statistical analysis of databases was identified as taaA(A/C)aATGGCt in highly expressed dicot plant genes. Removal of initiator ATG context sequence reduced the expression of recombinant alpha(1)-PI protein to fourfolds. The mutation of consensus base at +4 position to a pyrimidine either alone or with substitution at -3 position eliminated most of the alpha(1)-PI expression, while mutation at -3 alone resulted in about sevenfold reduction. The presence of steady-state levels of alpha(1)-PI transcript in transgenic plants indicated that the variation in expression is entirely due to the point mutations incorporated in translation initiation context. These results indicated the significance of conserved nucleotide sequence around initiator ATG codon in augmenting post-transcriptional events and high-level expression of heterologous genes in transgenic plants.