Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells

We have shown that highly purified vacuoles of suspension-cultured tomato (Lycopersicon esculentum) cells contain RNA-oligonucleotides, using two different approaches to label and detect RNA: (a) in vivo labeling of cellular RNA with [5-³H]uridine, followed by preparation of vacuoles from protoplasts and by quantification of radioactively labeled material; and (b) in vitro labeling and analysis on sequencing gels of nucleic acids prepared from tomato vacuoles and their identification as RNA. The intravacuolar location of the RNA found in vacuolar preparations was concluded from analyzing for RNA intact organelles after repeated flotation steps as well as ribonuclease A treatment. About 3% of the RNA in protoplasts was localized within vacuoles, exceeding by severalfold the contribution made by contamination with unlysed protoplasts and subcellular organelles. Investigation of the size distribution of vacuolar RNA revealed an oligonucleotide pattern strikingly different from that which would arise from contaminating protoplasts; vacuolar RNA fragments are considerably shorter than 80 nucleotides. Characterization of these oligoribonucleotides (3′-phosphorylated termini; relatively rich in pyrimidines) as possible products of tomato vacuolar ribonuclease I action, and, in addition, enzymatic hydrolysis of vacuolar RNA by inherent enzyme activities in lysed vacuole preparations support the hypothesis that plant vacuoles are involved in cellular nucleolytic processes.     

Tissue and Cellular Localization of Proteinase Inhibitors I and II in the Fruit of the Wild Tomato, Lycopersicon peruvianum (L.) Mill

The cellular and subcellular localization of proteinase inhibitor I and inhibitor II proteins in the fruit of the wild tomato species Lycopersicon peruvianum (L.) Mill., LA 107 was determined by immunoanalysis of tissue blots and protein-A gold immunocytochemistry. Tissue blot analysis showed that the proteinase inhibitor I proteins were located throughout the fruit tissue, with the exception of the seeds. Light microscopy, using immunocytochemical labeling, indicated that all the parenchyma cells of the pericarp contained inhibitor I and II proteins in dense vacuolar protein aggregates that were not membrane bound. The size, number, and morphology of the aggregates within individual cells varied greatly. The funiculus, ovule, and early embryonic tissues were devoid of inhibitor I and II. Immunocytochemical analysis using transmission electron microscopy confirmed that the proteinase inhibitor I proteins were principally located and stored in protein aggregates within the vacuole of the fruit parenchyma cells. Some cytoplasmic protein-A gold immunolabeling of inhibitor I proteins was evident, which may be related to the synthesis and intermediate transport steps preceding storage of the inhibitor I proteins in the vacuoles.     

Studies on Isolated Protoplasts and Vacuoles: I. GENERAL PROPERTIES

Protoplasts and vacuoles have been isolated in large quantitiesfrom the parenchymatous placental tissue of immature tomatofruit. Their mechanical stability and osmotic stability havebeen investigated. A small proportion of initially discretebut adjacent vacuoles were seen to coalesce; the possible significanceof this phenomenon in the growth of plant cells is discussed.The overall chemical composition of isolated protoplasts hasbeen determined, and their lipids have been analysed by thin-layerchromatography.     

Proteinase Inhibitors in Plant Seed

Three different types of proteinase inhibitors, I, II and III, were fractionated from Japanese radish seed by repeated column chromatographies on SE- and CM-cellulose. The finally purified preparation of inhibitor III was found to be homogeneous by both chromatographic and electrophoretic analyses. All three of them strongly and stoichiometrically inhibited trypsin whereas they showed weak inhibition on other proteinases, such as chymotrypsin, Nagarse and Pronase. From nitrogen content and ultraviolet absorption spectra, each of the inhibitors I and III was confirmed to be a protein. The molecular weights of inhibitors I and III were calculated to be 8000 and 12,000, respectively. These inhibitors were stable at temperatures above 90°C in an acidic pH.     

Proteinase Inhibitors in Plant Seeds

The proteinase inhibitors I (R-I) and III (R-III) isolated from Japanese radish seed were characterized in terms of their N-terminal amino acids, amino acid composition and reacting groups. The amino acid composition of two proteins differed from each other, while histidine, methionine and tryptophan contents were all low. N-Terminal amino acids of these inhibitors determined by Edman degradation were the same; valine.

By modifying free amino groups in the inhibitors with trinitrobenzenesulfonic acid, R-III was greatly inactivated in proportion to the modification of amino groups, but the activity of R-I was not affected.

However, modification of arginyl residues of R-I by cyclohexanedione reduced its activity. These results indicate that R-I is an arginine-type and R-III is a lysine-type inhibitor.     

Functional Specialization of Vacuoles in Sugarcane Leaf and Stem

Plant vacuoles are frequently targeted as a storage site for novel products. We have used environment-sensitive fluorescent dyes and the expression of vacuolar marker proteins to characterize the vacuoles in different organs and cell types of sugarcane. The results demonstrated that the lumen of the vacuole in the parenchyma cells of the stem is acidic (<pH 5) and contains active proteases, characteristic of lytic vacuoles. Western blots and tissue labelling with antibodies to vacuolar H⁺-ATPase suggest that this proton pump is involved in acidification of the vacuolar lumen. Quantitative real-time PCR was used to show that the expression of vacuolar proteases and a vacuolar sorting receptor is also coordinately regulated. In contrast to the stem parenchyma cells, the cells of sugarcane leaves contain diverse types of vacuoles. The pH of these vacuoles and their capacity to hydrolyze protease substrates varies according to cell type and developmental stage. Sugarcane suspension-cultures contain cells with vacuoles that resemble those of stem parenchyma cells and are thus a useful model system for investigating the properties of the vacuole. Understanding the growth and development of storage capacity will be useful in designing strategies to maximize the production of sucrose or alternative bioproducts.     

Proteinase Inhibitors in Germinating Barley Embryos

The activities of trypsin inhibitors and Aspergillus-proteinase inhibitors have been assayed in barley embryos germinating in different conditions. The Aspergillus-proteinase inhibitor activity increased about 20-fold during 6 days at 20°C both in separated embryos germinating on nutrient agar and in the embryos of undissected grains germinating on agar with or without mineral salts. Significant inhibitor activity was also present in root tips at later stages of vegetative growth. Whether any changes occurred in the molecular weight distribution of the inhibitors present in the seedlings was studied by gel filtration on Sephadex G-75; only minor changes were observed. The trypsin inhibitor activity per seedling remained roughly constant in all cultures. In gel filtration experiments all the activity appeared to be due to the inhibitor species present in resting embryos.     

Studies on Isolated Protoplasts and Vacuoles: II. THE ACTION OF GROWTH SUBSTANCES

The response of isolated protoplasts to indol-3-yl acetic acidwas investigated, and they were found to undergo a rapid water-uptakewith ultimate rupture of the plasmalemma and release of thelarge central vacuole. The use of a photomicrographic methodshowed that this response was optimal at 10^-5 M indol-3-yl aceticacid. No such response could be detected for isolated vacuoles.¹⁴C-labelled indol-3-yl acetic acid was used to obtain furtherinsight into the site of action of this growth substance. Evidenceis presented which suggests that the site of action of indol-3-ylacetic acid, for this response, is the plasmalemma, where itfacilitates an increased uptake of solutes which is followedby an osmotic water uptake.     

Tomato (Lycopersicon esculentum cv. Pik-Red) Leaf Carboxypeptidase: Identification, N-terminal Sequence, Stress-Regulation, and Specific Localization in the Paraveinal Mesophyll Vacuoles

Wounding of tomato (Lycopersicon esculentum L.) leaves causessystemic induction of a serine-type carboxypeptidase activity.We find this activity to be present in several isoforms. Antibodiesraised against the leaf carboxypeptidase inhibited the enzymeactivity and the immunoprecipitates were resolved into a 69-kDapolypeptide and a doublet of 35/37-kDa proteins on SDS-PAGE.Immunoblot analysis of the leaf proteins also immunodecoratedthe 69-kDa and 35/37-kDa proteins. Amino acid sequence analysisof the amino-terminus of the tomato leaf 69-kDa carboxypeptidaseshowed it to be similar to the barley A-chain carboxypeptidaseI [Sorenson et al. (1986) Carlsberg Res. Commun. 51: 475], sharingAla as the N-terminus and the sequences, AlaProGln and LeuProGlyPhe.Superimposition of a chemical stress (copper treatment) on woundingapparently lowered wound-induced carboxypeptidase activity inthe leaf, suggesting that cupric ions might interact with thewound signal. Immunogold electron microscopy indicated thatthe leaf carboxypeptidase was specifically localized withinthe inclusions of vacuoles of vascular parenchyma cells. Incupric ion-treated tissues, carboxypeptidase was found redistributedto other parts of the cell, indicating that this treatment,but not wounding, causes general vacuolar membrane damage. ⁴Deceased.     

慈菇蛋白酶抑制剂研究进展

蛋白酶抑制剂是一类能够抑制蛋白水解酶活性的物质。根据它们抑制的蛋白酶类型可分为丝氨酸、半胱氨酸、天冬氨酸、和金属蛋白酶抑制剂[1] 。由于它们能抑制昆虫肠道内以及一些病原微生物体内的蛋白酶[2～ 6 ] ,因此蛋白酶抑制剂在植物对昆虫和病原体的侵染防御系统中具有重要的作用。慈菇蛋白酶抑制剂Ａ、Ｂ是从慈菇球茎中分离纯化的双头多功能蛋白酶抑制剂 ,除了具备其他蛋白酶抑制剂在抗虫抗病方面的特点外 ,还有很多独特的优点。如 ,含量丰富、比活力高而且稳定 ;广谱性强 ;对胰蛋白酶、胰凝乳蛋白酶、激肽释放酶等多种蛋白酶有较强的抑…     

蛋白酶抑制剂及其在抗虫基因工程中的应用

蛋白酶抑制剂可以抑制昆虫的生长和发育,近年来在抗虫基因工程得广泛的应用。本文综述了蛋白酶抑制剂及其抗虫性,蛋白酶抑制剂转基因植物的研究概况,同时探讨了蛋白酶抑制剂在抗虫基因工程中的利用前景、存在问题和解决途径。     

利用多重PCR反应同时筛选番茄Cf-9和Tm-1基因

利用同一PCR反应体系,对分别与番茄抗叶霉病的Cf-9基因和抗番茄烟草花叶病毒病的Tm-1基因紧密连锁的PCR标记进行了同时扩增筛选,扩增的特异性片段与单引物扩增片段吻合。其中与Cf-9基因紧密连锁的CAPs标记在抗感试材均可扩增出560bp的特异片段,且都存在TaqⅠ酶切位点,抗病基因型酶切后分别产生了450bp、330bp和290bp的不同特异性片段,而感病基因型试材酶切后产生450bp和290bp的特异性片段;与Tm-1基因紧密连锁的SCAR标记为显性标记,只有抗病试材产生750bp的特异片段,不能被TaqⅠ酶切。经反复验证,结果稳定准确,可用于在同一PCR反应体系中对两个抗病基因进行同时筛选鉴定。该体系的建立不仅省时、省工、节省费用,而且可用于苗期辅助选育,加快番茄抗病育种进程。     

Purification and Properties of Neutral Proteinase I from Aspergillus oryzaePurification and Properties of Neutral Proteinase II from Aspergillus oryzae

Neutral proteinase I (the first peak in DEAE-cellulose chromatogrraphy) was purified from the Amberlite IRC-50 adsorbed fraction by chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. It shows an optimum pH of 7.0 for milk casein. The enzyme was found to be stable in the pH range of 5.5 to 12.0. The molecular weight of the enzyme was estimated to be about 41,000 by gel filtration. The enzyme had neither aminopeptidase nor carboxypeptidase activity, but degraded carbobenzoxy-glycyl-phenyl-alanine amide, poly-l-lysine and poly-l,α-glutamic acid. The enzyme was inhibited by ethylenediaminetetraacetate, but not inhibited by diisopropylphosphorofluoridate and potato inhibitor.     

Proteinase Inhibitors as Antistress Proteins in Higher Plants

Physicochemical and functional characteristics of plant protein proteinase inhibitors as antistress biopolymers were studied to determine the mechanisms for plant resistance to phytopathogens and to obtain disease-resistant cereal and leguminous cultures. The activity of trypsin, chymotrypsin, and subtilisin inhibitors varied in monocotyledonous and dicotyledonous cultures. Study varieties of leguminous and cereal cultures were shown to contain endogenous inhibitors specific to proteinases of phytopathogenic fungi Fusarium, Colletotrichum, Helminthosporium, and Botrytis. These inhibitors were characterized by species specificity and variety specificity. Protease inhibitors from buckwheat seeds inhibited proteases of fungal pathogens and suppressed germination of spores and growth of the fungal mycelium. Our results suggest that proteinaceous inhibitors of proteinases are involved in the protective reaction of plants under stress conditions.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 392–396.Original Russian Text Copyright © 2005 by Dunaevskii, Tsybina, Belyakova, Domash, Sharpio, Zabreiko, Belozerskii.     

Kazal型蛋白酶抑制剂结构与功能研究进展

蛋白酶抑制剂广泛存在于生物体内,在许多生命活动过程中发挥必不可少的作用,特别是对蛋白酶活性进行精确调控。其中Kazal型蛋白酶抑制剂是最重要的、研究最为广泛的酶抑制剂之一,该类抑制剂一般由一个或几个结构域组成,每一个结构域具有保守的序列和分子构象,同时发现该类抑制剂与蛋白酶作用的结合部位高度易变,它们大多数暴露于与溶剂接触的环上,其中P1部位是抑制作用的关键部位,抑制剂的专一性由P1部位氨基酸残基的性质决定,其它残基取代结合部位残基对抑制剂-酶的结合常数有显著的影响。Laskowski算法可直接从Kazal型丝氨酸蛋白酶抑制剂的序列推测其与6种丝氨酸蛋白酶之间的抑制常数(Ki)。目前在生物体内发现大量的Kazal型蛋白酶抑制剂,并证实其有重要的生物学功能。     

Proteinase Inhibitor II Gene Expression Induced by Electrical Stimulation and Control of Photosynthetic Activity in Tomato Plants

Mechanical damage and heat stimulation were used to activateproteinase inhibitor II (Pin2) gene expression in tomato plantsin both treated (local induction) and non-treated tissues (systemicinduction). Both stimuli have been shown to generate electricalsignals, leading to a systemic activation of gene expression.Treatment of tomato leaves with electrical current resultedin the accumulation of Pin2 mRNA in the local and systemic leaves.Additionally, all treatments inducing Pin2 gene activity gaverise to a significant alteration of stomatal aperture. However,heat stimulation provoked a different response in the stomatalparameters than mechanical wounding or electric treatment. Bothmechanical damage and electrical stimulation activated two characteristictime constants in the gas exchange relaxation kinetics. Conversely,heat stimulation resulted in only one major time constant. Theresults clearly show that direct current application to tomatoleaves initiates Pin2 mRNA accumulation locally and systemically.In addition, they suggest the participation of a second slowelectrical/hydraulic component in the wound response mechanismof tomato plants and a possible alternative pathway regulatingheat-induced Pin2 gene expression. (Received February 13, 1995; Accepted April 14, 1995)     

Proteinase inhibitors from Vigna unguiculata subsp. cylindrica: II. Inhibitors of subtilisin and trypsin

Two subtilisin inhibitors and two trypsin-chymotrypsin inhibitors were purified from seeds of Vigna unguiculata subsp. cylindrica. A third subtilisin inhibitor was partially purified. The subtilisin isoinhibitors were present in very small amounts in the seeds and the degree of purification of the three inhibitors was 20,000- to 48,000-fold. The purified inhibitors were found to be homogeneous on ultracentrifugation and polyacrylamide gel electrophoresis with or without dodecyl sulfate. The subtilisin inhibitors had no action on papain, ficin, chymopapain, bromelain, trypsin, chymotrypsin, or papain and the trypsin-chymotrypsin inhibitors were also inactive with other enzymes.     

Examination of Isolated Yeast Cell Vacuoles for Active Transport

Isolated vacuoles of the yeast Candida utilis did not show active transport of S-adenosylmethionine, uric acid, and several amino acids which they concentrate in vivo.