Silicon carbide fiber-mediated DNA delivery into cells of wheat (Triticum acstivum L.) mature embryos

We have demonstrated that foreign DNA can be delivered into cells of mature embryos of wheat (Triticum aestivum L.) using silicon carbide fibers (SCF). The highest transient expression of thegusA (GUS) gene was detected when dry embryos were vortexed for 10–30 min in a SCF-DNA solution containing 90–120 g/l of sucrose. Up to 100 (on average 20–40) blue expression units per embryo were observed. Scutellum side and epiblast of the intact wheat embryos are preferentially transformed. When embryos with the coleoptilar tip removed were treated and allowed to germinate, GUS staining was observed in emerging leaf tissues. The potential of this new approach for stable transformation of wheat is under investigation. It has been found that callus tissues induced from the SCF treated embryos contain GUS-expressing sectors one month after treatment.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)     

Weak Light-Induced Oxygen Consumption Observed during Photoreactivation Is Coupled to the Recovery of Oxygen Evolving Activity

During photoreactivation of the O₂-evolving center in Tris-inactivated/Mn-depletedthylakoids, a slow O₂-consumption occurred. This O₂-consumptionbecame detectable when the O₂-evolving activity of thylakoidswas inactivated by Tris-treatment and decreased as photoreactivationproceeded. The O₂-consumption and photoreactivation similarlyrequired Mn²⁺ at µM levels in addition to PSII electrondonors and shared severa common characteristics. Stimulationof O₂-consumption and photoreactivation by these cofactors werealways accompanied by enhancement in chlorophyll fluorescenceinduction, suggesting the involvement of a Mehler-type reactionin photoreactivation. Although the electron transport due tothis O₂-consumption was rapid enough to oxidize 4 Mn²⁺ ionsto reconstitute the tetranuclear Mn-cluster in each O₂-evolvingcenter in a few seconds, actual recovery of O₂-evolving activityoccurred more slowly in a few minutes. It was inferred thatphotoreactivation in Tris-inactivated thylakoids is not a simplephotooxidation of Mn2²⁺ but involves more complicated processeswhich are coupled to the Mehlertype electron transport fromPSII to oxygen via PSI. (Received July 11, 1994; Accepted August 23, 1996)     

Changes in organic matter composition of forest soil treated with a large amount of urea to promote ammonia fungi and the abilities of these fungi to decompose organic matter

Organic matter composition (lignin, holocellulose, 50% (v/v) methanol extract, water-soluble carbohydrate (WSC) and phenolics (WSP), petroleum ether extract, and ash) of A₀ layer soil treated with 700 g/m² of urea to promote ammonia fungi was investigated in a Japanese red pine (Pinus densiflora) forest. Nine species of fungi were found during the study period of 18 months after the treatment. Of these, seven species belong to the ammonia fungi. WSC content of the treated soil was lower than that of the control. Methanol extract content increased initially after the treatment, then decreased to below the control level. There were no consistent differences in other components between the treated plot and the control. The abilities to decompose cellulose, lignin, chitin, protein and lipid in 18 strains in 10 species of the ammonia fungi were also screened. Cellulose was not lysed byPseudombrophila deerata, Hebeloma spp. andLaccaria bicolor. Strong lignolytic activity was shown byLyophyllum tylicolor, Coprinus echinosporus andP. deerata. Chitin was decomposed byAmblyosporium botrytis, L. tylicolor, C. echinosporus andHebeloma vinosophyllum. All strains possessed proteolytic and lipolytic activities. Supply of glucose to the culture media resulted in weaker enzyme activities except for lignolytic ability.