Localization of proteasomes in plant cells

Summary Proteasomes, also known as multicatalytic proteinase complexes, were localized in suspension cells of potato (Solanum tuberosum) by direct immunofluorescence using polyclonal antibodies labelled with fluorescein isothiocyanate. The method used allows an estimate of relative amounts of proteasomal antigens in different cell components. Proteasomes are present in the nuclei and the cytoplasm. The nucleoplasm contains small areas of weak fluorescence. The peripheral cytoplasm and possibly elements of the cytoskeleton show higher fluorescence than other parts of the cytoplasm. This indicates a localization of proteasomes similar to that known from animal cells.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetra acetic acid - FITC fluorescein isothiocyanate - PBS phosphate buffered saline - PIPES piperazine-1,4-bis-(2-ethanesulfonic acid)     

Rye chromosome arm 3RS encodes a homodimeric inhibitor of insect α-amylase

A new inhibitor of insect -amylase, designated RDAI-1, has been purified from rye (Secale cereale L.) endosperm. RDAI-1 is homologous to wheat homodimeric inhibitors. This homology is supported by their similar N-terminal amino-acid sequences, inhibitory activities towards amylases from Tenebrio molitor (Coleoptera) and human saliva, and aggregative properties in gel-filtration chromatography. The gene encoding RDAI-1, IdhaR1, is located on the short arm of chromosome 3R, which is homoeologous with wheat chromosome arms 3BS and 3DS, where the genes for homodimeric inhibitors have been previously mapped.     

Cysteine proteinase forms in sprouting potato tuber

Transformation of plants with exogenous proteinase inhibitor genes represents an attractive strategy for the biological control of insect pests. However, such a strategy necessitates a thorough characterization of endogenous proteinases. which represent potential target enzymes for the exogenous inhibitors produced. In the present study. changes in general endoproteolytic activity were monitored during sprouting of potato ( Solanum tuberosum L. cv. Kennebec) tuber. Quantitative data obtained using standard procedures showed that an increase in cysteine proteinase (EC 3.4.22) activity occurs during sprouting. This increased activity results from the gradual appearance of new cysteine proteinase forms, as demonstrated by the use of class-specific proteinase activity gels. While only one cysteine proteinase form was present during early sprouting, at least six new active forms of the same class were shown to appear gradually after the mature tuber was sown, suggesting the involvement of a complex cysteine proteolytic system in the last stages of tuber protein breakdown. Interestingly, oryzacystatins I and II. two cysteine proleinase inhibitors potentially useful for insect control, had no effect on any tuber proteinase delected. Similar results were obtained with leaf, stem and stolon proteinases. This apparent absence of direct interference supports the potential of oryzacystatin genes for production of insect-tolerant transgenie potato plants.     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

Effects of low- and high-dose tranylcypromine on [3H]tryptamine binding sites in the rat hippocampus and striatum

Chronic studies were initiated in rats to determine the effects of high- and low-dose tranylcypromine (TCP) on [³H]tryptamine (³H-T) binding sites. Male Sprague-Dawley rats were administered TCP (0.5 or 2.5 mg/kg/day) or vehicle (distilled water) for 4, 10 or 28 days via Alzet minipumps. After decapitation, the hippocampus and striatum were used to prepare membrane fragments for single point³H-T binding. Hippocampal³H-T binding was reduced after 10 and 28 days with the low dose and after 4, 10 and 28 days with the high dose. Striatal³H-T binding was reduced by both doses at all time intervals. The high dose resulted in a significantly greater reduction in striatal³H-T binding than did the low-dose after 4, 10, and 28 days. These results suggest that a more rapid reduction of³H-T binding in the hippocampus and/or a greater reduction of³H-T binding in the striatum by high-dose TCP than by low-dose TCP may be contributing factors in the reported efficacy of the former in refractory depression.     

Midgut proteinase activities of three keratinolytic larvae,Hofmannophila pseudospretella,Tineola bisselliella,and Anthrenocerus australis,and the effect of proteinase inhibitors on proteolysis

The midgut proteinase activities were characterized from the keratinolytic larvae of two lepidopterans, Hofmannophila pseudospretella (Stainton) (Oecophoridae) and Tineola bisselliella (Hummel) (Tineidae), and one coleopteran, Anthrenocerus australis (Hope) (Dermestidae). The major endopeptidase activities, characterized using specific enzyme inhibitors, were serine proteinases with hydrolytic activity against N-benzoyl-DL-arginine-p-nitroanilide and against N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-leucine-p-nitroanilide. No significant levels of metalloendopeptidase or cysteine endopeptidase activities were detected. Aminopeptidase activity was present in all larvae. The enzyme levels and properties of the two moth larvae were similar to each other and to those of phytophagous lepidopteran larvae but different from those of the beetle larva. Whereas only a limited number of serine proteinase inhibitors inhibited the midgut proteolysis of the lepidopteran larvae, most inhibitors inhibited the midgut proteolysis of the beetle larva. © 1994 Wiley-Liss, Inc.     

Chromium-induced inhibition of ethylene evolution in bean (Phaseolus vulgaris) leaves

The influence of chromium concentration on ethylene production in bean plants ( Phaseolus vulgaris L. cv. Contender) was investigated. A Cr ion-induced inhibition of ethylene synthesis from endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) was observed within both leaf discs floated on 2 m M CrO²⁻₄ or Cr³⁺ and leaf discs from plants cultured in nutrient solutions containing 10, 20 or 40 μ M CrO²⁻₄. However, Cr ions supplied either to plants with the nutrient solution or to discs with the incubation medium rather increased the conversion of exogenous ACC to ethylene. Primary leaves of plants exposed to CrO²⁻₄-containing nutrient solutions showed a statistically insignificant decrease of ACC-synthase activity. In the trifoliolate leaves of plants exposed to 10 μ M CrO²⁻₄, in which a significant decrease of ethylene production from endogenous ACC was observed, a substantial increase of ACC synthase was found. These results indicate that Cr ion-induced inhibition of ethylene production is not due to a breakdown of membrane integrity, which is necessary for ethylene forming enzyme activity, but caused by metabolic alterations leading to decreased ACC availability. Chromium ions may act by inhibiting ACC synthase activity or by diverting a metabolic step prior to the ACC synthase catalyzed reaction.     

WIP1, a wound-inducible gene from maize with homology to Bowman-Birk proteinase inhibitors

We have cloned and sequenced a wound-inducible cDNA clone designated WIP1 (for wound-induced protein) from maize coleoptiles. It was isolated by differential screening of a cDNA library prepared from excised maize coleoptile segments. The deduced amino acid sequence predicts a secretory, cysteine-rich protein of 102 residues with a calculated molecular mass of 11 kDa and a typical N-terminal signal sequence. The protein has about 30% identity with various Bowman-Birk type proteinase inhibitors. Most interestingly, it is novel in that it is double-headed with exclusive specificity for chymotrypsin. WIP1 is strongly wound-induced in contrast to other members of the Bowman-Birk proteinase inhibitor family, which occur in seeds and are regulated during development. The response is fast, similar to defenceinduced genes, and measurable as early as 30 min after wounding. Induction can also be evoked in the intact coleoptiles and the signal is systemically transmitted in the coleoptile to adjacent regions of the wounded area. Isolation and analysis of the corresponding genomic clone reveals that WIP1 contains an intron of 90 nucleotides.     

5′-Isothiocyanato-N-1-naphthylphthalamic acid: a chemically reactive site-directed irreversible ligand for phytotropin receptors

A chemically reactive analog of the phytotropin N-1-naphthylphthalamic acid (NPA) was synthesized and evaluated as a site-directed irreversible ligand for the NPA receptor. The NPA analog (5-isothiocyanato-N-1-naphthylphthalamic acid; NCS-NPA) was synthesized in two steps. Pretreatment of etiolated Helianthus hypocotyl segments with NCS-NPA at concentrations in excess of 1 M resulted in a dose-dependent inhibition of basipetal [¹⁴C]IAA transport. Net uptake of IAA by hypocotyl segments was stimulated by NCS-NPA at concentrations of 1 M or greater. NCS-NPA inhibited the saturable binding of [³H]NPA in Helianthus microsomes in a dose-dependent fashion with 50% inhibition occurring at NCS-NPA concentrations of 3 to 10 nM. The binding affinity of [³H]NPA in microsomes pretreated with NCS-NPA followed by extensive washing was substantially reduced. These results demonstrate that NCS-NPA is a site-directed irreversible ligand for the NPA receptor and suggest that it may be of use in the purification and characterization of this biologically important receptor.Abbreviations ANPA 5-amino-naphthylphthalamic acid - IAA indole-3-acetic acid - NCS-NPA 5-isothiocyanato-N-1-naphthylphthalamic acid - NPA N-1-naphthylphthalamic acid - TLC thin-layer chromatography