Post Elicitation Research Articles

Lipopolysaccharide perception in Arabidopsis thaliana: Diverse LPS chemotypes from Burkholderia cepacia, Pseudomonas syringae and Xanthomonas campestris trigger differential defence-related perturbations in the metabolome

Lipopolysaccharides (LPSs) are microbe-associated molecular pattern molecules (MAMPs) from Gram-negative bacterial pathogens that potentially contain three different MAMPs (the O-polysaccharide chain, the oligosaccharide core and lipid A). LPSs was purified from Burkholderia cepacia, Pseudomonas syringae and Xanthomonas campestris and electrophoretically profiled. Outcomes of the interactions of the three different LPS chemotypes with Arabidopsis thaliana, as reflected in the induced defence metabolites, profiled at 12 h and 24 h post elicitation, were investigated. Plants were pressure-infiltrated with LPS solutions and methanol-based extractions at different time points were performed for untargeted metabolomics using ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Multivariate data modelling and chemometric analysis were applied to generate interpretable biochemical information from the multidimensional data sets. The three LPSs triggered differential metabolome changes in the plants as apparent from chromatographically distinct MS chromatograms. Unsupervised and supervised multivariate data models exhibited time- and treatment-related variations, and revealed discriminating metabolite variables. Heat map models comparatively displayed the up-regulated pathways affecting the metabolomes and Venn diagrams indicated up-regulated and shared metabolites among the three LPS treatments. The altered metabolomes reflect the up-regulation of metabolites from not only the glucosinolate pathway, but also from the shikimate-phenylpropanoid-flavonoid -, terpenoid - and indolic/alkaloid pathways, as well as oxygenated fatty acids. Distinct phytochemical profiles, especially at the earlier time point, suggest differences in the perception of the three LPS chemotypes, associated with the molecular patterns within the tripartite lipoglycans.

Proteome analysis and differential expression by JA driven elicitation in Andrographis paniculata (Burm. f.) Wall. ex Nees using Q-TOF–LC–MS/MS

Andrographis paniculata plantlets were elicited with 50 µM jasmonic acid (JA) in vitro for 1 week and proteome analysis was carried out. HPLC analysis revealed enhanced andrographolide content in JA treated plantlets. Isolated protein samples from both JA treated and untreated control plantlets were analysed by Q-TOF–LC–MS/MS. JA elicitation influenced expression of 22 proteins involved in the isoprenoid pathway in treated and three with untreated plantlets. 3308 proteins in control and 3994 from JA elicited were identified with 1393 found common in both. The differential expression study of proteins revealed a pattern with 621 decreased and 201 enhanced. About 40 metabolic processes identified by functional annotation of proteins and highly elevated (5.7%) post-translational modifications were observed in JA treated. KEGG analysis unveiled appearance of additional secondary metabolism related proteins; primarily phenylpropanoids, isoprenoids and flavonoids from both elicited and untreated. Various transcription factors such as DREB’s, MYC’s, WRKY, and MED which uniquely influenced terpenoid biosynthesis were identified through differential expression study following JA elicitation. The total proteome and differential expression of proteins in JA elicited and untreated in vitro grown A. paniculata plantlets are reported for the first time. These finding will be of significance in understanding elicitation mechanism, gene regulation, metabolic network changes in post elicitation scenario, search of new/novel compounds and developing strategies for manipulating yield enhancement of plant-derived bioactive molecules in general and diterpene lactones in particular. Andrographis paniculata proteome showed 3308 control proteins, 3994 in JA elicited tissue. Differential expression study indicated 621 downregulated, 201 upregulated proteins. KEGG revealed terpenoid biosynthesis upregulation attributed to enhanced andrographolide.

Benzoate-CoA ligase contributes to the biosynthesis of biphenyl phytoalexins in elicitor-treated pear cell cultures.

Benzoate-Coenzyme A ligase enzyme activity catalyzing the conversion of free benzoic acid to benzoyl-CoA was detected and biochemically characterized inthe elicitor-treated pear cell cultures. Asian pear (Pyrus pyrifolia) is an economically and nutritionally important fruit-bearing tree of the subtribe Malinae. Upon pathogen attack, pears produce unique benzoate-derived biphenyl phytoalexins. The upstream biosynthesis of the biphenyl in Malinae is still incomplete. Previously, protein preparations from yeast extract-treated pear cultures were able to convert L-phenylalanine to cinnamic acid catalyzed by the activity of the phenylalanine ammonia lyase. The same extract was able to perform a C2 side-chain cleavage of cinnamic acid to benzaldehyde followed by oxidation of the latter to benzoic acid owing to the molecularly-undefined benzaldehyde synthase and benzaldehyde dehydrogenase activities, respectively. The biosynthesis of biphenyls starts with benzoate-Coenzyme A ligase (BZL), which converts benzoic acid to benzoyl-CoA. Subsequently, the previously-defined biphenyl synthase uses benzoyl-CoA to form the biphenyls. Thecurrent study reports the first time detection and characterization of BZL activity in elicitor-treated pear cell cultures. The preferred substrate was benzoic acid (Km = 62 ± 4µM). Magnesium or manganese was prerequisite for the activity, which was enhanced by ~ 70% in the presence of potassium. Maximum BZL activity was observed 18h post elicitation, which is in agreement with the coordinate induction reported for the enzymes in the same pathway. The induced BZL activity preceded the accumulation of biphenyls supporting its involvement in their biosynthesis.

Effect of Oxylipins, Terpenoid Precursors and Wounding on Soft Corals' Secondary Metabolism as Analyzed via UPLC/MS and Chemometrics.

The effect of three oxylipin analogues, a terpenoid intermediate and wounding on the secondary metabolism of the soft corals Sarcophyton glaucum and Lobophyton pauciflorum was assessed. Examined oxylipins included prostaglandin (PG-E1), methyl jasmonate (MeJA), and arachidonic acid (AA) in addition to the diterpene precursor geranylgeranylpyrophosphate (GGP). Post-elicitation, metabolites were extracted from coral heads and analyzed via UPLC-MS followed by multivariate data analyses. Both supervised and unsupervised data analyses were used for sample classification. Multivariate data analysis revealed clear segregation of PG-E1 and MeJA elicited S. glaucum at 24 and 48 h post elicitation from other elicitor samples and unelicited control group. PG-E1 was found more effective in upregulating S. glaucum terpene/sterol levels compared to MeJA. Metabolites showing upregulation in S. glaucum include campestene-triol and a cembranoid, detected at ca. 30- and 2-fold higher levels compared to unelicited corals. Such an elicitation effect was less notable in the other coral species L. pauciflorum, suggesting a differential oxylipin response in soft corals. Compared to MeJA and PG, no elicitation effect was observed for GGP, AA or wounding on the metabolism of either coral species.

Metabolomics reveals biotic and abiotic elicitor effects on the soft coral Sarcophyton ehrenbergi terpenoid content

The effects of six biotic and abiotic elicitors, i.e. MeJA (methyl jasmonate), SA (salicylic acid), ZnCl2, glutathione and β-glucan BG (fungal elicitor), and wounding, on the secondary metabolite accumulation in the soft coral Sarcophyton ehrenbergi were assessed. Upon elicitation, metabolites were extracted and analysed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Except for MeJA, no differences in photosynthetic efficiency were observed after treatments, suggesting the absence of a remarkable stress on primary production. Chemometric analyses of UPLC-MS data showed clear segregation of SA and ZnCl2 elicited samples at 24 and 48 h post elicitation. Levels of acetylated diterpene and sterol viz., sarcophytonolide I and cholesteryl acetate, was increased in ZnCl2 and SA groups, respectively, suggesting an activation of specific acetyl transferases. Post elicitation, sarcophytonolide I level increased 132 and 17-folds at 48 h in 0.1 mM SA and 1 mM ZnCl2 groups, respectively. Interestingly, decrease in sarcophine, a major diterpene was observed only in response to ZnCl2, whereas no change was observed in sesquiterpene content following treatments. To the best of our knowledge, this study provides the first documentation for elicitation effects on a soft corals secondary metabolome and suggests that SA could be applied to increase diterpenoid levels in corals.

A FUZZY BASED APPROACH FOR EARLY REQUIREMENT PRIORITIZATION

The importance of the prioritization in commercial software development has been analyzed by many researchers. The gathered requirements are required to be put into an order of some priority. In other words we can say that there is a need to prioritize the requirements. It is evident that most of the approaches and techniques proposed in recent research to prioritize the requirements have not been widely adopted. These approaches are too complex, time consuming, or inconsistent and difficult to implement In this paper we propose a fuzzy based approach for requirement prioritization in which requirement are prioritized in early phase of requirement engineering as post elicitation step. This category of prioritization is known as early requirement prioritization. The proposed fuzzy based approach considers the nature of requirements by modeling their attributes as fuzzy variables. As such, these variables are integrated into a fuzzy based inference system in which the requirements represented as input attributes and ranked via the expected value operator of a fuzzy variable.

The NAC transcription factor gene ANAC072 is differentially expressed in Arabidopsis thaliana in response to microbe-associated molecular pattern (MAMP) molecules

NAC (NAM, ATAF1/2 and CUC2) transcription factors (TFs) play important roles in plant development and environmental stresses, though more recently involvement in biotic stresses has also been observed, but not explored in detail. Based on a differential display library obtained from lipopolysaccharide (LPS)-elicited cells of Arabidopsis thaliana, a differentially expressed sequence tag (EST) was identified, sequenced and found to code for a NAC protein with a N-terminal NAM domain. The corresponding gene, At4g27410.3, encodes a known NAC TF, ANAC072/RD26. A. thaliana callus, cell suspensions and seedlings were used to determine the expression levels of ANAC072 after treatment with Burkholderia cepacia and associated microbe-associated molecular pattern (MAMPs) molecules. ANAC072 showed early responsiveness towards B. cepacia and its LPS and was also responsive to peptidoglycan (PGN) and the flagellin-derived peptide, flg22. The response of cultured cells was characterised by the rapid up-regulation at 10 min post elicitation. Depending on the MAMP, the response was either transient and returned to basal levels, or sustained up to 90 min. The general trend of expression was similar in seedlings compared to that of the undifferentiated cells. Transcript levels of ANAC072 were positively regulated by abscisic acid and hydrogen peroxide, but ethylene, salicylic acid, methyl jasmonate and indole acetic acid had little effect on its expression. Our data indicate that ANAC072 is an early MAMP-responsive TF, part of the altered transcriptome following MAMP perception events.

Alternative oxidase (AOX) and phenolic metabolism in methyl jasmonate-treated hairy root cultures of Daucus carota L.

Methyl-jasmonate (MJ)-treated hairy roots of Daucus carota L. were used to study the influence of alternative oxidase (AOX) in phenylpropanoid metabolism. Phenolic acid accumulation, as well as total flavonoids and lignin content of the MJ-treated hairy roots were decreased by treatment with salicylhydroxamic acid (SHAM), a known inhibitor of AOX. The inhibitory effect of SHAM was concentration dependent. Treatment with propyl gallate (PG), another inhibitor of AOX, also had a similar inhibitory effect on accumulation of phenolic acid, total flavonoids and lignin. The transcript levels of two DcAOX genes (DcAOX2a and DcAOX1a) were monitored at selected post-elicitation time points. A notable rise in the transcript levels of both DcAOX genes was observed preceding the MJ-induced enhanced accumulation of phenolics, flavonoids and lignin. An appreciable increase in phenylalanine ammonia-lyase (PAL) transcript level was also observed prior to enhanced phenolics accumulation. Both DcAOX genes showed differential transcript accumulation patterns after the onset of elicitation. The transcript levels of DcAOX1a and DcAOX2a attained peak at 6hours post elicitation (hpe) and 12hpe, respectively. An increase in the transcript levels of both DcAOX genes preceding the accumulation of phenylpropanoid-derivatives and lignin showed a positive correlation between AOX activity and phenylpropanoid biosynthesis. The results provide important new insight about the influence of AOX in phenylpropanoid biosynthesis.

Jasmonic acid elicits oxidative defense and detoxification systems in Cucumis melo L. cells

This study investigated whether a jasmonic acid (JA) elicitation strategy developed in a conventional cell suspension culture could evoke melon resistance mechanisms, including secondary metabolite production. Twenty one day cultured melon cell suspensions grown in MS1 medium were supplemented with JA at the concentrations of 0.5, 5.0 and 10 µmol. Melon cultures were sampled 24, 48 and 72 h post elicitation to evaluate different defense related factors such as antioxidant enzymes, ascorbate metabolism and phenolic compounds. Results suggest that melon cells respond to JA reprogramming the primary and secondary metabolism which will result in melon plantlets with enhanced resistance against diverse stress conditions through the production of specific bioactive molecules. Jasmonic acid elicited melon cells exhibited enhanced oxidative enzymes activities and ascorbic acid, coumarin and p-coumaric amounts were found without growth retardation. Induced intracellular JA functions as a signal transducer acting upstream to H2O2, which is a secondary messenger triggering jasmonic signaling cascades by activating certain late genes that regulate the activity of catalase, peroxidase and de novo synthesis of five isozymes, ascorbic peroxidase detoxifying enzymes concomitant with ascorbate compound. Secondary metabolite production in melon cells seems to be activated upon JA exposure suggesting that this cell culture could be used as a source for rapid and increased production of coumarin, p-coumaric, ascorbic acid and likely other specific phenylpropanoids. These data provide further evidences for a role of jasmonic acid in the intracellular signal cascade that results in the accumulation of secondary compounds and ultimately induced melon resistance. This approach could assist further in understanding the metabolic mechanisms operating in melon cells under stress, and thus how to control them.

The case for the defense: plants versus Pseudomonas syringae

Incredible progress has been made over the last 20 years in understanding the components and mechanisms governing plant innate immunity. The most important discoveries concern pathogen recognition mechanisms, which divide perception of conserved elicitors at the cell periphery, and recognition of variable elicitors within the host cytoplasm. The underlying mechanisms of immunity post elicitation are complex and poorly defined. This review highlights emergent themes in plant–microbe interactions with a particular focus on the plant immune responses against infection by the bacterium Pseudomonas syringae.

Defense gene expression induced by a coffee-leaf extract formulation in tomato

Plant extracts have the potential to activate defense-related genes with microarray technology an effective means to probe the mechanism of action. A coffee-leaf extract formulation (NEFID) is shown to be effective in controlling bacterial spot caused by Xanthomonas vesicatoria in tomato ( Solanum lycopersicum). Transcriptional changes in tomato leaves elicited with NEFID were evaluated by using a tomato gene chip. A total of 268 genes were found to be differentially regulated with a majority up-regulated and encoding signal transduction, defense responses, and transcription factors. Chitinases, glucanases and peroxidases (PR-4), with reported activity against pathogens, were transcriptionally up-regulated and corresponding enzyme activities were over-expressed as early as 24 h post elicitation and remained elevated for up to five days after NEFID exposure. Since salicylic- and jasmonic-acid signaling components were not differentially transcribed with elicitor treatment while mitogen-activated proteins (MAP3K and MAPKK) as well as calcium dependent (calmodulin and phosphatidylinositol) signaling components were up-regulated, a SA/JA-independent transduction sequence for PR accumulation is postulated. These results demonstrate the ability of the coffee-leaf extract NEFID to differentially regulate gene expression in tomato.

Reinforcement of cell wall in roots of Lycopersicon esculentum through induction of phenolic compounds and lignin by elicitors

Changes in phenolic metabolism and lignin deposition have been studied in roots of tomato plants after elicitation with four elicitors which are Fusarium mycelium extract (FME), chitosan (CHT), Fusarium culture filtrate (FCF) and Trichoderma mycelium extract (TME). Most profound effect of elicitors was observed on ferulic acid among the phenolic compounds. After 24 h elicitation, the increase in ferulic acid content of root cell wall was 3.71 and 3.30 times by FME and CHT, respectively. The increase of 4-hydroxybenzoic acid was 2.71 and 2.16 times by these two elicitors. The level of 4-coumaric acid was little more than double by these two elicitors after 24 h elicitation. Most pronounced increase in lignin synthesis was also effected by FME followed by CHT. Lignin deposition in the root cell wall was increased 3.6, 5.4 and 7.1 times by FME during 12, 24 and 36 h after elicitation, respectively. Similarly, CHT increased lignin deposition by 2.8, 5.1 and 6.8 times at 12, 24 and 36 h after elicitation, respectively. FCF and TME also increased lignin deposition significantly in the cell walls of tomato roots during the above time periods of elicitation. Activity of phenylalanine ammonia lyase reached highest level at 24 h post elicitation under the influence of the elicitors. Peroxidase activity registered a sharp increase at 24 h post elicitation. Markedly increased level of polyphenol oxidase activity was found at 12 h post elicitation. Cinnamyl alcohol dehydrogenase activity was observed to reach highest level at 48 h post elicitation. Cell wall strengthening, through the deposition of lignin, preceded by the induction of the synthesizing enzymes appears to play an important role in the defense response of Lycopersicon esculentum in reaction to elicitors, including one derived from Fusarium oxysporum f. sp. lycopersici, the causal organism of Fusarium wilt of tomato.

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4'-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8'-linked lignan or 8-5'-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens.

Early Responses in Methyl Jasmonate-Preconditioned Cells toward Pathogen-Derived Elicitors

Early, signal transduction-related responses in cultured tobacco cells due to methyl jasmonate (MeJa), a cell-wall-derived elicitor from Phytophthora nicotianae and chitosan, were investigated. MeJa was an effective inducer of lipid peroxidation and lipoxygenase (LOX) activity with maximum levels reached within 2 h and 4–8 h, respectively. Chitosan and the elicitor induced a transient increase (1–4 h) in lipid peroxidation. Conditioning with MeJA, followed by secondary elicitation, led to a significant increase in malondialdehyde concentration after 1 h. Chitosan and the elicitor induced transient activation of LOX with maximal values between 8 and 12 h, with preconditioning resulting in a rapid increase in LOX activity at 4 h post elicitation. MeJA did not effect phosphoprotein accumulation but conditioning led to the potentiation and differential induction of phosphoproteins due to chitosan and elicitor. The results indicate that cells are sensitized by the exposure to MeJa to respond more intensely and rapidly toward secondary elicitation by fungal pathogen derived elicitors.