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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

Genus Psoralea: A review of the traditional and modern uses, phytochemistry and pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Psoralea (Fabaceae) harbours 105 accepted species that are extensively used by local peoples and medicinal practitioners of China, India, and other countries for treatment of tooth decay, psoriasis, leucoderma, leprosy, kidney problems, tuberculosis, indigestion, constipation and impotence. Presently, pharmacological research reports are available on only few species namely Bituminaria bituminosa (Syn: P. bituminosa), P. canescens, P. corylifolia, P. esculenta, P. plicata and P. glandulosa which are valued for their chemical constituents and traditional uses. AIM OF THE REVIEW: This review article provides explicit information on traditional uses, phytochemistry, and pharmacological activities of selected Psoralea species. The possible trends and perspectives for future research on these plants are also discussed. MATERIALS AND METHODS: An extensive and systematic review of the extant literature was carried out, and the data under various sections were identified using a computerized bibliographic search via the PubMed, Web of Science and Google Scholar, CAB Abstracts, MEDLINE, EMBASE, INMEDPLAN, NATTS as well as several websites. KEY FINDINGS: A total of 291 bioactive compounds from 06 species of genus Psoralea have been isolated and characterized. However, P. bituminosa alone possess nearly 150 compounds. These bioactive compounds belong to different chemical classes, including flavonoids, coumarins, furanocoumarins, chalcones, quinines, terpenoids and some others due to which these species exhibit significant anti-oxidant, anti-bacterial, anti-fungal, anti-viral, anti-helmintic, anti-diabetic, diuretic, hepatoprotective, anti-cancer and anti-tumor activities. P. corylifolia L. (Babchi), a Chinese traditional medicinal plant has been used in traditional medicine for many decades for its healing properties against numerous skin diseases such as leprosy, psoriasis and leucoderma. CONCLUSIONS: The in vitro studies and in vivo models have provided a simple bio-scientific justification for various ethnopharmacological uses of Psoralea species. From the toxicological perspective, the root, leaf, and seed extracts and their preparations have been proven to be safe when consumed in the recommended doses. But, meticulous studies on the pharmaceutical standardization, mode of action of the active constituents, and sustainable conservation of Psoralea species are needed, to meet the growing demands of the pharmaceutical industries, and to fully exploit their preventive and therapeutic potentials.

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.

Improvement in Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) by the inhibition of polyphenolics released during wounding of cotyledonary node explants.

Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) has been performed using cotyledonary node explants (CNs), which release phenolics upon excision that are detrimental to the viability of Agrobacterium tumefaciens and result in low transformation frequency. Twelve low molecular weight phenolic compounds and salicylic acid were identified in the exudates released upon excision during the preparation of cotyledonary nodes by reverse phase high-performance liquid chromatography (RP-HPLC). Zone inhibition assays performed with the explant exudates released at periodic intervals after excision showed the inhibition of A. tumefaciens. Agroinoculation of freshly excised cotyledonary nodes of chickpea showed 98-99 % inhibition of colony forming units (cfu). Osmium tetraoxide fixation of excised tissues showed enhanced accumulation of phenolics in the sub-epidermal regions causing enzymatic browning, affecting the viability and performance of A. tumefaciens for T-DNA delivery. The periodic analysis of exudates released from excised CNs showed enhanced levels of gallic acid (0.2945 ± 0.014 µg/g), chlorogenic acid (0.0978 ± 0.0046 µg/g), and quercetin (0.0971 ± 0.0046 µg/g) fresh weight, which were detrimental to A. tumefaciens. Quantitative assays and the elution profile showed the maximum leaching of phenolics, flavonoids, and salicylic acid immediately after the excision of explants and continued till 4 to 8 h post-excision. Pre-treatment of excised explants with inhibitors of polyphenol oxidase like L-cysteine, DTT, and sodium thiosulfate before co-cultivation showed the recovery of A. tumefaciens cfu, decreased the accumulation of phenolics, and improved transformation frequency. Our results show the hypersensitive response of excision stress for the expression of defense response-related genes and synthesis of metabolites in grain legume chickpea against pathogen infestation including Agrobacterium.

Single-Step Purification and Characterization of A Recombinant Serine Proteinase Inhibitor from Transgenic Plants.

Expression of recombinant therapeutic proteins in transgenic plants has a tremendous impact on safe and economical production of biomolecules for biopharmaceutical industry. The major limitation in their production is downstream processing of recombinant protein to obtain higher yield and purity of the final product. In this study, a simple and rapid process has been developed for purification of therapeutic recombinant α1-proteinase inhibitor (rα1-PI) from transgenic tomato plants, which is an abundant serine protease inhibitor in human serum and chiefly inhibits the activity of neutrophil elastase in lungs. We have expressed rα1-PI with modified synthetic gene in transgenic tomato plants at a very high level (≃3.2 % of total soluble protein). The heterologous protein was extracted with (NH4)2SO4 precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of rα1-PI with 54 % recovery. The plant-purified rα1-PI showed molecular mass and structural conformation comparable to native serum α1-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified rα1-PI protein. This work demonstrates a simple and efficient one-step purification of rα1-PI from transgenic plants, which is an essential prerequisite for further therapeutic development.

AtMYB12 expression in tomato leads to large scale differential modulation in transcriptome and flavonoid content in leaf and fruit tissues.

Plants synthesize secondary metabolites, including flavonoids, which play important role during various stresses for their survival. These metabolites are also considered as health-protective components in functional foods. Flavonols, one of the important groups of flavonoids, apart from performing several roles in plants have been recognized as potent phytoceuticals for human health. Tomato fruits are deficient in this group of flavonoids and have been an important target for enhancing the accumulation of flavonols through genetic manipulations. In the present study, AtMYB12 transcription factor of the Arabidopsis has been expressed under constitutive promoter in tomato. Transgenic tomato lines exhibited enhanced accumulation of flavonols and chlorogenic acid (CGA) in leaf and fruit accompanied with elevated expression of phenylpropanoid pathway genes involved in flavonol biosynthesis. In addition, global gene expression analysis in leaf and fruit suggested that AtMYB12 modulates number of molecular processes including aromatic amino acid biosynthesis, phytohormone signaling and stress responses. Besides this, a differential modulation of the genes in fruits and leaves is reported in this study. Taken together, results demonstrate that modulation of primary carbon metabolism and other pathways by AtMYB12 in tomato may lead to sufficient substrate supply for enhanced content of phenolics in general and flavonols in particular.