Characterization of SMV resistance of soybean produced by genetic transformation of SMV-CP gene in RNAi

Soybean mosaic virus (SMV), a species of the Potyvirus genus in the Potyviridae family, is one of the most typical viral diseases and results in yield and quality loss of cultivated soybean. Due to the depletion of genetic resources for resistance breeding, a trial of genetic transformation to improve disease resistance has been performed by introducing the SMV-CP gene by the RNA interference (RNAi) method via Agrobacterium-mediated transformation. Among 30 transgenic plants produced, 7 lines with enough seeds were infected with SMV and two lines (3 and 4) showed viral resistance to SMV infection. In genomic Southern blot analysis, all the lines tested contained at least one T-DNA insertion. Subsequent investigation confirmed that no viral CP gene expression was detected in two SMV-resistant lines after artificial inoculation of SMV, while non-transgenic control and other transgenic lines expressed substantial amounts of the viral gene. Viral symptoms affected seed morphology, and clean seeds were harvested from the resistant lines. Also, strong viral gene expression was detected from the seeds of susceptible lines. In further generations, the same phenotypic appearance was maintained among non-transgenic and transgenic plants. Finally, the presence of helper component-proteinase (HC-Pro), known as a suppressor of gene silencing apparatus, was checked among transgenic lines. No expression of HC-Pro in resistant lines indicated that the viral CP-RNAi transformation into soybean somehow created a functional gene silencing system and resulted in a viral-resistant phenotype.     

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells

The GRB2 associated binder 1 (GAB1) is an essential docking/adaptor protein for transmitting intracellular signals of the MET tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). We found that in response to hours of HGF/SF treatment, the GAB1 protein level is degraded by a mechanism involving MET activity and the proteasomal machinery. We also showed that GAB1 is both multi- and poly-ubiquitinated in a CBL-dependent manner. A long term exposure to HGF/SF caused a more sustained down-regulation of GAB1 than of MET, associated with a loss of reactivation of the ERK MAP kinases to subsequent acute ligand treatment. These data demonstrate that GAB1 is ubiquitinated by CBL and degraded by the proteasome, and plays a role in negative-feedback regulation of HGF/SF–MET signaling.     

Genetic Modification of the Soybean to Enhance the β-Carotene Content through Seed-Specific Expression

The carotenoid biosynthetic pathway was genetically manipulated using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). The PAC gene was linked to either the β-conglycinin (β) or CaMV-35S (35S) promoter to generate β-PAC and 35S-PAC constructs, respectively. A total of 37 transgenic lines (19 for β-PAC and 18 for 35S-PAC) were obtained through Agrobacterium-mediated transformation using the modified half-seed method. The multi-copy insertion of the transgene was determined by genomic Southern blot analysis. Four lines for β-PAC were selected by visual inspection to confirm an orange endosperm, which was not found in the seeds of the 35S-PAC lines. The strong expression of PAC gene was detected in the seeds of the β-PAC lines and in the leaves of the 35S-PAC lines by RT-PCR and qRT-PCR analyses, suggesting that these two different promoters function distinctively. HPLC analysis of the seeds and leaves of the T₂ generation plants revealed that the best line among the β-PAC transgenic seeds accumulated 146 µg/g of total carotenoids (approximately 62-fold higher than non-transgenic seeds), of which 112 µg/g (77%) was β-carotene. In contrast, the level and composition of the leaf carotenoids showed little difference between transgenic and non-transgenic soybean plants. We have therefore demonstrated the production of a high β-carotene soybean through the seed-specific overexpression of two carotenoid biosynthetic genes, Capsicum phytoene synthase and Pantoea carotene desaturase. This nutritional enhancement of soybean seeds through the elevation of the provitamin A content to produce biofortified food may have practical health benefits in the future in both humans and livestock.     

Anti-inflammatory activity of Camellia japonica oil

Camellia japonica oil (CJ oil) has been used traditionally in East Asia to nourish and soothe the skin as well as help restore the elasticity of skin. CJ oil has also been used on all types of bleeding instances. However, little is known about its anti-inflammatory effects. Therefore, the anti-inflammatory effects of CJ oil and its mechanisms of action were investigated. CJ oil inhibited LPS-induced production of NO, PGE(2), and TNF-α in RAW264.7 cells. In addition, expression of COX-2 and iNOS genes was reduced. To evaluate the mechanism of the anti-inflammatory activity of CJ oil, LPS-induced activation of AP-1 and NF-κB promoters was found to be significantly reduced by CJ oil. LPS-induced phosphorylation of IκBα, ERK, p38, and JNK was also attenuated. Our results indicate that CJ oil exerts anti-inflammatory effects by downregulating the expression of iNOS and COX-2 genes through inhibition of NF-κB and AP-1 signaling. [BMB reports 2012; 45(3): 177-182].     

RNAi-mediated <Emphasis Type="Italic">Soybean mosaic virus</Emphasis> (SMV) resistance of a Korean Soybean cultivar

Soybean [Glycine max (L.) Merr.] is an important crop for vegetable oil production, and is a major protein source worldwide. Because of its importance as a crop, genetic transformation has been used extensively to improve its valuable traits. Soybean mosaic virus (SMV) is one of the most well-known viral diseases affecting soybean. Transgenic soybean plants with improved resistance to SMV were produced by introducing HC-Pro coding sequences within RNA interference (RNAi) inducing hairpin construct via Agrobacterium-mediated transformation. During an experiment to confirm the response of transgenic plants (T₂) to SMV infection, no T₂ plants from lines #2 (31/31), #5 (35/35) or #6 (37/37) exhibited any SMV symptoms, indicating strong viral resistance (R), whereas NT (non-transgenic wild type) plants and those from lines #1, #3 and #4 exhibited mild mosaic (mM) or mosaic (M) symptoms. The northern blot analysis showed that three resistant lines (#2, #5 and #6) did not show the detection of viral RNA accumulation while NT, EV (transformed with empty vector carrying only Bar) and lines #1, #3 and #4 plants were detected. T₃ seeds from SMV-inoculated T₂ plants were harvested and checked for changes in seed morphology due to viral infection. T₃ seeds of lines #2, #5 and #6 were clear and seed coat mottling was not present, which is indicative of SMV resistance. RT-PCR and quantitative real-time PCR showed that T₃ seeds from the SMV-resistant lines #2, #5 and #6 did not exhibit any detection of viral RNA accumulation (HC-Pro, CP and CI), while the viral RNA accumulation was detected in SMV-susceptible lines #1, #3 and #4 plants. During the greenhouse test for viral resistance and yield components, T₃ plants from the SMV-inoculated transgenic lines #2, #5 and #6 showed viral resistance (R) and exhibited a more favorable average plant height, number of nodes per plant, number of branches per plant, number of pods per plant and total seed weight with statistical significance during strong artificial SMV infection than did other plant lines. In particular, the SMV-resistant line #2 exhibited superior average plant height, pod number and total seed weight with highly significance. According to our results, RNAi induced by the hairpin construct of the SMV HC-Pro sequence effectively confers much stronger viral resistance than did the methods used during previous trials, and has the potential to increase yields significantly. Because of its efficiency, the induction of RNAi-mediated resistance will likely be used more frequently as part of the genetic engineering of plants for crop improvement.     

Molecular characterization of the soybean L-asparaginase gene induced by low temperature stress

L-asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of the amide group of L-asparagine, releasing aspartate and NH4+. We isolated a low temperature-inducible cDNA sequence encoding L-asparaginase from soybean leaves. The full-length L-asparaginase cDNA, designated GmASP1, contains an open reading frame of 1,258 bp coding for a protein of 326 amino acids. Genomic DNA blotting and fluorescence in situ hybridization showed that the soybean genome has two copies of GmASP1. GmASP1 mRNA was induced by low temperature, ABA and NaCl, but not by heat shock or drought stress. E. coli cells expressing recombinant GmASP1 had 3-fold increased L-asparaginase activity. A possible function of L-asparaginase in the early response to low temperature stress is discussed.     

Egg survival is related to the colour matching of eggs to nest background in Black-tailed Gulls

A long-standing hypothesis posits that, for species with exposed nests, a close match between the colour of the eggs and that of the nest in which they are laid should enhance egg survival, but this has rarely been tested in a rigorous manner. Here, we demonstrate the effects of egg–nest colour matching on egg survival in Black-tailed Gulls (Larus crassirostris) on Hongdo Island, Korea. We quantified the ground colour of eggshells and that of the nest background using a digital camera and computerized RGB and greyscale colour systems. We show that a close match of eggshell ground colour and nest background colour was associated with increased chances of eggs surviving through to hatching. In particular, there were strong survival advantages for eggs matching the nest colour in sites with poor concealment, whereas there was no effect of eggshell ground colour in nests that were more concealed by vegetation. Our findings support the hypothesis that egg colour functions to make eggs cryptic and that egg colouration may be a significant factor affecting egg loss.     

Regulation of castasterone level in primary roots of maize, Zea mays

Gas chromatography–mass spectrometry analysis revealed that primary roots of maize contain 28-norcastasterone (28-norCS) and its biosynthetic precursors, cholesterol, and cholestanol, which suggests that the C₂₇-brassinosteroid (C₂₇-BR) biosynthetic pathway to generate 28-norCS is operative in the roots. A cell-free enzyme solution prepared from maize roots successfully mediated C24-methylation of 28-norCS to produce castasterone (CS) with the aid of S-adenosyl- l -methionine, which indicates that CS can be generated through C₂₇-BR biosynthesis, as well as C₂₈-BR biosynthesis, in maize roots. Enzymatic conversion study using the cell-free enzyme solution demonstrated that CS is converted into 26-norCS in the enzyme solution. Exogenously applied 28-norCS and 26-norCS showed less activity than CS in the activation of gravitropic curvature and inhibition of root elongation. Taken together, a steady-state level of CS, the active BR in maize roots, seems to be strictly controlled by complicated processes such as C₂₈- and C₂₇-BR biosynthesis and biodegradation by C26-demethylation to exert its biological activity.     

Effect of denervation on ATP consumption rate of diaphragm muscle fibers.

Denervation (DNV) of rat diaphragm muscle (DIAm) decreases myosin heavy chain (MHC) content in fibers expressing MHC(2X) isoform but not in fibers expressing MHC(slow) and MHC(2A). Since MHC is the site of ATP hydrolysis during muscle contraction, we hypothesized that ATP consumption rate during maximum isometric activation (ATP(iso)) is reduced following unilateral DIAm DNV and that this effect is most pronounced in fibers expressing MHC(2X). In single-type-identified, permeabilized DIAm fibers, ATP(iso) was measured using NADH-linked fluorometry. The maximum velocity of the actomyosin ATPase reaction (V(max) ATPase) was determined using quantitative histochemistry. The effect of DNV on maximum unloaded shortening velocity (V(o)) and cross-bridge cycling rate [estimated from the rate constant for force redevelopment (k(TR)) following quick release and restretch] was also examined. Two weeks after DNV, ATP(iso) was significantly reduced in fibers expressing MHC(2X), but unaffected in fibers expressing MHC(slow) and MHC(2A). This effect of DNV on fibers expressing MHC(2X) persisted even after normalization for DNV-induced reduction in MHC content. With DNV, V(o) and k(TR) were slowed in fibers expressing MHC(2X), consistent with the effect on ATP(iso). The difference between V(max) ATPase and ATP(iso) reflects reserve capacity for ATP consumption, which was reduced across all fibers following DNV; however, this effect was most pronounced in fibers expressing MHC(2X). DNV-induced reductions in ATP(iso) and V(max) ATPase of fibers expressing MHC(2X) reflect the underlying decrease in MHC content, while reduction in ATP(iso) also reflects a slowing of cross-bridge cycling rate.     

Changes in cardiovascular beta-adrenoceptor responses during hypothermia

The purpose of this study was to determine cardiovascular β-adrenergic responses during hypothermia. In the present study, we used isoproterenol (Iso), a nonselective, potent β-adrenoceptor agonist, well known for its positive chronotropic and inotropic pharmacologic actions at normothermia. Rats were instrumented to measure mean arterial pressure (MAP) and left ventricular (LV) pressure–volume changes using a Millar pressure–volume conductance catheter. Core temperature was manipulated from 37 (normothermia) to 24 °C (hypothermia) and back to 37 °C (rewarming) using both internal and external heat exchangers. During cooling at each temperature (33, 30, 27, and 24 °C), central hemodynamic variables and MAP were measured while intravenously infusing Iso (doses of 1.7, 5, 10, and 20 ng/min). Seven animals underwent all phases of the protocol. At normothermia Iso infusion resulted in a significant, dose-dependent increase in heart rate (HR), stroke volume (SV), cardiac output (CO), LV dP/dt_max (left ventricular maximum derivative of systolic pressure over time) but no change in MAP. During cooling Iso infusion caused no dose-dependent change in any of the hemodynamic variables. After rewarming, baseline HR and LV dP/dt_max were increased, whereas SV was significantly reduced when compared with their pre-hypothermic baseline values. This study shows that physiological cardiovascular responses mediated by the β-adrenoceptor are significantly diminished during core hypothermia.