• Despite recent advances in elucidation of natural products in root exudates, there are significant gaps in our understanding of the ecological significance of products in the rhizosphere. • Here, we investigated the potential of barley (Hordeum vulgare) to secrete defense root exudates when challenged by the soilborne pathogen Fusarium graminearum. • Liquid chromatography with photodiode array detection (LC-DAD) was used to profile induced small-molecular-weight exudates. Thus, t-cinnamic, p-coumaric, ferulic, syringic and vanillic acids were assigned to plant metabolism and were induced within 2 d after Fusarium inoculation. Biological tests demonstrated the ability of those induced root exudates to inhibit the germination of F. graminearum macroconidia. In vivo labeling experiments with ¹³CO₂ revealed that the secreted t-cinnamic acid was synthesized de novo within 2 d of fungal infection. Simultaneously to its root exudation, t-cinnamic acid was accumulated in the roots. Microscopic analysis showed that nonlignin cell wall phenolics were induced not only in necrosed zones but in all root tissues. • Results suggest that barley plants under attack respond by de novo biosynthesis and secretion of compounds with antimicrobial functions that may mediate natural disease resistance.

Background: The first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine β-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine. The resulting unstable aglycon is rapidly converted into a highly reactive dialdehyde, from which more than 2,000 MIAs are derived. Many studies were conducted to elucidate the biosynthesis and regulation of pharmacologically valuable MIAs such as vinblastine and vincristine in Catharanthus roseus or ajmaline in Rauvolfia serpentina. However, very few reports focused on the MIA physiological functions. Results: In this study we showed that a strictosidine pool existed in planta and that the strictosidine deglucosylation product(s) was (were) specifically responsible for in vitro protein cross-linking and precipitation suggesting a potential role for strictosidine activation in plant defence. The spatial feasibility of such an activation process was evaluated in planta. On the one hand, in situ hybridisation studies showed that CrSTR and CrSGD were coexpressed in the epidermal first barrier of C. roseus aerial organs. However, a combination of GFP-imaging, bimolecular fluorescence complementation and electromobility shift-zymogram experiments revealed that STR from both C. roseus and R. serpentina were localised to the vacuole whereas SGD from both species were shown to accumulate as highly stable supramolecular aggregates within the nucleus. Deletion and fusion studies allowed us to identify and to demonstrate the functionality of CrSTR and CrSGD targeting sequences. Conclusions: A spatial model was drawn to explain the role of the subcellular sequestration of STR and SGD to control the MIA metabolic flux under normal physiological conditions. The model also illustrates the possible mechanism of massive activation of the strictosidine vacuolar pool upon enzyme-substrate reunion occurring during potential herbivore feeding constituting a so-called "nuclear time bomb" in reference to the "mustard oil bomb" commonly used to describe the myrosinase-glucosinolate defence system in Brassicaceae.

Plant health and fitness widely depend on interactions with soil microorganisms. Some bacteria such as pseudomonads can inhibit pathogens by producing antibiotics, and controlling these bacteria could help improve plant fitness. In the present study, we tested whether plants induce changes in the antifungal activity of root-associated bacteria as a response to root pathogens. We grew barley plants in a split-root system with one side of the root system challenged by the pathogen Pythium ultimum and the other side inoculated with the biocontrol strain Pseudomonas fluorescens CHA0. We used reporter genes to follow the expression of ribosomal RNA indicative of the metabolic state and of the gene phlA, required for production of 2,4-diacetylphloroglucinol, a key component of antifungal activity. Infection increased the expression of the antifungal gene phlA. No contact with the pathogen was required, indicating that barley influenced gene expression by the bacteria in a systemic way. This effect relied on increased exudation of diffusible molecules increasing phlA expression, suggesting that communication with rhizosphere bacteria is part of the pathogen response of plants. Tripartite interactions among plants, pathogens, and bacteria appear as a novel determinant of plant response to root pathogens.

Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis.

The oomycete Aphanomyces euteiches causes up to 80 % crop loss in pea (Pisum sativum). Aphanomyces euteiches invades the root system leading to a complete arrest of root growth and ultimately to plant death. To date, disease control measures are limited to crop rotation and no resistant pea lines are available. The present study aims to get a deeper understanding of the early oomycete–plant interaction at the tissue and cellular levels. Here, the process of root infection by A. euteiches on pea is investigated using flow cytometry and microscopic techniques. Dynamic changes in secondary metabolism are analysed with high-performance liquid chromatography with diode-array detection. Root infection is initiated in the elongation zone but not in the root cap and border cells. Border-cell production is significantly enhanced in response to root inoculation with changes in their size and morphology. The stimulatory effect of A. euteiches on border-cell production is dependent on the number of oospores inoculated. Interestingly, border cells respond to pathogen challenge by increasing the synthesis of the phytoalexin pisatin. Distinctive responses to A. euteiches inoculation occur at the root tissue level. The findings suggest that root border cells in pea are involved in local defence of the root tip against A. euteiches. Root border cells constitute a convenient quantitative model to measure the molecular and cellular basis of plant–microbe interactions.