Annick Stintzi

Pathogenesis-related PR-1 proteins are antifungal. Isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans.

Three distinct basic 14-kD proteins, P14a, P14b, and P14c, were isolated from tomato (Lycopersicon esculentum Mill. cv Baby) leaves infected with Phytophthora infestans. They exhibited antifungal activity against P. infestans both in vitro (inhibition of zoo-spore germination) and in vivo with a tomato leaf disc assay (decrease in infected leaf surface). Serological cross-reactions and amino acid sequence comparisons showed that the three proteins are members of the PR-1 group of pathogenesis-related (PR) proteins. P14a and P14b showed high similarity to a previously characterized P14, whereas P14c was found to be very similar to a putative basic-type PR-1 from tobacco predicted from isolated DNA clones. This protein, named PR-1g, was purified from virus-infected tobacco (Nicotiana tabacum Samsun NN) leaves and characterized by amino acid microsequencing, along with the well-known acidic tobacco PR-1a, PR-1b, and PR-1c. The various tomato and tobacco PR-1 proteins were compared for their biological activity and found to display differential fungicidal activity against P. infestans in both the in vitro and in vivo assays, the most efficient being the newly characterized tomato P14c and tobacco PR-1g.

Enzymes in jasmonate biosynthesis - structure, function, regulation.

Jasmonates are a growing class of lipid-derived signaling molecules with diverse functions ranging from the initiation of biotic and abiotic stress responses to the regulation of plant growth and development. Jasmonate biosynthesis originates from polyunsaturated fatty acids in chloroplast membranes. In a first lipoxygenase-catalyzed reaction molecular oxygen is introduced to yield their 13-hydroperoxy derivatives. These fatty acid hydroperoxides are converted by allene oxide synthase and allene oxide cyclase to 12-oxophytodienoic acid (OPDA) and dinor-OPDA, i.e. the first cyclic intermediates of the pathway. In the subsequent step, the characteristic cyclopentanone ring structure of jasmonates is established by OPDA reductase. Until recently, jasmonic acid has been viewed as the end product of the pathway and as the bioactive hormone. It becomes increasingly clear, however, that biological activity extends to and may even differ between the various jasmonic acid metabolites and conjugates as well as its biosynthetic precursors. It has also become clear that oxygenated fatty acids give rise to a vast variety of bioactive compounds including but not limited to jasmonates. Recent insights into the structure, function, and regulation of the enzymes involved in jasmonate biosynthesis help to explain how this variety is generated while specificity is maintained.

Subtilases - versatile tools for protein turnover, plant development, and interactions with the environment.

Subtilases (SBTs) constitute a large family of serine peptidases. They are commonly found in Archaea, Bacteria and Eukarya, with many more SBTs in plants as compared to other organisms. The expansion of the SBT family in plants was accompanied by functional diversification, and novel, plant-specific physiological roles were acquired in the course of evolution. In addition to their contribution to general protein turnover, plant SBTs are involved in the development of seeds and fruits, the manipulation of the cell wall, the processing of peptide growth factors, epidermal development and pattern formation, plant responses to their biotic and abiotic environment, and in programmed cell death. Plant SBTs share many properties with their bacterial and mammalian homologs, but the adoption of specific roles in plant physiology is also reflected in the acquisition of unique biochemical and structural features that distinguish SBTs in plants from those in other organisms. In this article we provide an overview of the earlier literature on the discovery of the first SBTs in plants, and highlight recent findings with respect to their physiological relevance, structure and function.

The Protease-associated Domain and C-terminal Extension Are Required for Zymogen Processing, Sorting within the Secretory Pathway, and Activity of Tomato Subtilase 3 (SlSBT3)

A transgenic plant cell suspension culture was established as a versatile and efficient expression system for the subtilase SlSBT3 from tomato. The recombinant protease was purified to homogeneity from culture supernatants by fractionated ammonium sulfate precipitation, batch adsorption to cation exchange material, and anion exchange chromatography. Purified SlSBT3 was identified as a 79-kDa glycoprotein with both complex and paucimannosidic type glycan chains at Asn177, Asn203, Asn376, Asn697, and Asn745. SlSBT3 was found to be a very stable enzyme, being fully active at 60 °C and showing highest activity at alkaline conditions with a maximum between pH 7.5 and 8.0. Substrate specificity of SlSBT3 was analyzed in detail, revealing a preference for Gln and Lys in the P1 and P2 positions of oligopeptide substrates, respectively. Similar to bacterial, yeast, and mammalian subtilases, SlSBT3 is synthesized as a preproenzyme, and processing of the prodomain in the endoplasmic reticulum is a prerequisite for passage through the secretory pathway. SlSBT3 S538A and S538C active site mutants accumulated intracellularly as unprocessed zymogens, indicating that prodomain cleavage occurs autocatalytically. The wild-type SlSBT3 protein failed to cleave the prodomain of the S538A mutant in trans, demonstrating that zymogen maturation is an intramolecular process. Distinguishing features of plant as compared with mammalian subtilases include the insertion of a large protease-associated domain between the His and Ser residues of the catalytic triad and the C-terminal extension to the catalytic domain. Both features were found to be required for SlSBT3 activity and, consequently, for prodomain processing and secretion.

Jasmonic acid and its precursor 12-oxophytodienoic acid control different aspects of constitutive and induced herbivore defenses in tomato

Local induction of defense gene expression on wounding is mediated by 12-oxophytodienoic acid, in contrast with constitutive herbivore defense traits that rely on the biosynthesis of jasmonic acid and its isoleucine conjugate. The jasmonate family of growth regulators includes the isoleucine (Ile) conjugate of jasmonic acid (JA-Ile) and its biosynthetic precursor 12-oxophytodienoic acid (OPDA) as signaling molecules. To assess the relative contribution of JA/JA-Ile and OPDA to insect resistance in tomato (Solanum lycopersicum), we silenced the expression of OPDA reductase3 (OPR3) by RNA interference (RNAi). Consistent with a block in the biosynthetic pathway downstream of OPDA, OPR3-RNAi plants contained wild-type levels of OPDA but failed to accumulate JA or JA-Ile after wounding. JA/JA-Ile deficiency in OPR3-RNAi plants resulted in reduced trichome formation and impaired monoterpene and sesquiterpene production. The loss of these JA/JA-Ile -dependent defense traits rendered them more attractive to the specialist herbivore Manduca sexta with respect to feeding and oviposition. Oviposition preference resulted from reduced levels of repellant monoterpenes and sesquiterpenes. Feeding preference, on the other hand, was caused by increased production of cis-3-hexenal acting as a feeding stimulant for M. sexta larvae in OPR3-RNAi plants. Despite impaired constitutive defenses and increased palatability of OPR3-RNAi leaves, larval development was indistinguishable on OPR3-RNAi and wild-type plants, and was much delayed compared with development on the jasmonic acid-insensitive1 (jai1) mutant. Apparently, signaling through JAI1, the tomato ortholog of the ubiquitin ligase CORONATINE INSENSITIVE1 in Arabidopsis (Arabidopsis thaliana), is required for defense, whereas the conversion of OPDA to JA/JA-Ile is not. Comparing the signaling activities of OPDA and JA/JA-Ile, we found that OPDA can substitute for JA/JA-Ile in the local induction of defense gene expression, but the production of JA/JA-Ile is required for a systemic response.

Structural basis of substrate specificity of plant 12-oxophytodienoate reductases

12-Oxophytodienoate reductase 3 (OPR3) is a FMN-dependent oxidoreductase that catalyzes the reduction of the cyclopentenone (9S,13S)-12-oxophytodienoate [(9S,13S)-OPDA] to the corresponding cyclopentanone in the biosynthesis of the plant hormone jasmonic acid. In vitro, however, OPR3 reduces the jasmonic acid precursor (9S,13S)-OPDA as well as the enantiomeric (9R,13R)-OPDA, while its isozyme OPR1 is highly selective, accepting only (9R,13R)-OPDA as a substrate. To uncover the molecular determinants of this remarkable enantioselectivity, we determined the crystal structures of OPR1 and OPR3 in complex with the ligand p-hydroxybenzaldehyde. Structural comparison with the OPR1:(9R,13R)-OPDA complex and further biochemical and mutational analyses revealed that two active-site residues, Tyr78 and Tyr246 in OPR1 and Phe74 and His244 in OPR3, are critical for substrate filtering. The relatively smaller OPR3 residues allow formation of a wider substrate binding pocket that is less enantio-restrictive. Substitution of Phe74 and His244 by the corresponding OPR1 tyrosines resulted in an OPR3 mutant showing enhanced, OPR1-like substrate selectivity. Moreover, sequence analysis of the OPR family supports the filtering function of Tyr78 and Tyr246 and allows predictions with respect to substrate specificity and biological function of thus far uncharacterized OPR isozymes. The discovered structural features may also be relevant for other stereoselective proteins and guide the rational design of stereospecific enzymes for biotechnological applications.

Precursor processing for plant peptide hormone maturation by subtilisin-like serine proteinases

Prohormone processing by subtilases A flower that has gone to seed will drop its petals in a regulated process called abscission. Schardon et al. analyzed the production of the peptide hormone that regulates floral organ abscission in the model plant Arabidopsis thaliana. They used tissue-specific expression of proteinase inhibitors to identify the subtilisin-like proteinases that act as prohormone convertases required for peptide hormone production in plants. Science, this issue p. 1594 Redundant proteases mediate the formation of a peptide signal for the abscission of floral organs in Arabidopsis. Peptide hormones that regulate plant growth and development are derived from larger precursor proteins by proteolytic processing. Our study addressed the role of subtilisin-like proteinases (SBTs) in this process. Using tissue-specific expression of proteinase inhibitors as a tool to overcome functional redundancy, we found that SBT activity was required for the maturation of IDA (INFLORESCENCE DEFICIENT IN ABSCISSION), a peptide signal for the abscission of floral organs in Arabidopsis. We identified three SBTs that process the IDA precursor in vitro, and this processing was shown to be required for the formation of mIDA (the mature and bioactive form of IDA) as the endogenous signaling peptide in vivo. Hence, SBTs act as prohormone convertases in plants, and several functionally redundant SBTs contribute to signal biogenesis.

Jasmonate-dependent induction of polyphenol oxidase activity in tomato foliage is important for defense against Spodoptera exigua but not against Manduca sexta.

BackgroundJasmonates are involved in plant defense, participating in the timely induction of defense responses against insect herbivores from different feeding guilds and with different degrees of host specialization. It is less clear to what extent the induction of plant defense is controlled by different members of the jasmonate family and how specificity of the response is achieved. Using transgenic plants blocked in jasmonic acid (JA) biosynthesis, we previously showed that JA is required for the formation of glandular trichomes and trichome-borne metabolites as constitutive defense traits in tomato, affecting oviposition and feeding behavior of the specialist Manduca sexta. In contrast, JA was not required for the local induction of defense gene expression after wounding. In JA-deficient plants, the JA precursor oxophytodienoic acid (OPDA) substituted as a regulator of defense gene expression maintaining considerable resistance against M. sexta larvae. In this study, we investigate the contribution of JA and OPDA to defense against the generalist herbivore Spodoptera exigua.ResultsS. exigua preferred JA-deficient over wild-type tomato plants as a host for both oviposition and feeding. Feeding preference for JA-deficient plants was caused by constitutively reduced levels of repellent terpenes. Growth and development of the larvae, on the other hand, were controlled by additional JA-dependent defense traits, including the JA-mediated induction of foliar polyphenol oxidase (PPO) activity. PPO induction was more pronounced after S. exigua herbivory as compared to mechanical wounding or M. sexta feeding. The difference was attributed to an elicitor exclusively present in S. exigua oral secretions.ConclusionsThe behavior of M. sexta and S. exigua during oviposition and feeding is controlled by constitutive JA/JA-Ile-dependent defense traits involving mono- and sesquiterpenes in both species, and cis-3-hexenal as an additional chemical cue for M. sexta. The requirement of jasmonates for resistance of tomato plants against caterpillar feeding differs for the two species. While the OPDA-mediated induction of local defense is sufficient to restrict growth and development of M. sexta larvae in absence of JA/JA-Ile, defense against S. exigua relied on additional JA/JA-Ile dependent factors, including the induction of foliar polyphenol oxidase activity in response to S. exigua oral secretions.

Jasmonate Biosynthesis and Signaling for Induced Plant Defense against Herbivory

Jasmonates are a growing class of signaling molecules and plant hormones which are derived from polyunsaturated fatty acids via the octadecanoid pathway, and characterized by a pentacyclic ring structure. Until recently, jasmonic acid has been viewed as the end product of the pathway and as the bioactive hormone. It becomes increasingly clear, however, that biological activity is not limited to jasmonic acid, but extends to, and may even differ between its many metabolites and conjugates as well as its biosynthetic precursors. Like other plant hormones, jasmonates exhibit a broad spectrum of physiological activities, ranging from seed germination, over reproductive development, all the way to senescence. Jasmonates also serve important roles as signaling molecules in plant defense, particularly defense against insect herbivores and necrotrophic patghogens. In this chapter, we will briefly discuss each step of the octadecanoid pathway, emphasizing on those that are relevant for the regulation of jasmonic acid biosynthesis, and on insights derived from the recently solved crystal structures of two of the pathway’s enzymes. With respect to jasmonate signaling, we will focus on their role as signal molecules in the systemic defense response against insect herbivores, and on jasmonate-dependent activation of defense gene expression.

Arabidopsis PECTIN METHYLESTERASE17 is co-expressed with and processed by SBT3.5, a subtilisin-like serine protease

BACKGROUND AND AIMS In Arabidopsis thaliana, the degree of methylesterification (DM) of homogalacturonans (HGs), the main pectic constituent of the cell wall, can be modified by pectin methylesterases (PMEs). In all organisms, two types of protein structure have been reported for PMEs: group 1 and group 2. In group 2 PMEs, the active part (PME domain, Pfam01095) is preceded by an N-terminal extension (PRO part), which shows similarities to PME inhibitors (PMEI domain, Pfam04043). This PRO part mediates retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating PME activity through a post-translational mechanism. This study investigated the roles of a subtilisin-type serine protease (SBT) in the processing of a PME isoform. METHODS Using a combination of functional genomics, biochemistry and proteomic approaches, the role of a specific SBT in the processing of a group 2 PME was assessed together with its consequences for plant development. KEY RESULTS A group 2 PME, AtPME17 (At2g45220), was identified, which was highly co-expressed, both spatially and temporally, with AtSBT3.5 (At1g32940), a subtilisin-type serine protease (subtilase, SBT), during root development. PME activity was modified in roots of knockout mutants for both proteins with consequent effects on root growth. This suggested a role for SBT3.5 in the processing of PME17 in planta. Using transient expression in Nicotiana benthamiana, it was indeed shown that SBT3.5 can process PME17 at a specific single processing motif, releasing a mature isoform in the apoplasm. CONCLUSIONS By revealing the potential role of SBT3.5 in the processing of PME17, this study brings new evidence of the complexity of the regulation of PMEs in plants, and highlights the need for identifying specific PME-SBT pairs.