Assay and Biochemical Properties of the Proteinase Inhibitor-inducing Factor, a Wound Hormone

An assay has been developed for the proteinase inhibitor-inducing factor (PIIF), a wound hormone. PIIF is present in tomato (Lycopersicum esculentum var. Bonnie Best) leaf extracts and induces accumulation of proteinase Inhibitor I when the extracts are supplied briefly to excised leaves that are subsequently incubated in water under constant light. An active water-soluble crude PIIF solution was conveniently prepared from autoclaved and lyophilized tomato leaves. Accumulation of Inhibitor I, induced by crude PIIF, is linear, commencing at about 8 to 10 hours after feeding and continues for several hours. Evidence is presented that the PIIF-induced accumulation of Inhibitor I, determined immunologically, is accompanied by the accumulation of other trypsin and chymotrypsin inhibitors, determined enzymatically. The accumulation of Inhibitor I is inhibited by actinomycin D and cycloheximide but not by chloramphenicol or rifampin. PIIF cannot be replaced by traumatin, indoleacetic acid, gibberellic acid, kinetin, ethylene, or abscisic acid. PIIF activity was not destroyed by incubation with a number of proteolytic, carbohydrase, phosphatase, or pyrophosphatase enzymes. The active substance is insoluble in lipid solvents.     

Surface Printing of Plant Leaves for Phylogenetic Studies

A solution of plastic consisting of toluene, 720 ml; methanol, 180 ml; ethyl cellulose (Ethocel, standard 7 CPS), 250 gm; and Dow resin 276 V-2, 75 gm is applied to a leaf surface which has been dampened with toluene. The plastic is spread to a thin film with the edge of a card and allowed to dry. After drying, the plastic may be peeled from the leaf surface and either stored dry in a small envelope or mounted permanently on a microscope slide. Permanent mounts are prepared by placing a small section of the peel from the upper and lower surfaces of the leaf on a microscope slide and covering with a No. 1 cover glass. A small spot of balsam on each corner of the cover glass secures the glass in position. This air mount has proved to be superior to water, glycerol or balsam mounts. Fresh leaves are washed with a mild detergent before application of the plastic. Herbarium specimens are soaked in water overnight to restore the leaf to a semiturgid condition. Five species of different plant families have been illustrated to show the diagnostic features of the surface of the cuticle. An isolated layer of epidermal cells obtained by chemical maceration permitted cell and imprint comparison. The remarkable amount of detail shown by the prints is an aid for phylogenetic studies and may make the recognition of fossil cuticles possible.     

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.     

Wound-induced Accumulation of Trypsin Inhibitor Activities in Plant Leaves: Survey of Several Plant Genera

Asexual embryogenesis in Daucus carota L. `Queen Anne's Lace' callus was suppressed by Ethephon, ethylene, and 2,4-dichlorophenoxyacetic acid (2,4-D). The Ethephon effect could be attributed to volatile and nonvolatile substances. The volatile component was probably entirely ethylene. Ethylene was liberated in the cultures in direct proportion to Ethephon added to the medium. Autoclaving of Ethephon caused a substantial decrease of measurable ethylene. Continuous exposure of callus to 5 μl/l ethylene depressed somatic cell embryogenesis, but not markedly. Depression of embryogenesis by 2,4-D was unrelated to ethylene evolution.     

Plant Photoreceptors: Phylogenetic Overview

Plants possess photoreceptors to perceive light which controls most aspects of their lives. Three photoreceptor families are well characterized: cryptochromes (crys), phototropins (phots), and phytochromes (phys). Two putative families have been identified more recently: Zeitlupes (ZTLs) and UV-B photoreceptors (ULI). Using Arabidopsis thaliana and Oryza sativa photoreceptor sequences as references, we have searched for photoreceptor encoding genes in the major phyla of plant kingdom. For each photoreceptor family, using a phylogenetic tree based on the alignment of conserved amino acid sequences, we have tried to trace back the evolution and the emergence of the diverse photoreceptor ancestral sequences. The green alga Chlamydomonas contains one cry and one phot sequence, probably close to the corresponding ancestral sequences, and no phy-related sequence. The putative UV-B photoreceptors seem to be restricted to the Brassicacae. Except for mosses and ferns, which contain divergent photoreceptor numbers, the composition of the diverse photoreceptor families is conserved between species. A high conservation of the residues within domains is observed in each photoreceptor family. The complete phylogenic analysis of the photoreceptor families in plants has confirmed the existence of crucial evolutionary nodes between the major phyla. For each photoreceptor class, a major duplication occurred before the separation between Mono- and Eudicotyledons. This allowed postulating on the putative ancestral function of the photoreceptors. [Reviewing Editor: Dr. Rudiger Cerff]     

植物天冬氨酸蛋白酶的研究进展

天冬氨酸蛋白酶是四大类蛋白水解酶之一,广泛存在于多种生物中,参与了很多重要的生理过程。相对于其他生物而言,对植物天冬氨酸蛋白酶的研究相对滞后。自20世纪末以来,这一领域的工作取得了重大进展。经典的植物天冬氨酸蛋白酶除了具有保守的蛋白酶结构域外还有一段植物特有的插入片段;在动植物分离之后,植物天冬氨酸蛋白酶基因家族进行了大规模的扩增;该类蛋白酶在植物的整个生长发育过程中发挥重要的功能,尤其是胁迫反应、有性生殖、衰老和程序性死亡以及蛋白质的加工和降解。对近些年植物天冬氨酸蛋白酶的研究进展进行综述,并对其未来的发展进行展望。     

Purification and Characterization of Thiol Proteinase as a Nitrate Reductase-Inactivating Factor from Leaves of Hordeum distichum L.

A thiol proteinase was purified 6,400-fold from leaves of Hordeumdistichum L. by a sequence of ammonium sulfate fractionation,gel filtration, ion exchange chromatography, hydrophobic chromatographyand chromatofocusing. This enzyme also had nitrate reductase(NR)-inactivating activity, which was associated with proteolyticactivity in almost constant proportions during the purificationprocedures. Its molecular weight was estimated as 74,000 bygel filtration, and it had an isoelectric point of 4.05 andan apparent K_m of 0.83 mg ml^–1 for azocasein. The respectiveoptimum pH for proteolytic and NR-inactivating activities were6.0 and 7.0. On electrophoresis, the proteinase gave a majorband that coincided with both activities; it also produced afaint band associated with no activity. Our purified thiol proteinase inactivated FMNH₂-NR and MVH-NRas well as NADH-NR, but it had only a slight effect on NADHcytochrome c reductase activity. This enzyme also inactivatedglutamine synthetase. (Received September 16, 1983; Accepted January 26, 1984)     

系统发育多样性与系统发育结构在岛屿植物群落保护中的意义——以蜈支洲岛为例

为探讨系统发育多样性和系统发育结构在岛屿植物群落保护决策制定中的作用,以海南三亚蜈支洲岛被子植物群系为例,分析物种丰富度和系统发育多样性的相关性。结果表明,蜈支洲岛植被可分为10种群系类型,22个样方的物种丰富度与系统发育多样性呈显著正相关,但系统发育多样性与物种丰富度对植物多样性保护优先顺序的指示有差异。森林群系的物种丰富度和系统发育多样性普遍高于灌木和草本群系,4个森林群系样方表现为系统发育结构发散,3个森林群系样方表现为系统发育结构聚集。灌木群系中露兜树(Pandanus tectorius)灌丛和草海桐(Scaevola sericea)灌丛系统发育多样性较高,草本群系中厚藤(Vitex trifolia)灌草丛系统发育多样性较高,灌木和草本群系大多倾向于系统发育结构聚集。距海或人类活动区较近的群系可能受环境过滤的作用,而远离海和人类活动区的群系主要受竞争排斥作用影响。因此,在岛屿植物多样性保护策略制定中应当综合考虑物种和系统发育维度,以及环境、演替阶段及用岛方式等因素的影响。     

Cell Walls of Phaseolus vulgaris Leaves Contain the Azocoll-Digesting Proteinase

The leaves of Phaseolus vulgaris L. cv Greensleeves contain an endopeptidase with a pH optimum of 9.0 and an isoelectric point between 10.0 and 10.5. This endopeptidase is the only abundant Azocoll-digesting proteinase in the leaves. The activity of this enzyme is highest in immature leaves and declines as the leaf matures and senesces. Enzymically isolated protoplasts contain very little of this proteinase. The proteinase can be recovered readily from the extracellular fluid obtained by gentle centrifugation of leaf strips vacuum-infiltrated with a buffered solution. These experiments indicate that the Azocoll digesting proteinase is located in the periplasmic space and/or the cell wall.     

A Survey of Methods for Constructing Rooted Phylogenetic Networks

Rooted phylogenetic networks are primarily used to represent conflicting evolutionary information and describe the reticulate evolutionary events in phylogeny. So far a lot of methods have been presented for constructing rooted phylogenetic networks, of which the methods based on the decomposition property of networks and by means of the incompatible graph (such as the CASS, the LNETWORK and the BIMLR) are more efficient than other available methods. The paper will discuss and compare these methods by both the practical and artificial datasets, in the aspect of the running time of the methods and the effective of constructed phylogenetic networks. The results show that the LNETWORK can construct much simper networks than the others.     

Inverse Correlation of Thiol Proteinase with Nitrate Reductase Activities in Barley Leaves

Experiments conducted to determine the effects of leupeptin,a specific inhibitor of thiol proteinase, on extractable nitratereductase (NR) activity in leaves of Hordeum distichum duringdarkness revealed that leupeptin (0.01 mg.ml^–1) appliedto detached leaves significantly reduced the loss of NR activity.At the same time it also reduced the formation of small cytochromec reductase species, which is a degradation product of NR complex,Upon nitrate induction, extractable NR activity increased butthe content of thiol proteinase decreased. This inverse correlationwas also observed upon transfer of nitrate-grown barley seedlingsto nitrate-free nutrient solution. Furthermore, cycloheximide(0.1 mg.ml^–1) treatment of barley seedlings reduced thecontent of thiol proteinase and retarded the loss of NR activityunder noninducing conditions. These results suggest that invivo changes in NR content in leaves of Hordeum distichum arethe result of proteolysis by an endogenous thiol proteinase. (Received May 16, 1985; Accepted July 22, 1985)     

A Parvalbumin-Like Calcium-Binding Protein Is Present in Plant Leaves

A protein with relatively high homology in its N-terminal aminoacid sequence to animal parvalbumin and oncomodulin has beenidentified in leaves of rice (Oryza sativa L.). The PV-likeprotein has a relative molecular mass of 27,000 and an isoelectricpoint of 5.0. This protein was partially purified by ion-exchangechromatography, and the purified protein was found to have Ca²⁺-bindingactivity in a microscale Ca²⁺-binding assay. Furthermore, anantiserum raised against a synthetic oligopeptide based on theN-terminal amino acid sequence of this protein cross-reactedwith protein not only from rice but also from other monocotyledonousplants. (Received October 17, 1990; Accepted October 17, 1991)     

Proteinase inhibitors in plant biotechnology: A review

Possible utilities for natural inhibitors of proteolytic enzymes in plant biotechnology have been reviewed. Among the potential areas of use of the inhibitors are (1) construction of transgenic plants with increased resistance to insects and other pests and (2) development of procedures for biosynthesis of recombinant proteins. In the latter case, the inhibitors will serve to prevent the protein degradation by proteinases.     

Active Iron in Plant Leaves

Tomato plants were grown with limited or luxury water supplyand given nitrogen either as the ammonium ion or the nitrateion. The ‘active’ iron fractions of the leaves asextracted by etherized tenth molar hydrochloric acid showedno relationship with the total iron but a very significant linearrelationship was found between the active iron fraction andthe ratio of total phosphorus to total iron. Recalculation of results presented by other investigators formaize plants grown in soil with various levels of moisture,phosphorus and iron also showed a highly significant linearrelationship between the active iron fraction and the ratioof total phosphorus to total iron. Lycopersicon esculentum L., tomato, ‘active iron’, phosphorus, Zea mays     

半胱氨酸蛋白酶在植物细胞程序性死亡中的作用

介绍了参与执行与控制植物细胞程序性死亡（PCD）的半胱氨酸蛋白酶的研究进展。