Overexpression of the alfalfa DnaJ-like protein (MsDJLP) gene enhances tolerance to chilling and heat stresses in transgenic tobacco plants.

Heat shock proteins (HSPs) are generally considered as important molecular chaperones; they are known to perform critical functions in plant development and abiotic stress response processes. In this study, we examined the role of a HSP, the Medicago sativa DnaJ-like protein (MsDJLP), in alfalfa and its potential application for the development of abiotic stress tolerance in plants. We found that expression of the MsDJLP gene was induced by chilling (4 °C) and heat (42 °C), but not by cadmium (500 µM) or arsenic (500 µM) stresses. We then cloned the MsDJLP gene downstream of the strong constitutive CaMV 35S promoter and transformed it into tobacco plants. Ectopic expression of MsDJLP conferred enhanced tolerance to both chilling and heat stresses in transgenic tobacco plants. Under chilling stress, the transgenic tobacco plants showed lower H2O2 accumulation and electrolyte leakage (EL) activity, and better photosystem II efficiency than wild-type (WT) plants, indicating that photoinhibition was less severe in transgenic compared to WT plants. Following heat treatment, the transgenic plants showed better relative chlorophyll and water contents, and lower malondialdehyde accumulation than WT plants. Our study provides evidence for a pivotal role of MsDJLP for chilling and heat stress tolerance in transgenic tobacco plants.

Trigonelline attenuates the adipocyte differentiation and lipid accumulation in 3T3-L1 cells.

Trigonelline is a natural alkaloid mainly found in Trigonella Foenum Graecum (fenugreek) Fabaceae and other edible plants with a variety of medicinal applications. Therefore, we investigated the molecular mechanism of trigonelline (TG) on the inhibition of adipocyte differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline suppressed lipid droplet accumulation in a concentration (75 and 100 µM) dependent manner. Treatment of adipocyte with of TG down regulates the peroxisome proliferator-activated receptor (PPARγ) and CCAAT element binding protein (C/EBP-α) mRNA expression, which leads to further down regulation of other gene such as adiponectin, adipogenin, leptin, resistin and adipocyte fatty acid binding protein (aP2) as compared with respective control cells on 5th and 10th day of differentiation. Further, addition of triognelline along with troglitazone to the adipocyte attenuated the troglitazone effects on PPARγ mediated differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline might compete against troglitazone for its binding to the PPARγ. In addition, adipocyte treated with trigonelline and isoproterenol separately. Isoproterenol, a lipolytic agent which inhibits the fatty acid synthase and GLUT-4 transporter expression via cAMP mediated pathway, we found that similar magnitude response of fatty acid synthase and GLUT-4 transporter expression in trigonelline treated adipocyte. These results suggest that the trigonelline inhibits the adipogenesis by its influences on the expression PPARγ, which leads to subsequent down regulation of PPAR-γ mediated pathway during adipogenesis. Our findings provide key approach to the mechanism underlying the anti-adipogenic activity of trigonelline.

Chloroform extract of alfalfa (Medicago sativa) inhibits lipopolysaccharide-induced inflammation by downregulating ERK/NF-κB signaling and cytokine production.

Alfalfa (Medicago sativa L.) is commonly used as a traditional medicine and functional food. This study investigated the anti-inflammatory potential of alfalfa and the mechanisms involved. The chloroform extract of alfalfa aerial parts inhibited lipopolysaccharide (LPS)-stimulated immune responses more than ether, butanol, or water soluble extracts. Treatment with 1 µg/mL LPS increased nitrite concentrations to 44.3 µM in RAW267.4 macrophages, but it was reduced to 10.6 µM by adding 100 µg/mL chloroform extract. LPS treatment also increased the concentrations of tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß to 41.3, 11.6, and 0.78 ng/mL in culture supernatants of the cells, but these cytokine levels decreased to 12.5, 3.1, and 0.19 ng/mL, respectively, by pretreating with 100 µg/mL of the extract. ICR mice injected with LPS (30 mg/kg body weight) alone showed a 0% survival rate after 48 h of the injection, but 48-h survival of the mice increased to 60% after oral administration of the extract. Subfractions of the chloroform extract markedly suppressed LPS-mediated activation of the extracellular signal-regulated kinase and nuclear factor kappa-B. Cinnamic acid derivatives and fatty acids were found to be active constituents of the extract. This research demonstrated that alfalfa aerial parts exert anti-inflammatory activity and may be useful as a functional food for the prevention of inflammatory disorders.

Transgenic Expression of MsHsp23 Confers Enhanced Tolerance to Abiotic Stresses in Tall Fescue.

Tall fescue (Festuca arundinacea Schreb.) is an important cool season forage plant that is not well suited to extreme heat, salts, or heavy metals. To develop transgenic tall fescue plants with enhanced tolerance to abiotic stress, we introduced an alfalfa Hsp23 gene expression vector construct through Agrobacterium-mediated transformation. Integration and expression of the transgene were confirmed by polymerase chain reaction, northern blot, and western blot analyses. Under normal growth conditions, there was no significant difference in the growth of the transgenic plants and the non-transgenic controls. However, when exposed to various stresses such as salt or arsenic, transgenic plants showed a significantly lower accumulation of hydrogen peroxide and thiobarbituric acid reactive substances than control plants. The reduced accumulation of thiobarbituric acid reactive substances indicates that the transgenic plants possessed a more efficient reactive oxygen species-scavenging system. We speculate that the high levels of MsHsp23 proteins in the transgenic plants protect leaves from oxidative damage through chaperon and antioxidant activities. These results suggest that MsHsp23 confers abiotic stress tolerance in transgenic tall fescue and may be useful in developing stress tolerance in other crops.

Genotypic variation of Agrobacterium-mediated transformation of Italian ryegrass

In the present study, genotypic variation of Agrobacterium-mediated transformation of Korean Italian ryegrass has been evaluated. Mature seed-derived calli of seven cultivars were inoculated and co-cultured with Agrobacterium tumefaciens carrying the binary vector pCAMBIA1301, which contains a reporter gene (gus) and a plant selectable marker gene conferring resistance to hygromycin (hpt) in the T-DNA region. The effects of several factors such as callus type and callus age on transformation effectiveness and the expression of the GUS gene were investigated. The highest transformation effectiveness (6.7 percent) was obtained with the Hwasan 101 cultivar when 9-week-old calli (type-I) were inoculated with Agrobacterium. The overall transformation rates of the examined cultivars ranged from 0.4 percent to 6.7 percent. GUS histochemical assays, PCR, and southern analysis of transgenic plants demonstrated that transgenes were successfully integrated into the genome of Italian ryegrass. Thus, evaluation of transformation effectiveness and selection of a suitable cultivar of Italian ryegrass may improve molecular breeding of this species.