Clarence A. Ryan

A Polypeptide from Tomato Leaves Induces Wound-Inducible Proteinase Inhibitor Proteins

Defensive genes in plants can be activated by several different types of nonpeptide signaling molecules. An endogenous polypeptide, consisting of 18 amino acids, was isolated from tomato leaves and was able at very low concentrations to induce the synthesis of two wound-inducible proteinase inhibitor proteins when supplied to young tomato plants. The sequence of the polypeptide was determined, and an identical polypeptide was synthesized that possessed full inducing activity. These data establish that a polypeptide factor can initiate signal transduction to regulate the synthesis of defensive proteins in plant tissues.

The systemin receptor SR160 from Lycopersicon peruvianum is a member of the LRR receptor kinase family

The isolation to homogeneity of the 160-kDa systemin cell-surface receptor (SR160) from plasma membranes of suspension cultured cells of Lycopersicon peruvianum is reported. The purification procedure resulted in recovery of 13 μg of pure receptor protein, representing an 8,200-fold purification. Gel blot analyses using SR160-specific antibodies confirmed that a cross-reacting protein in the membranes of suspension-cultured cells comigrates with both the native and a deglycosylated form of the radiolabeled receptor. Internal amino acid sequences of the purified protein facilitated the isolation of a cDNA clone encoding the 160-kDa receptor. The identity of the encoded protein as SR160 was further confirmed by a comparison of its sequence with a mass spectral fingerprint of the SR160 protein. The deduced amino acid sequence of SR160 revealed that it is a member of the leucine-rich repeat (LRR) receptor kinase family, closely related to the brassinolide receptor kinase, BRI1.

Endogenous peptide defense signals in Arabidopsis differentially amplify signaling for the innate immune response

AtPep1, a 23-aa peptide encoded by Arabidopsis PROPEP1, a member of a small, six-member gene family, activates expression of the defense gene PDF1.2 (encoding defensin) and its own precursor gene, PROPEP1, through the jasmonate/ethylene signaling pathway, mediated by a cell-surface receptor, PEPR1. Overexpression of two family members, PROPEP1 and PROPEP2, enhances resistance of Arabidopsis plants against the pathogen Pythium irregulare, and PROPEP2 and PROPEP3 are expressed at highly elevated levels in Arabidopsis in response to pathogen infections and to several pathogen-associated molecules (general elicitors). Here, we report that PDF1.2, PR-1 (pathogenesis protein), and PROPEP genes were differentially expressed in the leaves of intact plants sprayed with methyl jasmonate and methyl salicylate and in excised leaves supplied through cut petioles with peptides derived from the C terminus of each of the encoded proteins. The expression of PDF1.2 and PR-1 elicited by the peptides was blocked in mutant plants deficient in the jasmonate/ethylene and salicylate pathways, and in wild-type plants by treatment with diphenylene iodonium chloride, an inhibitor of hydrogen peroxide production. PROPEP1, PROPEP 2, and PROPEP3 genes appear to have roles in a feedback loop that amplifies defense signaling pathways initiated by pathogens.

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.

Wound-inducible potato inhibitor II genes: enhancement of expression by sucrose.

Expression of a chimeric potato Inhibitor II-CAT gene in transgenic tobacco plants was enhanced 50-fold when leaf tissue was floated on solutions containing 1% sucrose. The expression of the chimeric gene was also enhanced when leaf sections were floated on solutions of glucose, fructose, and maltose, but not when floated on solutions of mannitol. The increased expression due to sucrose was found to be correlated with an increase in CAT mRNA. Thus, the carbohydrates or their metabolic products may control Inhibitor II gene expression by regulating transcription. Levels of potato Inhibitor II proteins in leaf sections from young potato plants floated on 3% sucrose solutions increased 3-fold compared to leaf sections floated on water, supporting the possibility that sucrose may have a role in regulating or enhancing the expression of wound-inducible proteinase inhibitor genes in potato tissues. It is suggested that the Inhibitor II gene and perhaps other genes regulated by sucrose may contain a specific ‘sucrose enhancer’ that strongly increases transcription of genes already active at low levels.

Systemic wound signaling in plants: A new perception

Over 100 species of plants exhibit systemic wound signaling that induces the production of defensive chemicals in leaves and stems (1). The signaling is caused by herbivore attacks and also occurs in response to some pathogens (2–5). The most intensively studied systemic signaling response is that found in species of the Solanaceae family, where a systemic wound signal that is graft transmissible regulates the expression of defensive proteinase inhibitors (PIs) and polyphenol oxidase genes (6) (Fig. 1). On wounding, an 18-aa polypeptide, called systemin (7), interacts with a cell-surface receptor (8, 9) to initiate a signaling cascade that includes the release of linolenic acid (18 carbon atoms) from plant cell membranes and its subsequent conversion to 12 oxo-phytodienoic acid (OPDA; 18 carbon atoms) and jasmonic acid (JA; 12 carbon atoms) (10) through the well known octadecanoid pathway (11). JA activates the expression of several signaling pathway genes that up-regulate JA synthesis and the production of H2O2 (12) leading to the synthesis of PIs. OPDA and JA synthesis is analogous to elements of the inflammatory pathway in animals in which arachidonic acid (20 carbon atoms) is converted to prostaglandins (13). Systemin is considered a mobile long-distance signal, and OPDA isomers (14) and JA have been considered to be localized signals produced in target cells. Li et al., in a recent issue of PNAS (15), use two mutants defective in the wound-signaling pathway to provide evidence that JA or a derivative may also act as a long-distance transmissible signal for wound signaling. Jasmonic acid or a derivative may act as a long-distance transmissible signal for wound signaling.

The organization of the prosystemin gene

The organization of the gene encoding tomato prosystemin, a 200 amino acid protein precursor of the 18 amino acid polypeptide inducer of proteinase inhibitor synthesis in tomato and potato plants, is reported. The prosystemin sequence reveals that the gene, which is composed of five homologous pairs of exons plus a non-homologous exon at the C-terminus containing the systemin sequence, has evolved by several gene duplication-elongation events from a much smaller ancestral gene. The nucleotide and amino acid sequence homologies among the exons suggest that a small ancestral gene was duplicated to form at least two tandem repeats, followed by subsequent duplication-elongation events that resulted in five tandemly repeated nucleotide sequences and three duplicated amino acid sequence elements. Since the systemin nucleotide or amino acid sequence was not duplicated, it was either not part of the gene duplication-elongation events or its coding region evolved separately and may even have been added to the tandemly repeated part of the gene at a later time.

Three hydroxyproline-rich glycopeptides derived from a single petunia polyprotein precursor activate defensin I, a pathogen defense response gene

Hydroxyproline-rich glycopeptides (HypSys peptides) are recently discovered 16–20-amino acid defense signals in tobacco and tomato leaves that are derived from cell wall-associated precursors. The peptides are powerful wound signals that activate the expression of defensive genes in tobacco and tomato leaves in response to herbivore attacks. We have isolated a cDNA from petunia (Petunia hybrida) leaves encoding a putative protein of 214 amino acids that is a homolog of tobacco and tomato HypSys peptide precursors and is inducible by wounding and MeJA. The deduced protein contains a leader sequence and four predicted proline-rich peptides of 18–21 amino acids. Three of the four peptides were isolated from leaves, and each peptide contained hydroxylated prolines and glycosyl residues. Each of the peptides has a -GR- motif at its N terminus, indicating that it may be the substrate site for a processing enzyme. The peptides were active in a petunia suspension culture bioassay at nanomolar concentrations, but they did not induce the expression of defense genes that are directed against herbivores, as found in tobacco and tomato leaves. They did, however, activate expression of defensin 1, a gene associated with inducible defense responses against pathogens.

Accumulation of a metallo-carboxypeptidase inhibitor in leaves of wounded potato plants☆

Abstract Mechanical wounding of young potato plants induces over a two fold increase in inhibitory activity against the bovine pancreatic metalloexopeptidase carboxypeptidase A. This increase in inhibitory activity in both wounded and unwounded leaves parallels the increases of two inhibitors of bovine serine endopeptidases, trypsin and chymotrypsin. This suggests that the Proteinase Inhibitor Inducing Factor is regulating the synthesis and accumulation of inhibitors of two different mechanistic classes of proteases found in animals and microorganisms. These increases in antiproteolytic activities due to wounding support the hypothesis that this response is part of a defense mechanism directed against plant pests.

Differential expression of a chimeric CaMV-tomato proteinase Inhibitor I gene in leaves of transformed nightshade, tobacco and alfalfa plants.

The open reading frame and terminator region of a wound-inducible tomato Inhibitor I gene, regulated by the CaMV 35S promoter, was stably integrated into the genomes of nightshade (Solanum nigrum), tobacco (Nicotiana tabacum), and alfalfa (Medicago sativa), using an Agrobacterium-mediated transformation system. The expression of the foreign Inhibitor I gene in leaves of each species was studied at the mRNA and protein levels. The levels of Inhibitor I protein present in leaves of each species correlated with the levels of mRNA. The average levels of both mRNA and Inhibitor I protein were highest in leaves of transgenic nightshade plants (over 125 μg of Inhibitor I per g tissue), less in tobacco plants (about 75 μg/g tissue), and lowest in leaves of transgenic alfalfa plants (below 20 μg/g tissue). Inhibitor I protein was observed in all tissues throughout transgenic plant species, but inhibitor concentration per gram of tissue was 2–3 times higher in young developing leaf tissues and floral organs. The differences in the expression of the CaMV-tomato Inhibitor I gene among the different plant genera suggests that either the rate of transcription of the foreign gene or the rate of degradation of the nascent Inhibitor I mRNA varies among genera. Using electron microscopy techniques, the newly synthesized pre-pro-Inhibitor I protein was shown to be correctly processed and stored as a mature Inhibitor I protein within the central vacuoles of leaves of transgenic nightshade and alfalfa. The results of these experiments suggest that maximal expression of foreign proteinase inhibitor genes, and perhaps other foreign defense genes, may require gene constructs that are fashioned with promoters and terminators that allow maximum expression in the selected plant species.