Synthesis Of Proteinase Inhibitors Research Articles

Regulation of synthesis of proteinase inhibitors I and II mRNAs in leaves of wounded tomato plants

Levels of two wound-inducible serine proteinase inhibitors, called Inhibitor I and Inhibitor II, and their mRNAs were quantified in leaves of tomato (Lycopersicon escululentum (L.) Mill.) plants after wounding the leaves with a hemostat. A single wound on a lower leaf of 25-old tomato plants caused the accumulation of the two inhibitor proteins in wounded and non-wounded leaves beginning about 4-6 h following wounding. The rate of inhibitor accumulation was maximal in leaves for the next 4 h and then declined. By 20 h the accumulation had nearly ceased. Following a single wound, Inhibitor I mRNA [600 bases in length] and Inhibitor II mRNA (760 bases) began to accumulate in wounded leaves about 2 h before the inhibitor proteins could be detected. The levels of mRNA for both inhibitors reached a maximum at about 8 h following wounding and then decayed, both with apparent half lives of about 10 h. Four consecutive wounds, inflicted hourly, increased the levels of mRNA for both inhibitors to over twice the levels induced by a single wound. Within 4 h following multiple wounds, Inhibitor I mRNA represented about 0.5% of the total polyadenylated mRNA (poly(A(+))mRNA) and Inhibitor II mRNA about 0.15% of the total. The rates of accumulation of the two inhibitor proteins varied depending upon the age of the plants and their environment during growth, and ranged between 3 and 10 μg Inhibitor I·h(-1)·(g tissue)(-1) for Inhibitor I and about half of these rates for Inhibitor II. Nuclei were isolated from leaves of wounded and non-wounded plants, and in mRNA runoff experiments using specific inhibitor copy DNAs (cDNAs) as probes the synthesis of Inhibitor I and II mRNAs were shown to be regulated, at least in part, at the level of transcription.

THE PHYTOALEXIN RESPONSE: ELICITATION, SIGNALLING AND CONTROL OF HOST GENE EXPRESSION

Summary1. Induced resistance of plants to incompatible races of their microbial pathogens is often characterized by rapid cell death (the hypersensitive response) and the accumulation of low‐Mr antimicrobial compounds termed phytoalexins. There is much indirect evidence to support a major role for phytoalexin induction as an event determining host resistance, and genetical analysis of race‐specific pathogen‐host interactions predicts that induced resistance occurs via interaction between a pathogen avirulence gene product and a host resistance gene product. Elicitors (inducing agents) of phytoalexin accumulation are possible candidates for the active products directly or indirectly resulting from the expression of microbial avirulence genes. The present review considers the process of phytoalexin induction, at the molecular level, from the fungal elicitor to the early changes in host gene expression associated with its action.2. A number of microbial polysaccharides, glycoproteins, pectic enzymes, peptides and fatty acids have potent elicitor activity.3. The results of studies on the purification and characterization of microbial elicitors depend on the nature of the bioassay used. Methods for elicitor extraction may lead to artifacts or loss of race specificity.4. Elicitors isolated from plant pathogens may be race‐specific or race‐non‐specific. In cases where only race‐non‐specific elicitors can be shown, race‐specific induction of phytoalexins may result from the action of enhancer or suppressor molecules. Fungal glucans have been proposed as candidates to act in both these roles.5. Molecular genetic approaches to the identification of phytopathogenic bacterial avirulence genes may help to prove or disprove the role of elicitors of the phytoalexin response as agents responsible for the induction of host resistance. Similar analyses, involving genetic transformation, should soon be possible for phytopathogenic fungi.6. Studies on the nature of host receptors for microbial elicitors are still in their infancy. Such receptors are probably localized in the plant plasma membrane, and elicitation results in often striking changes in host membrane properties.7. Cyclic 3′,5′‐adenosine monophosphate and polyamines do not appear to act in plants as intracellular transducers of the phytoalexin response. Interest is now being shown in a possible role for calcium in intracellular signalling.8. Plant cells contain endogenous elicitor molecules whose synthesis or release may play a role in the intercellular transmission of the phytoalexin response. The main candidates for endogenous elicitors are pectic fragments of the host cell wall, although no direct evidence for their involvement in plant‐pathogen interactions is available. Pectic fragments may act as synergists in parallel with, rather than as couplers in series with, microbial elicitors. The role of ethylene as a response coupler for induced resistance phenomena is difficult to assess.9. Some elicitors may themselves move to the host cell nucleus, although whether this is their effective site of action remains unclear.10. Induction of the phytoalexin response is associated with specific changes in host gene expression related to the selective induction of new mRNA species and enzyme activities.11. Progress is now being made in the characterization of enzymes specific for phytoalexin biosynthesis. Enzyme induction and/or infection appears to involve rapid modulation of gene transcription, although some post‐translational events may also be involved in determining induction patterns.12. Work is now commencing on the sequencing of the genes encoding elicitor‐inducible enzymes. Multigene families have been identified for phenylalanine ammonia‐lyase and chalcone synthase.13. Phytoalexin accumulation may be accompanied by the rapid induction of ethylene biosynthesis and the expression of genes encoding activities involved in the synthesis of hydroxyproline‐rich glycoproteins.14. Pectic endogenous elicitors of phytoalexin accumulation are also active as inducers of systemic proteinase inhibitor synthesis in some solanaceous species.15. Results of studies on the mechanisms of signal transmission and differential gene expression in relation to active defence reactions should be of relevance to many other aspects of the plant's response to environmental stimuli at the physiological, biochemical and molecular genetical levels.

Regulation of proteinase inhibitor genes in food plants.

Proteinase inhibitor proteins in the families Solanaceae, Leguminosae and Graminae are stored in seeds and tubers and are also found to accumulate in leaves in response to pest attacks. The isolation of the proteinase Inhibitor I cDNA and its gene from tomato has provided information concerning proteinase inhibitor synthesis, processing and compartmentation in leaves under pest attacks. Strategies are now being developed using proteinase inhibitor genes, to genetically engineer the quantity and quality of these potentially defensive and highly nutritional proteins of important crop plants.

Wound-induced trypsin inhibitor in alfalfa leaves: identity as a member of the Bowman-Birk inhibitor family.

The primary structure of the wound-inducible trypsin inhibitor from alfalfa (ATI) establishes it as a member of the Bowman-Birk proteinase inhibitor family. The time course of induction of ATI in alfalfa following wounding is similar to the induction of the nonhomologous proteinase inhibitors I and II in tomato and potato leaves, and, like inhibitors I and II, ATI is induced to accumulate in excised leaves supplied with the proteinase inhibitor inducing factor from tomato leaves. The similarity of the wound induction of ATI to that of inhibitors I and II indicates that wound-regulated systems are present in Solanaceae and Leguminosae plant families that possess a common fundamental recognition system regulating synthesis of proteinase inhibitors in response to pest attacks. ATI is the first Bowman-Birk inhibitor that has been found in leaves and is the only member of this family known to be regulated by wounding.

Chitosans and pectic polysaccharides both induce the accumulation of the antifungal phytoalexin pisatin in pea pods and antinutrient proteinase inhibitors in tomato leaves

The Proteinase Inhibitor Inducing Factor, PIIF, a pectic polysaccharide that induces synthesis and accumulation of proteinase inhibitor proteins in tomato and potato leaves, is an effective elicitor of the phytoalexin pisatin in pea pod tissues. The levels of pisatin induced by PIIF, and the time course of elicitation, are similar to those induced by chitosans, β-1,4 glucosamine polymers, which are potent elicitors of pisatin in pea pods. Similarly, the chitosans, found in both insect and fungal cell walls, are the most potent inducers yet found of proteinase inhibitor accumulation in excised tomato cotyledons. The similarity in the induction of synthesis of proteinase inhibitors in tomato cotyledons and of pisatin in pea pods by pectic polysaccharides and chitosans suggests that the two polysaccharide types may be triggering a similar fundamental system present in pea and tomato plants that regulates the expression of genes for natural protection systems.

Proteinase inhibitor-inducing factor activity in tomato leaves resides in oligosaccharides enzymically released from cell walls

The synthesis and accumulation of proteinase inhibitor I in excised tomato leaves can be induced with oligosaccharides obtained by fungal endo-alpha-1,4-polygalacturonase digestion of a pectic polysaccharide (M(r) 5000-10,000) isolated from tomato leaves. Active oligosaccharides were also released from isolated tomato leaf cell walls by endopolygalacturonases partially purified from tomato plants. It is suggested that oligosaccharides, released from plant cell wall pectic polysaccharides by either endogenous or exogenous endopolygalacturonases at a wound or infection site, may have hormone-like roles in regulating plant defense responses in unwounded tissues many centimeters away from the site of release.

Regulation of Proteinase Inhibitor Synthesis in Tomato Leaves

Messenger RNA was isolated from young excised tomato leaves, induced to accumulate proteinase Inhibitors I and II with the proteinase inhibitor inducing factor (PIIF), and translated in vitro in a rabbit reticulocyte lysate system. Translatable messenger RNAs specific for Inhibitors I and II were present in PIIF-induced leaves but were not present without PIIF induction. The nascent in vitro-synthesized inhibitors migrated with an apparent molecular weight 2,000 to 3,000 daltons larger than that of the two inhibitors isolated from leaves. The molecular weights of the preinhibitors were identical whether translated from mRNA from PIIF-induced leaves or translated from mRNA isolated from wounded leaves. Incubation of excised PIIF-induced plants in CO(2)-free air doubled the rate of in vivo synthesis of Inhibitor I over that in normal air (Ryan CA 1977 Biochem Biophys Res Commun 77: 1004-1008) but did not affect the rate of in vivo Inhibitor II accumulation. The rate of incorporation of (35)SO(4) (2-) into soluble proteins was 70% less when leaves were incubated in CO(2)-free air rather than normal air. Messenger RNA isolated from PIIF-induced plants incubated in the presence or absence of CO(2) was translated in vitro. The amount of in vitro-translatable mRNA present for each inhibitor (per microgram total mRNA) was the same in leaves incubated in either atmosphere. Therefore, the increased rate of synthesis and accumulation of Inhibitor I in a CO(2)-free atmosphere does not appear to result from an increased level of mRNA but appears to be controlled at a posttranscriptional level.

Chemical characterization, synthesis and distribution of proteinase inhibitor in newborn rat epidermis.

A protein solubilized in Tris-HCl/saline buffer from keratinized cells of newborn rat epidermis exhibited inhibitor activity to papain and ficin, but not to trypsin, cathepsin D and pepsin. This protein was purified from keratinized cells as well as nonkeratinized and germinative cells by means of IgG affinity chromatography. The inhibitors extracted from all cell layers were immunologically identical and had a molecular weight of approximately 12,500 +/- 500. Since amino acid analysis showed that the inhibitor contains about 35 residues of glycine per mol, [3H]glycine was used to investigate synthesis of the protein. The inhibitor from nonkeratinized and germinative cells was radioactively labeled by 2 h after injection and appeared in keratinized cells by 48 h after injection. Indirect immunofluorescence microscopy demonstrated in situ distribution of the protein in the entire epidermis, and the protein localized by the plasma membrane in granular cells and diffusely in keratinized cells was shown to be insoluble in Tris-HCl saline buffer. The results indicate that a thiol-proteinase inhibitor is synthesized in epidermal cells during keratinization and is retained as part of the cytoplasmic structure

Wound-regulated synthesis and vacuolar compartmentation of proteinase inhibitors in plant leaves.

The loss of agricultural crops because of plant pests is enormous. Estimates have been presented that over 30% of the potential yield of many crops is lost, either in the field or in storage, due to pest attacks. The recovery of even 5–10% of these losses could have major impacts on the political, social and economic aspects of food production in both developed and underdeveloped nations.

Studies on the synthesis of proteinase inhibitors. I. Synthesis and activity of nonapeptide fragments of soybean Bowman-Birk inhibitor.

Two heterodetic cyclic nonapeptides, X-Cys-Thr-Lys-Ser-Asn-Pro-Pro-Gln-Cys-Y (Ia: X = Ac, Y = NH2; Ib: X = H, Y = OH), which correspond to residues 14-22 in the sequence of Bowman-Birk inhibitor, have been synthesized by Merrifield's solid-phase method. Inhibitory activities of Ia and Ib on tryptic hydrolysis of amide and ester substrates were examined. When Gly2-Lys-Gly3 and Tos-Arg-OMe were used as substrates, the values of I50 for the peptide Ia were calculated to be 3.6 micron and 40 micron, respectively. When Gly2-Lys-Gly3 was used as a substrate, the value of Ki was calculated to be 1.5 micron. Ia was hydrolyzed slowly by trypsin, losing the inhibitory activity. When the Lys-Ser bond of Ia was cleved with trypsin, the modified Ia could not be regenerated by trypsin. The linear peptide S, S'-dicarboxamidomethyl-Ia also was inactive and appeared to be a good substrate. Optical rotatory dispersion studies showed that the active fragments have characteristic conformations which were lost upon modification to inactive derivatives.

The regulation by carbon dioxide of protein synthesis in tomato leaves

The presence of atmospheric carbon dioxide supresses the light-dependent synthesis of proteinase Inhibitor I in leaves of intact, wounded tomato plants and in detached leaves induced with the proteinase inhibitor inducing factor, PIIF. In carbon dioxide-free air, or in 100% oxygen, the rate of synthesis of Inhibitor I is over two times the rate in the presence of normal air. The results suggest that either CO2 directly or through a product or products of photorespiration is regulating protein synthesis in tomato leaves.