Search

Research into the relationship between epigenetic regulation and resistance to biotic stresses provides alternatives for plant protection and crop improvement. To unravel the mechanisms underlying tomato responses to Botrytis cinerea, we performed a chromatin immunoprecipitation (ChIP) analysis showing the increase in H3K9ac mark along the early induced genes SlyDES, SlyDOX1, and SlyLoxD encoding oxylipin-pathway enzymes, and SlyWRKY75 coding for a transcriptional regulator of hormonal signaling. This histone mark showed a more distinct distribution than the previously studied H3K4me3. The RNAPol-ChIP analysis reflected the actual gene transcription associated with increased histone modifications. A different pattern of marks in the oxylipin-related genes against P. syringae supported a pathogen-specific profile, while no significant differences occurred in SlyWRKY75. The epigenetic regulation of SlyWRKY75 by the intron-binding miR1127-3p was supported by the presence of SlyWRKY75 pre-mRNA in control plants. Interestingly, mRNA was found to be accumulated in response to B. cinerea and P. syringae, while reduction in miRNA only occurred against B. cinerea. The intronic region presented a similar pattern of marks than the rest of the gene in both pathosystems, except for H3K4me3 in the miRNA binding site upon B. cinerea. We located the gene encoding Sly-miR1127-3p, which presented reduced H3K4me3 on its promoter against B. cinerea., This work was supported by grants from the Spanish Ministry of Science and Innovation (AGL2013-49023-C3) and Economy (AGL2017-85987-C3), co-funded by the European Regional Development Funds (ERDF). Óscar Crespo-Salvador was the recipient of a research contract from AGL2013-49023-C3-1-R; AGL2017-85987-C3-2-R. M.J. López-Galiano was awarded with a University of Valencia Ph. D. fellowship.

Priming by natural compounds is an interesting alternative for sustainable agriculture, which also contributes to explore the molecular mechanisms associated with stress tolerance. Although hosts and stress types eventually determine the mode of action of plant-priming agents, it highlights that many of them act on redox signalling. These include vitamins thiamine, riboflavin and quercetin; organic acids like pipecolic, azelaic and hexanoic; volatile organic compounds such as methyl jasmonate; cell wall components like chitosans and oligogalacturonides; H2O2, etc. This review provides data on how priming inducers promote stronger and faster responses to stress by modulating the oxidative environment, and interacting with signalling pathways mediated by salycilic acid, jasmonic acid and ethylene. The histone modifications involved in priming that affect the transcription of defence-related genes are also discussed. Despite the evolutionary distance between plants and animals, and the fact that the plant innate immunity takes place in each plant cell, they show many similarities in the molecular mechanisms that underlie pathogen perception and further signalling to activate defence responses. This review highlights the similarities between priming through redox signalling in plants and in mammalian cells. The strategies used by pathogens to manipulate the host´s recognition and the further activation of defences also show similarities in both kingdoms. Moreover, phytochemicals like sulforaphane and 12-oxo-phytodienoic acid prime both plant and mammalian responses by activating redox-sensitive genes. Hence research data into the priming of plant defences can provide additional information and a new viewpoint for priming mammalian defence, and vice versa.

Determination of histone epigenetic marks in Arabidopsis and tomato genes in the early response to Botrytis cinerea may contribute to find biomarkers of the early detection of this devastating pathogen. Recent studies have linked epigenetic modifications with plant responses to biotic stresses. Information about specific histone marks upon necrotrophic pathogens is scarce. Here we wondered whether the altered responsiveness of specific genes in plants infected with Botrytis cinerea was associated with changes in chromatin structure. We performed a chromatin immunoprecipitation analysis that obtained differential epigenetic signature of activating marks H3K4me3, H3K9ac, and the repressor one H3K27me3 on both the promoter and the body of the highly induced PR1 in Arabidopsis plants infected with B. cinerea at 24 and 33 h after inoculation. We also determined the histone marks' profile in two differentially expressed genes in response to B. cinerea, as well as to oxidative stress, given its relevance in this infection. These are both the induced CYP71A13, which encodes a cytochrome P450 involved in camalexin synthesis, and is essential against this necrotroph and the repressed EXL7 (Exordium-like 1). We also adapted our protocol in tomato plants infected with B. cinerea. At 24 hpi, H3K4me3 level increased on the promoter and at different locations of the body of the genes induced upon B. cinerea, including DES (divinyl ethyl synthase), LoxD (lipoxygenase D), DOX1 (α-dioxygenase 1), PR2 (pathogenesis-related protein2), WRKY53 and WRKY33. The histone modifications determined herein will allow future studies on epigenetic marks and their transgenerational inheritance in plants infected with B. cinerea. In addition, the analyzed genes are potential biomarkers of B. cinerea infection that could contribute to its early detection in tomato and related crops.

Research into the relationship between epigenetic regulation and resistance to biotic stresses provides alternatives for plant protection and crop improvement. To unravel the mechanisms underlying tomato responses to Botrytis cinerea, we performed a chromatin immunoprecipitation (ChIP) analysis showing the increase in H3K9ac mark along the early induced genes SlyDES, SlyDOX1, and SlyLoxD encoding oxylipin-pathway enzymes, and SlyWRKY75 coding for a transcriptional regulator of hormonal signaling. This histone mark showed a more distinct distribution than the previously studied H3K4me3. The RNAPol-ChIP analysis reflected the actual gene transcription associated with increased histone modifications. A different pattern of marks in the oxylipin-related genes against P. syringae supported a pathogen-specific profile, while no significant differences occurred in SlyWRKY75. The epigenetic regulation of SlyWRKY75 by the intron-binding miR1127-3p was supported by the presence of SlyWRKY75 pre-mRNA in control plants. Interestingly, mRNA was found to be accumulated in response to B. cinerea and P. syringae, while reduction in miRNA only occurred against B. cinerea. The intronic region presented a similar pattern of marks than the rest of the gene in both pathosystems, except for H3K4me3 in the miRNA binding site upon B. cinerea. We located the gene encoding Sly-miR1127-3p, which presented reduced H3K4me3 on its promoter against B. cinerea.

Insect-plant interactions are governed by a complex equilibrium between the mechanisms through which plant recognize insect attack and orchestrate downstream signaling events that trigger plant defense responses, and the mechanisms by which insects overcome plant defenses. Due to this tight and dynamic interplay, insight into the nature of the plant defense response can be gained by analyzing changes in the insect herbivores digestive system upon plant feeding. In this work we have identified a Solanum melongena miraculin-like protease inhibitor in the midgut juice of Colorado potato larvae feeding on eggplant plants treated with the natural inducer of plant defenses hexanoic acid. We analyzed the corresponding gene expression by qRT-PCR and our results showed that this eggplant miraculin-like gene enhanced induction contributes to the hexanoic acid priming effect in this Solanaceae species. Moreover, our data evidencing that OPDA might be involved in this gene regulation highlights its potential as biomarker in eggplant plant responses to stress mediated this oxylipin signaling pathway.

Key message SlyWRKY75: gene expression was induced in response to biotic stresses, especially in Botrytis cinerea-infected tomato plants, in which Sly-miR1127-3p is a putative SlyWRKY75 regulator and epigenetic marks were detected. Abstract WRKY75 transcription factor involved in Pi homeostasis was recently found also induced in defense against necrotrophic pathogens. In this study, we analyzed by RT-qPCR the expression of SlyWRKY75 gene in tomato plants in response to abiotic stresses (drought or heat) and biotic stresses (Colorado potato beetle larvae infestation, Pseudomonas syringae or Botrytis cinerea infection) being only differentially expressed following biotic stresses, especially upon B. cinerea infection (55-fold induction). JA and JA-Ile levels were significantly increased in tomato plants under biotic stresses compared with control plants, indicating that SlyWRKY75 might be a transcriptional regulator of the JA pathway. The contribution of miRNAs and epigenetic molecular mechanisms to the regulation of this gene in B. cinerea-infected tomato plants was explored. We identified a putative Sly-miR1127-3p miRNA predicted to bind the intronic region of the SlyWRKY75 genomic sequence. Sly-miR1127-3p miRNA was repressed in infected plants (0.4-fold) supporting that it might act as an epigenetic regulation factor of SlyWRKY75 gene expression rather than via the post-transcriptional mechanisms of canonical miRNAs. It has been proposed that certain miRNAs can mediate DNA methylation in the plant nucleus broadening miRNA functions with transcriptional gene silencing by targeting intron-containing pre-mRNAs. Histone modifications analysis by chromatin immunoprecipitation (ChIP) demonstrated the presence of the activator histone modification H3K4me3 on SlyWRKY75 transcription start site and gene body. The induction of this gene in response to B. cinerea correlates with the presence of an activator mark. Thus, miRNAs and chromatin modifications might cooperate as epigenetic factors to modulate SlyWRKY75 gene expression.

During plant-pathogen interactions, the plant cell wall forms part of active defence against invaders. In recent years, cell wall-editing enzymes, associated with growth and development, have been related to plant susceptibility or resistance. Our previous work identified a role for several tomato and Arabidopsis endo-1,4-β-glucanases (EGs) in plant-pathogen interactions. Here we studied the response of the Arabidopsis thaliana T-DNA insertion mutant lacking EG Korrigan1 (KOR1) infected with Pseudomonas syringae. KOR1 is predicted to be an EG which is thought to participate in cellulose biosynthesis. We found that kor1-1 plants were more susceptible to P. syringae, and displayed severe disease symptoms and enhanced bacterial growth if compared to Wassilewskija (Ws) wild-type plants. Hormonal and gene expression analyses revealed that the jasmonic acid (JA) pathway was activated more in kor1-1 plants with an increase in the JA-biosynthesis gene LOX3 and a greater accumulation of JA. Upon infection the accumulation of JA and JA-isoleucine (JA-Ile) was higher than in wild-type plants and increased the induction of LOX3 and the JA-responsive PDF1.2 gene. In addition, the increase of salicylic acid (SA) in healthy and infected kor1-1 may reflect the complex interaction between JA and SA, which results in the more susceptible phenotype displayed by the infected mutant plants. Callose deposition was enhanced in infected kor1-1 and an increase in pathogen-induced hydrogen peroxide took place. The susceptible phenotype displayed by KOR1-deficient plants was coronatine-independent. No significant changes were detected in the hormonal profile of the kor1-1 plants infected by coronatine-deficient P. syringae cmaA, which supports that absence of EG KOR1 alters per se the plant response to infection. We previously reported increased resistance of kor1-1 to B. cinerea, hence, the lack of this EG alters cell wall properties and plant responses in such a way that benefits P. syringae colonisation but restricts B. cinerea invasion.

Summary Treatment with the resistance priming inducer hexanoic acid (Hx) protects tomato plants from Botrytis cinerea by activating defence responses. To investigate the molecular mechanisms underlying hexanoic acid-induced resistance (Hx-IR), we compared the expression profiles of three different conditions: Botrytis-infected plants (Inf), Hx-treated plants (Hx) and Hx-treated + infected plants (Hx+Inf). The microarray analysis at 24 h post-inoculation showed that Hx and Hx+Inf plants exhibited the differential expression and priming of many Botrytis-induced genes. Interestingly, we found that the activation by Hx of other genes was not altered by the fungus at this time point. These genes may be considered to be specific targets of the Hx priming effect and may help to elucidate its mechanisms of action. It is noteworthy that, in Hx and Hx+Inf plants, there was up-regulation of proteinase inhibitor genes, DNA-binding factors, enzymes involved in plant hormone signalling and synthesis, and, remarkably, the genes involved in oxidative stress. Given the relevance of the oxidative burst occurring in plant–pathogen interactions, the effect of Hx on this process was studied in depth. We showed by specific staining that reactive oxygen species (ROS) accumulation in Hx+Inf plants was reduced and more restricted around infection sites. In addition, these plants showed higher ratios of reduced to oxidized glutathione and ascorbate, and normal levels of antioxidant activities. The results obtained indicate that Hx protects tomato plants from B. cinerea by regulating and priming Botrytis-specific and non-specific genes, preventing the harmful effects of oxidative stress produced by infection.

Interaction between insect herbivores and host plants can be modulated by endogenous and exogenous compounds present in the source of food and might be successfully exploited in Colorado potato beetle (CPB) pest management. Feeding tests with CPB larvae reared on three solanaceous plants (potato, eggplant and tomato) resulted in variable larval growth rates and differential susceptibility to Bacillus thuringiensis Cry3Aa toxin as a function of the host plant. An inverse correlation with toxicity was observed in Cry3Aa proteolytic patterns generated by CPB midgut brush-border membrane vesicles (BBMV) from Solanaceae-fed larvae, being the toxin most extensively proteolyzed on potato, followed by eggplant and tomato. We found that CPB cysteine proteases intestains may interact with Cry3Aa toxin and, in CPB BBMV from larvae fed all three Solanaceae, the toxin was able to compete for the hydrolysis of a papain substrate. In response to treatment with the JA-dependent plant inducer Hexanoic acid (Hx), we showed that eggplant reduced OPDA basal levels and both, potato and eggplant induced JA-Ile. CPB larvae feeding on Hx-induced plants exhibited enhanced Cry3Aa toxicity, which correlated with altered papain activity. Results indicated host-mediated effects on B. thuringiensis efficacy against CPB that can be enhanced in combination with Hx plant induction.

Summary Hexanoic acid-induced resistance (Hx-IR) is effective against several pathogens in tomato plants. Our study of the mechanisms implicated in Hx-IR against Pseudomonas syringae pv. tomato DC3000 suggests that hexanoic acid (Hx) treatment counteracts the negative effect of coronatine (COR) and jasmonyl-isoleucine (JA-Ile) on the salicylic acid (SA) pathway. In Hx-treated plants, an increase in the expression of jasmonic acid carboxyl methyltransferase (JMT) and the SA marker genes PR1 and PR5 indicates a boost in this signalling pathway at the expense of a decrease in JA-Ile. Moreover, Hx treatment potentiates 12-oxo-phytodienoic acid accumulation, which suggests that this molecule might play a role per se in Hx-IR. These results support a positive relationship between the SA and JA pathways in Hx-primed plants. Furthermore, one of the mechanisms of virulence mediated by COR is stomatal re-opening on infection with P. syringae. In this work, we observed that Hx seems to inhibit stomatal opening in planta in the presence of COR, which suggests that, on infection in tomato, this treatment suppresses effector action to prevent bacterial entry into the mesophyll.

Our previous studies into plants have demonstrated a correlation linking priming plant defense, callose accumulation and control of oxidative burst. We have also shown that lack of Cu–Zn superoxide dismutase BCSOD1 reduces the fungal infection associated with the interplay between O 2 - /H 2 O 2 and plant responses. Herein we studied the effect on Arabidopsis and tomato responses of the absence of the B. cinerea NADPH oxidase (Nox) complex, a major source of O 2 - production. Absence of the BcNnox complex (∆bcnoxAB) showed significantly impaired virulence to WT in both cultures. This was associated with an increase in pathogen-induced callose, an O 2 - /H 2 O 2 imbalance with increased O 2 - and reduced H 2 O 2, and the early induction of the PTI-marker WRKY53. However, ∆bcnoxAB produced different effects on the hormone-signaling pathways and redox-sensitive genes in both hosts. Our results indicated that the BcNox constitutes a virulence factor that might contribute to oxidative burst and perform additional roles with different impacts on Arabidopsis and tomato plants. The analysis of the mutants lacking catalytic subunits ∆BcNoxA and ∆BcNoxB, and the regulatory subunit ∆BcNoxR, revealed that callose always correlated with the infection phenotype in both cultures, while oxidative burst did not.

The antifungal activity of hexanoic acid on the phytopathogen Botrytis cinerea was studied. This chemical inhibited both spore germination and mycelial growth in vitro in a concentration- and pH-dependent manner, and stopped spore germination at a very early stage, preventing germ-tube development. The minimum fungicidal concentration (MFC) for in vitro spore germination was 16 m m . Hexanoic acid also inhibited in vitro mycelial growth of germinated spores at an MFC of 12 m m . Studies performed to characterize the mechanisms underlying the antimicrobial effect of hexanoic acid showed that it alters fungal membrane permeability. In addition, hexanoic acid treatment increased the levels of spermine, spermidine, putrescine and cadaverine in B. cinerea mycelia. Spray application of hexanoic acid at fungicidal concentrations on 4-week-old tomato plants prior to fungal inoculation reduced necrosis diameter by approximately 60%. Application of the same hexanoic acid concentrations on previously infected plants reduced further necrosis expansion by around 30%. The results suggest that this chemical acts as a preventive and curative fungicide. Interestingly, treatments with hexanoic acid at concentrations below the MFC in hydroponic solution prior to fungal inoculation significantly reduced necrosis area. These results suggest an inducer effect of plant responses for hexanoic acid treatments at these concentrations. Hexanoic acid is a good candidate for safe antifungal treatments for the control of B. cinerea , which is responsible for many economic losses on fruits, vegetables and flowers.

Three novel amides of adipic acid, 5-carbamoil ethyl pentanoate (N1), 5-(2-furfurylmethylcarbamoil) ethyl pentanoate (N2) and 5-(3-aminopropylcarbamoil) ethyl pentanoate (N3) were synthesized and their resistance-inducing activity was studied on pepper (Capsicum annuum) plants. Treatment with low doses of each amide protected pepper plants against the pathogen Alternaria solani, N1 being the most effective. Although their mode of action is still unknown, prechallenge studies demonstrated the induction of the phenylpropanoid pathway and an antisenescence effect. These effects were previously shown for other mixtures and derivatives of adipic acid. These new chemicals showed no antimicrobial activity at doses much higher than those used for plant treatments. This fact supports the idea that they are acting mainly via planta by inducing natural resistance pathways. Treatments based on these novel chemicals produce no phytotoxic effects and improve plant growth and development. For that reason, they could constitute an attractive alternative to fungicides. It is interesting to note that they are adipic acid derivatives, a compound used as alimentary additive with non toxic properties. Obviously a complete safety study is necessary before these chemicals could be commerzialized.

Differential display was used to isolate tomato genes responding to fungal infection. Here we describe the isolation and characterization of a gene that is down-regulated in tomato fruits infected with the phytopathogen Botrytis cinerea. The cDNA identified encodes a protein that shares sequence similarity to the amino terminal region of CCH, a copper chaperone from Arabidopsis thaliana, that participates in intracellular copper homeostasis by delivering Cu to the secretory pathway. The fact that this newly characterized tomato gene, referred to as LeCCH (Lycopersicon esculentum copper chaperone), be differentially expressed after fungal infection, suggests an interesting relationship between copper homeostasis and plant defense responses. LeCCH contains the conserved metal-binding domain MXCXGC but interestingly, lacks the C-terminal extension present in previously described plant members of this copper chaperone family, that seems to be involved in metallochaperone intercellular transport. This fact indicates that LeCCH is a novel plant copper chaperone that could act locally at the infection site, affecting the copper homeostasis in this particular stress situation.

1,2,3,4-tetra-O-acetyl-6-ethyladipate-beta- D-glucopyranose (TOGE), a glycoside derivative of adipic acid monoethyl ester and 1,2,3,4-tetra-O-acetyl-beta- D-glucopyranose, was synthesized and the resistance-inducing activity shown by TOGE-1 (TOGE plus furfurylamine) and TOGE-2 (TOGE plus 1,3-diaminepropane) was assayed. TOGE-1 and TOGE-2 protected tomato plants against two different fungal pathogens Phytophthora citrophthora and Alternaria solani. Foliar treatments at very low concentrations (2.5 mg l(-1) TOGE, 0.5 mg l(-1) amine) clearly reduced the disease incidence for both pathogens. TOGE-2 application was the most effective on intact plants as well as on detached leaves, reducing fungal growth by more than 46% with respect to control plants. On the other hand TOGE-1 treatment reduced fungal advance by 21%. These results demonstrate a high protective effect against fungal infections for both chemicals. A possible direct antimicrobial effect was discounted due to the weak activity observed in vitro against these pathogens at the low concentrations used in plants. TOGE-2 treatment clearly activates resistance against both pathogens and improves the protective effect previously shown by FGA mixture (adipic acid monoethyl ester, 1,2,3,4-tetra-O-acetyl-beta- D-glucopyranose and furfurylamine) [V. Flors et al. (2001) J Agric Food Chem 49:2569-2575]. The obtained results indicate that TOGE-1 and TOGE-2 act as resistance inducers. Although their mode of action is still unknown, pre-challenge studies have demonstrated the induction of the phenylpropanoid pathway and antioxidant activities. Both chemicals have demonstrated a beneficial effect on plant development, increasing chlorophyll and protein contents, photosynthetic rate and water-use efficiency. The improvement of plant growth and development produced by these treatments suggests crop tolerance to these chemicals, although effective formulations that are safe to humans must be developed before this technology can be used commercially.

n this study, we have used untargeted global metabolomic analysis to determine and compare the chemi-cal nature of the metabolites altered during the infection of tomato plants (cv. Ailsa Craig) with Botry-tis cinerea (Bot)orPseudomonas syringae pv. tomato DC3000 (Pst), pathogens that have different invasionmechanisms and lifestyles. We also obtained the metabolome of tomato plants primed using the naturalresistance inducer hexanoic acid and then infected with these pathogens. By contrasting the metabolomicprofiles of infected, primed, and primed + infected plants, we determined not only the processes or compo-nents related directly to plant defense responses, but also inferred the metabolic mechanisms by whichpathogen resistance is primed. The data show that basal resistance and hexanoic acid-induced resistance toBot and Pst are associated with a marked metabolic reprogramming. This includes significant changes inamino acids, sugars and free fatty acids, and in primary and secondary metabolism. Comparison of themetabolic profiles of the infections indicated clear differences, reflecting the fact that the plant’s chemicalresponses are highly adapted to specific attackers. The data also indicate involvement of signaling mole-cules, including pipecolic and azelaic acids, in response to Pst and, interestingly, to Bot. The compound 1-methyltryptophan was shown to be associated with the tomato–Pst and tomato–Bot interactions as well aswith hexanoic acid-induced resistance. Root application of this Trp-derived metabolite also demonstratedits ability to protect tomato plants against both pathogens. This work was supported by grants from the Spanish Ministry ofScience and Innovation (AGL2010-22300-C03-01-02 and AGL2013-49023-C03-01-02-R), co-funded by the European Regional Develop-ment Fund, the Generalitat Valenciana Grupos de Excelencia (PROMETEO/2012-066) and P1.1B2013-75 Pla de Promoció de la Investigació, Universitat Jaume I.

Some alternative control strategies of currently emerging plant diseases are based on the use of resistance inducers. This review highlights the recent advances made in the characterization of natural compounds that induce resistance by a priming mechanism. These include vitamins, chitosans, oligogalacturonides, volatile organic compounds, azelaic and pipecolic acid, among others. Overall, other than providing novel disease control strategies that meet environmental regulations, natural priming agents are valuable tools to help unravel the complex mechanisms underlying the induced resistance (IR) phenomenon. The data presented in this review reflect the novel contributions made from studying these natural plant inducers, with special emphasis placed on hexanoic acid (Hx), proposed herein as a model tool for this research field. Hx is a potent natural priming agent of proven efficiency in a wide range of host plants and pathogens. It can early activate broad-spectrum defenses by inducing callose deposition and the salicylic acid (SA) and jasmonic acid (JA) pathways. Later it can prime pathogen-specific responses according to the pathogen’s lifestyle. Interestingly, Hx primes redox-related genes to produce an anti-oxidant protective effect, which might be critical for limiting the infection of necrotrophs. Our Hx-IR findings also strongly suggest that it is an attractive tool for the molecular characterization of the plant alarmed state, with the added advantage of it being a natural compound., This work was supported by grants from the Spanish Ministry of Science and Innovation (AGL2010-22300-C03-01-02-03) co-funded by European Regional Development Fund (ERDF) and Generalitat Valenciana Grupos de Excelencia (PROMETEO/2012/066). Maria de la O Leyva and Ivan Finiti were recipients of a research contract from AGL2010-22300-C03-01.

Treatment with the resistance priming inducer hexanoic acid (Hx) protects tomato plants from Botrytis cinerea by activating defence responses. To investigate the molecular mechanisms underlying hexanoic acid‐induced resistance (Hx‐IR), we compared the expression profiles of three different conditions: Botrytis‐infected plants (Inf), Hx‐treated plants (Hx) and Hx‐treated + infected plants (Hx+Inf). The microarray analysis at 24 h post‐inoculation showed that Hx and Hx+Inf plants exhibited the differential expression and priming of many Botrytis‐induced genes. Interestingly, we found that the activation by Hx of other genes was not altered by the fungus at this time point. These genes may be considered to be specific targets of the Hx priming effect and may help to elucidate its mechanisms of action. It is noteworthy that, in Hx and Hx+Inf plants, there was up‐regulation of proteinase inhibitor genes, DNA‐binding factors, enzymes involved in plant hormone signalling and synthesis, and, remarkably, the genes involved in oxidative stress. Given the relevance of the oxidative burst occurring in plant–pathogen interactions, the effect of Hx on this process was studied in depth. We showed by specific staining that reactive oxygen species (ROS) accumulation in Hx+Inf plants was reduced and more restricted around infection sites. In addition, these plants showed higher ratios of reduced to oxidized glutathione and ascorbate, and normal levels of antioxidant activities. The results obtained indicate that Hx protects tomato plants from B. cinerea by regulating and priming Botrytis‐specific and non‐specific genes, preventing the harmful effects of oxidative stress produced by infection.

Hexanoic acid‐induced resistance (Hx‐IR) is effective against several pathogens in tomato plants. Our study of the mechanisms implicated in Hx‐IR against Pseudomonas syringae pv. tomato DC3000 suggests that hexanoic acid (Hx) treatment counteracts the negative effect of coronatine (COR) and jasmonyl‐isoleucine (JA‐Ile) on the salicylic acid (SA) pathway. In Hx‐treated plants, an increase in the expression of jasmonic acid carboxyl methyltransferase (JMT) and the SA marker genes PR1 and PR5 indicates a boost in this signalling pathway at the expense of a decrease in JA‐Ile. Moreover, Hx treatment potentiates 12‐oxo‐phytodienoic acid accumulation, which suggests that this molecule might play a role per se in Hx‐IR. These results support a positive relationship between the SA and JA pathways in Hx‐primed plants. Furthermore, one of the mechanisms of virulence mediated by COR is stomatal re‐opening on infection with P. syringae. In this work, we observed that Hx seems to inhibit stomatal opening in planta in the presence of COR, which suggests that, on infection in tomato, this treatment suppresses effector action to prevent bacterial entry into the mesophyll.

Plant cell wall modification is a critical component in stress responses. Endo-1,4-β-glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence-signalling network. A study of a set of Arabidopsis EG T-DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant-pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant-pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.

Polyamines induce the transport in vitro of the precursor of ornithine transcarbamylase (pOTC) into isolated rat liver mitochondria by facilitating its functional binding to these organelles. Comparative studies of the effect on the in vitro transport of pOTC of polyamine derivatives and related compounds have allowed us to establish that: (i) at least two protonated amino groups per molecule are necessary to induce the pOTC transport; (ii) a distance of three -CH2- groups between the amino groups in diamines is enough to induce this effect, although no differences were observed with diamines having distances of three to eight -CH2- groups. Longer distances resulted in a marked decrease of the effect.

Soil drench treatments with hexanoic acid can effectively protect Arabidopsis plants against Botrytis cinerea through a mechanism based on a stronger and faster accumulation of JA-dependent defenses. Plants impaired in ethylene, salicylic acid, abscisic acid or glutathion pathways showed intact protection by hexanoic acid upon B. cinerea infection. Accordingly, no significant changes in the SA marker gene PR-1 in either the SA or ABA hormone balance were observed in the infected and treated plants. In contrast, the JA signaling pathway showed dramatic changes after hexanoic acid treatment, mainly when the pathogen was present. The impaired JA mutants, jin1-2 and jar1 , were unable to display hexanoic acid priming against the necrotroph. In addition, hexanoic acid-treated plants infected with B. cinerea showed priming in the expression of the PDF1.2 , PR-4 and VSP1 genes implicated in the JA pathways. Moreover, JA and OPDA levels were primed at early stages by hexanoic acid. Treatments also stimulated increased callose accumulation in response to the pathogen. Although callose accumulation has proved an effective IR mechanism against B. cinerea , it is apparently not essential to express hexanoic acid-induced resistance (HxAc-IR) because the mutant pmr4.1 (callose synthesis defective mutant) is protected by treatment. We recently described how hexanoic acid treatments can protect tomato plants against B. cinerea by stimulating ABA-dependent callose deposition and by priming OPDA and JA-Ile production. We clearly demonstrate here that Hx-IR is a dependent plant species, since this acid protects Arabidopsis plants against the same necrotroph by priming JA-dependent defenses without enhancing callose accumulation.

The Saccharomyces cerevisiae POT1 gene is, as are other yeast peroxisomal protein genes, inducible by fatty acids and repressible by glucose. We have now found that it is also induced during the stationary phase of the culture. To investigate these three regulatory circuits, we have studied the mRNA levels of regulatory mutants as well as the changes in chromatin structure upon gene activation. We conclude that the regulation of transcriptional activity in glucose repression, oleate induction, and stationary phase induction follow different molecular mechanisms. We suggest that this multiplicity of regulatory mechanisms may represent a general rule for the yeast peroxisomal protein genes.

We have demonstrated that root treatment with hexanoic acid protects tomato plants against Botrytis cinerea. Hexanoic acid-induced resistance (Hx-IR) was blocked in the jasmonic acid (JA)-insensitive mutant jai1 (a coi1 homolog) and in the abscisic acid (ABA)-deficient mutant flacca (flc). Upon infection, the LoxD gene as well as the oxylipin 12-oxo-phytodienoic acid and the bioactive molecule JA-Ile were clearly induced in treated plants. However, the basal ABA levels were not altered. Hexanoic acid primed callose deposition against B. cinerea in a cultivar-dependent manner. Treated plants from Ailsa Craig, Moneymaker, and Rheinlands Ruhm showed increased callose deposition but not from Castlemart. Hexanoic acid did not prime callose accumulation in flc plants upon B. cinerea infection; therefore, ABA could act as a positive regulator of Hx-IR by enhancing callose deposition. Furthermore, although hexanoic acid protected the JA-deficient mutant defensless1 (def1), the priming for callose was higher than in the wild type. This suggests a link between JA and callose deposition in tomato. Hence, the obtained results support the idea that callose, oxylipins, and the JA-signaling pathway are involved in Hx-IR against B. cinerea. Moreover our data support the relevance of JA-signaling for basal defense against this necrotroph in tomato. Hexanoic acid also protected against Pseudomonas syringae, indicating a broad-spectrum effect for this new inducer.

Polyamines induce the transport in vitro of the rat liver precursor of ornithine transcarbamylase (pOTC) into isolated rat liver mitochondria. The accumulation of this precursor at the level of binding to the mitochondrial surface has allowed us to establish that polyamines are involved in the interaction of the precursor with the mitochondrial surface. Transport of a chimeric protein having the signal sequence of pOTC fused to a fragment of the cytosolic protein human arginosuccinate lyase was also induced by polyamines. The sensitivity of the pOTC synthesized in vitro and of the chimeric protein to proteinases decreases in the presence of polyamines. This result suggests that polyamines may play a role in modulating the folding of precursors to favour their binding to mitochondria.

Cel1 and Cel2 are members of the tomato (Solanum lycopersicum Mill) endo-beta-1,4-glucanase (EGase) family that may play a role in fruit ripening and organ abscission. This work demonstrates that Cel1 protein is present in other vegetative tissues and accumulates during leaf development. We recently reported the downregulation of both the Cel1 mRNA and protein upon fungal infection, suggesting the involvement of EGases in plant-pathogen interactions. This hypothesis was confirmed by assessing the resistance to Botrytis cinerea infection of transgenic plants expressing both genes in an antisense orientation (Anti-Cel1, Anti-Cel2 and Anti-Cel1-Cel2). The Anti-Cel1-Cel2 plants showed enhanced resistance to this fungal necrotroph. Microscopical analysis of infected leaves revealed that tomato plants accumulated pathogen-inducible callose within the expanding lesion. Anti-Cel1-Cel2 plants presented a faster and enhanced callose accumulation against B. cinerea than wild-type plants. The inhibitor 2-deoxy-d-glucose, a callose synthesis inhibitor, showed a direct relationship between faster callose accumulation and enhanced resistance to B. cinerea. EGase activity appears to negatively modulate callose deposition. The absence of both EGase genes was associated with changes in the expression of the pathogen-related genes PR1 and LoxD. Interestingly, Anti-Cel1-Cel2 plants were more susceptible to Pseudomonas syringae, displaying severe disease symptoms and enhanced bacterial growth relative to wild-type plants. Analysis of the involvement of Cel1 and Cel2 in the susceptibility to B. cinerea in fruits was done with the ripening-impaired mutants Never ripe (Nr) and Ripening inhibitor (rin). The data reported in this work support the idea that enzymes involved in cell wall metabolism play a role in susceptibility to pathogens.

The in vitro and in vivo antifungal activity of adipic acid monoethyl ester (AAME) on the necrotrophic pathogen Botrytis cinerea has been studied. This chemical effectively controlled this important phytopathogen, inhibited spore germination and mycelium development at non-phytotoxic concentrations. The effectiveness of AAME treatment is concentration-dependent and influenced by pH. Spore germination in the presence of AAME is stopped at a very early stage, preventing germ tube development. In addition, cytological changes such as retraction of the conidial cytoplasm in the fungus are observed. AAME was also found to act on membrane integrity, affecting permeability without exhibiting lytic activity, as described previously for other antifungal compounds. Polyamine content in the mycelium of B. cinerea was also affected in response to AAME treatment, resulting in putrescine reduction and spermine accumulation similar to a number of antifungal agents. Microscopic observation of treated conidia after inoculation on tomato leaves suggested that inhibited spores are not able to attach to and penetrate the leaf. Finally, AAME completely suppressed the grey mould disease of tomato fruits under controlled inoculation conditions, providing evidence for its efficacy in a biological context and for the potential use of this chemical as an alternative fungicide treatment.

The effects of exogenous application of a chemical mixture consisting of adipic acid monoethyl ester, furfurylamine, and 1,2,3,4-tetra-O-acetyl-beta-D-glucopyranose (FGA) on various metabolic pathways and the plant-fungus interaction have been studied in Solanaceae plants. Tomato and pepper plants were sprayed with the FGA mixture, and different biochemical parameters such as gas exchange, chlorophyll concentration, protein, cell wall sugar and phenolics contents, and peroxidase and phenylalanine ammonia lyase (PAL) activities were measured. FGA-treated plants showed, in general, an increase in cell wall sugar content and decreases in the chlorophyll degrading rate and the peroxidase activity. These results suggest that FGA (a possible synthetic regulator) could act as a retardant--antisenescence agent in Solanaceae plants. The FGA mixture increased the PAL activity and promoted an overall rise in the concentration of flavonoids and phenolic compounds. Therefore, FGA induced the synthesis of compounds that could give protection to plants against pathogens or insects. To further verify this putative protection, several fungi were inoculated in intact plants. Exogenous FGA applications on intact plants delayed fungus-provoked lesion development. In addition, data also showed that applications of 1,2,3,4-tetra-O-acetyl-beta-D-glucopyranose inhibited fungal growth in vitro. These results confirm that FGA can activate protective mechanisms in plants upon contact with invaders such as fungi.

The thiolase family is a widespread group of proteins present in prokaryotes and three cellular compartments of eukaryotes. This fact makes this family interesting in order to study the evolutionary process of eukaryotes. Using the sequence of peroxisomal thiolase from Saccharomyces cerevisiae recently obtained by us and the other known thiolase sequences, a phylogenetic analysis has been carried out. It shows that all these proteins derived from a primitive enzyme, present in the common ancestor of eubacteria and eukaryotes, which evolved into different specialized thiolases confined to various cell compartments. The evolutionary tree obtained is compatible with the endosymbiotic theory for the origin of peroxisomes. © 1992, Springer-Verlag New York Inc. All rights reserved.

We have previously shown that some changes occur in the chromatin structure of the 3' flank of the yeast SUC2 gene in going from a repressed to an active state. In an attempt to find out the causes of these changes, we have carried out experiments in which mutant copies of SUC2 locus lacking either 5' or 3' flanks have been analysed for their transcriptional activity and chromatin structure. These experiments allowed us to discard any relationship between SUC2 transcription and chromatin changes within its 3'flank. Sequencing of this flank and mRNA analysis, however, resulted in the location of a putative peroxisomal 3-oxoacyl-CoA thiolase gene (POT1), which is repressible by glucose. The disruption of the gene produced a yeast strain unable to use oleic acid as a carbon source. This is the first time that chromatin structure analysis has permitted the identification of a new gene.

The polyamines spermidine, spermine and putrescine, by themselves, at physiological concentrations, induce the transport of the precursor of ornithine carbamoyltransferase into isolated rat liver mitochondria. The presence of polyamines in the transport medium results in the approach of both mitochondrial membranes, suggesting a possible role of these molecules in the transport of the precursor of ornithine carbamoyltransferase into mitochondria, by the formation and/or stabilization of mitochondrial structures involved in the transport system.

Apocytochrome c, the cytosolic precursor of cytochrome c, competes with the precursor of ornithine carbamoyltransferase (OCT) for entry into isolated rat liver mitochondria.

We have examined the effect of low molecular weight components of the transport mixture generally used for the import of rat liver pre-ornithine carbamoyltransferase by isolated rat liver mitochondria. These studies revealed that spermidine and spermine, at physiological concentrations, stimulate the transport of the precursor of ornithine carbamoyltransferase into mitochondria. This stimulatory effect of spermidine and spermine is concentration-dependent and is completely inhibited at higher than physiological concentrations (20 mM for spermidine and 4 mM for spermine). Magnesium ions, which also have a stimulatory effect, inhibit the stimulatory effect of spermidine.

Most mitochondrial proteins are coded by nuclear genes, synthesized outside mitochondria, and imported into these organelles in a post-translational event (1). The import process usually involves the following steps: 1) synthesis of a precursor polypeptides with an amino-terminal extension (signal peptide), on free cytoplasmic polysomes, 2) binding of the precursor to a receptor-like component on the mitochondrial surface, 3) energy-dependent translocation across both mitochondrial membranes, possibly through “contact sites” between outer and inner membranes, 4) removal of the amino-terminal extension by a chelator-sensitive protease in the mitochondrial matrix, and 5) mature proteins location into their functional sites. Exceptions to this sequence of events include apocytochrome c and all proteins of the outer membrane which are synthesized without a leading peptide and transported into mitochondria without need of a membrane potential.

The precursor of ornithine carbamoyltransferase can be transported in vitro into rat liver mitochondria using the postmitochondrial supernatant from rat liver, a more homologous medium than the commonly used rabbit reticulocyte lysate. The transport of the precursor in the case of reticulocyte lysate requires a standard translation mixture. In the presence of the postmitochondrial supernatant the same is true. However, when the components of the translation mixture were added individually to the postmitochondrial supernatant, it was found that spermidine or spermine, at physiological concentrations, sufficed for the transport of the precursor of ornithine carbamoyltransferase. The activity of the postmitochondrial supernatant was inactivated by trypsin and slightly decreased by RNase treatment; it was not lost by dialysis or by heating at 100 degrees C.

Lycopersicon esculentum plants exhibit increased salt stress tolerance following treat‑ ment with adipic acid monoethylester and 1,3‑diaminepropane (DAAME), known as an inducer of resistance against biotic stress in tomato and pepper. For an efficient water and nutrient uptake, plants should adapt their water potential to compensate a decrease in water soil potential produced by salt stress. DAAME‑treated plants showed a faster and stronger water potential reduction and an enhanced proline accumulation. Salinity‑induced oxidative stress was also ameliorated by DAAME treatments. Oxidative membrane damage and ethylene emission were both reduced in DAAME‑treated plants. This effect is probably a consequence of an increase of both non‑enzymatic antioxidant activity as well as peroxidase activity. DAAME‑mediated tolerance resulted in an unaltered photosynthetic rate and a stimulation of the decrease in transpiration under stress conditions without a cost in growth due to salt stress. The reduction in transpiration rate was concomitant with a reduction in phytotoxic Na + and Cl ‑ accumulation under saline stress. Interestingly, the ABA deficient tomato mutant sitiens was insensitive to DAAME‑induced tolerance following NaCl stress exposure. Additionally, DAAME treatments increased the ABA content of leaves, therefore, an intact ABA signalling pathway seems to be important to express DAAME‑induced salt tolerance. Here, we show a possibility of enhance tomato stress tolerance by chemical induction of the major plant defences against salt stress. DAAME‑induced tolerance against salt stress could be complementary to or share elements with induced resistance against biotic stress. This might be the reason for the observed wide spectrum of effectiveness of this compound.