Pseudomonas fluorescens imparts cadmium stress tolerance in Arabidopsis thaliana via induction of AtPCR2 gene expression.

BACKGROUND: Cadmium is a non-essential, third largest heavy metal contaminant with long retention time that poses environmental hazards. It emanating majorly from industrial processes and phosphate fertilizers. Cadmium is effortlessly assimilated by plants and leads to yield loss. Henceforth, identification of mechanisms to attenuate the heavy metal toxicity in crops is beneficial for enhanced yields. RESULTS: Beneficial soil bacteria have been known to combat both biotic and abiotic stress, thereby promoting plant growth. Amongst them, Pseudomonas fluorescens has been shown to enhance abiotic stress resistance in umpteen crops for instance maize and groundnut. Here, we investigated the role of P. fluorescens in conferring cadmium stress resistance in Arabidopsis thaliana. In silico analysis of PCR2 gene and promoter revealed the role, in cadmium stress resistance of A. thaliana. Real-time expression analysis employing qRT-PCR ratified the upregulation of AtPCR2 transcript under cadmium stress up to 6 folds. Total leaf (50%), biomass (23%), chlorophyll content (chlorophyll-a and b 40%, and 36 %) silique number (50%), and other growth parameters significantly improved on bacterial treatment of the 2mM Cd-stressed plants. CONCLUSION: Moreover, generated 35s-promoter driven AtPCR2 over-expressing transgenic lines that exhibited resistance to cadmium and other heavy metal stress. Taken together, a crucial interplay of P. fluorscens mediated enhanced expression of AtPCR2 significantly induced cadmium stress resistance in Arabidopsis plants.

Chitin Biosynthesis Inhibition of Meloidogyne incognita by RNAi-Mediated Gene Silencing Increases Resistance to Transgenic Tobacco Plants.

Meloidogyne incognita is a devastating plant parasitic nematode that causes root knot disease in a wide range of plants. In the present study, we investigated host-induced RNA interference (RNAi) gene silencing of chitin biosynthesis pathway genes (chitin synthase, glucose-6-phosphate isomerase, and trehalase) in transgenic tobacco plants. To develop an RNAi vector, ubiquitin (UBQ1) promoter was directly cloned, and to generate an RNAi construct, expression of three genes was suppressed using the GATEWAY system. Further, transgenic Nicotiana benthamiana lines expressing dsRNA for chitin synthase (CS), glucose-6-phosphate isomerase (GPI), and trehalase 1 (TH1) were generated. Quantitative PCR analysis confirmed endogenous mRNA expression of root knot nematode (RKN) and revealed that all three genes were more highly expressed in the female stage than in eggs and in the parasitic stage. In vivo, transformed roots were challenged with M. incognita. The number of eggs and root knots were significantly decreased by 60-90% in RNAi transgenic lines. As evident, root galls obtained from transgenic RNAi lines exhibited 0.01- to 0.70-fold downregulation of transcript levels of targeted genes compared with galls isolated from control plants. Furthermore, phenotypic characteristics such as female size and width were also marginally altered, while effect of egg mass per egg number in RNAi transgenic lines was reduced. These results indicate the relevance and significance of targeting chitin biosynthesis genes during the nematode lifespan. Overall, our results suggest that further developments in RNAi efficiency in commercially valued crops can be applied to employ RNAi against other plant parasitic nematodes.

Natural Korean Medicine Dang-Gui: Biosynthesis, Effective Extraction and Formulations of Major Active Pyranocoumarins, Their Molecular Action Mechanism in Cancer, and Other Biological Activities.

Angelica gigas Nakai (AGN) is a crucial oriental medicinal herb that grows especially in Korea and the Far-East countries. It contains chemically active compounds like pyranocoumarins, polyacetylenes and essential oils, which might be useful for treatment of several chronic diseases. It has been used for centuries as a traditional medicine in Southeast Asia, but in Western countries is used as a functional food and a major ingredient of several herbal products. The genus Angelica is also known as 'female ginseng' due to its critical therapeutic role in female afflictions, such as gynecological problems. However, it is well-documented that the AGN pyranocoumarins may play vital beneficial roles against cancer, neurodisorders, inflammation, osteoporosis, amnesia, allergies, depression, fungi, diabetes, ischemia, dermatitis, reactive oxygen species (ROS) and androgen. Though numerous studies revealed the role of AGN pyranocoumarins as therapeutic agents, none of the reviews have published their molecular mechanism of action. To the best of our knowledge, this would be the first review that aims to appraise the biosynthesis of AGN's major active pyranocoumarins, discuss effective extraction and formulation methods, and detail the molecular action mechanism of decursin (D), decursinol angelate (DA) and decursinol (DOH) in chronic diseases, which would further help extension of research in this area.

Development of Genome-Wide SSR Markers from Angelica gigas Nakai Using Next Generation Sequencing.

Angelica gigas Nakai is an important medicinal herb, widely utilized in Asian countries especially in Korea, Japan, and China. Although it is a vital medicinal herb, the lack of sequencing data and efficient molecular markers has limited the application of a genetic approach for horticultural improvements. Simple sequence repeats (SSRs) are universally accepted molecular markers for population structure study. In this study, we found over 130,000 SSRs, ranging from di- to deca-nucleotide motifs, using the genome sequence of Manchu variety (MV) of A. gigas, derived from next generation sequencing (NGS). From the putative SSR regions identified, a total of 16,496 primer sets were successfully designed. Among them, we selected 848 SSR markers that showed polymorphism from in silico analysis and contained tri- to hexa-nucleotide motifs. We tested 36 SSR primer sets for polymorphism in 16 A. gigas accessions. The average polymorphism information content (PIC) was 0.69; the average observed heterozygosity (HO) values, and the expected heterozygosity (HE) values were 0.53 and 0.73, respectively. These newly developed SSR markers would be useful tools for molecular genetics, genotype identification, genetic mapping, molecular breeding, and studying species relationships of the Angelica genus.

Molecular Cloning and Characterization of Carotenoid Pathway Genes and Carotenoid Content in Ixeris dentata var. albiflora.

Ixeris dentata var. albiflora is considered as a potential therapeutic agent against mithridatism, calculous, indigestion, pneumonia, hepatitis, and tumors as well as good seasoned vegetable in Far East countries. Phytoene synthase (PSY), phytoene desaturase (PDS) ξ-carotene desaturase (ZDS), lycopene ß-cyclase (LCYB), lycopene ε-cyclase (LCYE), ε-ring carotene hydroxylase (CHXB), and zeaxanthin epoxidase (ZDS) are vital enzymes in the carotenoid biosynthesis pathway. We have examined these seven genes from I. dentata that are participated in carotenoid biosynthesis utilizing an Illumina/Solexa HiSeq 2000 platform. In silico analysis of the seven deduced amino acid sequences were revealed its closest homology with other Asteracea plants. Further, we explored transcript levels and carotenoid accumulation in various organs of I. dentata using quantitative real time PCR and high-performance liquid chromatography, respectively. The highest transcript levels were noticed in the leaf for all the genes while minimal levels were noticed in the root. The maximal carotenoid accumulation was also detected in the leaf. We proposed that these genes expressions are associated with the accumulation of carotenoids. Our findings may suggest the fundamental clues to unravel the molecular insights of carotenoid biosynthesis in various organs of I. dentata.

Genetically modified phytase crops role in sustainable plant and animal nutrition and ecological development: a review.

Globally, plant-derivatives especially cereals and legumes are the major staple food sources for animals. The seeds of these crops comprise of phytic acid, the major repository form of the phosphorus, which is not digestible by simple-stomached animals. However, it is the most important factor responsible for impeding the absorption of minerals by plants that eventually results in less use of fertilizers that ultimately cause eutrophication in water bodies. Although abundant phosphorus (P) exists in the soils, plants cannot absorb most of the P due to its conversion to unavailable forms. Hence, additional P supplementation is indispensable to the soil to promote crop yields which not only leads to soil infertility but also rapid depletion of non-renewable P reservoirs. Phytase/phosphatase enzyme is essential to liberate P from soils by plants and from seeds by monogastric animals. Phytases are kind of phosphatases which can hydrolyse the indigestible phytate into inorganic Phosphate (Pi) and lower myo-inositol. There are several approaches to mitigate the problems associated with phytate indigestibility. One of the best possible solutions is engineering crops to produce heterologous phytase to improve P utilization by monogastric animals, plant nutrition and sustainable ecological developments. Previously published reviews were focused on either soil phytate or seed-phytate, related issues, but this review will address both the problems as well as phytate related ecological problems. This review summarizes the overall view of engineered phytase crops and their role in sustainable agriculture, animal nutrition and ecological development.

Improving flavour and quality of tomatoes by expression of synthetic gene encoding sweet protein monellin.

Monellin a sweet-tasting protein exists naturally as a heterodimer of two non-covalently linked subunits chain A and B, which loses its sweetness on denaturation. In this study, we validated the expression of a synthetic monellin gene encoding a single polypeptide chain covalently linking the two subunits under T7 and fruit-ripening-specific promoters in Escherichia coli and tomato fruits, respectively. Purified recombinant monellin protein retained its sweet flavour at 70 °C and pH 2. We developed 15 transgenic T0 tomato plants overexpressing monellin, which were devoid of any growth penalty or phenotypic abnormalities during greenhouse conditions. T-DNA integration and fruit-specific heterologous expression of monellin had occurred in these transgenic tomato lines. ELISA revealed that expression of monellin was 4.5% of the total soluble fruit protein. Functional analyses of transgenic tomatoes of T2-5 and T2-14 lines revealed distinctly strong sweetness compared with wild type. Monellin a potential non-carbohydrate sweetener, if expressed in high amounts in fruits and vegetables, would enhance their flavour and quality.

Isolation and molecular characterization of thermostable phytase from Bacillus subtilis (BSPhyARRMK33).

The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its ß-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg(-1) obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74% of its original activity after incubation at 95 °C for 10 min. In the presence of Ca(2+), the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is achieved at 7.0 and 55 °C, respectively. Thermostable nature and wide range of pH are promising features of recombinant BsPhyARRMK33 protein that may be employed as an efficient alternative to commercially known phytases and thereby alleviate environmental eutrophication.