Isolation and characterization of cold-regulated transcriptional activator <Emphasis Type="Italic">LpCBF3</Emphasis> gene from perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

In plants, low temperatures can activate the CBF cold response pathway playing a prominent role in cold acclimation by triggering a set of cold-related gene expressions. CBF homologous gene, designated as LpCBF3, from a cold-tolerant perennial ryegrass (Lolium perenne L.) accession was identified. It carries the sequences for nuclear localization signal (NLS), AP2 DNA-binding domains and an acidic activation present in most of the plant CBF proteins. Southern analysis indicated the presence of three homologs of LpCBF3 gene in perennial ryegrass genome, and only one amino acid variation in LpCBF3 protein between cold-tolerant and -sensitive perennial ryegrass accessions. In their putative promoter regions, some differential regions were found. Northern blotting and RT-PCR analysis found that LpCBF3 reached the highest expression after 1.5 h of cold treatment (4 degrees C). The COR homologous gene, a downstream gene of CBF, can be expressed in the plant stem of cold-tolerant perennial ryegrass accessions without cold treatment. Without cold treatment, the COR gene cannot be activated in cold-sensitive perennial ryegrass accessions. Cold treatment can prompt expression levels of COR homologous genes in both perennial ryegrass accessions. In transgenic Arabidopsis, the overexpression of LpCBF3 with the 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance.     

Metabolic activity of Siberian permafrost isolates, <Emphasis Type="Italic">Psychrobacter arcticus</Emphasis> and <Emphasis Type="Italic">Exiguobacterium sibiricum</Emphasis>, at low water activities

The Siberian permafrost is an extreme, yet stable environment due to its continuously frozen state. Microbes maintain membrane potential and respiratory activity at average temperatures of -10 to -12 degrees C that concentrate solutes to an a (w) = 0.90 (5 osm), The isolation of viable Psychrobacter arcticus sp. 273-4 and Exiguobacterium sibiricum sp. 255-15 from ancient permafrost suggests that these bacteria have maintained some level of metabolic activity for thousands of years. Permafrost water activity was simulated using (1/2) TSB + 2.79 m NaCl (5 osm) at and cells were held at 22 and 4 degrees C. Many cells reduced cyano-tetrazolium chloride (CTC) indicating functioning electron transport systems. Increased membrane permeability was not responsible for this lack of electron transport, as more cells were determined to be intact by LIVE/DEAD staining than were reducing CTC. Low rates of aerobic respiration were determined by the slope of the reduced resazurin line for P. arcticus, and E. sibiricum. Tritiated leucine was incorporated into new proteins at rates indicating basal level metabolism. The continued membrane potential, electron transport and aerobic respiration, coupled with incorporation of radio-labeled leucine into cell material when incubated in high osmolarity media, show that some of the population is metabolically active under simulated in situ conditions.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): gene integration,expression and inheritance

A reproducible method of Agrobacterium-mediated transformation was developed for Cicer arietinum (chickpea). Initial explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. The plasmid contained a bi-functional fusion gene conferring both -glucuronidase and neomycin phosphotransferase activities, under the control of a 35S35SAMV promoter. Using a series of tissue culture media for co-cultivation, shoot initiation and rooting, we recovered transgenic plants from approximately 1.3% of the sliced embryo axes. The addition of a shoot elongation medium to the protocol improved the success rate to 3.1% but increased the time in tissue culture. Inheritance of the gus gene was followed through four generations, both through expression and Southern hybridization assays, and showed the expected Mendelian inheritance pattern.NRCC Grant No. 46589.     

Hypocotyl-based <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) and application for RNA interference

An efficient system of gene transformation is necessary for soybean [Glycine max (L.) Merrill] functional genomics and gene modification by using RNA interference (RNAi) technology. To establish such system, we improved the conditions of tissue culture and transformation for increasing the frequency of adventitious shoots and decreasing the browning and necrosis of hypocotyls. Adding N(6)-benzylaminopurine (BAP) and silver nitrate in culture medium enhanced the shoot formation on hypocotyls. BAP increased the frequency of the hypocotyls containing adventitious shoots, while silver nitrate increased the number of shoots on the hypocotyls. As a result, the number of adventitious shoots on hypocotyls cultured in medium containing both BAP and silver nitrate was 5-fold higher than the controls. Adding antioxidants in co-cultivation medium resulted in a significant decrease in occurrence of browning and necrosis of hypocotyls and increase in levels of beta-Glucuronidase (GUS) gene expression. Histochemical assays showed that the apical meristem of hypocotyls was the "target tissue" for Agrobacterium tumefaciens transformation of soybean. Gene silencing of functional gene by using RNAi technology was carried out under above conditions. A silencing construct containing an inverted-repeat fragment of the GmFAD2 gene was introduced into soybean by using the A. tumefaciens-mediated transformation. Several lines with high oleic acid were obtained, in which mean oleic acid content ranged from 71.5 to 81.9%. Our study demonstrates that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics.     

Temperature-dependent changes of cell shape during heterophyllous leaf formation in <Emphasis Type="Italic">Ludwigia </Emphasis><Emphasis Type="Italic">arcuata</Emphasis> (Onagraceae)

Although elongation of epidermal cells in submerged leaves is thought to be a common feature of heterophyllous aquatic plants, such elongation has not been observed in Ludwigia arcuata Walt. (Onagraceae). In this study we found that reduced culture temperature induced the elongation of epidermal cells of submerged leaves in L. arcuata. Since such submerged leaves also showed a reduction in the number of epidermal cells aligned across the leaf transverse axis, these data indicate that heterophyllous leaf formation in L. arcuata is partially temperature sensitive, i.e., the elongation of epidermal cells was temperature sensitive while the reduction in the number of epidermal cells did not show such temperature sensitivity. To clarify the mechanisms that cause such temperature sensitivity, we examined the effects of ethylene, which induced the formation of submerged-type leaves on aerial shoots at the relatively high culture-temperature of 28 degrees C. At 23 degrees C, ethylene induced both cell elongation and reduction in the number of epidermal cells across the leaf transverse axis, while cell elongation was not observed at 28 degrees C. Moreover, both submergence and ethylene treatment induced a change in the arrangement of cortical microtubules (MTs) in epidermal cells of developing leaves at 23 degrees C. Such changes in the arrangement of MTs was not induced at 28 degrees C. Factors involved in the temperature-sensitive response to ethylene would be critical for temperature-sensitive heterophyllous leaf formation in L. arcuata.     

<Emphasis Type="Italic">Stachybotrys atra</Emphasis> BP-A produces alkali-resistant and thermostable cellulases

A cellulose-degrading fungal strain has been isolated from a rotten rag. Morphological characterization and ITS1, 5.8S and ITS2 rDNA sequencing showed that the strain is a new isolate of Stachybotrys atra. The strain secreted high cellulase activity in media supplemented with rice straw. However, cellulases were not produced in glucose-supplemented media. The crude cellulase showed the highest activity on amorphous celluloses such as carboxymethyl cellulose, while activity on crystalline celluloses such as Avicel was lower. The optimal temperature and pH for CMCase activity were 70 degrees C and pH 5 respectively, although a second peak of activity was found at pH 8. Activity was strongly inhibited by Cu(2+), Mn(2+) and Hg(2+). Analysis by SDS-PAGE, isoelectric focusing and zymography showed that the strain secretes a complex cellulase system comprising several enzymes. Most of these enzymes are alkali-resistant CMCases that remained stable at pH 9 and 65 degrees C for at least 1 h. Cellulose binding assays showed notable differences among the CMCases. While some CMCase bands did not bind Avicel, other bands bound to this polymer and were eluted either with NaCl or by boiling with SDS. Analysis by two-dimensional electrophoresis showed that the band eluted by SDS boiling contained at least 4 different polypeptides. The complex set of cellulases produced by the strain, and their activity and stability at alkaline pH and a high temperature indicate that both the isolated strain and the cellulases identified are good candidates for biotechnological applications involving cellulose modification.     

Mineralization of chlorpyrifos by co-culture of <Emphasis Type="Italic">Serratia</Emphasis> and <Emphasis Type="Italic">Trichosporon</Emphasis> spp.

A bacterial strain (Serratia sp.) that could transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol (TCP) and a TCP-mineralizing fungal strain (Trichosporon sp.) were isolated from activated sludge by enrichment culture technique. The fungus could also degrade 50 mg chlorpyrifos l(-1) within 7 days. Co-cultures completely mineralized 50 mg chlorpyrifos l(-1) within 18 h at 30 degrees C and pH 8 using a total inocula of 0.15 g biomass l(-1).     

Time Course of <Emphasis Type="Italic">c-fos</Emphasis>, <Emphasis Type="Italic">vasopressin</Emphasis> and <Emphasis Type="Italic">oxytocin</Emphasis> mRNA Expression in the Hypothalamus Following Long-Term Dehydration

Summary 1. This study presents a time course analysis of the messenger RNA (mRNA) levels of c-fos, vasopressin (VP), and oxytocin (OT) in the paraventricular (PVN) and supraoptic nucleus (SON), following acute and chronic dehydration by water deprivation. 2. Male Wistar rats were separated into five groups: nondehydrated (control group) and dehydrated for 6, 24, 48 and 72 h. Following water deprivation, animals were decapitated, their blood was collected for hematocrit, osmolality, and plasma sodium measurements, and brains were removed for dissection of both PVN and SON. 3. As expected, the hematocrit, osmolality, plasma sodium, and weight loss were increased after water deprivation. In SON, a significant increase in both VP and OT mRNA expression was observed 6 h after dehydration reaching a peak at 24 h and returning to basal levels of expression at 72 h. In the PVN, an increase in both VP and OT mRNA expression occurred 24 h after dehydration. At 72 h the VP and OT mRNA expression levels had decreased but they were still at higher levels than those detected in control animals. 4. These results suggest that SON is the first nucleus to respond to the dehydration stimulus. Additionally, we also observed an increase in c-fos mRNA expression in both PVN and SON 6 h after water deprivation, which progressively decreased 24, 48, and 72 h after the onset of water deprivation. Therefore, it is possible that c-fos may be involved in the modulation of VP and OT genes, regulating the mRNA expression levels on a temporally distinct basis within the PVN and SON.