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Peanut cultivars are known to produce stilbene compounds. Transcriptional control plays a key role in the early stages of the stress response mechanism, involving both PR-proteins and stilbene compounds. In this study, the production of stilbenoid compounds, especially prenylated, was investigated in two cultivars of peanut hairy root lines, designated as K2-K599 and T9-K599 elicited with a combination of chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD): CHT + MeJA + CD. The antioxidant activities and stilbenoid content of both K2-K599 and T9-K599 hairy root lines increased significantly during the elicitation period. The T9-K599 hairy root line expressed higher ABTS and FRAP antioxidant activities than the K2-K599 line while the latter exhibited greater total phenolic content than the former at all-time points. Additionally, the K2-K599 line exhibited more stilbene compounds, including trans-resveratrol, trans-arachidin-1, and trans-arachidin-3 than the T9-K599 line, which showed statistically significant differences at all-time points. Gene expression of the enzyme involved in the stilbene biosynthesis pathway (PAL, RS, RS3) was observed, responding early to elicitor treatment and the metabolic production of a high level of stilbenoid compounds at a later stage. The antioxidant enzyme (CuZn-SOD, APX, GPX) and pathogenesis-related protein (PR; PR4A, PR5, PR10, chitinase) genes were strongly expressed after elicitor treatment at 24 h and decreased with an increasing elicitation time. Investigation of the response mechanism illustrates that the elicitor treatment can affect various plant responses, including plant cell wall structure and integrity, antioxidant system, PR-proteins, and secondary plant metabolites at different time points after facing external environmental stimuli.

Main conclusion OsJAZ11 regulates phosphate homeostasis by suppressing jasmonic acid signaling and biosynthesis in rice roots. Abstract Jasmonic Acid (JA) is a key plant signaling molecule which negatively regulates growth processes including root elongation. JAZ (JASMONATE ZIM-DOMAIN) proteins function as transcriptional repressors of JA signaling. Therefore, targeting JA signaling by deploying JAZ repressors may enhance root length in crops. In this study, we overexpressed JAZ repressor OsJAZ11 in rice to alleviate the root growth inhibitory action of JA. OsJAZ11 is a low phosphate (Pi) responsive gene which is transcriptionally regulated by OsPHR2. We report that OsJAZ11 overexpression promoted primary and seminal root elongation which enhanced Pi foraging. Expression studies revealed that overexpression of OsJAZ11 also reduced Pi starvation response (PSR) under Pi limiting conditions. Moreover, OsJAZ11 overexpression also suppressed JA signaling and biosynthesis as compared to wild type (WT). We further demonstrated that the C-terminal region of OsJAZ11 was crucial for stimulating root elongation in overexpression lines. Rice transgenics overexpressing truncated OsJAZ11ΔC transgene (i.e., missing C-terminal region) exhibited reduced root length and Pi uptake. Interestingly, OsJAZ11 also regulates Pi homeostasis via physical interaction with a key Pi sensing protein, OsSPX1. Our study highlights the functional connections between JA and Pi signaling and reveals JAZ repressors as a promising candidate for improving low Pi tolerance of elite rice genotypes.

BmG10H-1 transcript from B. monnieri was functionally active. BmG10H-1 promoter drives GUS activity in response to MeJA and wounding. BmMYB35 regulates BmG10H-1 transcript by binding to its promoter. Geraniol 10-hydroxylase (G10H) is one of the important regulatory cytochrome P450 monooxygenase, which is involved in the biosynthesis of monoterpene alkaloids. However, G10H is not characterized at the enzymatic or at the regulatory aspect in B. monnieri. In the present study, we have identified two transcripts of BmG10H (BmG10H-1and BmG10H-2) and characterized the methyl jasmonate (MeJA) and wound responsive BmG10H-1 transcript from B. monnieri. BmG10H-1 showed induced expression after 3 h of MeJA and wounding treatment in the shoot. Yeast purified recombinant BmG10H-1 protein is enzymatically active, having Vmax of 0.16 µMsec−1 μg−1 protein and catalyzes the hydroxylation of geraniol to 10-hydroxy geraniol. The BmG10H-1 promoter was isolated by using the genome walking method. BmG10H-1 promoter can drive GUS expression in transgenic Arabidopsis thaliana. GUS activity of MeJA and wound-treated Arabidopsis seedlings were found to be increased as compared to the control untreated seedlings, whereas no GUS activity was found in deleted MeJA responsive and W-box cis-elements. This shows that the BmG10H-1 promoter contains functional MeJA (TGACG) and wound responsive (TGACCT) cis-elements. Further, shoot specific and MeJA responsive recombinant BmMYB35 protein was purified, which binds with the MYB recognition cis-element (TGGTTA) present in the BmG10H-1 promoter and transcriptionally activates the reporter gene in yeast. In conclusion, the characterization of MeJA and wound responsive BmG10H-1 provides novel information about its transcriptional regulation by binding with MYB transcription factor in B. monnieri.

Genome-wide identification, expression analysis of the MYC family in Camellia sinensis, and potential functional characterization of CsMYC2.1 have laid a solid foundation for further research on CsMYC2.1 in jasmonate (JA)-mediated response. Myelocytomatosis (MYC) of basic helix-loop-helix (bHLH) plays a major role in JA-mediated plant growth and developmental processes through specifically binding to the G-box in the promoters of their target genes. In Camellia sinensis, studies on the MYC gene family are limited. Here, we identified 14 C. sinensis MYC (CsMYC) genes, and further analyzed the evolutionary relationship, gene structure, and motif pattern among them. The expression patterns of these CsMYC genes in different tissues suggested their important roles in diverse function in tea plant. Four MYC transcription factors with the highest homology to MYC2 in Arabidopsis were localized in the nucleus. Two of them, named CsMYC2.1 and CsMYC2.2, exhibited transcriptional self-activating activity, and, therefore, could significantly activate the promoter containing G-box motif, whereas CsJAM1.1 and CsJAM1.2 lack the transcriptional self-activating activity, indirectly mediating the JA pathway through interacting with CsMYC2.1 and CsMYC2.2. Furthermore, Yeast Two-Hybrid (Y2H) and Bimolecular Fluorescent Complimentary (BiFC) assays showed that CsMYC2.1 could interact with CsJAZ3/7/8 proteins. Genetically, the complementation of CsMYC2.1 in myc2 mutants conferred the ability to restore the sensitivity to JA signals. The results provide a comprehensive characterization of the 14 CsMYCs in C. sinensis, establishing a solid foundation for further research on CsMYCs in JA-mediated response.

Jasmonic acid positively modulates vitamin E accumulation, but the latter can also partly influence the capacity to accumulate the jasmonic acid precursor, 12-oxo-phytodienoic acid, in white-leaved rockrose (Cistus albidus L.) plants growing in their natural habitat. This study suggests a bidirectional link between chloroplastic antioxidants and lipid peroxidation-derived hormones in plants. While vitamin E is well known for its antioxidant properties being involved in plant responses to abiotic stress, jasmonates are generally related to biotic stress responses in plants. Studying them in non-model plants under natural conditions is crucial for the knowledge on their relationship, which will help us to better understand mechanisms and limits of stress tolerance to implement better conservation strategies in vulnerable ecosystems. We studied a typical Mediterranean shrub, white-leaved rockrose (Cistus albidus) under natural conditions during three winters and we analyzed both α and γ-tocopherol, and the three main jasmonates forms 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and jasmonoyl-isoleucine (JA-Ile). We found that JA contents positively correlated with vitamin E accumulation, most particularly with γ-tocopherol, the precursor of α-tocopherol (the most active vitamin E form). This finding was confirmed by exogenous application of methyl jasmonate (MeJA) in leaf discs under controlled conditions, which increased γ-tocopherol when applied at 0.1 mM MeJA and α-tocopherol at 1 mM MeJA. Furthermore, a complementary meta-analysis study with previously published reports revealed a positive correlation between JA and vitamin E, although this relationship turned to be strongly species specific. A strong negative correlation was observed, however, between total tocopherols and OPDA (a JA precursor located in chloroplasts). This antagonistic effect was observed between α-tocopherol and OPDA, but not between γ-tocopherol and OPDA. It is concluded that (i) variations in jasmonates and vitamin E due to yearly, inter-individual and sun orientation-driven variability are compatible with a partial regulation of vitamin E accumulation by jasmonates, (ii) vitamin E may also exert a role in the modulation of the biosynthesis of OPDA, with a much smaller effect, if any, on other jasmonates, and (iii) a trade-off in the accumulation of vitamin E and jasmonates might occur in the regulation of biotic and abiotic stress responses in plants.

Main conclusion Methyl jasmonate promotes the synthesis of rosmarinic acid in Salvia miltiorrhiza Bunge and Salvia castanea f. tomentosa Stib, and it promotes the latter more strongly. Abstract Salvia miltiorrhiza Bunge (SMB) is a traditional Chinese medicinal material, its water-soluble phenolic acid component rosmarinic acid has very important medicinal value. Salvia castanea f. tomentosa Stib (SCT) mainly distributed in Nyingchi, Tibet. Its pharmacological effects are similar to SMB, but its rosmarinic acid is significantly higher than the former. Methyl jasmonate (MJ) as an inducer can induce the synthesis of phenolic acids in SMB and SCT. However, the role of MJ on rosmarinic acid in SMB is controversial. Therefore, this study used SMB and SCT hair root as an experimental material and MJ as a variable. On one hand, exploring the controversial reports in SMB; on the other hand, comparing the differences in the mechanism of action of MJ on the phenolic acids in SMB and SCT. The content of related metabolites and the expression of key genes in the synthesis pathway of rosmarinic acid was analyzed by 1H-NMR combined with qRT-PCR technology. Our research has reached the following conclusions: first of all, MJ promotes the accumulation of rosmarinic acid and related phenolic acids in the metabolic pathways of SMB and SCT. After MJ treatment, the content of related components and gene expression are increased. Second, compared to SMB, SCT has a stronger response to MJ. It is speculated that the different responses of secondary metabolism-related genes to MJ may lead to different metabolic responses of salvianolic acid between the two. Electronic supplementary material The online version of this article (10.1007/s00425-020-03506-y) contains supplementary material, which is available to authorized users.

Transcriptomic studies in resistant and susceptible tea cultivars have been performed to reveal the different defense molecular mechanisms of tea after E. onukii feeding. The molecular mechanism by which tea plants respond to small green leafhopper Empoasca onukii (Matsuda) damage is unclear. Using the resistant tea plant cultivar Juyan (JY) and the susceptible tea plant cultivar Enbiao (EB) as materials, this study performed RNA-seq on tea leaf samples collected at three time points (6 h, 12 h, 24 h) during exposure of the plants to leafhopper to reveal the molecular mechanisms that are activated in susceptible and resistant tea plant cultivars in response to leafhopper damage. The numbers of DEGs in the susceptible tea cultivar during early (6 h) and late (24 h) stages of leafhopper induction were higher than those in the resistant cultivar at the same time points. The stress responses to leafhopper were most intense at 12 h in both tea cultivars. Pathway enrichment analysis showed that most up-regulated DEGs and their related metabolic pathways were similar in the two tea cultivars. However, during the early stage of leafhopper induction (6 h), jasmonic acid (JA)-related genes were significantly up-regulated in the resistant cultivar. The terpenoid biosynthetic pathway and the α-linolenic acid metabolic pathway were activated earlier in the resistant cultivar and remained activated until the late stage of leafhopper damage. Our results confirmed that after leafhopper damage, the resistant tea cultivar activated its defense responses earlier than the susceptible cultivar, and these defense responses were mainly related to terpenoid metabolism and JA biosynthetic pathway. The results provide important clues for further studies on resistance strategy of tea plants to pest.

Ubiquitin ligase VpRH2 is a negative regulator in the grape ABA pathway by inhibiting ABL1, PYR1 and GRP2A expressions, and its promoter is inhibited by ABA treatment. In higher plants, ubiquitin ligases play key roles in various cellular processes. As in our previous study (Wang et al. in J Exp Bot 68:1669-1687, 2017), grape RING-H2-type ubiquitin ligase gene VpRH2 and its promoter was induced by powdery mildew and showed resistance to the disease. Diverse small-molecule hormones, like salicylic acid (SA), methyl jasmonate (MeJA) or abscisic acid (ABA), play pivotal roles in plant resistance. Here we found that VpRH2 expression could be induced by SA and MeJA treatment, but inhibited by ABA treatment. The promoter of VpRH2 revealed a similar variation trend under exogenous hormone treatments as the gene expression by GUS activity assay. By a series of deletion fragments, the promoter fragment of VpRH2-P656 to VpRH2-P513 was necessary in response to MeJA treatment, and the inhibition of ABA treatment to the VpRH2 promoter was independent of the ABRE motif. Over-expression of VpRH2 in Arabidopsis thaliana plants displayed ABA-insensitive phenotypes at the germination stage compared to wild type plants. In VpRH2 over-expressing Vitis vinifera cv. Thompson Seedless plants after ABA treatments, the expression of the ABA pathway related genes ABL1 and PYR1 showed a suppresive trend. Moreover, VpGRP2A (an VpRH2-interacting protein) also showed a suppresive trend in response to ABA treatment in VpRH2-overexpressing plants. Our results demonstrate that VpRH2 is a negative regulator in the grape ABA signal pathway by inhibiting ABL1, PYR1 and GRP2A expressions, and its promoter was also inhibited by ABA treatment.

In the current study, we showed that exogenous GABA supplementation increases the endogenous GABA level, several amino acids, and phytohormones in citrus plants, suggesting that GABA works in harmony with phytohormones. Gamma-aminobutyric acid (GABA) plays a key role in cytosolic regulation of pH, controlling of carbon and nitrogen metabolism, and protection against biotic and abiotic stresses. Although it is well-known that GABA is implicated in plant defense and it could act as a signaling molecule, its effect on phytohormones is not completely understood. In this study, we investigated the effect of exogenous GABA on citrus phytohormones using gas chromatography-mass spectrometry. A significant increase in endogenous GABA was observed in GABA-treated plants. The highest increase in GABA was recorded in plants treated with 10 mM 7 days post-treatment. In addition, we observed a moderate increase in several amino acids including glycine, L-alanine, L-proline, L-asparagine, and L-glutamine. The levels of benzoic acid, cinnamic acid, salicylic acid, trans-jasmonic acid, indole acetic acid, indole propionic acid, and abscisic acid were significantly increased in GABA-treated plants compared to the control. The gene expression showed that GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) were induced in GABA-treated plants, indicating a conversion of GABA to succinate. In addition, the gene expression of the regulatory enzymes of the TCA cycle (malate dehydrogenase and succinic dehydrogenase) was upregulated in GABA-treated plants, indicating an induction of respiration. In agreement with the chemical analysis, the gene expression results showed that most of the genes implicated in the biosynthesis of phytohormones were also upregulated in GABA-treated plants. Our results indicated that GABA works in harmony with phytohormones and suggested that regulation of phytohormones by exogenous GABA could play a key role in reducing plant stress.

The jasmonic acid (JA)-responsive transcription factor OsMYC2 acts as a positive regulator of leaf senescence by direct regulation of some senescence-associated genes in rice. OsMYC2, a transcription factor (TF), acts as a positive regulator of jasmonic acid (JA) signaling involved in development and defense in rice. Here, we report that OsMYC2 plays an important role in leaf senescence under dark-induced senescence (DIS) conditions. Overexpression of OsMYC2 significantly promoted leaf senescence, indicated by reduction of chlorophyll content under DIS conditions in rice. Leaf senescence under the DIS conditions was negatively regulated by OsJAZ8, a rice jasmonate ZIM-domain protein involved in the JA signaling pathway. OsMYC2 upregulated the expression of some senescence-associated genes (SAGs) and selectively bound to the G-box/G-box-like motifs in the promoters of some SAGs in vivo. These results suggest that OsMYC2 acts as a positive regulator of leaf senescence by direct- or indirect-regulation of SAGs in rice.

This study describes a new role for hydroperoxide lyase branch of oxylipin biosynthesis pathway in protecting photosynthetic apparatus under high light conditions. Lipid-derived signaling molecules, oxylipins, produced by a multi-branch pathway are central in regulation of a wide range of functions. The two most known branches, allene oxide synthase (AOS) and 13-hydroperoxide lyase (HPL) pathways, are best recognized as producers of defense compounds against biotic challenges. In the present work, we examine the role of these two oxylipin branches in plant tolerance to the abiotic stress, namely excessive light. Towards this goal, we have analyzed variable chlorophyll fluorescence parameters of intact leaves of Arabidopsis thaliana genotypes with altered oxylipin profile, followed by examining the impact of exogenous application of selected oxylipins on functional activity of photosynthetic apparatus in intact leaves and isolated thylakoid membranes. Our findings unequivocally bridge the function of oxylipins to photosynthetic processes. Specifically, HPL overexpressing lines display enhanced adaptability in response to high light treatment as evidenced by lower rate constant of photosystem 2 (PS2) photoinhibition and higher rate constant of PS2 recovery after photoinhibition. In addition, exogenous application of linolenic acid, 13-hydroperoxy linolenic acid, 12-oxophytodienoic acid, and methyl jasmonate individually, suppresses photochemical activity of PS2 in intact plants and isolated thylakoid membranes, while application of HPL-branch metabolites—does not. Collectively these data implicate function of HPL branch of oxylipin biosynthesis pathway in guarding PS2 under high light conditions, potentially exerted through tight regulation of free linolenic acid and 13-hydroperoxy linolenic acid levels, as well as competition with production of metabolites by AOS-branch of the oxylipin pathway.

Co-expression and regulatory networks yield important insights into the growth-defense tradeoffs mechanism under jasmonic acid (JA) signals in Arabidopsis. Elevated defense is commonly associated with growth inhibition. However, a comprehensive atlas of the genes associated with the plant growth-defense tradeoffs under JA signaling is lacking. To gain an insight into the dynamic architecture of growth-defense tradeoffs, a coexpression network analysis was employed on publicly available high-resolution transcriptomes of Arabidopsis treated with coronatine (COR), a mimic of jasmonoyl-l-isoleucine. The genes involved in JA-mediated growth-defense tradeoffs were systematically revealed. Promoter enrichment analysis revealed the core regulatory module in which the genes underwent rapid activation, sustained upregulation after COR treatment, and mediated the growth-defense tradeoffs. Several transcription factors (TFs), including RAP2.6L, MYB44, WRKY40, and WRKY18, were identified as instantly activated components associated with pathogen and insect resistance. JA might rapidly activate RAV1 and KAN1 to repress brassinosteroid (BR) response genes, upregulate KAN1, the C2H2 TF families ZF2, ZF3, ZAT6, and STZ/ZAT10 to repress the biosynthesis, transport, and signaling of auxin to arrest growth. Independent datasets and preserved analyses validated the reproducibility of the results. Our study provided a comprehensive snapshot of genes that respond to JA signals and provided valuable resources for functional studies on the genetic modification of breeding population that exhibit robust growth and defense simultaneously.

Main conclusion: Jasmonic acid (JA) negatively regulates stomatal development by promoting LCD expression and hydrogen sulfide (H2S) biosynthesis. H2S inhibits the initiation of stomata formation and acts upstream of SPEECHLESS. Abstract: Stomatal development is strictly regulated by endogenous signals and environmental cues. We recently revealed that jasmonic acid (JA) negatively regulates stomatal development in Arabidopsis thaliana cotyledons (Han et al., Plant Physiol 176:2871-2885, 2018), but the underlying molecular mechanism remains largely unknown. Here, we uncovered a role for H2S in regulating stomatal development. The H2S scavenger hypotaurine reversed the JA-induced repression of stomatal development in the epidermis of wild-type Arabidopsis. The H2S-deficient mutant lcd displayed increased stomatal density and stomatal index values, which were rescued by treatment with sodium hydrosulfide (NaHS; an H2S donor) but not JA, suggesting that JA-mediated repression of stomatal development is dependent on H2S biosynthesis. The high stomatal density of JA-deficient mutants was rescued by exogenous NaHS treatment. Further analysis indicated that JA positively regulates LCD expression, L-cysteine desulfhydrases (L-CDes) activity, and endogenous H2S content. Furthermore, H2S represses the expression of stomate-associated genes and functions downstream of stomate-related signaling pathway components TOO MANY MOUTHS (TMM) and STOMATAL DENSITY AND DISTRIBUTION1 (SDD1) and upstream of SPEECHLESS (SPCH). Therefore, H2S acts downstream of JA signaling to regulate stomatal development in Arabidopsis cotyledons.

Pseudomonas fluorescens induces gibberellin and ethylene signaling via hydrogen peroxide in planta . Ethylene activates abscisic acid signaling. Hormones increase sesquiterpenoid biosynthesis gene expression and enzyme activity, inducing essential oil accumulation. Atractylodes lancea is a famous Chinese medicinal plant, whose main active components are essential oils. Wild A. lancea has become endangered due to habitat destruction and over-exploitation. Although cultivation can ensure production of the medicinal material, the essential oil content in cultivated A. lancea is significantly lower than that in the wild herb. The application of microbes as elicitors has become an effective strategy to increase essential oil accumulation in cultivated A. lancea. Our previous study identified an endophytic bacterium, Pseudomonas fluorescens ALEB7B, which can increase essential oil accumulation in A. lancea more efficiently than other endophytes; however, the underlying mechanisms remain unknown (Physiol Plantarum 153:30-42, 2015; Appl Environ Microb 82:1577-1585, 2016). This study demonstrates that P. fluorescens ALEB7B firstly induces hydrogen peroxide (H2O2) signaling in A. lancea, which then simultaneously activates gibberellin (GA) and ethylene (ET) signaling. Subsequently, ET activates abscisic acid (ABA) signaling. GA and ABA signaling increase expression of HMGR and DXR, which encode key enzymes involved in sesquiterpenoid biosynthesis, leading to increased levels of the corresponding enzymes and then an accumulation of essential oils. Specific reactive oxygen species and hormone signaling cascades induced by P. fluorescens ALEB7B may contribute to high-efficiency essential oil accumulation in A. lancea. Illustrating the regulation mechanisms underlying P. fluorescens ALEB7B-induced essential oil accumulation not only provides the theoretical basis for the inducible synthesis of terpenoids in many medicinal plants, but also further reveals the complex and diverse interactions among different plants and their endophytes.

Main conclusion A metabolic profiling including calculation of energy cost of amino acids biosynthesis in cultured cells ofScrophularia striatashowed that methyl jasmonate-inducible oxidative stress elicited secondary metabolites formation derived from phenylalanine and tyrosine and increased energy cost for these amino acids biosynthesis. Understanding of the metabolic pathways in cell culture of Scrophularia striata, an aromatic plant species, facilitates means of production of pharmaceutical metabolites under oxidative stress. In this study, we evaluated the effects of MeJA on the S. striata metabolic pathway and the responses to oxidative stress. Exposure to methyl jasmonate (MeJA) affects plant growth, effectively induces production of reactive oxygen species (ROS) and inserts oxidative stress at the cellular level which results in alteration of primary metabolites and production of phenylepropanoid compounds. Cells treated with MeJA indicated increase in the activities of three antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPx) as well as intracellular H2O2 and MDA contents compared with mock-treated cells. High performance liquid chromatography (HPLC)-based metabolome analysis revealed dynamic metabolic changes in oxidatively stressed S. striata cells, e.g., general phenylpropanoid pathway, phenylethanoid-glycosides, lignans, and increased energy cost of biosynthesis and accumulation of amino acids. Furthermore, principal component analysis (PCA)—derived score plots demonstrated that MeJA affects cellular metabolism in S. striata cells and significantly alters metabolite composition under MeJA-inducible oxidative stress. These observations suggest that MeJA-elicited cell suspension cultures of S. striata balanced the production of primary and secondary metabolites in coordination with ROS-scavenging system.

OsPGIP4 overexpression enhances resistance to bacterial leaf streak in rice. Polygalacturonase-inhibiting proteins are thought to play important roles in the innate immunity of rice against fungi. Here, we show that the chromosomal location of OsPGIP4 coincides with the major bacterial leaf streak resistance quantitative trait locus qBlsr5a on the short arm of chromosome 5. OsPGIP4 expression was up-regulated upon inoculation with the pathogen Xanthomonas oryzae pv. oryzicola strain RS105. OsPGIP4 overexpression enhanced the resistance of the susceptible rice variety Zhonghua 11 to RS105. In contrast, repressing OsPGIP4 expression resulted in an increase in disease lesions caused by RS105 in Zhonghua 11 and in Acc8558, a qBlsr5a resistance donor. More interestingly, upon inoculation, the activated expression of pathogenesis-related genes was attenuated for those genes involved in the salicylic acid pathway, while the activated expression of jasmonic acid pathway markers was increased in the overexpression lines. Our results not only provide the first report that rice PGIP could enhance resistant against a bacterial pathogen but also indicate that OsPGIP4 is a potential component of the qBlsr5a locus for bacterial leaf streak in rice.

MYB15 promoter of Vitis quinquangularis has potential as a target for disease resistance breeding, and its involvement in PTI is associated with a range of defense mechanisms. China is a center of origin for Vitis and is home to diverse wild Vitis genotypes, some of which show superior pathogen resistance, although the underlying molecular basis for this has not yet been elucidated. In the current study, we identified a transcription factor, MYB15, from the Chinese wild grape, Vitis quinquangularis, whose promoter region (pVqMYB15) was shown to be induced by basal immunity (also called PAMP-triggered immunity, PTI) triggered by flg22, following heterologous expression in Nicotiana benthamiana and homologous expression in grapevine. By analyzing the promoter structure and activity, we identified a unique 283 bp sequence that plays a key role in the activation of basal immunity. In addition, we showed that activation of the MYB15 promoter correlates with differences in the expression of MYB15 and RESVERATROL SYNTHASE (RS) induced by the flg22 elicitor. We further tested whether the MYB15 induction triggered by flg22 was consistent with MYB15 and RS expression following inoculation with Plasmopara viticola in grape (V. quinquangularis and Vitis vinifera) leaves. Mapping upstream signals, we found that calcium influx, an RboH-dependent oxidative burst, an MAPK cascade, and jasmonate and salicylic acid co-contributed to flg22-triggered pVqMYB15 activation. Our data suggest that the MYB15 promoter has potential as a target for disease resistance breeding, and its involvement in PTI is associated with a range of defense mechanisms.

Main conclusion The results provide a significant verification of the activity of exogenously applied phytohormones: gibberellic acid, jasmonic acid, and brassinolide in the modulation of the plant’s response to nickel treatment. Abstract The study investigated nickel accumulation and its toxicity to Daphne jasminea shoots cultured in vitro with or without exogenous supplementation with phytohormones: gibberellic acid (GA3), jasmonic acid (JA), and brassinolide (BL). The aim was to verify the modulatory effect of exogenous plant growth regulators (PGRs) on plant reaction to Ni excess. The combined action of Ni and PGRs was evaluated at the anatomical, ultrastructural, and biochemical levels. Nickel toxicity was manifested in decreased biomass accretion and growth tolerance index (83–53.6%), attributed to enhanced synthesis of growth inhibitors, mainly abscisic acid. As a defence reaction, endogenous gibberellins accumulated. Exogenous GA3 ameliorated the plant reaction to Ni stress, inducing proliferation and growth rate. Ni tolerance in the presence of GA3 was attributed to peroxisomal reactions that stimulated the synthesis of endogenous JA. In contrast, the application of BL caused enhanced Ni accumulation. Plants suffered from pronounced stress due to massive oxidation. The defence strategy of plants subjected to Ni and BL involved cell wall rearrangements. Exogenous JA stimulated the synthesis of active auxins and salicylic acid, contributing to enhanced mitotic activity within explants. However, JA disturbed the integrity of chloroplasts and lamellar compartments. Our study revealed that an action of exogenous PGRs may either enhance tolerance to Ni or increase metal toxicity in D. jasminea. Particularly in in vitro culture, where explants are subjected to external phytohormonal stimuli, the combined effects of supplemental PGRs may enhance stress and substantially affect plant development. Our results provide a significant verification of exogenous PGRs activity in the modulation of plant response to nickel. Electronic supplementary material The online version of this article (10.1007/s00425-018-2979-6) contains supplementary material, which is available to authorized users.

Silencing of miR156 in rice confers enhanced resistance to brown planthopper through reducing JA and JA-Ile biosynthesis. Rice brown planthopper (BPH, Nilaparvata lugens Stal) threatens the sustainability of rice production and global food security. Due to the rapid adaptation of BPH to current germplasms in rice, development of novel types of resistant germplasms becomes increasingly important. Plant ontogenetic defense against pathogen and herbivores offers a broad spectrum and durable resistance, and has been experimentally tested in many plants; however, the underlying molecular mechanism remains unclear. miR156 is the master regulator of ontogeny in plants; modulation of miR156 is, therefore, expected to cause corresponding changes in BPH resistance. To test this hypothesis, we silenced miR156 using a target mimicry method in rice, and analyzed the resistance of miR156-silenced plants (MIM156) to BPH. MIM156 plants exhibited enhanced resistance to BPH based on analyses of honeydew excretion, nymph survival, fecundity of BPH, and the survival ratio of rice plants after BPH infestation. Molecular analysis indicated that the expression of MPK3, MPK6, and WRKY70, three genes involved in BPH resistance and jasmonic acid (JA) signaling, was altered in MIM156 plants. The JA and bioactive jasmonoyl-isoleucine levels and the expression of genes involved in JA biosynthesis were significantly reduced in MIM156 plants. Restoration of JA level by exogenous application increased the number of BPH feeding on MIM156 plants and reduced its resistance to BPH. Our findings suggest that miR156 negatively regulates BPH resistance by increasing JA level in rice; therefore, modulation of miR156-SPLs’ pathway may offer a promising way to breed rice varieties with enhanced resistance against BPH and elite agronomically important traits.

Biotic stresses induce the expression of mulberry cystatins. MaCPI-4 protein is stable in silkworm digestive fluid and accumulates in gut food debris and frass. Plant cystatins are considered to be involved in defense responses to insect herbivores though little is known about how cystatins from the natural host respond to a specialist herbivory and the following postingestive interaction is also poorly understood. Here, we studied the biotic stress-mediated inductions of cystatins from mulberry tree, and examined the stability of mulberry cystatin proteins in the gut of silkworm, Bombyx mori, a specialist insect feeding on mulberry leaf. First, we cloned and characterized six cystatin genes from a mulberry cultivar, Morus atropurpurea Roxb., named as MaCPI-1 to MaCPI-6. The recombinant MaCPI-1, MaCPI-3 and MaCPI-4 proteins, which showed inhibitory effects against papain in vitro, were produced. Silkworm herbivory as well as methyl jasmonate (MeJA) treatment induced the expression of five mulberry cystatin genes, and the highest inductions were observed from MaCPI-1 and MaCPI-6. Mechanical wounding led to the inductions of four cystatin genes. The differential induction occurred in MaCPI-2. The induced protein changes were detected from three mulberry cystatins comprising MaCPI-1, MaCPI-3 and MaCPI-4. In vivo and in vitro assays showed that MaCPI-1 and MaCPI-3 proteins were susceptible to silkworm digestive fluid and MaCPI-4 had an antidigestive stability, and was detected in silkworm gut and frass. Collectively, our data indicated that biotic stresses resulted in the transcriptional inductions and protein changes of mulberry cystatins (MaCPIs), and identified MaCPI-4 with stability in the gut of its specialist herbivore.

The application of jasmonic acid results in an increased secondary growth, as well as additional secondary phloem fibres and higher lignin content in the hypocotyl of textile hemp (Cannabis sativa L.). Secondary growth provides most of the wood in lignocellulosic biomass. Textile hemp (Cannabis sativa L.) is cultivated for its phloem fibres, whose secondary cell wall is rich in crystalline cellulose with a limited amount of lignin. Mature hemp stems and older hypocotyls are characterised by large blocks of secondary phloem fibres which originate from the cambium. This study aims at investigating the role of exogenously applied jasmonic acid on the differentiation of secondary phloem fibres. We show indeed that the exogenous application of this plant growth regulator on young hemp plantlets promotes secondary growth, differentiation of secondary phloem fibres, expression of lignin-related genes, and lignification of the hypocotyl. This work paves the way to future investigations focusing on the molecular network underlying phloem fibre development.

N-decanoyl-homoserine lactone activates plant systemic resistance against Botrytis cinerea in tomato plants, which is largely dependent on jasmonic acid biosynthesis and signal transduction pathways. Rhizosphere bacteria secrete N-acylated-homoserine lactones (AHLs), a type of specialized quorum-sensing signal molecule, to coordinate their population density during communication with their eukaryotic hosts. AHLs behave as low molecular weight ligands that are sensed by plants and promote the host’s resistance against foliar pathogens. In this study, we report on N-decanoyl-homoserine lactone (DHL), which is a type of AHL that induces systemic immunity in tomato plants and protects the host organism against the necrotrophic fungus Botrytis cinerea. Upon DHL treatment, tomato endogenous jasmonic acid (JA) biosynthesis (rather than salicylic acid biosynthesis) and signal transduction were significantly activated. Strikingly, the DHL-induced systemic resistance against B. cinerea was blocked in the tomato JA biosynthesis mutant spr2 and JA signaling gene-silenced plants. Our findings highlight the role of DHL in systemic resistance against economically important necrotrophic pathogens and suggest that DHL-induced immunity against B. cinerea is largely dependent on the JA signaling pathway. Manipulation of DHL-induced resistance is an attractive disease management strategy that could potentially be used to enhance disease resistance in diverse plant species.

Pyrroloquinazoline alkaloids are medicinally important compounds, determined by HPLC from cell cultures of Adhatoda vasica . The maximum production of vasicinone (12-fold) and vasicine (8.3-fold) was enhanced by stimulating the anthranilate synthase activity via feeding of tryptophan and sorbitol. The decoction of Adhatoda vasica leaves is used for the treatment of throat irritations, inflammations and recommended as expectorant. The plant species contains pyrroloquinazoline alkaloids and has been reported to demonstrate various biological activities. To investigate the effect of elicitors to increase the production of alkaloids, five groups (auxins and cytokinins, biotic elicitors, polysaccharides, amino acids and salts) of elicitors were evaluated. Maximum production of vasicinone (72.74 ± 0.74 mg/g DW; 12-fold) and vasicine (99.44 ± 0.28 mg/g DW; 8.3-fold) was enhanced by feeding of tryptophan and sorbitol at 50 mM concentration in cell cultures. Fourteen free amino acids were estimated from the elicited cells. Sorbitol stimulated up to a maximum accumulation of serine (8.2-fold). The maximal anthranilate synthase (AS) activity (7.5 ± 0.47 pkat/mg protein; 2.9-fold) was induced by salicylic acid and sorbitol. Anthranilate synthase functions as rate-limiting factor for the biosynthesis of pyrroloquinazoline alkaloids. Our results support the widespread use of tryptophan and sorbitol as elicitors to raise the production of vasicinone, vasicine, 2-acetyl benzyl amine and other pyrroloquinazoline alkaloids in cell cultures of A. vasica.

Transgenic mustard plants ( Brassica juncea ) expressing non-allergenic and biologically safe RiD peptide show higher tolerance against Lipaphis erysimi. Rorippa indica defensin (RiD) has previously been reported as a novel insecticidal protein derived from a wild crucifer Rorippa indica. RiD was found to have an effective insecticidal property against mustard aphid, Lipaphis erysimi. In the present study, RiD was highly upregulated in R. indica during aphid infestation initiating a defense system mediated by jasmonic acid (JA), but not by salicylic acid (SA)/abscisic acid (ABA). RiD has also been assessed for biosafety according to the FAO/WHO guideline (allergenicity of genetically modified foods; Food And Agriculture Organisation of the United Nations, Rome, Italy, 2001) and Codex Alimentarius Guideline (Guidelines for the design and implementation of national regulatory food safety assurance programme associated with the use of veterinary drugs in food producing animals. Codex Alimentarius Commission. GL, pp 71–2009, 2009). The purified protein was used to sensitize BALB/c mice and they showed normal histopathology of lung and no elevated IgE level in their sera. As the protein was found to be biologically safe and non-allergenic, it was used to develop transgenic Brassica juncea plants with enhanced aphid tolerance, which is one of the most important oilseed crops and is mostly affected by the devastating pest—L. erysimi. The transgene integration was monitored by Southern hybridization, and the positive B. juncea lines were further analyzed by Western blot, ELISA, immunohistolocalization assays and in planta insect bioassay. Transgenic plants expressing RiD conferred a higher level of tolerance against L. erysimi. All these results demonstrated that RiD is a novel, biologically safe, effective insecticidal agent and B. juncea plants expressing RiD are important components of integrated pest management.

Main conclusion The ERF022 gene was found to affect embryogenic transition in somatic cells in Arabidopsis via the ethylene-related pathway. The study provides evidence that ERF022 - LEC2 interaction is involved in the auxin–ethylene crosstalk that operates in somatic embryogenesis induction. The ERF022 gene of the ERF family was previously identified among the transcription factor genes that were differentially expressed in an embryogenic culture of Arabidopsis. A strong inhibition of the gene was found to be associated with the induction of somatic embryogenesis (SE) and an erf022 mutant was indicated to display a substantially impaired capacity for SE. Therefore, the molecular function of ERF022 in the induction of SE was studied in the present work. A phenotype of an erf022 mutant was indicated as being related to an increased content of ethylene. The results further suggest that the ERF022 controls the genes that are involved in both the biosynthesis (ACS7) and signalling (ERF1, ETR1) of ethylene and indicate that the ERF022 is a new regulatory element in ethylene-related responses that negatively control the ethylene content and perception. It is proposed that the negative impact of ethylene on the induction of SE may result from a modulation of the auxin-related genes that control the embryogenic transition in somatic cells. Among them, the LEC2, which is a key regulator of the induction of SE through the stimulation of auxin synthesis, was possibly related to ERF022. The results of the study provide new hormone-related clues to define the genetic network that governs SE. A putative model of the regulatory pathway is proposed that is involved in the induction of SE in which the auxin–ethylene interactions are controlled by ERF022 and LEC2 and their targets. Electronic supplementary material The online version of this article (doi:10.1007/s00425-014-2225-9) contains supplementary material, which is available to authorized users.

Multiplicity of protease inhibitors induced by predators may increase the understanding of a plant’s intelligent behavior toward environmental challenges. Information about defense mechanisms of non-genomic model plant passion fruit (Passiflora edulis Sims) in response to predator attack is still limited. Here, via biochemical approaches, we showed its flexibility to build-up a broad repertoire of potent Kunitz-type trypsin inhibitors (KTIs) in response to methyl jasmonate. Seven inhibitors (20–25 kDa) were purified from exposed leaves by chromatographic techniques. Interestingly, the KTIs possessed truncated Kunitz motif in their N-terminus and some of them also presented non-consensus residues. Gelatin-Native-PAGE established multiple isoforms for each inhibitor. Significant differences regarding inhibitors’ activity toward trypsin and chymotrypsin were observed, indicating functional polymorphism. Despite its rarity, two of them also inhibited papain, and such bifunctionality suggests a recruiting process onto another mechanistic class of target protease (cysteine-type). All inhibitors acted strongly on midgut proteases from sugarcane borer, Diatraea saccharalis (a lepidopteran insect) while in vivo assays supported their insecticide properties. Moreover, the bifunctional inhibitors displayed activity toward midgut proteases from cowpea weevil, Callosobruchus maculatus (a coleopteran insect). Unexpectedly, all inhibitors were highly effective against midgut proteases from Aedes aegypti a dipteran insect (vector of neglected tropical diseases) opening new avenues for plant-derived PIs for vector control-oriented research. Our results reflect the KTIs’ complexities in passion fruit which could be wisely exploited by influencing plant defense conditions. Therefore, the potential of passion fruit as source of bioactive compounds with diversified biotechnological application was strengthened.

The productivity of Brassica oilseeds is severely affected by its major pest: aphids. Unavailability of resistance source within the crossable germplasms has stalled the breeding efforts to derive aphid resistant cultivars. In this study, jasmonate-mediated host defense in Indian mustard Brassica juncea (L.) Czern. was evaluated and compared with regard to its elicitation in response to mustard aphid Lipaphis erysimi (Kalt.) and the defense elicitor methyl jasmonate (MeJ). Identification of jasmonate-induced unigenes in B. juncea revealed that most are orthologous to aphid-responsive genes, identified in taxonomically diverse plant-aphid interactions. The unigenes largely represented genes related to signal transduction, response to biotic and abiotic stimuli and homeostasis of reactive oxygen species (ROS), in addition to genes related to cellular and metabolic processes involved in cell organization, biogenesis, and development. Gene expression studies revealed induction of the key jasmonate biosynthetic genes (LOX, AOC, 12-OPDR), redox genes (CAT3 and GST6), and other downstream defense genes (PAL, ELI3, MYR, and TPI) by several folds, both in response to MeJ and plant-wounding. However, interestingly aphid infestation even after 24 h did not elicit any activation of these genes. In contrast, when the jasmonate-mediated host defense was elicited by exogenous application of MeJ the treated B. juncea plants showed a strong antibiosis effect on the infesting aphids and reduced the growth of aphid populations. The level of redox enzymes CAT, APX, and SOD, involved in ROS homeostasis in defense signaling, and several defense enzymes viz. POD, PPO, and PAL, remained high in treated plants. We conclude that in B. juncea, the jasmonate activated endogenous-defense, which is not effectively activated in response to mustard aphids, has the potential to reduce population growth of mustard aphids.

Soil-borne fungal pathogen, Fusarium oxysporum causes major economic losses by inducing necrosis and wilting symptoms in many crop plants. Management of fusarium wilt is achieved mainly by the use of chemical fungicides which affect the soil health and their efficiency is often limited by pathogenic variability. Hence understanding the nature of interaction between pathogen and host may help to select and improve better cultivars. Current research evidences highlight the role of oxidative burst and antioxidant enzymes indicating that ROS act as an important signaling molecule in banana defense response against Fusarium oxysporum f.sp. cubense. The role of jasmonic acid signaling in plant defense against necrotrophic pathogens is well recognized. But recent studies show that the role of salicylic acid is complex and ambiguous against necrotrophic pathogens like Fusarium oxysporum, leading to many intriguing questions about its relationship between other signaling compounds. In case of banana, a major challenge is to identify specific receptors for effector proteins like SIX proteins and also the components of various signal transduction pathways. Significant progress has been made to uncover the role of defense genes but is limited to only model plants such as Arabidopsis and tomato. Keeping this in view, we review the host response, pathogen diversity, current understanding of biochemical and molecular changes that occur during host and pathogen interaction. Developing resistant cultivars through mutation, breeding, transgenic and cisgenic approaches have been discussed. This would help us to understand host defenses against Fusarium oxysporum and to formulate strategies to develop tolerant cultivars.

Inducible defenses that provide enhanced resistance to insect attack are nearly universal in plants. The defense-signaling cascade is mediated by the synthesis, movement, and perception of jasmonate (JA) and the interaction of this signaling molecule with other plant hormones and messengers. To explore how the interaction of JA and ethylene influences induced defenses, we employed the never-ripe (Nr) tomato mutant, which exhibits a partial block in ethylene perception, and the defenseless (def1) mutant, which is deficient in JA biosynthesis. The defense gene proteinase inhibitor (PIN2) was used as marker to compare plant responses. The Nr mutant showed a normal wounding response with PIN2 induction, but the def1 mutant did not. As expected, methyl JA (MeJA) treatment restored the normal wound response in the def1 mutant. Exogenous application of MeJA increased resistance to Helicoverpa zea, induced defense gene expression, and increased glandular trichome density on systemic leaves. Exogenous application of ethephon, which penetrates tissues and decomposes to ethylene, resulted in increased H. zea growth and interfered with the wounding response. Ethephon treatment also increased salicylic acid in systemic leaves. These results indicate that while JA plays the main role in systemic induced defense, ethylene acts antagonistically in this system to regulate systemic defense.

Main conclusion Representational difference analysis of cDNA was performed and differential products were sequenced and annotated. Candidate genes involved in biosynthesis of diosgenin in fenugreek were identified. Detailed mechanism of diosgenin synthesis was proposed. Fenugreek (Trigonella foenum-graecum L.) is a valuable medicinal and crop plant. It belongs to Fabaceae family and has a unique potential to synthesize valuable steroidal saponins, e.g., diosgenin. Elicitation (methyl jasmonate) and precursor feeding (cholesterol and squalene) were used to enhance the content of sterols and steroidal sapogenins in in vitro grown plants for representational difference analysis of cDNA (cDNA-RDA). To identify candidate genes involved in diosgenin biosynthesis, differential, factor-specific libraries were subject to the next-generation sequencing. Approximately 9.9 million reads were obtained, trimmed, and assembled into 31,491 unigenes with an average length of 291 bp. Then, functional annotation and gene ontogeny enrichment analysis was performed by aligning all-unigenes with public databases. Within the transcripts related to sterol and steroidal saponin biosynthesis, we discovered novel candidate genes of diosgenin biosynthesis and validated their expression using quantitative RT-PCR analysis. Based on these findings, we supported the idea that diosgenin is biosynthesized from cycloartenol via cholesterol. This is the first report on the next-generation sequencing of cDNA-RDA products. Analysis of the transcriptomes enriched in low copy sequences contributed substantially to our understanding of the biochemical pathways of steroid synthesis in fenugreek. Electronic supplementary material The online version of this article (doi:10.1007/s00425-017-2657-0) contains supplementary material, which is available to authorized users.

A key tree species for the forest industry in Europe is Norway spruce [Picea abies (L.) Karst.]. One of its major diseases is stem and butt rot caused by Heterobasidion parviporum (Fr.) Niemela & Korhonen, which causes extensive revenue losses every year. In this study, we investigated the parallel induction of Norway spruce genes presumably associated with salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways previously observed in response to H. parviporum. Relative gene expression levels in bark samples of genes involved in the salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways after wounding and inoculation with either the saprotrophic biocontrol fungus Phlebiopsis gigantea or with H. parviporum were analysed with quantitative PCR at the site of the wound and at two distal locations from the wound/inoculation site to evaluate their roles in the induced defence response to H. parviporum in Norway spruce. Treatment of Norway spruce seedlings with methylsalicylate, methyljasmonate and inhibitors of the jasmonic acid/ethylene signalling pathway, as well as the Phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid were conducted to determine the responsiveness of genes characteristic of the different pathways to different hormonal stimuli. The data suggest that jasmonic acid-mediated signalling plays a central role in the induction of the genes analysed in this study irrespective of their responsiveness to salicylic acid. This may suggest that jasmonic acid-mediated signalling is the prioritized module in the Norway spruce defence signalling network against H. parviporum and that there seems to be no immediate antagonism between the modules in this interaction.

The role of defence gene expression triggered by Cd toxicity in the plant's response to Botrytis cinerea was investigated in Arabidopsis thaliana Columbia 0. Silicon (0 or 1.5 mM) and Cd (0, 1 or 10 μM) were supplied to 3-month-old solution-cultured plants. After 3 days, half of the plants of each treatment were inoculated with Botrytis. Supplied Cd concentrations were below the toxicity threshold and did not cause shoot growth inhibition or evidence of oxidative stress, while Botrytis infection severely decreased plant growth in all treatments. The expression of marker genes PR1 and BGL2 for the salicylic acid (SA) and the PDF1.2 for the jasmonic acid-ethylene (JA-ET) signalling pathways was enhanced in 10 μM Cd-treated non-infected plants. Twenty hours after inoculation, PDF1.2 expression showed a strong increase in all treatments, while enhanced PR1, BGL2, and CHIB expression was only found 7 days after infection. A great synergistic effect of Cd and Botrytis on PDF1.2 expression was found in 10 μM Cd-treated plants. Silicon decreased PR1, BGL2, and CHIB, while increasing PDF1.2 expression, which indicates its role as a modulator of the signalling pathways involved in the plant's response to fungal infection. Botrytis growth decreased in 10 μM Cd-treated plants, which could be due to the combined effects of Cd and Botrytis activating the SA and JA-ET-mediated signalling pathways. Taken together, our results provide support for the view that Cd concentrations close to the toxicity threshold induce defence signalling pathways which potentiate the plant's response against fungal infection.

Trichomes contribute to plant resistance against herbivory by physical and chemical deterrents. To better understand their role in plant defense, we systemically studied trichome morphology, chemical composition and the response of the insect herbivores Helicoverpa zea and Leptinotarsa decemlineata (Colorado potato beetle = CPB) on the tomato hairless (hl), hairy (woolly) mutants and wild-type Rutgers (RU) and Alisa Craig (AC) plants. Hairless mutants showed reduced number of twisted glandular trichomes (types I, IV, VI and VII) on leaf and stem compared to wild-type Rutgers (RU), while woolly mutants showed high density of non-glandular trichomes (types II, III and V) but only on the leaf. In both mutants, trichome numbers were increased by methyl jasmonate (MeJA), but the types of trichomes present were not affected by MeJA treatment. Glandular trichomes contained high levels of monoterpenes and sesquiterpenes. A similar pattern of transcript accumulation was observed for monoterpene MTS1 (=TPS5) and sesquiterpene synthase SST1 (=TPS9) genes in trichomes. While high density of non-glandular trichome on leaves negatively influenced CPB feeding behavior and growth, it stimulated H. zea growth. High glandular trichome density impaired H. zea growth, but had no effect on CPB. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that glandular trichomes highly express protein inhibitors (PIN2), polyphenol oxidase (PPOF) and hydroperoxide lyase (HPL) when compared to non-glandular trichomes. The SlCycB2 gene, which participates in woolly trichome formation, was highly expressed in the woolly mutant trichomes. PIN2 in trichomes was highly induced by insect feeding in both mutant and wild-type plants. Thus, both the densities of trichomes and the chemical defenses residing in the trichomes are inducible.

Dehydrins are highly hydrophilic proteins involved in playing key adaptive roles in response to abiotic stress conditions having dehydration as a common component. In the present study, a novel banana SK(3)-type dehydrin, MusaDHN-1, was identified and later characterized using transgenic banana plants to investigate its functions in abiotic stress tolerance. Expression profiling in native banana plants demonstrated that MusaDHN-1 was induced in leaves by drought, salinity, cold, oxidative and heavy metal stress as well as by treatment with signalling molecules like abscisic acid, ethylene and methyl jasmonate. Promoter analysis carried out by making a MusaDHN-1 promoter: β-glucuronidase fusion construct reconfirmed the abiotic stress inducibility of MusaDHN-1. Transgenic banana plants constitutively overexpressing MusaDHN-1 were phenotypically normal and displayed improved tolerance to drought and salt-stress treatments in both in vitro and ex vitro assays. Enhanced accumulation of proline and reduced malondialdehyde levels in drought and salt-stressed MusaDHN-1 overexpressing plants further established their superior performance in stressed conditions. This study is the first to report generation of transgenic banana plants engineered for improved drought and salt-stress tolerance.

Expression of a class V chitinase gene (At4g19810, AtChiC) in Arabidopsis thaliana was examined by quantitative real-time PCR and by analyzing microarray data available at Genevestigator. The gene expression was induced by the plant stress-related hormones abscisic acid (ABA) and jasmonic acid (JA) and by the stress resulting from the elicitor flagellin, NaCl, and osmosis. The recombinant AtChiC protein was produced in E. coli, purified, and characterized with respect to the structure and function. The recombinant AtChiC hydrolyzed N-acetylglucosamine oligomers producing dimers from the non-reducing end of the substrates. The crystal structure of AtChiC was determined by the molecular replacement method at 2.0 Å resolution. AtChiC was found to adopt an (β/α)(8) fold with a small insertion domain composed of an α-helix and a five-stranded β-sheet. From docking simulation of AtChiC with pentameric substrate, the amino acid residues responsible for substrate binding were found to be well conserved when compared with those of the class V chitinase from Nicotiana tabacum (NtChiV). All of the structural and functional properties of AtChiC are quite similar to those obtained for NtChiV, and seem to be common to class V chitinases from higher plants.

The elaborate networks and the crosstalk of established signaling molecules like salicylic acid (SA), jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), reactive oxygen species (ROS) and glutathione (GSH) play key role in plant defense response. To obtain further insight into the mechanism through which GSH is involved in this crosstalk to mitigate biotic stress, transgenic Nicotiana tabacum overexpressing Lycopersicon esculentum gamma-glutamylcysteine synthetase (LeECS) gene (NtGB lines) were generated with enhanced level of GSH in comparison with wild-type plants exhibiting resistance to pathogenesis as well. The expression levels of non-expressor of pathogenesis-related genes 1 (NPR1)-dependent genes like pathogenesis-related gene 1 (NtPR1), mitogen-activated protein kinase kinase (NtMAPKK), glutamine synthetase (NtGLS) were significantly enhanced along with NtNPR1. However, the expression levels of NPR1-independent genes like NtPR2, NtPR5 and short-chain dehydrogenase/reductase family protein (NtSDRLP) were either insignificant or were downregulated. Additionally, increase in expression of thioredoxin (NtTRXh), S-nitrosoglutathione reductase 1 (NtGSNOR1) and suppression of isochorismate synthase 1 (NtICS1) was noted. Comprehensive analysis of GSH-fed tobacco BY2 cell line in a time-dependent manner reciprocated the in planta results. Better tolerance of NtGB lines against biotrophic Pseudomonas syringae pv. tabaci was noted as compared to necrotrophic Alternaria alternata. Through two-dimensional gel electrophoresis (2-DE) and image analysis, 48 differentially expressed spots were identified and through identification as well as functional categorization, ten proteins were found to be SA-related. Collectively, our results suggest GSH to be a member in cross-communication with other signaling molecules in mitigating biotic stress likely through NPR1-dependent SA-mediated pathway.

Transgenic tobacco plants capable of over-expressing Xenopus PPARα (xPPARα), a transcription factor known to be required for peroxisome proliferation in animals, were recently generated. These plants (herewith referred to as PPAR-OE) were found to have increased peroxisome abundance, higher peroxisomal acyl-CoA oxidase and catalase activity and modified fatty acid metabolism. Further characterization of PPAR-OE plants revealed a higher susceptibility to virulent and a partial loss of resistance to avirulent Pseudomonas syringae pathogens, whereas the basal resistance response remained unaffected. Biochemical- and defense-related gene expression analyses showed that increased susceptibility to bacterial invasion coincided with the generalized reduction in H(2)O(2) and salicylic acid (SA) levels observed within the first 24 h of bacterial contact. Decreased H(2)O(2) levels were correlated with modified activity levels of catalase and other antioxidant enzymes. A correspondence between a rapid (within 1-24 hpi; ACCO and AOC) and sustained increase (up to 6 days pi; ACCO) in the expression levels of ethylene (ACCO) and jasmonic acid (AOC) biosynthetic genes and a higher susceptibility to virulent bacterial invasion was also observed in PPAR-OE plants. Conversely, no apparent differences in the short- and/or long-term expression levels of markers for the hypersensitive-response, oxidative burst and systemic-acquired resistance were observed between wild type and PPAR-OE plants. The results suggest that peroxisome proliferation could lead to increased susceptibility to bacterial pathogens in tobacco by altering the redox balance of the plant and the expression pattern of key defense signaling pathway genes.

The root of Bupleurum falcatum L. (Apiaceae) has long been one of the most important traditional herbal medicines in Asian countries. A group of triterpene saponins (saikosaponins) are the major constituents of this plant. Squalene synthase (SS) may play a regulatory role in directing triterpene intermediates and sterol pathways. Here, we investigated the regulatory role of the squalene synthase (BfSS1) gene in the biosynthesis of phytosterol and triterpene in B. falcatum. BfSS1 mRNA accumulated ubiquitously in plant organs and markedly increased in roots after treatment with methyl jasmonate (MeJA), ABA and ethephon. Transgenic B. falcatum constructs overexpressing BfSS1 in the sense and antisense orientations were assembled using the Agrobacterium-mediated method. Transgenic roots overexpressing BfSS1 in the sense orientation resulted in enhanced production of both phytosterol and saikosaponins. Overexpression of the BfSS1 gene in the sense orientation increased the mRNA accumulation of downstream genes such as squalene epoxidase and cycloartenol synthase but unexpectedly decreased the mRNA levels of β-amyrin synthase (β-AS), a triterpene synthase mRNA. MeJA treatment of wild-type roots strongly stimulated β-AS mRNA accumulation and saikosaponin production but suppressed phytosterol production. MeJA treatment of transgenic roots overexpressing BfSS1 in the sense orientation failed to stimulate β-AS mRNA accumulation but still enhanced saikosaponin and phytosterol production. These results indicate that overexpression of BfSS1 in B. falcatum regulates more powerfully the downstream genes than elicitor (MeJA) treatment in triterpene and phytosterol biosynthesis.

Jasmonoyl-L-isoleucine (JA-Ile) is a key jasmonate signal that probably functions in all plant species. The JASMONATE RESISTANT 1 (JAR1) enzyme synthesizes JA-Ile in Arabidopsis [Arabidopsis thaliana (L.) Heynh.], but a similar enzyme from tomato [Solanum lycopersicum (L.)] was not previously described. Tomato SlJAR1 has 66% sequence identity with Arabidopsis JAR1 and the SlJAR1-GST fusion protein purified from Escherichia coli catalyzed the formation of JA-amino acid conjugates in vitro. Kinetic analysis showed the enzyme has a strong preference for Ile over Leu and Val and it was about 10-fold more active with (+)-7-iso-JA than with its epimer (-)-JA. Leaf wounding rapidly increased JA-Ile 50-fold to about 450 pmol g(-1) FW at 30 min after wounding, while conjugates with Leu, Phe, Val and Met were only marginally increased or not detected. Nearly all of the endogenous JA-Ile was the bioactive epimer (+)-7-iso-JA-Ile and there was no evidence for its conversion to (-)-JA-Ile up to 6 h after wounding. A transgenic RNAi approach was used to suppress SlJAR1 transcript that reduced JA-Ile accumulation by 50-75%, suggesting that other JA conjugating enzymes may be present. These results show that SlJAR1 synthesizes the bioactive conjugate (+)-7-iso-JA-Ile and this is the predominant isomer accumulated in wounded tomato leaves.

Adventitious roots (ARs) are induced by auxins. Jasmonic acid (JA) and methyl jasmonate (MeJA) are also plant growth regulators with many effects on development, but their role on ARs needs investigation. To this aim, we analyzed AR formation in tobacco thin cell layers (TCLs) cultured with 0.01-10 microM MeJA, either under root-inductive conditions, i.e., on medium containing 10 microM indole-3-butyric acid (IBA) and 0.1 microM kinetin, or without hormones. The explants were excised from the cultivars Samsun, Xanthii and Petite Havana, and from genotypes with altered AR-forming ability in response to auxin, namely the non-rooting rac mutant and the over-rooting Agrobacterium rhizogenes rolB transgenic line. Results show that NtRNR1 (G1/S) and Ntcyc29 (G2/M) gene activity, cell proliferation and meristemoid formation were stimulated in hormone-cultured TCLs by submicromolar MeJA concentrations. The meristemoids developed either into ARs and xylogenic nodules, or into xylogenic nodules only (rac TCLs). MeJA-induced meristemoid over-production characterized rolB TCLs. No rooting or xylogenesis occurred under hormone-free conditions, independently of MeJA and genotype. Endogenous JA progressively (days 1-4) increased in hormone-cultured TCLs in the absence of MeJA. JA levels were enhanced by 0.1 microM MeJA, on both days 1 and 4. Endogenous IBA was the only auxin detected, both in the free form and as IBA-glucose. Free IBA increased up to day 2, remaining constant thereafter (day 4). Its level was enhanced by 0.1 microM MeJA only on day 1, while IBA conjugation was not affected by MeJA. Taken together, these results show that an interplay between jasmonates and auxins regulates AR formation and xylogenesis in tobacco TCLs.

Polyethylene glycol sorbitan monoacylates (Tween) are detergents of widespread use in plant sciences. However, little is known about the plant response to these compounds. Interestingly, the structure of Tweens’ detergents (especially from Tween 20) resembles the lipid A structure from gram-negative bacteria polysaccharides (a backbone with short saturated fatty acids). Thus, different assays (microarray, GC–MS, RT–PCR, Northern blots, alkalinization and mutant analyses) were conducted in order to elucidate physiological changes in the plant response to Tween 20 detergent. Tween 20 causes a rapid and complex change in transcript abundance which bears all characteristics of a pathogenesis-associated molecular pattern (PAMP)/elicitor-induced defense response, and they do so at concentrations which cause no detectable deleterious effects on plant cellular integrity. In the present work, it is shown that the PAMP/elicitor-induced defense responses are caused by medium-chain fatty acids which are efficiently released from the Tween backbone by the plant, notably lauric acid (12:0) and methyl lauric acid. These compounds induce the production of ethylene, medium alkalinization and gene activation in a jasmonateindependent manner. Medium-chain fatty acids are thus novel elicitors/regulators of plant pathogen defense as they have being proved in animals. Fil: Hunzicker, Gretel Mara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina

Analysis of the promoters of defense-related genes is valuable for determining stress signaling and transcriptional activation during pathogen infection. Here, we have isolated and functionally characterized the promoter region of the pepper (Capsicum annuum) pectin methylesterase inhibitor 1 (CaPMEI1) gene in transiently transformed tobacco plants and stably transformed Arabidopsis plants. Among four 5' deletion constructs analyzed, the -958-bp CaPMEI1 promoter induced a high level of GUS reporter activity in tobacco leaf tissue, driven by pathogen infection as well as by ethylene and methyl jasmonate (MeJA) treatment. The 204-bp region from -958 bp to -754 bp of the CaPMEI1 promoter is responsible for the stress-responsive expression. In addition, the pepper transcription factor CARAV1 activated the CaPMEI1 promoter in tobacco leaves, whereas the transcription factor CAbZIP1 did not. In the transgenic Arabidopsis plants, the -958 bp CaPMEI1 promoter was functionally regulated by developmental cues, bacterial and oomycete pathogen infections, and treatment with ethylene and MeJA. Histochemical GUS staining analyses of Arabidopsis tissues revealed that the CaPMEI1 promoter was mainly activated in leaf veins in response to various biotic and abiotic stimuli. Together, these results suggest that CaPMEI1 promoter activation may be a critical molecular event for host defense response and ethylene- and MeJA-mediated CaPMEI1 gene expression.

We have previously isolated a Brassica juncea cDNA encoding a novel chitinase BjCHI1 with two chitin-binding domains (Zhao and Chye in Plant Mol Biol 40:1009-1018, 1999). The expression of BjCHI1 was highly inducible by methyl jasmonate (MeJA) treatment, wounding, caterpillar feeding, and pathogenic fungal infection. These observations suggest that the promoter of BjCHI1 gene might contain specific cis-acting elements for stress responses. Here, we report the cloning and characterization of the BjCHI1 promoter. A 1,098 bp BjCHI1 genomic DNA fragment upstream of the ATG start codon was isolated by PCR walking and various constructs were made by fusing the BjCHI1 promoter or its derivatives to beta-glucuronidase reporter gene. The transgenic Arabidopsis plants showed that the BjCHI1 promoter responded to wounding and MeJA treatment, and to treatments with either NaCl or polyethyleneglycol (PEG 6000), indicating that the BjCHI1 promoter responses to both biotic and abiotic stresses. A transient gene expression system of Nicotiana benthamiana leaves was adopted for promoter deletion analysis, and the results showed that a 76 bp region from -695 to -620 in the BjCHI1 promoter was necessary for MeJA-responsive expression. Furthermore, removal of a conserved T/G-box (AACGTG) at -353 to -348 of the promoter greatly reduced the induction by MeJA. This is the first T/G-box element identified in a chitinase gene promoter. Gain-of-function analysis demonstrated that the cis-acting element present in the 76 bp region requires coupling with the T/G-box to confer full magnitude of BjCHI1 induction by MeJA.

The plant hormone, jasmonic acid (JA), is known to have a critical role in both resistance and susceptibility against bacterial and fungal pathogen attack. However, little is known about the involvement of JA in the interactions between plants and toxigenic necrotrophic fungal pathogens. Using the tomato pathotype of Alternaria alternata (Aa) and its AAL-toxin/tomato interaction as a model system, we demonstrate a possible role for JA in susceptibility of plants against pathogens, which utilize host-specific toxins as virulence effectors. Disease development and in planta growth of the tomato pathotype of Aa were decreased in the def1 mutant, defective in biosynthesis of JA, compared with the wild-type (WT) cultivar. Exogenous methyl jasmonate (MeJA) application restored pathogen disease symptoms to the def1 mutant and led to increased disease in the WT. On the other hand, necrotic cell death was similarly induced by AAL-toxin both on def1 and WT, and MeJA application to the tomatoes did not affect the degree of cell death by the toxin. These results indicate that the JA-dependent signaling pathway is not involved in host basal defense responses against the tomato pathotype of Aa, but rather might affect pathogen acceptability via a toxin-independent manner. Data further suggest that JA has a promotional effect on susceptibility of tomato to toxigenic and necrotrophic pathogens, such that pathogens might utilize the JA signaling pathway for successful infection.

The role of plant pleiotropic drug resistance (PDR) type ATP-binding cassette (ABC) transporters remains poorly understood. We characterized the expression of the rice pleiotropic drug resistance (PDR) gene family in roots, where PDR transporters are believed to have major functions. A prototypical oligonucleotide array was developed containing 70-mers chosen in the gene-specific 3' untranslated regions of the rice PDR genes, other full-molecule rice ABC transporter genes and relevant marker genes. Jasmonates, which are involved in plant defense and secondary metabolism, proved major inducers of PDR gene expression. Over half of the PDR genes were JA-induced in roots of rice; OsPDR9 to the highest level. Salicylic acid, involved in plant pathogen defense, markedly induced the expression of OsPDR20. OsPDR20 was cDNA cloned and characterized. Abscisic acid, typically involved in water deficit responses, particularly induced OsPDR3 in roots and shoot and OsPDR6 in rice leaves. OsPDR9 and OsPDR20 were furthermore up-regulated in response to dithiothreitol- or glutathione-induced redox perturbations. Exogenous application of the weak organic acids lactic acid, malic acid, and citric acid differentially induced the expression of OsPDR3, OsPDR8, OsPDR9 and OsPDR20 in rice seedling roots. This transcriptional survey represents a guide for the further functional analysis of individual PDR transporters in roots of rice.

Most plants encode a limited set of polygalacturonase inhibitor (PGIP) genes that may be involved in aspects of plant development, but more importantly in the inactivation of polygalacturonases (PG) secreted by pathogens. Previously, we characterized two Brassica napus PGIP genes, BnPgip1 and BnPgip2, which were differentially expressed in response to pathogen infection and wounding. Here we report that the B. napus genome encodes a set of at least 16 PGIP genes that are similar to BnPgip1 or BnPgip2. This is the largest Pgip gene family reported to date. Comparison of the BnPGIPs revealed several sites within the xxLxLxx region of leucine rich repeats that form beta-sheets along the interacting face of the PGIP that are hypervariable and represent good candidates for generating PGIP diversity. Characterization of the regulatory regions and RT-PCR studies with gene-specific primers revealed that individual genes were differentially responsive to pathogen infection, mechanical wounding and signaling molecules. Many of the BnPgip genes responded to infection by the necrotic pathogen, Sclerotinia sclerotiorum; however, these genes were also induced either by jasmonic acid, wounding and salicylic acid or some combination thereof. The large number of PGIPs and the differential manner in which they are regulated likely ensures that B. napus can respond to attack from a broad spectrum of pathogens and pests.