Determination of alpha-helix and beta-sheet stability in the solid state: a solid-state NMR investigation of poly(L-alanine)

The relative stability of alpha-helix and beta-sheet secondary structure in the solid state was investigated using poly(L-alanine) (PLA) as a model system. Protein folding and stability has been well studied in solution, but little is known about solid-state environments, such as the core of a folded protein, where peptide packing interactions are the dominant factor in determining structural stability. (13)C cross-polarization with magic angle spinning (CPMAS) NMR spectroscopy was used to determine the backbone conformation of solid powder samples of 15-kDa and 21.4-kDa PLA before and after various sample treatments. Reprecipitation from helix-inducing solvents traps the alpha-helical conformation of PLA, although the method of reprecipitation also affects the conformational distribution. Grinding converts the secondary structure of PLA to a final steady-state mixture of 55% beta-sheet and 45% alpha-helix at room temperature regardless of the initial secondary structure. Grinding PLA at liquid nitrogen temperatures leads to a similar steady-state mixture with 60% beta-sheet and 40% alpha-helix, indicating that mechanical shear force is sufficient to induce secondary structure interconversion. Cooling the sample in liquid nitrogen or subjecting it to high pressure has no effect on secondary structure. Heating the sample without grinding results in equilibration of secondary structure to 50% alpha-helix/50% beta-sheet at 100 degrees C when starting from a mostly alpha-helical state. No change was observed upon heating a beta-sheet sample, perhaps due to kinetic effects and the different heating rate used in the experiments. These results are consistent with beta-sheet approximately 260 J/mol more stable than alpha-helix in solid-state PLA.     

Involvement of chloroplasts in the programmed death of plant cells

The effect of cyanide, an apoptosis inducer, on pea leaf epidermal peels was investigated. Illumination stimulated the CN^–-induced destruction of guard cells (containing chloroplasts and mitochondria) but not of epidermal cells (containing mitochondria only). The process was prevented by antioxidants (-tocopherol, 2,5-di-tret-butyl-4-hydroxytoluene, and mannitol), by anaerobiosis, by the protein kinase C inhibitor staurosporine, and by cysteine and serine protease inhibitors. Electron acceptors (menadione, p-benzoquinone, diaminodurene, TMPD, DCPIP, and methyl viologen) suppressed CN^–-induced apoptosis of guard cells, but not epidermal cells. Methyl viologen had no influence on the removal of CN^–-induced nucleus destruction in guard cells under anaerobic conditions. The light activation of CN^–-induced apoptosis of guard cells was suppressed by DCMU (an inhibitor of the electron transfer in Photosystem II) and by DNP-INT (an antagonist of plastoquinol at the Q_o site of the chloroplast cytochrome b ₆ f complex). It is concluded that apoptosis initiation in guard cells depends on the simultaneous availability of two factors, ROS and reduced quinones of the electron transfer chain. The conditions for manifestation of programmed cell death in guard and epidermal cells of the pea leaf were significantly different.     

Migration of nerve growth cones requires detergent-resistant membranes in a spatially defined and substrate-dependent manner

Motility of nerve growth cones (GCs) is regulated by region-specific activities of cell adhesion molecules (CAMs). CAM activities could be modified by their localization to detergent-resistant membranes (DRMs), specialized microdomains enriched in signaling molecules. This paper deals with a question of whether DRMs are involved in GC migration stimulated by three CAMs; L1, N-cadherin (Ncad), and beta1 integrin. We demonstrate that L1 and Ncad are present in DRMs, whereas beta1 integrin is exclusively detected in non-DRMs of neurons and that localization of L1 and Ncad to DRMs is developmentally regulated. GC migration mediated by L1 and Ncad but not by beta1 integrin is inhibited after DRM disruption by micro-scale chromophore-assisted laser inactivation (micro-CALI) of GM1 gangliosides or by pharmacological treatments that deplete cellular cholesterol or sphingolipids, essential components for DRMs. Characteristic morphology of GCs induced by L1 and Ncad is also affected by micro-CALI-mediated DRM disruption. Micro-CALI within the peripheral domain of GCs, or even within smaller areas such as the filopodia and the lamellipodia, is sufficient to impair their migration. However, micro-CALI within the central domain does not affect GC migration. These results demonstrate the region-specific involvement of DRMs in CAM-dependent GC behavior.     

Effect of high temperature on fine structure of amylopectin in rice endosperm by reducing the activity of the starch branching enzyme

Rice (Oryza sativa L.) grain quality is affected by the environmental temperature it experiences. To investigate the physiological molecular mechanisms of the effect of high temperatures on rice grain, a non-waxy indica rice was grown under two temperature conditions, (29/35 degrees C) and (22/28 degrees C), during the ripening stage in two phytotrons. The activities and gene expression of key enzymes for the biosynthesis of amylose and amylopectin were examined. The activity and expression levels of soluble endosperm starch synthase I were higher at 29/35 degrees C than that at 22/28 degrees C. In contrast, the activities and expression levels of the rice branching enzyme1, the branching enzyme3 and the granule bound starch synthase of the endosperm were lower at 29/35 degrees C than those at 22/28 degrees C. These results suggest that the decreased activity of starch branching enzyme reduces the branching frequency of the branches of amylopectin, which results in the increased amount of long chains of amylopectin of endosperm in rice grain at high temperature.     

Effects of arsenate and phosphate on their accumulation by an arsenic-hyperaccumulator Pteris vittata L.

Arsenate and phosphate interactions are important for better understanding their uptake and accumulation by plant due to their similarities in chemical behaviors. The present study examined the effects of arsenate and phosphate on plant biomass and uptake of arsenate and phosphate by Chinese brake (Pteris vittata L.), a newly-discovered arsenic hyperaccumulator. The plants were grown for 20 weeks in a soil, which received the combinations of 670, 2670, or 5340 mol kg^–1 arsenate and 800, 1600, or 3200 mol kg^–1 phosphate, respectively. Interactions between arsenate and phosphate influenced their availability in the soil, and thus plant growth and uptake of arsenate and phosphate. At low and medium arsenate levels (670 and 2670 mol kg^–1), phosphate had slight effects on arsenate uptake by and growth of Chinese brake. However, phosphate substantially increased plant biomass and arsenate accumulation by alleviating arsenate phytotoxicity at high arsenate levels (5340 mol kg^–1). Moderate doses of arsenate increased plant phosphate uptake, but decreased phosphate concentrations at high doses because of its phytotoxicity. Based on our results, the minimum P/As molar ratios should be at least 1.2 in soil solution or 1.0 in fern fronds for the growth of Chinese brake. Our findings suggest that phosphate application may be an important strategy for efficient use of Chinese brake to phytoremediate arsenic contaminated soils. Further study is needed on the mechanisms of interactive effects of arsenate and phosphate on Chinese brake in hydroponic systems.     

Glycosides of 2-C-methyl-D-erythritol from the fruits of anise,coriander and cumin

Eight glycosides of 2-C-methyl-D-erythritol (1) were isolated from the fruit of anise, and their structures were clarified as 1-O-beta-D-glucopyranoside, 3-O-beta-D-glucopyranoside, 4-O-beta-D-glucopyranoside, 1-O-beta-D-fructofuranoside, 3-O-beta-D-fructofuranoside, 4-O-beta-D-fructofuranoside, 1-O-beta-D-(6-O-4-hydroxybenzoyl)-glucopyranoside and 1-O-beta-D-(6-O-4-methoxybenzoyl)-glucopyranoside of 2-C-methyl-D-erythritol (2-9), respectively. Furthermore, 2 and 4 were isolated from the fruit of coriander, and 2, 3 and 4 were isolated from the fruit of cumin. Though the phosphate of 1 was known to be one of the first precursors of isoprenoids in the non-mevalonate pathway, and 1 is considered to be a common constituent in Umbelliferous plants, the glycosides of 1 are found for the first time.     

Efficient fine mapping of the naked caryopsis gene (<Emphasis Type="Italic">nud</Emphasis>) by HEGS (High Efficiency Genome Scanning)/AFLP in barley

The hulled or naked caryopsis character of barley (Hordeum vulgare L.) is an important trait for edibility and to follow its domestication process. A single recessive gene, nud, controls the naked caryopsis character, and is located on the long arm of chromosome 7H. To develop a fine map around the nud locus efficiently, the HEGS (High Efficiency Genome Scanning) electrophoresis system was combined with amplified fragment length polymorphism (AFLP). From bulked segregant analysis of 1,894 primer combinations, 12 AFLP fragments were selected as linked markers. For mapping, an F₂ population of 151 individuals derived from a cross between Kobinkatagi (naked type) and Triumph (hulled type) was used. Seven AFLP markers were localized near the nud region. A fine map was developed with one-order higher resolution than before, along with the seven anchor markers. Among the seven linked AFLP markers (KT1–7), KT1, KT2 and KT6 were co-dominant, and the former two were detected for their single-nucleotide polymorphisms (SNPs) in the same length of fragments after electrophoresis with the non-denaturing gels of HEGS. The nud locus has co-segregated with KT3 and KT7, and was flanked by KT2 and KT4, at the 0.3-cM proximal and the 1.2-cM distal side, respectively. Four of these AFLP markers were converted into sequence-characterized amplified region (SCAR) markers, one of which was a dominant marker co-segregating with the nud gene.Communicated by G. Wenzel     

Fine linkage mapping enables dissection of closely linked quantitative trait loci for seed dormancy and heading in rice

Two quantitative trait loci (QTLs) for seed dormancy (tentatively designated Sdr1) and heading date (Hd8) have been mapped to approximately the same region on chromosome 3 by interval mapping of backcross inbred lines derived from crosses between the rice cultivars Nipponbare (japonica) and Kasalath (indica). To clarify whether Sdr1 and Hd8 could be dissected genetically, we carried out fine-scale mapping with an advanced backcross progeny. We selected a BC₄F₁ plant, in which a small chromosomal region including Sdr1 and Hd8, on the short arm of chromosome 3, remained heterozygous, whereas all the other chromosomal regions were homozygous for Nipponbare. Days-to-heading and seed germination rate in the BC₄F₂ plants showed continuous variation. Ten BC₄F₂ plants with recombination in the vicinity of Sdr1 and Hd8 were selected on the basis of the genotypes of the restriction fragment length polymorphism (RFLP) markers flanking both QTLs. Genotypes of those plants for Sdr1 and Hd8 were determined by advanced progeny testing of BC₄F₄ families. Sdr1 was mapped between the RFLP markers R10942 and C2045, and co-segregated with C1488. Hd8 was also mapped between C12534S and R10942. Six recombination events were detected between Sdr1 and Hd8. These results clearly demonstrate that Sdr1 and Hd8 were tightly linked. Nearly isogenic lines for Sdr1 and Hd8 were selected by marker-assisted selection.Communicated by D. Mackill     

Molecular marker dissection of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) plant architecture under temperate and tropical climates

Rice (Oryza sativa L.) plants develop vertically with shoot elongation and horizontally with tillering. The purpose of this study was to identify and characterize genomic regions influencing the rice plant architecture by quantitative trait locus (QTL) analysis for the component traits: culm length (CL), panicle length (PnL), panicle number (PnN) and tiller number (TN). For this QTL analysis, 191 recombinant inbred lines (F₇) derived from a cross of Milyang 23 (M23) and Akihikari (AK) were grown in 1995, 1996 and 1997 (May–Oct) in Joetsu, Japan (temperate climate), and in the 2000 dry season (Jan–Apr), the 2000 wet season (Jun–Oct) and the 2001 dry season in Los Baños, The Philippines (tropical climate). Results showed that rice plant architecture was influenced by 19 genomic regions categorized into five groups. In Group I, two regions (on chrs. 6 and 11) affected shoot elongation (CL and PnL) and tillering (PnN and TN) in opposite directions more significantly in Los Baños than in Joetsu. In Group II, two regions (chrs. 3 and 12) affected shoot elongation, whereas in Group III, five regions [chrs. 1 (two), 2, 3 and 9] affected only culm length (CL). Expressions of four regions of Group III were influenced by either tropical or temperate environments. In Group IV, seven regions (chrs. 1, 2, 4, 5, 6, 8 and 9) controlled panicle development (PnN or PnL), and in Group V, three regions (chrs. 1, 2 and 3) regulated tillering (PnN or TN). Characterizing these 19 genomic regions provided a detailed analysis of rice plant architecture with emphasis on the multiple effect and environmental responsive regions.Communicated by D. Mackill