Coagulansin-A has beneficial effects on the development of bovine embryos in?vitro via HSP70 induction

Coagulansin-A (withanolide) is the steroidal lactone obtained from Withania coagulans which belong to Solanaceae family. The present study investigated the effects of coagulansin-A on bovine oocyte maturation and embryo development in vitro. All these oocytes were aspirated from the ovaries obtained from Korean Hanwoo cows at a local abattoir. To determine whether coagulansin-A has beneficial effects on bovine oocyte maturation in vitro, 355 oocytes per group (control and treated) in seven replicates were subjected with different concentrations (1, 2.5, 5, 7.5 and 10 μM) of coagulansin-A. The coagulansin-A was added in the in vitro maturation (IVM) media followed by in vitro fertilization (IVF) and then in vitro culture (IVC). Only treatment with 5 μM coagulansin-A remarkably (P<0.05) improved embryos development (Day 8 blastocyst) having 27.30 and 40.01% for control and coagulansin-A treated groups respectively. Treatment with 5 μM coagulansin-A significantly induced activation of heat shock protein 70 (HSP70) (P<0.05). Immunofluorescence analysis revealed that 5 μM coagulansin-A treatment also significantly inhibited oxidative stress and inflammation during bovine embryo development in vitro by decreasing 8-oxoguanosine (8-OxoG) (P<0.05) and nuclear factor-κB (NF-κB) (P<0.05). The expressions of HSP70 and NF-κB were also conformed through real-time PCR (RT-PCR). Additionally, the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay confirmed that coagulansin-A treatment significantly improved the embryo quality and reduced bovine embryo DNA damage (P<0.05). The present study provides new information regarding the mechanisms by which coagulansin-A promotes bovine embryo development in vitro.     

Salinity Stress in Wheat: Effects,Mechanisms and Management Strategies

Salinity stress is a major threat to global food production and its intensity is continuously increasing because of anthropogenic activities. Wheat is a staple food and a source of carbohydrates and calories for the majority of people across the globe. However, wheat productivity is adversely affected by salt stress, which is associated with a reduction in germination, growth, altered reproductive behavior and enzymatic activity, disrupted photosynthesis, hormonal imbalance, oxidative stress, and yield reductions. Thus, a better understanding of wheat (plant) behavior to salinity stress has essential implications to devise counter and alleviation measures to cope with salt stress. Different approaches including the selection of suitable cultivars, conventional breeding, and molecular techniques can be used for facing salt stress tolerance. However, these techniques are tedious, costly, and labor-intensive. Management practices are still helpful to improve the wheat performance under salinity stress. Use of arbuscular mycorrhizal fungi, plant growth-promoting rhizobacteria, and exogenous application of phytohormones, seed priming, and nutrient management are important tools to improve wheat performance under salinity stress. In this paper, we discussed the effect of salinity stress on the wheat crop, possible mechanisms to deal with salinity stress, and management options to improve wheat performance under salinity conditions.     

Histo-morphological analysis of rice callus cultures reveals differential regeneration response with varying media combinations

Genetic transformation of most indica rice (Oryza sativa) cultivars is hampered by poor in vitro culture performance and low regeneration potential. Histological study of primary calli can provide substantial information on their regeneration potential and can be used for early grading of calli expected to develop plantlets on regeneration media. The study was aimed to undertake histological analysis of primary calli derived from mature seeds of five indica rice cultivars viz. KSK-133, KS-282, Shaheen Basmati, Super Basmati, and DilRosh in order to assess their regeneration potential on different media combinations supplemented with various hormone concentrations (N6 + 2 mg/L 2,4-Dichlorophenoxyacetic acid; N6 + 2 mg/L 2–4 D + 2 mg/L Benzylaminopurine and MS + 2 mg/L 2,4-D). Calli with regeneration capability were subjected to histological assays by examining toulidine blue stained 5–8 μm thin sections for the presence of meristematic zones exhibiting embryogenic callus features. Based on our observations, formation of embryoids or embryoid-like structures was pronounced in KSK-133 and KS-282 calli. However, DilRosh, Super Basmati and Shaheen Basmati did not show these characteristic features. Three-week-old calli of all rice cultivars were transferred into regeneration medium (MS + 2 mg/L BAP + 1 mg/L Naphthaleneacetic acid). KSK-133 and KS-282 showed the highest regeneration potential (81% and 76%, respectively). These data were supported by histological observations where characteristic embryogenic units (EU) were noticed in these genotypes. These meristematic regions displayed high mitotic activity and stained relatively dark. The embryogenic calli cells were found heavily cytoplasmic with prominent nuclei and were located on the callus surface or inside surrounded by parenchymal cells.

     

Isolation and functional analysis of cotton universal stress protein promoter in response to phytohormones and abiotic stresses

The 949 bp promoter fragment upstream from the translation initiation site of the GUSP gene encoding a universal stress protein was isolated from the genomic DNA of Gossypium arboreum. Some putative cis-acting elements involved in stress responses including E-box, ABRE, DPBF-box, and MYB-core elements were found in the promoter region. In an Agrobacterium-mediated transient expression assay, strong activation of the GUSP full promoter region occurred in tobacco leaves following dehydration, abscisic acid, salt, heavy metal, gibberellic acid and dark treatments. Deletion analysis of the promoter revealed that the dehydration, abscisic acid and salt responses were affected by the deletion between −208 and −949 bp and showed 2–4-fold induction. However, in response to dark, gibberellic acid and heavy metals the induction was only 2-fold. These findings further our understanding of the regulation of GUSP expression. This is an important study as no report of this universal stress protein promoter is available in literature.     

Characterization of pokeweed antiviral protein binding to mRNA cap analogs: competition with nucleotides and enhancement by translation initiation factor iso4G

Pokeweed antiviral protein (PAP) is a type I ribosomal inactivating protein (RIP). PAP binds to and depurinates the sarcin/ricin loop (SRL) of ribosomal RNA resulting in the cessation of protein synthesis. PAP has also been shown to bind to mRNA cap analogs and depurinate mRNA downstream of the cap structure. The biological role of cap binding and its possible role in PAP activity are not known. Here we show the first direct quantitative evidence for PAP binding to the cap analog m(7)GTP. We report a binding affinity of 43.3+/-0.1 nM at 25 degrees C as determined by fluorescence quenching experiments. This is similar to the values reported for wheat cap-binding proteins eIFiso4E and eIFiso4F. van't Hoff analysis of m(7)GTP-PAP equilibrium reveals a binding reaction that is enthalpy driven and entropy favored with TDeltaS degrees contributing 15% to the overall value of DeltaG degrees . This is in contrast to the wheat cap-binding proteins which are enthalpically driven in the DeltaG degrees for binding. Competition experiments indicate that ATP and GTP compete for the cap-binding site on PAP with slightly different affinities. Fluorescence studies of PAP-eIFiso4G binding reveal a protein-protein interaction with a K(d) of 108.4+/-0.3 nM. eIFiso4G was shown to enhance the interaction of PAP with m(7)GTP cap analog by 2.4-fold. These results suggest the involvement of PAP-translation initiation factor complexes in RNA selection and depurination.     

Kinetic Mechanism for the Binding of eIF4F and Tobacco Etch Virus Internal Ribosome Entry Site RNA: EFFECTS OF eIF4B AND POLY(A)-BINDING PROTEIN*

The wheat germ eukaryotic translation initiation factor (eIF) 4F binds tightly to the mRNA internal ribosome entry site (IRES) of tobacco etch virus (TEV) to promote translation initiation. When eIF4F is limiting, TEV is preferentially translated compared with host cell mRNA. To gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eIF4F binding to TEV IRES were examined. The association rate constant (k_on) and dissociation rate constant (k_off) for eIF4F binding to IRES were 59 ± 2.1 μm⁻¹ s⁻¹ and 12.9 ± 0.3 s⁻¹, respectively, comparable with the rates for capped RNA. Binding of eIF4E or eIF4F to the cap of mRNA is the rate-limiting step for initiation of cap-dependent protein synthesis. The concentration dependence of the reactions suggested a simple one-step association mechanism. However, the association rate was reduced more than 10-fold when KCl concentration was increased from 50 to 300 mm, whereas the dissociation rate constant was increased 2-fold. The addition of eIF4B and poly(A)-binding protein enhanced the association rate of eIF4F ∼3-fold. These results suggest a mechanism where eIF4F initially binds electrostatically, followed by a conformational change to further stabilize binding. Poly(A)-binding protein and eIF4B mainly affect the eIF4F/TEV association rate. These results demonstrate the first direct kinetic measurements of translation initiation factor binding to an IRES.     

Expression of <Emphasis Type="Italic">rol</Emphasis> genes in transgenic soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) leads to changes in plant phenotype,leaf morphology,and flowering time

Soybean (Glycine max L.) cultivar NARC-4 was transformed with constructs carrying rolA, rolB, or rolC genes, each under the control of the Cauliflower Mosaic Virus 70S promoter. Cotyledonary nodes of soybean seeds were infected with Agrobacterium tumefaciens strain LBA4404 carrying one of the three rol genes, along with nptII in the plasmid pLBR. The efficiency of the transformation varied slightly among the three constructs, with frequencies of 6, 7, and 5% for the rolA, rolB, and rolC genes, respectively, being observed. Southern blot analysis confirmed the integration of rol genes in the soybean genome with varying numbers of copies of the transgene. All transformed plants showed enhanced rooting, but the number of adventitious roots was higher for transformants carrying the rolB transgene. rolA and rolC transformants showed dwarf phenotypes, clustered branching, and variations in leaf morphology. Furthermore, these plants flowered within a short period of time and produced lower numbers of flowers. rolB transformants showed variations in phenotype, including dwarf to semi-dwarf and shrubby growth, abnormal stem growth, short internodes, variations in leaf morphology, and greenish to yellowish-green colored leaves. These plants also flowered early, but dwarf plants produced low numbers of flowers, while shrubby plants produced high numbers of flowers, but these were mostly infertile.     

Usefulness of powdered and fresh egg yolk for cryopreservation of Zebu bull spermatozoa

This experiment was designed to compare powdered egg yolk with fresh egg yolk in an extender for cryopreservation of Zebu bull semen. Sperm motility, plasma membrane integrity and viability were assessed at different stages of cryopreservation (post-dilution, pre-freezing and post-thawing). Sperm plasma membrane integrity remained similar at all the stages of cryopreservation. Sperm motility and viability were significantly higher after thawing in the extender containing powdered egg yolk. In conclusion, powdered egg yolk may be used in an extender for the cryopreservation of Zebu bull spermatozoa.     

Poly(A) tail affects equilibrium and thermodynamic behavior of tobacco etch virus mRNA with translation initiation factors eIF4F,eIF4B and PABP

We have investigated the effects of poly(A)-tail on binding of eIF4F, eIF4B and PABP with tobacco etch virus (TEV) IRES RNA. The fluorescence anisotropy data showed that the addition of poly(A)₂₀ increases the binding affinity of eIF4F·4B and eIF4F·PABP complexes to IRES RNA ~ 2- and 4-fold, respectively. However, the binding affinity of eIF4F with PK1 was enhanced ~ 11-fold with the addition of PABP, eIF4B, and poly(A)₂₀ together. Whereas, poly(A)₂₀ alone increases the binding affinity of eIF4F·4B·PABP with PK1 RNA about 3-fold, showing an additive effect rather than the large increase in affinity as shown for cap binding. Thermodynamic data showed that PK1 RNA binding to protein complexes in the presence of poly(A)₂₀ was enthalpy-driven and entropy-favorable. Poly(A)₂₀ decreased the entropic contribution 75% for binding of PK1 RNA to eIF4F·4B·PABP as compared to eIF4F alone, suggesting reduced hydrophobic interactions for complex formation and an overall conformational change. Overall, these results demonstrate the first direct effect of poly(A) on the equilibrium and thermodynamics of eIF4F and eIF4F·4B·PABP with IRES-RNA.