Aleš Svatoš

A glucosinolate metabolism pathway in living plant cells mediates broad-spectrum antifungal defense

Selection pressure exerted by insects and microorganisms shapes the diversity of plant secondary metabolites. We identified a metabolic pathway for glucosinolates, known insect deterrents, that differs from the pathway activated by chewing insects. This pathway is active in living plant cells, may contribute to glucosinolate turnover, and has been recruited for broad-spectrum antifungal defense responses. The Arabidopsis CYP81F2 gene encodes a P450 monooxygenase that is essential for the pathogen-induced accumulation of 4-methoxyindol-3-ylmethylglucosinolate, which in turn is activated by the atypical PEN2 myrosinase (a type of β-thioglucoside glucohydrolase) for antifungal defense. We propose that reiterated enzymatic cycles, controlling the generation of toxic molecules and their detoxification, enable the recruitment of glucosinolates in defense responses.

Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. VII. Changes in the plant's proteome

When Manduca sexta attacks Nicotiana attenuata, fatty acid-amino acid conjugates (FACs) in the larvaes oral secretions (OS) are introduced into feeding wounds. These FACs trigger a transcriptional response that is similar to the response induced by insect damage. Using two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time of flight, and liquid chromatography-tandem mass spectrometry, we characterized the proteins in phenolic extracts and in a nuclear fraction of leaves elicited by larval attack, and/or in leaves wounded and treated with OS, FAC-free OS, and synthetic FACs. Phenolic extracts yielded approximately 600 protein spots, many of which were altered by elicitation, whereas nuclear protein fractions yielded approximately 100 spots, most of which were unchanged by elicitation. Reproducible elicitor-induced changes in 90 spots were characterized. In general, proteins that increased were involved in primary metabolism, defense, and transcriptional and translational regulation; those that decreased were involved in photosynthesis. Like the transcriptional defense responses, proteomic changes were strongly elicited by the FACs in OS. A semiquantitative reverse transcription-PCR approach based on peptide sequences was used to compare transcript and protein accumulation patterns for 17 candidate proteins. In six cases the patterns of elicited transcript accumulation were consistent with those of elicited protein accumulation. Functional analysis of one of the identified proteins involved in photosynthesis, RuBPCase activase, was accomplished by virus-induced gene silencing. Plants with decreased levels of RuBPCase activase protein had reduced photosynthetic rates and RuBPCase activity, and less biomass, responses consistent with those of herbivore-attacked plants. We conclude that the response of the plants proteome to herbivore elicitation is complex, and integrated transcriptome-proteome-metabolome analysis is required to fully understand this ubiquitous ecological interaction.

Nonuniform distribution of glucosinolates in Arabidopsis thaliana leaves has important consequences for plant defense

The spatial distribution of plant defenses within a leaf may be critical in explaining patterns of herbivory. The generalist lepidopteran larvae, Helicoverpa armigera (the cotton bollworm), avoided the midvein and periphery of Arabidopsis thaliana rosette leaves and fed almost exclusively on the inner lamina. This feeding pattern was attributed to glucosinolates because it was not evident in a myrosinase mutant that lacks the ability to activate glucosinolate defenses by hydrolysis. To measure the spatial distribution of glucosinolates in A. thaliana leaves at a fine scale, we constructed ion intensity maps from MALDI-TOF (matrix assisted laser desorption/ionization-time of flight) mass spectra. The major glucosinolates were found to be more abundant in tissues of the midvein and the periphery of the leaf than the inner lamina, patterns that were validated by HPLC analyses of dissected leaves. In addition, there were differences in the proportions of the three major glucosinolates in different leaf regions. Hence, the distribution of glucosinolates within the leaf appears to control the feeding preference of H. armigera larvae. The preferential allocation of glucosinolates to the periphery may play a key role in the defense of leaves by creating a barrier to the feeding of chewing herbivores that frequently approach leaves from the edge.

CYP71B15 (PAD3) Catalyzes the Final Step in Camalexin Biosynthesis

Camalexin represents the main phytoalexin in Arabidopsis (Arabidopsis thaliana). The camalexin-deficient phytoalexin deficient 3 (pad3) mutant has been widely used to assess the biological role of camalexin, although the exact substrate of the cytochrome P450 enzyme 71B15 encoded by PAD3 remained elusive. 2-(Indol-3-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid (dihydrocamalexic acid) was identified as likely intermediate in camalexin biosynthesis downstream of indole-3-acetaldoxime, as it accumulated in leaves of silver nitrate-induced pad3 mutant plants and it complemented the camalexin-deficient phenotype of a cyp79b2/cyp79b3 double-knockout mutant. Recombinant CYP71B15 heterologously expressed in yeast catalyzed the conversion of dihydrocamalexic acid to camalexin with preference of the (S)-enantiomer. Arabidopsis microsomes isolated from leaves of CYP71B15-overexpressing and induced wild-type plants were capable of the same reaction but not microsomes from induced leaves of pad3 mutants. In conclusion, CYP71B15 catalyzes the final step in camalexin biosynthesis.

Siderophores mediate reduced and increased uptake of cadmium by Streptomyces tendae F4 and sunflower (Helianthus annuus), respectively

Aims:  As a toxic metal, cadmium (Cd) affects microbial and plant metabolic processes, thereby potentially reducing the efficiency of microbe or plant‐mediated remediation of Cd‐polluted soil. The role of siderophores produced by Streptomyces tendae F4 in the uptake of Cd by bacteria and plant was investigated to gain insight into the influence of siderophores on Cd availability to micro‐organisms and plants.

Involvement of siderophores in the reduction of metal-induced inhibition of auxin synthesis in Streptomyces spp

Unlike synthetic metal chelators, microbe-assisted phytoremediation provides plants with natural metal-solubilizing chelators which do not constitute a potential source of environmental pollution. Concurrently with microbial chelators, plant growth promotion can be enhanced through bacterially-produced phytohormones. In this work, the simultaneous production of siderophores and auxins by Streptomyces was studied to gain insight for future application in plant growth and phytoremediation in a metal-contaminated soil. Standard auxin and siderophore detection assays indicated that all of the investigated Streptomyces strains can produce these metabolites simultaneously. However, Al(3+), Cd(2+), Cu(2+), Fe(3+) and Ni(2+), or a combination of Fe(3+) and Cd(2+), and Fe(3+) and Ni(2+) affected auxin production negatively, as revealed by spectrophotometry and gas chromatography-mass spectrometry. This effect was more dramatic in a siderophore-deficient mutant. In contrast, except for Fe, all the metals stimulated siderophore production. Mass spectrometry showed that siderophore and auxin-containing supernatants from a representative Streptomyces species contain three different hydroxamate siderophores, revealing the individual binding responses of these siderophores to Cd(2+) and Ni(2+), and thus, showing their auxin-stimulating effects. We conclude that siderophores promote auxin synthesis in the presence of Al(3+), Cd(2+), Cu(2+) and Ni(2+) by chelating these metals. Chelation makes the metals less able to inhibit the synthesis of auxins, and potentially increases the plant growth-promoting effects of auxins, which in turn enhances the phytoremediation potential of plants.

Matrix-free UV-laser desorption/ionization (LDI) mass spectrometric imaging at the single-cell level: Distribution of secondary metabolites of Arabidopsis thaliana and Hypericum species

The present paper describes matrix-free laser desorption/ionisation mass spectrometric imaging (LDI-MSI) of highly localized UV-absorbing secondary metabolites in plant tissues at single-cell resolution. The scope and limitations of the method are discussed with regard to plants of the genus Hypericum. Naphthodianthrones such as hypericin and pseudohypericin are traceable in dark glands on Hypericum leaves, placenta, stamens and styli; biflavonoids are also traceable in the pollen of this important phytomedical plant. The highest spatial resolution achieved, 10 microm, was much higher than that achieved by commonly used matrix-assisted laser desorption/ionization (MALDI) imaging protocols. The data from imaging experiments were supported by independent LDI-TOF/MS analysis of cryo-sectioned, laser-microdissected and freshly cut plant material. The results confirmed the suitability of combining laser microdissection (LMD) and LDI-TOF/MS or LDI-MSI to analyse localized plant secondary metabolites. Furthermore, Arabidopsis thaliana was analysed to demonstrate the feasibility of LDI-MSI for other commonly occurring compounds such as flavonoids. The organ-specific distribution of kaempferol, quercetin and isorhamnetin, and their glycosides, was imaged at the cellular level.

MALDI‐imaging mass spectrometry – An emerging technique in plant biology

Recent advances in instrumentation and sample preparation have facilitated the mass spectrometric (MS) imaging of a large variety of biological molecules from small metabolites to large proteins. The technique can be applied at both the tissue and the single‐cell level, and provides information regarding the spatial distribution of specific molecules. Nevertheless, the use of MS imaging in plant science remains far from routine, and there is still a need to adapt protocols to suit specific tissues. We present an overview of MALDI‐imaging MS (MSI) technology and its use for the analysis of plant tissue. Recent methodological developments have been summarized, and the major challenges involved in using MALDI‐MSI, including sample preparation, the analysis of metabolites and peptides, and strategies for data evaluation are all discussed. Some attention is given to the identification of differentially distributed compounds. To date, the use of MALDI‐MSI in plant research has been limited. Examples include leaf surface metabolite maps, the characterization of soluble metabolite translocation in planta, and the profiling of protein/metabolite patterns in cereal grain cross‐sections. Improvements to both sample preparation strategies and analytical platforms (aimed at both spectrum acquisition and post‐acquisition analysis) will enhance the relevance of MALDI‐MSI technology in plant research.

Structural Complexity, Differential Response to Infection, and Tissue Specificity of Indolic and Phenylpropanoid Secondary Metabolism in Arabidopsis Roots

Levels of indolic and phenylpropanoid secondary metabolites in Arabidopsis (Arabidopsis thaliana) leaves undergo rapid and drastic changes during pathogen defense, yet little is known about this process in roots. Using Arabidopsis wild-type and mutant root cultures as an experimental system, and the root-pathogenic oomycete, Pythium sylvaticum, for infections, we analyzed the aromatic metabolite profiles in soluble extracts from uninfected and infected roots, as well as from the surrounding medium. A total of 16 indolic, one heterocyclic, and three phenylpropanoid compounds were structurally identified by mass spectrometry and nuclear magnetic resonance analyses. Most of the indolics increased strongly upon infection, whereas the three phenylpropanoids decreased. Concomitant increases in both indolic and phenylpropanoid biosynthetic mRNAs suggested that phenylpropanoids other than those examined here in “soluble extracts” were coinduced with the indolics. These and previous results indicate that roots differ greatly from leaves with regard to the nature and relative abundance of all major soluble phenylpropanoid constituents. For indolics, by contrast, our data reveal far-reaching similarities between roots and leaves and, beyond this comparative aspect, provide an insight into this highly diversified yet under-explored metabolic realm. The data point to metabolic interconnections among the compounds identified and suggest a partial revision of the previously proposed camalexin pathway.

Novel roles for genetically modified plants in environmental protection

Transgenic plants of environmental benefit typically consist of plants that either reduce the input of agrochemicals into the environment or make the biological remediation of contaminated areas more efficient. Examples include the construction of species that result in reduced pesticide use and of species that contain genes for either the degradation of organics or the increased accumulation of inorganics. Cutting-edge approaches, illustrated by our own work, focus on the applicability of genetically modified (GM) plants that produce insect pheromones or that are specifically tailored to the phytoremediation of cadmium or PCBs. This paper discusses the role that the next generation of GM plants might play in preventing and reducing chemical contamination and in converting contaminated sites into safe agricultural or recreational land.