Analysis of Defense-Related Gene Expression and Leaf Metabolome in Wheat During the Early Infection Stages of Blumeria graminis f. sp. tritici.

Blumeria graminis f. sp. tritici (Bgt) is an obligate biotrophic fungal pathogen responsible for powdery mildew in bread wheat (Triticum aestivum). Upon Bgt infection, the wheat plant activates basal defense mechanisms, namely PAMP-triggered immunity, in the leaves during the first few days. Understanding this early stage of quantitative resistance is crucial for developing new breeding tools and evaluating plant resistance inducers for sustainable agricultural practices. In this sense, we used a combination of transcriptomic and metabolomic approaches to analyze the early steps of the interaction between Bgt and the moderately susceptible wheat cultivar Pakito. Bgt infection resulted in an increasing expression of genes encoding pathogenesis-related (PR) proteins (PR1, PR4, PR5, and PR8) known to target the pathogen, during the first 48 h postinoculation. Moreover, RT-qPCR and metabolomic analyses pointed out the importance of the phenylpropanoid pathway in quantitative resistance against Bgt. Among metabolites linked to this pathway, hydroxycinnamic acid amides containing agmatine and putrescine as amine components accumulated from the second to the fourth day after inoculation. This suggests their involvement in quantitative resistance via cross-linking processes in cell walls for reinforcement, which is supported by the up-regulation of PAL (phenylalanine ammonia-lyase), PR15 (oxalate oxidase) and POX (peroxidase) after inoculation. Finally, pipecolic acid, which is considered a signal involved in systemic acquired resistance, accumulated after inoculation. These new insights lead to a better understanding of basal defense in wheat leaves after Bgt infection.

Function of hydroxycinnamoyl spermidines in seedling growth of Arabidopsis.

Hydroxycinnamic acid amides are involved in various developmental processes as well as in biotic and abiotic stress responses. Among them, the presence of spermidine derivatives, such as N1,N8-di(coumaroyl)-spermidine and N1,N8-di(sinapoyl)-spermidine, and their biosynthetic genes have been reported in Arabidopsis, but their functions in plants are still unknown. We chemically synthesized the above-mentioned spermidine derivatives to assess their physiological functions in Arabidopsis. We evaluated the growth and development of chemically treated Arabidopsis and demonstrated that these compounds inhibited seed germination, hypocotyl elongation, and primary root growth, which could be due to modulation of plant hormone homeostasis and signaling. The results suggest that these compounds are regulatory metabolites that modulate plant growth and development.

Facile Amidation of Non-Protected Hydroxycinnamic Acids for the Synthesis of Natural Phenol Amides.

Phenol amides are bioactive compounds naturally present in many plants. This class of compounds is known for antioxidant, anti-inflammatory, and anticancer activities. To better understand the reactivity and structure-bioactivity relationships of phenol amides, a large set of structurally diverse pure compounds are needed, however purification from plants is inefficient and laborious. Existing syntheses require multiple steps, including protection of functional groups and are generally overly complicated and only suitable for specific combinations of hydroxycinnamic acid and amine. Thus, to facilitate further studies on these promising compounds, we aimed to develop a facile general synthetic route to obtain phenol amides with a wide structural diversity. The result is a protocol for straightforward one-pot synthesis of phenol amides at room temperature within 25 h using equimolar amounts of N,N'-dicyclohexylcarbodiimide (DCC), amine, hydroxycinnamic acid, and sodium bicarbonate in aqueous acetone. Eight structurally diverse phenol amides were synthesized and fully chemically characterized. The facile synthetic route described in this work is suitable for a wide variety of biologically relevant phenol amides, consisting of different hydroxycinnamic acid subunits (coumaric acid, ferulic acid, and sinapic acid) and amine subunits (agmatine, anthranilic acid, putrescine, serotonin, tyramine, and tryptamine) with yields ranging between 14% and 24%.

Comprehensive profiling of semi-polar phytochemicals in whole wheat grains (Triticum aestivum) using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry.

INTRODUCTION: Wheat (Triticum aestivum) it is one of the most important staple food crops worldwide and represents an important resource for human nutrition. Besides starch, proteins and micronutrients wheat grains accumulate a highly diverse set of phytochemicals. OBJECTIVES: This work aimed at the development and validation of an analytical workflow for comprehensive profiling of semi-polar phytochemicals in whole wheat grains. METHOD: Reversed-phase ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC/ESI-QTOFMS) was used as analytical platform. For annotation of metabolites accurate mass collision-induced dissociation mass spectra were acquired and interpreted in conjunction with literature data, database queries and analyses of reference compounds. RESULTS: Based on reversed-phase UHPLC/ESI-QTOFMS an analytical workflow for comprehensive profiling of semi-polar phytochemicals in whole wheat grains was developed. For method development the extraction procedure and the chromatographic separation were optimized. Using whole grains of eight wheat cultivars a total of 248 metabolites were annotated and characterized by chromatographic and tandem mass spectral data. Annotated metabolites comprise hydroquinones, hydroxycinnamic acid amides, flavonoids, benzoxazinoids, lignans and other phenolics as well as numerous primary metabolites such as nucleosides, amino acids and derivatives, organic acids, saccharides and B vitamin derivatives. For method validation, recovery rates and matrix effects were determined for ten exogenous model compounds. Repeatability and linearity were assessed for 39 representative endogenous metabolites. In addition, the accuracy of relative quantification was evaluated for six exogenous model compounds. CONCLUSIONS: In conjunction with non-targeted and targeted data analysis strategies the developed analytical workflow was successfully applied to discern differences in the profiles of semi-polar phytochemicals accumulating in whole grains of eight wheat cultivars.

Sclerotinia sclerotiorum Infection Triggers Changes in Primary and Secondary Metabolism in Arabidopsis thaliana.

Sclerotinia sclerotiorum is a devastating plant pathogen that causes substantial losses in various agricultural crops. Although plants have developed some well-known defense mechanisms against invasive fungi, much remains to be learned about plant responses to fungal pathogens. In this study, we investigated how S. sclerotiorum infection affects plant primary and secondary metabolism in the model plant Arabidopsis thaliana. Our results showed that soluble sugar and amino acid content changed significantly in A. thaliana leaves upon fungal colonization, with a decrease in sucrose and an increase in mannitol, attributed to fungal biosynthesis. Furthermore, the jasmonate signaling pathway was rapidly activated by S. sclerotiorum infection, and there was a striking accumulation of antifungal metabolites such as camalexin, p-coumaroyl agmatine, feruloyl agmatine, and Nδ-acetylornithine. On the other hand, the characteristic defense compounds of the Brassicaceae, the glucosinolates, were not induced in A. thaliana infected by S. sclerotiorum. Our study provides a better understanding of how A. thaliana primary and secondary metabolism is modified during infection by a fungal pathogen like S. sclerotiorum that has both hemibiotrophic and necrotrophic stages.

Separation and Characterization of Phenolamines and Flavonoids from Rape Bee Pollen, and Comparison of Their Antioxidant Activities and Protective Effects Against Oxidative Stress.

Phenolamines and flavonoids are two important components in bee pollen. There are many reports on the bioactivity of flavonoids in bee pollen, but few on phenolamines. This study aims to separate and characterize the flavonoids and phenolamines from rape bee pollen, and compare their antioxidant activities and protective effects against oxidative stress. The rape bee pollen was separated to obtain 35% and 50% fractions, which were characterized by HPLC-ESI-QTOF-MS/MS. The results showed that the compounds in 35% fraction were quercetin and kaempferol glycosides, while the compounds in 50% fraction were phenolamines, including di-p-coumaroyl spermidine, p-coumaroyl caffeoyl hydroxyferuloyl spermine, di-p-coumaroyl hydroxyferuloyl spermine, and tri-p-coumaroyl spermidine. The antioxidant activities of phenolamines and flavonoids were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and ferric reducing antioxidant power (FRAP) assays. It was found that the antioxidant activity of phenolamines was significantly higher than that of flavonoids. Moreover, phenolamines showed better protective effects than flavonoids on HepG2 cells injured by AAPH. Furthermore, phenolamines could significantly reduce the reactive oxygen species (ROS), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and increase the superoxide dismutase (SOD) and glutathione (GSH) levels. This study lays a foundation for the further understanding of phenolamines in rape bee pollen.

Jasmonic acid/ethylene signaling coordinates hydroxycinnamic acid amides biosynthesis through ORA59 transcription factor.

Hydroxycinnamic acid amides (HCAAs) are a class of antimicrobial metabolites involved in plant defense against necrotrophic pathogens, including Alternaria brassicicola and Botrytis cinerea. The agmatine coumaryl transferase (AtACT) is the key enzyme that catalyzes the last reaction in the biosynthesis of HCAAs, including p-coumaroylagmatine (CouAgm) and feruloylagmatine in Arabidopsis thaliana. However, the regulatory mechanism of AtACT gene expression is currently unknown. Yeast one-hybrid screening using the AtACT promoter as bait isolated the key positive regulator ORA59 that is involved in jasmonic acid/ethylene (JA/ET)-mediated plant defense responses. AtACT gene expression and HCAAs biosynthesis were synergistically induced by a combination of JA and ET. In the AtACT promoter, two GCC-boxes function equivalently for trans-activation by ORA59 in Arabidopsis protoplasts, and mutation of either GCC-box abolished AtACT mRNA accumulation in transgenic plants. Site-directed mutation analysis demonstrated that the specific Leu residue at position 228 of the ORA59 EDLL motif mainly contributed to its transcriptional activity on AtACT gene expression. Importantly, MEDIATOR25 (MED25) and ORA59 homodimer are also required for ORA59-dependent activation of the AtACT gene. These results suggest that ORA59 and two functionally equivalent GCC-boxes form the regulatory module together with MED25 that enables AtACT gene expression and HCAAs biosynthesis to respond to simultaneous activation of the JA/ET signaling pathways.

HvWRKY23 regulates flavonoid glycoside and hydroxycinnamic acid amide biosynthetic genes in barley to combat Fusarium head blight.

Crop plant resistance against pathogens is governed by dynamic molecular and biochemical responses driven by complex transcriptional networks. However, the underlying mechanisms are largely unclear. Here we report an interesting role of HvWRKY23 transcription factor (TF) in modulating defense response against Fusarium head blight (FHB) in barley. The combined approach of gene silencing, metabolomics, real time expression analysis and ab initio bioinformatics tools led to the identification of the HvWRKY23 role in FHB resistance. The knock-down of HvWRKY23 gene in the FHB resistant barley genotype CI9831, followed by inoculation with Fusarium graminearum, led to the down regulation of key flavonoid and hydroxycinnamic acid amide biosynthetic genes resulting in reduced accumulation of resistant related (RR) secondary metabolites such as pelargonidin 3-rutinoside, peonidin 3-rhamnoside-5-glucoside, kaempferol 3-O-arabinoside and other flavonoid glycosides. Reduced abundances of RR metabolites in TF silenced plants were also associated with an increased proportion of spikelets diseased and amount of fungal biomass in spikelets, depicting the role of HvWRKY23 in disease resistance. The luciferase reporter assay demonstrated binding of HvWRKY23 protein to promoters of key flavonoid and hydroxycinnamic acid amides (HCAA) biosynthetic genes, such as HvPAL2, HvCHS1, HvHCT, HvLAC15 and HvUDPGT. The accumulation of high abundances of HCAAs and flavonoid glycosides reinforce cell walls to contain the pathogen to initial infection area. This gene in commercial cultivars can be edited, if non-functional, to enhance resistance against FHB.

Metabolo-transcriptome profiling of barley reveals induction of chitin elicitor receptor kinase gene (HvCERK1) conferring resistance against Fusarium graminearum.

KEY MESSAGE: We report plausible disease resistance mechanisms induced by barley resistant genotype CI89831 against Fusarium head blight (FHB) based on metabolo-transcriptomics approach. We identified HvCERK1 as a candidate gene for FHB resistance, which is functional in resistant genotype CI9831 but non-functional in susceptible cultivars H106-371 and Zhedar-2. For the first time, we were able to show a hierarchy of regulatory genes that regulated downstream biosynthetic genes that eventually produced resistance related metabolites that reinforce the cell walls to contain the pathogen progress in plant. The HvCERK1 can be used for replacing in susceptible commercial cultivars, if non-functional, based on genome editing. Fusarium head blight (FHB) management is a great challenge in barley and wheat production worldwide. Though barley genome sequence and advanced omics technologies are available, till date none of the resistance mechanisms has been clearly deciphered. Hence, this study was aimed at identifying candidate gene(s) and elucidating resistance mechanisms induced by barley resistant genotype CI9831 based on integrated metabolomics and transcriptomics approach. Following Fusarium graminearum infection, we identified accumulation of specific set of induced secondary metabolites, belonging to phenylpropanoid, hydroxycinnamic acid (HCAA) and jasmonic acid pathways, and their biosynthetic genes. In association with these, receptor kinases such as chitin elicitor receptor kinase (HvCERK1) and protein kinases such as MAP kinase 3 (HvMPK3) and MAPK substrate 1 (HvMKS1), and transcription factors such as HvERF1/5, HvNAC42, HvWRKY23 and HvWRKY70 were also found upregulated with high fold change. Polymorphism studies across three barley genotypes confirmed the presence of mutations in HvCERK1 gene in two susceptible genotypes, isolating this gene as a potential candidate for FHB resistance. Further, the silencing of functional HvCERK1 gene in the resistant genotype CI9831, followed by gene expression and metabolite analysis revealed its role as an elicitor recognition receptor that triggered downstream regulatory genes, which in turn, regulated downstream metabolic pathway genes to biosynthesize resistance related (RR) metabolites to contain the pathogen to spikelet infection. A putative model on metabolic pathway regulation is proposed.

Synthesis and regulation of chlorogenic acid in potato: Rerouting phenylpropanoid flux in HQT-silenced lines.

Chlorogenic acid (CGA) is the major phenolic sink in potato tubers and can constitute over 90% of total phenylpropanoids. The regulation of CGA biosynthesis in potato and the role of the CGA biosynthetic gene hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) was characterized. A sucrose induced accumulation of CGA correlated with the increased expression of phenylalanine ammonia-lyase (PAL) rather than HQT. Transient expression of the potato MYB transcription factor StAN1 (anthocyanin 1) in tobacco increased CGA. RNAi suppression of HQT resulted in over a 90% reduction in CGA and resulted in early flowering. The reduction in total phenolics and antioxidant capacity was less than the reduction in CGA, suggesting flux was rerouted into other phenylpropanoids. Network analysis showed distinct patterns in different organs, with anthocyanins and phenolic acids showing negative correlations in leaves and flowers and positive in tubers. Some flavonols increased in flowers, but not in leaves or tubers. Anthocyanins increased in flowers and showed a trend to increase in leaves, but not tubers. HQT suppression increased biosynthesis of caffeoyl polyamines, some of which are not previously reported in potato. Decreased PAL expression and enzyme activity was observed in HQT suppressed lines, suggesting the existence of a regulatory loop between CGA and PAL. Electrophysiology detected no effect of CGA suppression on potato psyllid feeding. Collectively, this research showed that CGA in potatoes is synthesized through HQT and HQT suppression altered phenotype and redirected phenylpropanoid flux.

Transcription factor StWRKY1 regulates phenylpropanoid metabolites conferring late blight resistance in potato.

Quantitative resistance is polygenically controlled and durable, but the underlying molecular and biochemical mechanisms are poorly understood. Secondary cell wall thickening is a critical process in quantitative resistance, regulated by transcriptional networks. This paper provides compelling evidence on the functionality of StWRKY1 transcription factor, in a compatible interaction of potato-Phytophthora infestans, to extend our knowledge on the regulation of the metabolic pathway genes leading to strengthening the secondary cell wall. A metabolomics approach was used to identify resistance-related metabolites belonging to the phenylpropanoid pathway and their biosynthetic genes regulated by StWRKY1. The StWRKY1 gene in resistant potato was silenced to decipher its role in the regulation of phenylpropanoid pathway genes to strengthen the secondary cell wall. Sequencing of the promoter region of StWRKY1 in susceptible genotypes revealed the absence of heat shock elements (HSEs). Simultaneous induction of both the heat shock protein (sHSP17.8) and StWRKY1 following pathogen invasion enables functioning of the latter to interact with the HSE present in the resistant StWRKY1 promoter region. EMSA and luciferase transient expression assays further revealed direct binding of StWRKY1 to promoters of hydroxycinnamic acid amide (HCAA) biosynthetic genes encoding 4-coumarate:CoA ligase and tyramine hydroxycinnamoyl transferase. Silencing of the StWRKY1 gene was associated with signs of reduced late blight resistance by significantly increasing the pathogen biomass and decreasing the abundance of HCAAs. This study provides convincing evidence on the role of StWRKY1 in the regulation of downstream genes to biosynthesize HCAAs, which are deposited to reinforce secondary cell walls.

Characterization of a spermidine hydroxycinnamoyltransferase in Malus domestica highlights the evolutionary conservation of trihydroxycinnamoyl spermidines in pollen coat of core Eudicotyledons.

Phenolamides, so called hydroxycinnamic acid amides, are specialized metabolites produced in higher plants, involved in development, reproduction and serve as defence compounds in biotic interactions. Among them, trihydroxycinnamoyl spermidine derivatives were initially found to be synthetized by a spermidine hydroxycinnamoyltransferase (AtSHT) in Arabidopsis thaliana and to accumulate in the pollen coat. This study reports the identification, in Malus domestica, of an acyltransferase able to complement the sht mutant of Arabidopsis. The quantitative RT-PCR expression profile of MdSHT reveals a specific expression in flowers coordinated with anther development and tapetum cell activities. Three phenolamides including N (1),N (5),N (10)-tricoumaroyl spermidine and N (1),N (5)-dicoumaroyl-N (10)-caffeoyl spermidine identified by LC/MS, were shown to accumulate specifically in pollen grain coat of apple tree. Moreover, in vitro biochemical characterization confirmed MdSHT capacity to synthesize tri-substituted spermidine derivatives with a substrate specificity restricted to p-coumaroyl-CoA and caffeoyl-CoA as an acyl donor. Further investigations of the presence of tri-substituted hydroxycinnamoyl spermidine conjugates in higher plants were performed by targeted metabolic analyses in pollens coupled with bioinformatic analyses of putative SHT orthologues in a wide range of available plant genomes. This work highlights a probable early evolutionary appearance in the common ancestral core Eudicotyledons of a novel enzyme from the BAHD acyltransferase superfamily, dedicated to the synthesis of trihydroxycinnamoyl spermidines in pollen coat. This pathway was maintained in most species; however, recent evolutionary divergences have appeared among Eudicotyledons, such as an organ reallocation of SHT gene expression in Fabales and a loss of SHT in Malvales and Cucurbitales.

Exploring the formation of heterodimers of barley hydroxycinnamoylagmatines by oxidative enzymes.

Dimers of hydroxycinnamoylagmatines are phenolic compounds found in barley and beer. Although they are bioactive and sensory-active compounds, systematic reports on their structure-property relationships are missing. This is partly due to lack of protocols to obtain a diverse set of hydroxycinnamoylagmatine homo- and heterodimers. To better understand dimer formation in complex systems, combinations of the monomers coumaroylagmatine (CouAgm), feruloylagmatine (FerAgm), and sinapoylagmatine (SinAgm) were incubated with horseradish peroxidase. For all combinations, the main oxidative coupling products were homodimers. Additionally, minor amounts of heterodimers were formed, except for the combination of FerAgm and CouAgm. Oxidative coupling was also performed with laccases from Agaricus bisporus and Trametes versicolor, resulting in formation of the same coupling products and no formation of CouAgm-FerAgm heterodimers. Our protocol for oxidative coupling combinations of hydroxycinnamoylagmatines yielded a structurally diverse set of coupling products, facilitating production of dimers for future research on their structure-property relationships.

Maize hydroxycinnamic acids: unveiling their role in stress resilience and human health.

Maize production is pivotal in ensuring food security, particularly in developing countries. However, the crop encounters multiple challenges stemming from climatic changes that adversely affect its yield, including biotic and abiotic stresses during production and storage. A promising strategy for enhancing maize resilience to these challenges involves modulating its hydroxycinnamic acid amides (HCAAs) content. HCAAs are secondary metabolites present in plants that are essential in developmental processes, substantially contributing to defense mechanisms against environmental stressors, pests, and pathogens, and exhibiting beneficial effects on human health. This mini-review aims to provide a comprehensive overview of HCAAs in maize, including their biosynthesis, functions, distribution, and health potential applications.

Barley-derived beer brewing by-products contain a high diversity of hydroxycinnamoylagmatines and their dimers.

To aid valorisation of beer brewing by-products, more insight into their composition is essential. We have analysed the phenolic compound composition of four brewing by-products, namely barley rootlets, spent grain, hot trub, and cold trub. The main phenolics detected were hydroxycinnamoylagmatines and dimers thereof. Barley rootlets contained the highest hydroxycinnamoylagmatine content and cold trub the highest dimer content. Additionally, variations in (dimeric) hydroxycinnamoylagmatine composition and content were observed in fourteen barley rootlet samples. The most abundant compound in all rootlets was the glycosylated 4-O-7'/3-8'-linked heterodimer of coumaroylagmatine and feruloylagmatine, i.e. CouAgm-4-O-7'/3-8'-(4'Hex)-DFerAgm. Structures of glycosylated and hydroxylated derivatives of coumaroylagmatine were elucidated by NMR spectroscopy after their purification from a rootlet extract. An MS-based decision tree was developed, which aids in identifying hydroxycinnamoylagmatine dimers in complex mixtures. In conclusion, this study shows that the diversity of phenolamides and (neo)lignanamides in barley-derived by-products is larger than previously reported.

Lipopolysaccharides from Ralstonia solanacearum induce a broad metabolomic response in Solanum lycopersicum.

Ralstonia solanacearum, one of the most destructive crop pathogens worldwide, causes bacterial wilt disease in a wide range of host plants. The major component of the outer membrane of Gram-negative bacteria, lipopolysaccharides (LPS), has been shown to function as elicitors of plant defense leading to the activation of signaling and defense pathways in several plant species. LPS from a R. solanacearum strain virulent on tomato (LPSR. sol.), were purified, chemically characterized, and structurally elucidated. The lipid A moiety consisted of tetra- to hexa-acylated bis-phosphorylated disaccharide backbone, also decorated by aminoarabinose residues in minor species, while the O-polysaccharide chain consisted of either linear tetrasaccharide or branched pentasaccharide repeating units containing α-L-rhamnose, N-acetyl-ß-D-glucosamine, and ß-L-xylose. These properties might be associated with the evasion of host surveillance, aiding the establishment of the infection. Using untargeted metabolomics, the effect of LPSR. sol. elicitation on the metabolome of Solanum lycopersicum leaves was investigated across three incubation time intervals with the application of UHPLC-MS for metabolic profiling. The results revealed the production of oxylipins, e.g., trihydroxy octadecenoic acid and trihydroxy octadecadienoic acid, as well as several hydroxycinnamic acid amide derivatives, e.g., coumaroyl tyramine and feruloyl tyramine, as phytochemicals that exhibit a positive correlation to LPSR. sol. treatment. Although the chemical properties of these metabolite classes have been studied, the functional roles of these compounds have not been fully elucidated. Overall, the results suggest that the features of the LPSR. sol. chemotype aid in limiting or attenuating the full deployment of small molecular host defenses and contribute to the understanding of the perturbation and reprogramming of host metabolism during biotic immune responses.

Biomimetic Enzymatic Oxidative Coupling of Barley Phenolamides: Hydroxycinnamoylagmatines.

Oxidative coupling of hydroxycinnamoylagmatines in barley (Hordeum vulgare) and related Hordeum species is part of the plant defense mechanism. Three linkage types have been reported for hydroxycinnamoylagmatine dimers, but knowledge on oxidative coupling reactions underlying their formation is limited. In this study, the monomers coumaroylagmatine, feruloylagmatine, and sinapoylagmatine were each incubated with horseradish peroxidase. Their coupling reactivity was in line with the order of peak potentials measured: sinapoylagmatine (245 mV) > feruloylagmatine (341 mV) > coumaroylagmatine (506 mV). Structure elucidation of fourteen in vitro coupling products by NMR and MS revealed that the three main linkage types were identical to those naturally present in Hordeum species, namely, 4-O-7'/3-8', 2-7'/8-8', and 8-8'/9-N-7'. Furthermore, we identified two linkage types that were not previously reported for hydroxycinnamoylagmatine dimers, namely, 8-8' and 4-O-8'. We conclude that oxidative coupling by horseradish peroxidase can be used for biomimetic formation of natural antifungal hydroxycinnamoylagmatine dimers from barley.

The Role of Hydroxycinnamic Acid Amide Pathway in Plant Immunity.

The compounds involved in the hydroxycinnamic acid amide (HCAA) pathway are an important class of metabolites in plants. Extensive studies have reported that a variety of plant hydroxycinnamamides exhibit pivotal roles in plant-pathogen interactions, such as p-coumaroylagmatine and ferulic acid. The aim of this review is to discuss the emerging findings on the functions of hydroxycinnamic acid amides (HCAAs) accumulation associated with plant defenses against plant pathologies, antimicrobial activity of HCAAs, and the mechanism of HCAAs involved in plant immune responses (such as reactive oxygen species (ROS), cell wall response, plant defense hormones, and stomatal immunity). However, these advances have also revealed the complexity of HCAAs participation in plant defense reactions, and many mysteries remain to be revealed. This review provides an overview of the mechanistic and conceptual insights obtained so far and highlights areas for future exploration of phytochemical defense metabolites.

Broa, an Ethnic Maize Bread, as a Source of Phenolic Compounds.

Maize is an important source of phenolic compounds, specially hydroxycinnamic acids, which are widely known for their antioxidant activity and associated health benefits. However, these effects depend on their bioaccessibility, which is influenced by the different techniques used for food processing. Several traditional products can be obtained from maize and, in Portugal, it is used for the production of an ethnic bread called broa. In order to evaluate the effect of processing on maize phenolic composition, one commercial hybrid and five open-pollinated maize flours and broas were studied. The total phenolic content and antioxidant activity were evaluated by the Folin-Ciocalteu and ORAC assays, respectively. The major phenolics, namely ferulic and p-coumaric acids (in their soluble-free, soluble-conjugated and insoluble forms), insoluble ferulic acid dimers and soluble hydroxycinnamic acid amides were quantitated. Results show that the total phenolic content, antioxidant activity and hydroxycinnamic acids resisted traditional processing conditions used in the production of broas. The content in soluble-free phenolics increased after processing, meaning that their bioaccessibility improved. Portuguese traditional broas, produced with open-pollinated maize varieties, can be considered an interesting dietary source of antioxidant compounds due to the higher content in hydroxycinnamic acids and derivatives.

Dynamic metabolic and transcriptomic profiling reveals the biosynthetic characteristics of hydroxycinnamic acid amides (HCAAs) in sunflower pollen.

Sunflower pollen is a natural nutritious food with a long history and multiple functions, however, the main chemical components apart from flavonoids and their biosynthesis processes have not been thoroughly investigated. In this study, seven hydroxycinnamic acid amides (HCAAs) (1-7) abundant in sunflower pollen were isolated and identified as one type of the pollen's main chemicals. For a comprehensive understanding of HCAA biosynthesis in Helianthus annuus flowers, RNA-seq, metabolomics, and key genes related to biosynthesis in the sunflower were studied. A large number of compounds at different sunflower growth stages (the 7th, 14th, 21st, and 28th days) and high expression levels of related genes in the transcriptome were detected. A molecular network was constructed to clarify the synthetic pathway of HCAAs, which revealed high transcriptional levels of spermidine hydroxycinnamoyl transferase genes (HaSHT2795 and HaSHT2436) in 14-21-days-old flowers. HaSHT2795 enzymes catalyze tri-coumaroylspermidine formation, and virus-induced gene silencing to inhibit HaSHT2795 and HaSHT2436 could significantly reduce the synthesis of hydroxycinnamic acid amides in sunflower pollen. HCAAs were inferred to be related to the formation of pollen walls and the health effects of pollen. Analyzing HCAA biosynthesis and accumulation in H. annuus pollen will be helpful to understand the functions of HCAAs in the development of pollen and its nutritional value.