A fluorescent antibiotic resistance marker for rapid production of transgenic rice plants

Blasticidin S (BS) is an aminoacylnucleoside antibiotic used for the control of rice blast disease. To establish a new cereal transformation system, we constructed a visual marker gene designated gfbsd, encoding an enhanced green fluorescent protein (EGFP) fused to the N-terminus of BS deaminase (BSD). It was cloned into a monocot expression vector and introduced into rice (Oryza sativa L. cv. Nipponbare) calluses by microprojectile bombardment. Three to five weeks after the bombardment, multicellular clusters emitting bright-green EGFP fluorescence were obtained with 10 microg/ml BS, which is not sufficient to completely inhibit the growth of non-transformed tissues. Fluorescent sectors (approximately 2mm in diameter) excised from the calluses regenerated into transgenic plantlets (approximately 10 cm in height) as early as 51 (average 77+/-11) days after the bombardment. The visual antibiotic selection was more efficient and required less time than the bialaphos selection with bar. In addition, the small size (1.1 kb) of gfbsd is preferable for construction of transformation vectors. This new marker gene will make a significant contribution in molecular genetic studies of rice plants.     

Effects of charged peptides on electron transfer from [Fe(CN)6]4– to cytochrome c or plastocyanin

 Interactions of charged peptides, such as aspartic acid peptides (Aspptds) and lysine peptides (Lysptds), with cytochrome c (cyt c) or plastocyanin (PC) have been studied by measuring electron transfer between [Fe(CN)₆]^4– and cyt c or PC in the presence of these peptides. Aspptds, up to penta-aspartic acid, served as competitive inhibitors of electron transfer from [Fe(CN)₆]^4– to oxidized cyt c, while Lysptds, up to penta-lysine, promoted electron transfer from [Fe(CN)₆]^4– to oxidized PC. The electron transfer inhibitory effects of Aspptds are explained as competitive inhibition due to neutralization of the positively charged amino acid residues at the surface of cyt c by electrostatic interactions, whereas the electron transfer promoting effects of Lysptds may be due to formation of PC·Lysptd or Lysptd·[Fe(CN)₆]^4– complexes subsequently forming an electron transferring complex, PC·Lysptd·[Fe(CN)₆]^4–, without repulsion of the negative charges. The inhibitory effect of Aspptds and promotional effect of Lysptds became significant as the net charge or concentration of the peptides increased. The promotional effects of Lysptds decreased as the net charge of the PC negative patch was decreased by mutagenesis. Thus, charged peptides may serve as a probe for investigation of the molecular recognition character of proteins. Received: 19 May 1998 / Accepted: 27 July 1998     

Organocatalytic α-hydroxymethylation of cyclopentanone with aqueous formaldehyde: Easy access to chiral δ-lactones

Optically active lactones are important synthons in perfume and aroma manufacturing. Therefore, developments of efficient asymmetric syntheses are desired. Organocatalytic asymmetric α-hydroxymethylations of cyclopentanone with aqueous formaldehyde have been developed, to furnish the corresponding α-(hydroxymethyl)cyclopentanone with high enantioselectivity. Further chemical transformation of α-(hydroxymethyl)cyclopentanone gave the key intermediate for jasmine lactone, which is widely found in fruits and flowers.     

REGULATION OF BIOSYNTHESIS OF DIMETHYLSULFONIOPROPIONATE AND ITS UPTAKE IN STERILE MUTANT OF ULVA PERTUSA (CHLOROPHYTA)1

It has been shown that marine algae produce the compatible solute dimethylsulfoniopropionate (DMSP) from methionine (Met) via four enzymatic reactions in which the third step, synthesis of 4‐dimethylsulfonio‐2‐hydroxy‐butyrate (DMSHB) from 4‐methylthio‐2‐hydroxybutyrate (MTHB), is the committing step. However, regulation of the biosynthetic pathways and transport properties of DMSP is largely unknown. Here, the effects of sulfur and sodium concentrations on the uptake and synthesis of DMSHB and DMSP were examined in a sterile mutant of Ulva pertusa Kjellm. Sulfur deficiency increased the activity of the sulfur assimilation enzyme O‐acetyl serine sulfhydrylase but decreased the MTHB S‐methyltransferase activity, suggesting the preferential utilization of sulfur atoms for Met metabolites other than DMSP. Uptake of DMSP and DMSHB was enhanced by S deficiency. High salinity enhanced the MTHB S‐methyltransferase activity as well as the uptake of DMSHB. The MTHB S‐methyltransferase activity was inhibited by its product DMSP. These data demonstrate the importance of MTHB S‐methyltransferase activity and uptake of DMSHB for the regulation of DMSP.     

Isolation and characterization of a novel peroxisomal choline monooxygenase in barley

Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline → betaine aldehyde → GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [¹⁴C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [¹⁴C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.     

Optimization of intravascular shear stress assessment in vivo

The advent of microelectromechanical systems (MEMS) sensors has enabled real-time wall shear stress (WSS) measurements with high spatial and temporal resolution in a 3-D bifurcation model. To optimize intravascular shear stress assessment, we evaluated the feasibility of catheter/coaxial wire-based MEMS sensors in the abdominal aorta of the New Zealand white (NZW) rabbits. Theoretical and computational fluid dynamics (CFD) analyses were performed. Fluoroscope and angiogram provided the geometry of aorta, and the Doppler ultrasound system provided the pulsatile flow velocity for the boundary conditions. The physical parameters governing the shear stress assessment in NZW rabbits included (1) the position and distance from which the MEMS sensors were mounted to the terminal end of coaxial wire or the entrance length, (L_e), (2) diameter ratios of aorta to the coaxial wire (D_aorta /D_{coaxial wire}=1.5–9.5), and (3) the range of Reynolds numbers (116–1550). At an aortic diameter of 2.4 mm and a maximum Reynolds number of 212 (a mean Reynolds number of 64.2), the time-averaged shear stress (τ_ave) was computed to be 10.06 dyn cm^?2 with a systolic peak at 33.18 dyn cm^?2. In the presence of a coaxial wire (D_aorta /D_{coaxial wire}=6 and L_e=1.18 cm), the τ_ave value increased to 15.54 dyn cm^?2 with a systolic peak at 51.25 dyn cm^?2. Real-time intravascular shear stress assessment by the MEMS sensor revealed an τ_ave value of 11.92 dyn cm^?2 with a systolic peak at 47.04 dyn cm^?2. The difference between CFD and experimental τ_ave was 18.5%. These findings provided important insights into packaging the MEMS sensors to optimize in vivo shear stress assessment.     

Formation of Chlorophyll-Protein Complexes during Greening 1. Distribution of Newly Synthesized Chlorophyll among Apoproteins

The relationship between the accumulation of Chl and the apoproteinsof the light-harvesting Chl a/b-protein complex of PS II (LHCII)during the greening of cucumber cotyledons was studied. LHCIIapoproteins were not detected in etiolated cotyledons. Uponillumination, Chl a was formed as a result of photoconversionof protochlorophyllide (Pchlide) which had accumulated in thedark. During the lag period that preceded the accumulation ofChl, a small amount of LHCII apoproteins appeared. The amountof LHCII apoproteins increased with increases in levels of Chlb, though somewhat more rapidly during the first 10 h of greening.Treatment with benzyladenine (BA) or levulinic acid (LA) wasused to vary the supply of Chl a for apoproteins by promotingor inhibiting the synthesis of Chl a, respectively. LA decreasedbut BA increased the rate of accumulation of Chl b and LHCIIapoproteins. Only small amounts of Chl b and LHCII apoproteinswere formed under intermittent illumination. However, in thepresence of chloramphenicol (CAP), which inhibits the synthesisof plastome-coded proteins including apoproteins of the P700-Chla-protein complex (CP1) and a Chl a-protein complex of PS II(CPa), we observed the accumulation of Chl b and LHCII apoproteins,both of which are of nuclear origin. During incubation in thedark after intermittent exposure to light, CAP alone allowedneither destruction nor accumulation of Chl b and LHCII apoproteins,but it did enhance the effect of CaCl₂ in inducing both Chlb and these apoproteins. These results can be explained by assumingthat apoproteins of CP1 and CPa have a higher affinity for Chla than do LHCII apoproteins. When the availability of Chl ais limited, these apoproteins compete with one another for Chla, with the resultant preferential formation of CP1 and CPa.However, when the supply of Chl a becomes large enough for saturationof apoproteins of CP1 and CPa, some of the Chl a is incorporatedinto LHCII apoproteins either directly or after conversion toChl b. Thus, the formation of different Chl-protein complexes(CPs) is regulated by the relative rates of synthesis of Chla and apoproteins and by differential affinities of the apoproteinsfor Chl a. ⁴Present address: Kyowa Hakko Co., Ltd., 4041, Ami-machi, Inashiki,Ibaraki, 300-03 Japan (Received September 14, 1989; Accepted April 26, 1990)     

Formation of multiplex lamellae by equilibrium slow freezing of cortical parenchyma cells of mulberry and its possible relationship to freezing tolerance

Summary Cortical parenchyma cells of mulberry (Morus bombycis Koidz. cv. Goroji) become extremely cold hardy in winter and can tolerate equilibrium freezing below –30 °C and subsequent immersion into liquid nitrogen. We show in this ultrastructural study that, in these extremely cold hardy cortical parenchyma cells of mulberry collected in winter, initiation of freezing at –5 °C resulted in the formation of multiplex lamellae (MPL) that completely covered the area beneath the plasma membrane. The MPL were produced by fusion of pre-existing vesicular endoplasmic reticulum (ER), via a reticular ER network. The completed MPL were composed of a parallel array of sheet-like ER cisternae. This structural reorganization of the ER was completed within 10 min upon freezing at –5 °C and was quickly reversed upon thawing. The same structural reorganization of the ER was produced by osmotic dehydration of the cortical tissues with a 2.7 osmol sorbitol solution at 20 °C. Thus, the structural reorganization of the ER upon freezing was, in fact, produced by dehydration. In winter samples, the formation of MPL with the initiation of freezing completely inhibited close apposition of membranes upon deep freezing that has been reported to be a cause of freezing injury via the production of ultrastructural changes in the plasma membrane. Similar but more or less incomplete MPL were produced by freezing or osmotic dehydration in cortical parenchyma cells collected in spring and autumn, and these MPL partly inhibited close apposition of membranes. MPL were not produced in the cells of mulberry collected in summer and close apposition of membranes occurred upon deep freezing. We speculate that the formation of MPL with the initiation of freezing might play a specific role in inhibiting the close apposition of membranes due to the specific nature of the cisternal membranes and might, consequently, be responsible for the high freezing tolerance of winter cells.     

The formation and consumption of lipid droplets in the zygote and germinated cells ofClosterium ehrenbergii

The formation and consumption of lipid droplets was observed with an electron microscope in the zygote and the germinated cells of the green alga,Closterium ehrenbergii. The lipid droplets were formed in lysosomal vesicles during zygote maturation following conjugation. In the germinated cells, they were enclosed in ERs and gradually consumed in them. This consumption occurred in the cells at the early stages of expansion. The derivative substances may possibly be used for cell surface expansion.