Mass Spectrometry Imaging Disclosed Spatial Distribution of Defense-Related Metabolites in Triticum spp.

Fusarium Head Blight is the most common fungal disease that strongly affects Triticum spp., reducing crop yield and leading to the accumulation of toxic metabolites. Several studies have investigated the plant metabolic response to counteract mycotoxins accumulation. However, information on the precise location where the defense mechanism is taking place is scarce. Therefore, this study aimed to investigate the specific tissue distribution of defense metabolites in two Triticum species and use this information to postulate on the metabolites’ functional role, unlocking the “location-to-function” paradigm. To address this challenge, transversal cross-sections were obtained from the middle of the grains. They were analyzed using an atmospheric-pressure (AP) SMALDI MSI source (AP-SMALDI5 AF, TransMIT GmbH, Giessen, Germany) coupled to a Q Exactive HF (Thermo Fisher Scientific GmbH, Bremen, Germany) orbital trapping mass spectrometer. Our result revealed the capability of (AP)-SMALDI MSI instrumentation to finely investigate the spatial distribution of wheat defense metabolites, such as hydroxycinnamic acid amides, oxylipins, linoleic and α-linoleic acids, galactolipids, and glycerolipids.

Function of hydroxycinnamoyl spermidines in seedling growth of Arabidopsis

Hydroxycinnamic acid amides (HCAAs) 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.

Induced ligno-suberin vascular coating and tyramine-derived hydroxycinnamic acid amides restrict Ralstonia solanacearum colonization in resistant tomato roots

The soil borne pathogen Ralstonia solanacearum is the causing agent of bacterial wilt, a devastating disease affecting major agricultural crops. R. solanacearum enters plants through the roots and reaches the vasculature, causing rapid wilting. We recently showed that tomato varieties resistant to bacterial wilt restrict bacterial movement in the plant. In the present work we go a step forward by identifying the physico-chemical nature of the barriers induced in resistant tomato roots in response to R. solanacearum. We describe that resistant tomato specifically responds to infection by assembling de novo a structural barrier at the vasculature formed by a ligno-suberin coating and tyramine-derived hydroxycinnamic acid amides (HCAAs). On the contrary, susceptible tomato does not form these reinforcements in response to the pathogen but instead displays lignin structural changes compatible with its degradation. Further, we show that overexpressing genes of the ligno-suberin pathway in a commercial susceptible variety of tomato restricts R. solanacearum movement inside the plant and slows disease progression, enhancing resistance to the pathogen. We thus propose that the induced barrier in resistant plants does not only restrict the movement of the pathogen, but may also prevent cell wall degradation by the pathogen and confer anti-microbial properties.

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.

Clovamide, a Hydroxycinnamic Acid Amide, Is a Resistance Factor Against Phytophthora spp. in Theobroma cacao

The hydroxycinnamic acid amides (HCAAs) are a diverse group of plant-specialized phenylpropanoid metabolites distributed widely in the plant kingdom and are known to be involved in tolerance to abiotic and biotic stress. The HCAA clovamide is reported in a small number of distantly related species. To explore the contribution of specialized metabolites to disease resistance in cacao (Theobroma cacao L., chocolate tree), we performed untargeted metabolomics using liquid chromatography – tandem mass spectrometry (LC-MS/MS) and compared the basal metabolite profiles in leaves of two cacao genotypes with contrasting levels of susceptibility to Phytophthora spp. Leaves of the tolerant genotype ‘Scavina 6’ (‘Sca6’) were found to accumulate dramatically higher levels of clovamide and several other HCAAs compared to the susceptible ‘Imperial College Selection 1’ (‘ICS1’). Clovamide was the most abundant metabolite in ‘Sca6’ leaf extracts based on MS signal, and was up to 58-fold higher in ‘Sca6’ than in ‘ICS1’. In vitro assays demonstrated that clovamide inhibits growth of three pathogens of cacao in the genus Phytophthora, is a substrate for cacao polyphenol oxidase, and is a contributor to enzymatic browning. Furthermore, clovamide inhibited proteinase and pectinase in vitro, activities associated with defense in plant-pathogen interactions. Fruit epidermal peels from both genotypes contained substantial amounts of clovamide, but two sulfated HCAAs were present at high abundance exclusively in ‘Sca6’ suggesting a potential functional role of these compounds. The potential to breed cacao with increased HCAAs for improved agricultural performance is discussed.

Hydroxycinnamic Acids and Their Derivatives in Broa, a Traditional Ethnic Maize Bread

Maize is one of the most interesting dietary sources of hydroxycinnamic acids, widely known for their beneficial health effects, namely antioxidant properties. This work aims to identify hydroxycinnamic acids and their derivatives in broa, a Portuguese traditional ethnic maize bread, and corresponding maize flours. Soluble and insoluble phenolic fractions of diverse maize flours and corresponding broas were prepared and analysed by HPLC-DAD-MS/MS (high-performance liquid chromatography coupled with diode array detector and tandem mass spectrometry). Besides free hydroxycinnamic acids, mainly ferulic and p-coumaric acids, several structural isomers and stereoisomers of insoluble ferulic acid dehydrodimers (n = 18) and trimers (n = 11), were also identified. Hydroxycinnamic acid amides consisting of coumaroyl and feruloyl conjugates (n = 22) were present in both soluble and insoluble fractions of maize flours and breads, in different isomeric forms. A new compound was putatively identified as bis-N,N′-diferuloyl putrescine. Additionally, more complex and insoluble hydroxycinnamic acid amides, derived from ferulic acid dehydrodimers (n = 47) and trimers (n = 18), were also putatively identified for the first time, suggesting that hydroxycinnamic acid amides are also linked to maize cell walls. Since hydroxycinnamic derivatives were not only identified in maize flours, but also in broas, they can contribute to the antioxidant properties and beneficial health effects of maize-based foods.