Germin and germin-like proteins: evolution, structure, and function

Germin and germin-like proteins (GLPs) are encoded by a family of genes found in all plants. They are part of the cupin superfamily of biochemically diverse proteins, a superfamily that has a conserved tertiary structure, though with limited similarity in primary sequence. The subgroups of GLPs have different enzyme functions that include the two hydrogen peroxide-generating enzymes, oxalate oxidase (OxO) and superoxide dismutase. This review summarizes the sequence and structural details of GLPs and also discusses their evolutionary progression, particularly their amplification in gene number during the evolution of the land plants. In terms of function, the GLPs are known to be differentially expressed during specific periods of plant growth and development, a pattern of evolutionary subfunctionalization. They are also implicated in the response of plants to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic (salt, heat/cold, drought, nutrient, and metal) stress. Most detailed data come from studies of fungal pathogenesis in cereals. This involvement with the protection of plants from environmental stress of various types has led to numerous plant breeding studies that have found links between GLPs and QTLs for disease and stress resistance. In addition the OxO enzyme has considerable commercial significance, based principally on its use in the medical diagnosis of oxalate concentration in plasma and urine. Finally, this review provides information on the nutritional importance of these proteins in the human diet, as several members are known to be allergenic, a feature related to their thermal stability and evolutionary connection to the seed storage proteins, also members of the cupin superfamily

Multiple pulses improve electroporation efficiency in Agrobacterium tumefaciens

Electroporation entails brief, high intensity pulse to create transient pores in the cell membrane to facilitate the entry of exogenous macromolecules, which may otherwise be excluded. Removal of the external field leads to the resealing of the membrane electropores permitting the survival of the electrically stimulated recipient cells. Using this technique foreign deoxyribonucleic acid (DNA) has been successfully introduced into many cell types both from prokaryotes and eukaryotes. Increase in pulse voltage and length beyond a critical limit has been reported to decrease transformation efficiency, hence in this study we have investigated another strategy i.e. increase in the number of pulses at constant high voltage and pulse duration. Commonly used Agrobacterium strains LBA4404 and EHA101 and binary vector pCAMBIA1301 were used. Transformants were selected on a combination of hygromycin and kanamycin, and confirmed by polymerase chain reaction (PCR) and restriction analysis. Increase in the number of pulses was found to show a significant and linear increase in transformation efficiency

Protective effect of leaf extract of Ficus carica L. against carbon tetrachloride-induced hepatic toxicity in mice and HepG2 cell line

Purpose: To determine the in vivo and in vitro hepatoprotective effects of Ficus carica.Methods: The methanol leaf extract of Ficus carica L was further fractionated into n-hexane, ethyl acetate and aqueous fractions. For in vivo study, male albino mice were divided into twelve groups. Hepatotoxicity was induced in the mice using carbon tetrachloride (CCl4). The extract of F. carica and its fractions were administered at doses of 200 and 400 mg/kg. Silymarin was used as standardhepatoprotective drug. The protective effects of the extract and fractions were determined via assay of biochemical parameters and antioxidant enzymes in the liver. The histopathology of the liver was also studied. Moreover, the in vitro hepatoprotective effect of the extract and fractions against CCl4-induced damage was determined in HepG2 cell line.Results: There were significant increases in the serum levels of liver biomarkers in CCl4-treated group, whereas treatments with plant extract and fractions significantly reduced the levels of these parameters (p < 0.05). In addition, results from histopathology revealed evidence of protective effect of Ficus carica through reversal of CCl4-induced decreases in the activities of liver antioxidant enzymes.Conclusion: These results indicate that methanol leaf extract of Ficus carica L. and its fractions exert significant and dose-dependent hepatoprotective effects in vivo and in vitro. Keywords: Ficus carica, Hepatoprotection, Carbon tetrachloride, Liver biomarker

Multiple pulses improve electroporation efficiency in Agrobacterium tumefaciens

Electroporation entails brief, high intensity pulse to create transient pores in the cell membrane to facilitate the entry of exogenous macromolecules, which may otherwise be excluded. Removal of the external field leads to the resealing of the membrane electropores permitting the survival of the electrically stimulated recipient cells. Using this technique foreign deoxyribonucleic acid (DNA) has been successfully introduced into many cell types both from prokaryotes and eukaryotes. Increase in pulse voltage and length beyond a critical limit has been reported to decrease transformation efficiency, hence in this study we have investigated another strategy i.e. increase in the number of pulses at constant high voltage and pulse duration. Commonly used Agrobacterium strains LBA4404 and EHA101 and binary vector pCAMBIA1301 were used. Transformants were selected on a combination of hygromycin and kanamycin, and confirmed by polymerase chain reaction (PCR) and restriction analysis. Increase in the number of pulses was found to show a significant and linear increase in transformation efficiency

Genetic analysis of wheat grains using digital imaging and their relationship to enhance grain weight

Phenomic characterization through digital imaging (DI) can capture the three dimensional variation in wheat grain size and shape using different image orientations. Digital imaging may help identifying genomic regions controlling grain morphology using association mapping with simple sequence repeats (SSRs) markers. Accordingly, seed shape phenotypic data of a core collection of 55 wheat genotypes, previously characterized for osmotic and drought tolerance, were produced using computer based Smart grain software. Measured dimensions included seed volume, area, perimeters, length, width, length to width ratio, circularity, horizontal deviation from ellipse (HDEV), vertical deviation from ellipse (VDEV), factor form density (FFD) etc. The thousand grain weight (TGW) was positively correlated with grain size direct measurements; however, VDEV, FFD and other derived grain attributes showed no or negative correlation with TGW. Digital imaging divided the genotypes correctly into well-defined clusters. The wheat genotypes studied were further grouped into two sub-clusters by the Bayesian structure analysis using unlinked SSR markers. A number of loci over various chromosomal regions were found associated to grain morphology by the genome wide analysis using mixed linear model (MLM) approach. A considerable number of marker-trait associations (MTAs) on chromosomes 1D and 2D may carry new alleles with potential to enhance grain weight due to the use of untapped wild accessions of Aegilops tauschii. Conclusively, we demonstrated the application of multiple approaches including high throughput phenotyping using DI complemented with genome wide association studies to identify candidate genomic regions underlying these traits, which allows a better understanding on molecular genetics of wheat grain weight

Nectarin IV, a Potent Endoglucanase Inhibitor Secreted into the Nectar of Ornamental Tobacco Plants. Isolation, Cloning, and Characterization

We have isolated and characterized the Nectarin IV (NEC4) protein that accumulates in the nectar of ornamental tobacco plants (Nicotiana langsdorffii × Nicotiana sanderae var LxS8). This 60-kD protein has a blocked N terminus. Three tryptic peptides of the protein were isolated and sequenced using tandem mass spectroscopy. These unique peptides were found to be similar to the xyloglucan-specific fungal endoglucanase inhibitor protein (XEGIP) precursor in tomato (Lycopersicon esculentum) and its homolog in potato (Solanum tuberosum). A pair of oligonucleotide primers was designed based on the potato and tomato sequences that were used to clone a 1,018-bp internal piece of nec4 cDNA from a stage 6 nectary cDNA library. The remaining portions of the cDNA were subsequently captured by 5′ and 3′ rapid amplification of cDNA ends. Complete sequencing of the nec4 cDNA demonstrated that it belonged to a large family of homologous proteins from a wide variety of angiosperms. Related proteins include foliage proteins and seed storage proteins. Based upon conserved identity with the wheat (Triticum aestivum) xylanase inhibitor TAXI-1, we were able to develop a protein model that showed that NEC4 contains additional amino acid loops that are not found in TAXI-1 and that glycosylation sites are surface exposed. Both these loops and sites of glycosylation are on the opposite face of the NEC4 molecule from the site that interacts with fungal hemicellulases, as indicated by homology to TAXI-I. NEC4 also contains a region homologous to the TAXI-1 knottin domain; however, a deletion in this domain restructures the disulfide bridges of this domain, resulting in a pseudoknottin domain. Inhibition assays were performed to determine whether purified NEC4 was able to inhibit fungal endoglucanases and xylanases. These studies showed that NEC4 was a very effective inhibitor of a family GH12 xyloglucan-specific endoglucanase with a K(i) of 0.35 nm. However, no inhibitory activity was observed against other family GH10 or GH11 xylanases. The patterns of expression of the NEC4 protein indicate that, while expressed in nectar at anthesis, it is most strongly expressed in the nectary gland after fertilization, indicating that inhibition of fungal cell wall-degrading enzymes may be more important after fertilization than before

Characterization of olive oils obtained fromwild olive trees (Olea ferruginea Royle) in Pakistan

Pakistan is lacking in edible oils and large amounts of resources are being used to import these. Olea ferruginea Royle (Oleaceae), locally known as Kahu, is native to Northern part of the country, and the fruit of this tree is currently not being utilized for any useful purpose. The present study was conducted to exploit a new source of virgin olive oil (OWOT) based on chemical composition and quality parameters. The fruits from wild olive trees were collected from different locations in Pakistan (i.e. Bhara Kahu, Kotli Sattian and Dir Swat); whereas a reference sample (OCOT) of a local variety (Zaitoon II) Olea europaea L. was collected from Barani Agricultural Research Institute, Chakwal (BARIC) for comparison. The basic quality characteristics of oils such as free acidity, peroxide value, specific UV absorptions and sensory analysis demonstrated that the oils belong to the \u201clampante olive oil\u201d commercial category due to low quality of processed olives. Some minor discrepancies with respect to the standard olive oil composition (linoleic acid slight exceeding 1% and traces of erucic acid and brassicasterol) were found that should be further studied to understand their etiology. Concerning minor compounds, tocopherols were found in low quantities whereas higher amounts of f-carotene and lutein were observed in OWOT compared to OCOT. Finally, OWOTs showed a relatively low quantity of hydrophilic phenols that proportionally expressed three times less antioxidant activity compared with OCOT. Careful control of fruit quality and good practices before olive milling could improve not only quality of the product, but also provide a new promising source of edible virgin oils