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Resting seeds of Scots pine (Pinus sylvestris L.) contain inhibitors which inhibit the proteinase activity present in germinating seeds but have no effect on trypsin or chymotrypsin. When a crude inhibitor preparation was chromatographed on Sephadex G-75, the inhibitor activity separated into four peaks with elution volumes corresponding to the molecular weights 24,000, 14,600, 14,000, and 9000. Each of the inhibitors affected both the hydrolysis of haemoglobin at pH 3.7 and the hydrolysis of casein at pH 5.4 and 7.0 by proteinase extracts prepared from "germinating" endosperms. These results suggest that one major proteinase was possibly acting in all the assays. In resting seeds inhibitor activity was present in both the embryo and the endosperm, the activity (per mg dry weight) in the embryo being about 2-fold that in the endosperm. In the endosperms of germinating seeds the inhibitor activity per seed decreased at about the same rate as total N and dry weight. In the seedlings the activity per seedling remained approximately constant. The patterns of the activity changes suggest that the inhibitors do not control the breakdown of storage proteins; a more probable function is the protection of other cellular components from the high proteinase activities required for the rapid proteolysis during germination. [ABSTRACT FROM AUTHOR]

Extracts of resting pine seeds inhibited the proteinase activities present in extracts of endosperms of germinating seeds (hydrolysis of haemoglobin at pH 3.7 and hydrolysis of casein at pH 5.4 and 7.0). Heating the extracts of resting seeds at 60°C destroyed their own proteinase activity but their proteinase inhibitor activity decreased by only 25 to 30%. Some properties of the inhibitor(s) were studied using extracts treated at 60°C. The inhibitor activities were non-dialysable, the inhibition increased linearly with increasing inhibitor concentration up to 80% of total proteinase activity, and the maximal inhibition was 80% at pH 3.7, 90% at pH 5.4, and 97% at pH 7.0. The extracts of resting seeds did not inhibit the pepsin-like acid pine proteinase that accounts for a minor part of the proteolytic activity of endosperm extracts at pH 3.7. Neither did they have any effect on the acid pine carboxypeptidase or trypsin and chymotrypsin. Fresh extracts of endosperms of germinating seeds contained relatively high proteinase activity (assayed directly) and moderate inhibitor activity (assayed after treatment at 60°C). When fresh extracts were dialysed at 5°C for 48 h their proteinase activities increased considerably while the corresponding inhibitor activities disappeared. It is concluded that the decrease of inhibitors during dialysis is due to enzymatic inactivation and that the corresponding increase of proteinase activities is at least partly due to the destruction of the inhibitors. [ABSTRACT FROM AUTHOR]

A collection of cDNAs was isolated from a λgt11 potato (Solanum tuberosum L.) tuber library by using a soybean lipoxygenase (LOX; linoleate:oxygen oxidoreductase, EC 1.13.11.12) sequence as a hybridization probe. These cDNA clones were classified by restriction patterns and sizes and two clones, plox1 and plox2, were selected for sequencing and further analysis. These clones were found to be virtually identical and contain full-length cDNAs having a high degree of homology with other cloned LOX genes. plox1 contains a 2 806-bp insert, with an open reading frame that encodes a protein of 861 amino acids (96.9 kDa), consistent with molecular mass estimates of the principal tuber LOX deduced from SDS-PAGE. The deduced sequence contains iron-binding domains that are conserved among LOXs. Restriction analysis of potato genomic DNA confirmed that potato LOX is comprised of a gene family with at least three members. The plox1 sequence hybridized to a transcript of 2.8 kb in gel blot analyses of total RNA extracted from potato tuber. Treatment of potato disks and leaves with methyl jasmonate or jasmonic acid increased LOX enzyme activity, as well as abundance of LOX mRNA, consistent with jasmonate's reported effect on LOX expression in plants. The fungal elicitor arachidonic acid (AA) strongly induced LOX enzyme activity in leaves and induced LOX mRNA transcripts in potato leaves and tuber disks. Infection of potato leaves with Phytophthora infestans induced LOX activity as well as LOX mRNA accumulation in both incompatible and compatible interaction types. LOX expression was compared with that of a wound-inducible proteinase inhibitor (PINII) and a pathogenesis-related protein (P4). Jasmonates induced PINII but not P4 transcript accumulation in potato leaves. In contrast, AA induced P4 but not PINII transcript accumulation in potato leaves. Infection with P. infestans resulted in a gene expression pattern similar to that observed with AA. [ABSTRACT FROM AUTHOR]

Wounding induces expression of genes encoding defense-related proteins involved in wound healing. An intensive survey has been carried out to clarify the initial signal transduction pathways that mediate this stress to expression of genes. In this context, signal molecules that intermediate in the wound signal to cellular response have been actively searched for. Jasmonic acid (JA) has been considered to be a key signal molecule in this pathway. Systemin, ABA, ethylene, and electrical current have been suggested to function by transmitting the wound signal to JA. A mitogen-activated protein kinase has been shown to respond rapidly to wounding, and proposed to function as one of the key enzymes involved in JA biosynthesis. Transgenic plants overexpressing a gene encoding a Rab-type, small GTP-binding protein contained 6-fold higher levels of cytokinins than wild-type plants, and responded to wounding by rapidly producing JA and, uncommonly, accumulating salicylic acid (SA), a pathogenic signal. These phenomena observed in the transgenic plants were reproduced when wild-type plants were wounded in the presence of the synthetic cytokinin, benzylaminopurine, suggesting that cytokinins are indispensable in the control of endogenous levels of JA and SA. [ABSTRACT FROM AUTHOR]

Proteinase inhibitors can be induced by wounding in shoots of tomato (Lycopersicon esculentum [L.] Mill. cv. Moneymaker). These inhibitors are toxic to insects, but their ecological importance is not clear. Published work suggests that proteinase inhibitors may be wound‐inducible in tomato only while the plants are young (less than 30 days). In the present investigation the influence of plant age on wound‐inducible proteinase inhibitor was re‐assessed using tomato plants grown in an outdoor polythene tunnel, with natural lighting and without supplementary heat. In contrast to previous findings, proteinase inhibitor was shown to be induced by wounding in plants of all ages. However, the systemic efficacy of wounds was much reduced in mature plants, possibly because such plants have outgrown the range of the wound‐signalling system. [ABSTRACT FROM AUTHOR]

Proteinase inhibitors (PI) are expressed in several members of the Solanaceae. According to species, PI can be both developmentally and environmentally regulated. We investigated the presence and developmental regulation of PI in the flowers of Nicotiana plumbaginifolia. By trypsin affinity chromatography a small protein, Np‐PI‐II, was isolated. This protein had an N‐terminus similar to a part of a Nicotiana alata PI‐II, and an inhibitory activity towards trypsin. Inhibitory activity is expressed at very early stages of floral development. No inhibitor is present in the vegetative meristems. The inhibitory activity remains high throughout flower maturation and rapidly declines in the ovary after pollination. In the stigma of mature Nicotiana tabacum flowers trypsin inhibitory activity is detected extracellularly. Proteinase inhibitor activity is not a regular feature of the stigmas of several solanaceous genera. Proteinase inhibitors apparently have no function in the development of floral structures and probably act as protective agents against fungal infection of and insect damage to the floral parts and the developing fruit. [ABSTRACT FROM AUTHOR]

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 proteinase inhibitors potentially useful for insect control, had no effect on any tuber proteinase detected. 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 transgenic potato plants. [ABSTRACT FROM AUTHOR]

The early events of potato (Solanum tuberosum L. cv. Superior) tuberization were examined by using a model system of axillary bud tuber development from petiole- leaf single-node cuttings. Both fresh weight and starch accumulation were monitored to establish a developmental framework for morphological changes.. Fresh weight and starch content began to increase in axillary buds after 2 days. Visible changes in bud morphology could be detected 4 days after the start of incubation. Substantial increases in both total protein and total RNA were observed at the onset of tuber morphology. Immunoblot analysis showed that the major tuber protein, patatin, could be initially detected in day 4 buds and that a 22-kDa proteinase inhibitor could be initially detected at day 8. Northern blot analysis corroborated this pattern of accumulation at the RNA level for both protein types. Substantial accumulation of the two proteinase inhibitor mRNAs occurred later than patatin mRNA accumulation. The results of this study showed that there is considerable accumulation of both protein and mRNA occurring during the early stages of tuber development prior to the substantial accumulation of the major tuber storage proteins. [ABSTRACT FROM AUTHOR]