Ultrastructure of Protein Crystals in Potato and Tomato Trichomes

Large protein crystals were located in the leaf and stem trichomesof Solanum tuberosum L. and Lycopersicon esculentum Mill. Inpotato the crystals ranged from 1.05 to 4.5 µm (average2.3 µm) on a side and in tomato they ranged from 1.16to 3.5 µm (average 2.7 µm) on a side. The proteinnature of the crystals was determined by histochemical stainingwith Coumassie brilliant blue R250 and aniline blue black. Thecrystalline structure of the inclusions was observed in ultrathinsections using electron microscopy. In potato, in cleared areasof the cytoplasm, ribosomes were observed scattered among proteinfilaments. The filaments were approximately 7 nm in diameter.Morphologically similar crystals were observed in the tomatotrichomes but the protein filaments were smaller (approximately4 nm in diameter). Protein crystals were also observed in palisadeand spongy parenchyma and epidermal leaf cells in tomato. Protein crystals, trichomes, potato, Solanum tuberosum L., tomato, Lycopersicon esculentum Mill., ultrastructure     

Biochemical and Histochemical Localization of Monoterpene Biosynthesis in the Glandular Trichomes of Spearmint (Mentha spicata)

The primary monoterpene accumulated in the glandular trichomes of spearmint (Mentha spicata) is the ketone (−)-carvone which is formed by cyclization of the C₁₀ isoprenoid intermediate geranyl pyrophosphate to the olefin (−)-limonene, hydroxylation to (−)-trans-carveol and subsequent dehydrogenation. Selective extraction of the contents of the glandular trichomes indicated that essentially all of the cyclase and hydroxylase activities resided in these structures, whereas only about 30% of the carveol dehydrogenase was located here with the remainder located in the rest of the leaf. This distribution of carveol dehydrogenase activity was confirmed by histochemical methods. Electrophoretic analysis of the partially purified carveol dehydrogenase from extracts of both the glands and the leaves following gland removal indicated the presence of a unique carveol dehydrogenase species in the glandular trichomes, suggesting that the other dehydrogenase found throughout the leaf probably utilizes carveol only as an adventitious substrate. These results demonstrate that carvone biosynthesis takes place exclusively in the glandular trichomes in which this natural product accumulates.     

Multiple Discrete Transitions Underlie Voltage-Dependent Activation in CLC Cl/H Antiporters

Most mammalian chloride channels and transporters in the CLC family display pronounced voltage-dependent gating. Surprisingly, despite the complex nature of the gating process and the large contribution to it by the transport substrates, experimental investigations of the fast gating process usually produce canonical Boltzmann activation curves that correspond to a simple two-state activation. By using nonlinear capacitance measurements of two mutations in the ClC-5 transporter, here we are able to discriminate and visualize discrete transitions along the voltage-dependent activation pathway. The strong and specific dependence of these transitions on internal and external [Cl⁻] suggest that CLC gating involves voltage-dependent conformational changes as well as coordinated movement of transported substrates.     

Microbial Oxidation of Gaseous Hydrocarbons: Production of Methylketones from Corresponding n-Alkanes by Methane-Utilizing Bacteria

Cell suspensions of methane-utilizing bacteria grown on methane oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding methylketones (acetone, 2-butanone, 2-pentanone, 2-hexanone). The product methylketones accumulated extracellularly. The rate of production of methylketones varied with the organism used for oxidation; however, the average rate of acetone, 2-butanone, 2-pentanone, and 2-hexanone production was 1.2, 1.0, 0.15, and 0.025 μmol/h per 5.0 mg of protein in cell suspensions. Primary alcohols and aldehydes were also detected in low amounts as products of n-alkane (propane and butane) oxidation, but were rapidly metabolized further by cell suspensions. The optimal conditions for in vivo methylketone formation from n-alkanes were compared in Methylococcus capsulatus (Texas strain), Methylosinus sp. (CRL-15), and Methylobacterium sp. (CRL-26). The rate of acetone and 2-butanone production was linear for the first 60 min of incubation and directly increased with cell concentration up to 10 mg of protein per ml for all three cultures tested. The optimal temperatures for the production of acetone and 2-butanone were 35°C for Methylosinus trichosporium sp. (CRL-15) and Methylobacterium sp. (CRL-26) and 40°C for Methylcoccus capsulatus (Texas). Metal-chelating agents inhibited the production of methylketones, suggesting the involvement of a metal-containing enzymatic system in the oxidation of n-alkanes to the corresponding methylketones. The soluble crude extracts derived from methane-utilizing bacteria contained an oxidized nicotinamide adenine dinucleotide-dependent dehydrogenase which catalyzed the oxidation of secondary alcohols.     

Morphological and Biochemical Correlates in the Characterization of Sarcocystis spp.1

ABSTRACT. Isoenzyme electrophoretic techniques were applied to the characterization of seven Sarcocystis spp. that had been identified by conventional morphological studies. Cystozoites were harvested from macroscopic cysts from sheep, cattle, and mice and from microscopic cysts from sheep, cattle, and goats. Soluble cystozoite extracts were subjected to cellulose acetate gel electrophoresis and characterized at 15 of the 39 enzyme loci examined. Genetic relationships among isolates were examined by simple phenetic clustering. Two different morphological types of macroscopic cysts from sheep, identified as S. gigantea (syn. S. ovifelis) and S. medusiformis, consistently differed at 40% of the loci examined. Such genetic divergence confirms their separate morphotypic classification. Both differed from microscopic cyst isolates from sheep at 87% of the loci examined; however, two different morphotypes of microscopic cysts were found in the sheep sampled (thick-walled and thin-walled cysts). Until sufficient numbers of each type can be isolated and examined separately, both were regarded as belonging to the species S. tenella (syn. S. ovicanis). Macroscopic and microscopic cysts from cattle consistently differed at 80% of the loci thereby supporting their separate classification as S. hirsuta (syn. S. bovifelis) and S. cruzi (syn. S. bovicanis), respectively. Isolates from goats (microscopic cysts identified as S. capracanis) differed from S. tenella and S. cruzi at 20% and 47% of the loci, respectively. All macroscopic cyst isolates from the various host animal species (including S. muris from mice) differed from each other at nearly all loci. Isoenzyme electrophoretic techniques therefore provided genetic evidence supporting the classification of these various Sarcocystis spp. by their morphological characteristics.     

Tomato Transformation and Transgenic Plant Production

Tomato transformation and regeneration were analysed and optimized. Cotyledon explants from Lycopersicon esculentum cv. UC82B, were infected by Agrobacterium tumefaciens strain LBA4404 harbouring the neomycin phosphotransferase (NPTII) reporter gene. The effects of phenolic compounds, vitamins and growth regulators on plant transformation and regeneration were studied. Increasing the vitamin thiamine concentration from 0.1 mg l^–1 in standard medium to 0.4 mg l^–1 decreased the chlorophyll lost that accompanied the expansion of necrotic areas in cotyledon explants. Optimal shoot regeneration rate was obtained with a balanced concentration of 0.5 mg l^–1 auxin indolelacetic acid (IAA) and 0.5 mg l^–1 cytokinin zeatin riboside. Finally, when the phenolic acetosyringone was present in the co-culture medium at 200 µM, confirmed transgenic lines reached 50% of antibiotic resistant shoots. Under the above conditions, the transformation efficiency reached 12.5%.     

A Biochemical and Morphological Study of Rat Liver Microsomes

Microsomes isolated by differential centrifugation from a rat liver homogenate in 0.88 M sucrose solution have been studied from the biochemical and morphological point of view. 1. Under these experimental conditions, the "total microsome" fraction was obtained by centrifuging the cytoplasmic extract free of nuclei and mitochondria, for 3 hours at 145,000 g. Morphologically, the total microsomes consist mainly of "rough-surfaced membranes" and "smooth" ones. 2. The total microsomes have been divided into 2 subfractions so that the 1st microsomal fraction contains the "rough" vesicles (2 hours centrifugation at 40,000 g) while the 2nd microsomal fraction consists essentially of smooth vesicles, free particles, and ferritin (centrifugation of the supernatant at 145,000 g for 3 hours). 3. By the action of 0.4 per cent sodium deoxycholate in 0.88 M sucrose, it was possible to obtain a pellet for each of the 2 fractions which consisted of dense particles, rich in RNA, poor in lipids, and which represented about 50 to 60 percent of the RNA and 10 to 15 per cent of the proteins. The results have been discussed taking into consideration the hypothesis of the presence of RNA in the membranes of microsomal vesicles.     

Morphological Responses and Molecular Modifications in Tomato Plants after Mechanical Stimulation

Study of the growth responses of Lycopersicon escu-lentum (Mill.cv. VFN8) to mechanical stimulation applied to a single younginternode showed a rapid and sharp decrease in stem elongationand an inhibition of elongation of several internodes, indicativeof information transmission in the plant. A new tomato cDNApartial clone encoding calmodulin was isolated and used to studythe time course of the gene induction in response to the rubbingtreatment. Northern blot analysis showed a maximum accumulationof calmodulin mRNA 2 h after mechanical stimulation, not onlyin the rubbed internode, but also in upper and lower internodesand in young leaves. Treatment of the plant with calcium andEGTA showed the involvement of calcium and, in particular, intracellularcalcium in calmodulin gene expression and cellular response. (Received February 10, 1997; Accepted July 11, 1997)     

西藏核桃种质的形态特征和生化成分分析

研究了西藏不同地区核桃的形态特征,分析比较了西藏不同地区核桃的生化成分.结果表明:三径均值最大为4.04cm,最小为3.11cm.壳厚测定表明集中分布在3.1～3.9mm之间.蛋白质含量最高的是麻达嘎,为21.85%,其变化范围为15.1%～21.85%.脂肪含量最高的是八宿2号.为69.6%,其变化范围为61.7%～69.6%.据此筛选出8个优系.     

Shoot Inversion-induced Ethylene Production in the Diageotropica Tomato Mutant

Shoot inversion was found to inhibit shoot elongation over 24h in the diageotropica (dgt) mutant tomato and its isogenicparent, VNF8, by 55% and 51%, respectively. Previous studieswith normal tomato and other species would suggest that gravitystress-induced ethylene production in the inverted shoot retardselongation. Since exposure of the diageotropic shoot of thedgt mutant tomato to ethylene restores normal upward growth,it might appear that dgt is defective in its capacity to produceethylene. That shoot inversion did stimulate some ethylene productionand that the ethylene action inhibitor, AgNO₃, largely negatedthe inhibiting effect of shoot inversion on elongation in dgtstrongly suggest that the ethylene produced in the invertedshoot was responsible for its retardation of growth. Althoughethylene production in the slower growing dgt was much lessthan that in the faster-growing VNF8, the dgt shoot was foundto be much more sensitive to ethylene. Shoot inversion, ethylene, shoot elongation, diageotropica (dgt), auxin     

Stress-induced Ethylene Production in the Ethylene-requiring Tomato Mutant Diageotropica

Ethylene synthesis in vegetative tissues is thought to be controlled by indoleacetic acid (IAA). However, ethylene synthesis in the diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) was much less sensitive to IAA than in the normal variety (VFN8). Yet, mechanical wounding stimulated ethylene production by the mutant. The dgt tomato provides an opportunity to study the regulation of stress ethylene independent of IAA effects. Waterlogging (i.e. anaerobic stress) stimulated production of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), in the roots. The ACC was transported to the shoot where it was converted to ethylene. The dgt mutant efficiently utilized ACC for ethylene synthesis under aerobic conditions. The results confirm that the genetic lesion in dgt is located at a step prior to the formation of ACC. Furthermore, induction of ethylene synthesis by anaerobic or mechanical stresses in this mutant is independent of IAA action.     

Variation and Evolution of Leaf Trichomes in the Chinese Hamamelidaceae

Leaves of 25 species which cover 13 genera of the Chinese Hamamelidaceae (sensu lato ) were examined by light microscope (LM) and scanning electron microscope (SEM) to reveal the nature and variation of trichomes. The trichomes showed greater diversity under SEM than under LM and naked eyes. Based on Theobald’s scheme, they can be divided into four types: 1. Simple trichomes: Unbranched, curved or straight (Altingia, liquidambar, Semiliquidambar; 2. 2- 4 armed trichomes: Two to four branched (Corylopsis, Loropetalum, sinowilsonia); 3. Stellate trichomes: With more than five branches. They may be tufted (Forthunearia, Loropetalum, sinowilsonia, Hamamelis, Distylium) or storied (Eustigma); 4. Scales: Peltate and flattened. In the genus Rhodoleia, the scales are totally composed of small boat-shaped ones, whereas in the genus Sycopsis they are rotately branched on the verge but unbranched and flattened in the middle. The distribution of trichome types is of grest significance in hamamelidaceous phylogeny. The simple trichomes mainly exist in Liquidambaroideae and Exbucklandioideae, the stellate and 2-4 armed trichomes occur in Hamamelidoideae, and the scales are confined to Sycopsis (Hamamelidoideae) and Rhodoleia (Rhodoleioideae). From this fact, the authors consider that there are two evolutionary lines in the family, i. e. the Disanthoideae- Exbucklandioideae- Liquidambaroideae line and the Rhodoleioideae-Hamamelidoideae line though some subfamilies are isolated. Within Hamamelidoideae, the trichome types also have taxonomic significance. Corylopsis is a primitive group, whereas Eustigma, Distylium and Sycopsis are respectively on the top of evolutionary branches and the other genera are transitional groups. These genera might be separated as tribes, Corylopsideae, Eustigmateae, Distylieae, Sycopsideaeand Hamamelideae. The relationships among these tribes are given in Fig. l.     

Ethylene Production and Respiratory Behavior of the rin Tomato Mutant

Little or no change in ethylene or CO₂ production occurred in rin tomato mutant fruits monitored for up to 120 days after harvest. Of the abnormally ripening tomatoes investigated, including “Never ripe” (Nr Y a h, Nr c l₂ r), “Evergreen” (gf r) and “Green Flesh” (gf), only rin did not show a typical climacteric and ethylene rise.     

Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.