Glandular Trichomes and Isoprenoid Biosynthesis

In plant ecology, biochemical analyses of bryophytes and vascular plants are often conducted on dried herbarium specimen as species typically grow in distant and inaccessible locations. Here, we present an automated in silico compound classification framework to annotate metabolites using an untargeted data independent acquisition (DIA)–LC/MS–QToF-sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH) ecometabolomics analytical method. We perform a comparative investigation of the chemical diversity at the global level and the composition of metabolite families in ten different species of bryophytes using fresh samples collected on-site and dried specimen stored in a herbarium for half a year. Shannon and Pielou’s diversity indices, hierarchical clustering analysis (HCA), sparse partial least squares discriminant analysis (sPLS-DA), distance-based redundancy analysis (dbRDA), ANOVA with post-hoc Tukey honestly significant difference (HSD) test, and the Fisher’s exact test were used to determine differences in the richness and composition of metabolite families, with regard to herbarium conditions, ecological characteristics, and species. We functionally annotated metabolite families to biochemical processes related to the structural integrity of membranes and cell walls (proto-lignin, glycerophospholipids, carbohydrates), chemical defense (polyphenols, steroids), reactive oxygen species (ROS) protection (alkaloids, amino acids, flavonoids), nutrition (nitrogen- and phosphate-containing glycerophospholipids), and photosynthesis. Changes in the composition of metabolite families also explained variance related to ecological functioning like physiological adaptations of bryophytes to dry environments (proteins, peptides, flavonoids, terpenes), light availability (flavonoids, terpenes, carbohydrates), temperature (flavonoids), and biotic interactions (steroids, terpenes). The results from this study allow to construct chemical traits that can be attributed to biogeochemistry, habitat conditions, environmental changes and biotic interactions. Our classification framework accelerates the complex annotation process in metabolomics and can be used to simplify biochemical patterns. We show that compound classification is a powerful tool that allows to explore relationships in both molecular biology by “zooming in” and in ecology by “zooming out”. The insights revealed by our framework allow to construct new research hypotheses and to enable detailed follow-up studies.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant phospholipids in membranes. The biosynthesis of phospholipids occurs mainly via the Kennedy pathway. Recent studies have shown that through this pathway, choline (Cho) moieties are synthesized through the methylation of phosphoethanolamine (PEtn) to phosphocholine (PCho) by phospho-base N-methyltransferase. In Arabidopsis thaliana, the phosphoethanolamine/phosphocholine phosphatase1 (PECP1) is described as an enzyme that regulates the synthesis of PCho by decreasing the PEtn level during phosphate starvation to avoid the energy-consuming methylation step. By homology search, we identified a gene (At4g29530) encoding a putative PECP1 homolog from Arabidopsis with a currently unknown biological function in planta. We found that At4g29530 is not induced by phosphate starvation, and is mainly expressed in leaves and flowers. The analysis of null mutants and overexpression lines revealed that PEtn, rather than PCho, is the substrate in vivo, as in PECP1. Hydrophilic interaction chromatography-coupled mass spectrometry analysis of head group metabolites shows an increased PEtn level and decreased ethanolamine level in null mutants. At4g29530 null mutants have an early flowering phenotype, which is corroborated by a higher PC/PE ratio. Furthermore, we found an increased PCho level. The choline level was not changed, so the results corroborate that the PEtn-dependent pathway is the main route for the generation of Cho moieties. We assume that the PEtn-hydrolyzing enzyme participates in fine-tuning the metabolic pathway, and helps prevent the energy-consuming biosynthesis of PCho through the methylation pathway.

Nucleotide-binding domain–leucine-rich repeat-type immune receptors (NLRs) protect plants against pathogenic microbes through intracellular detection of effector proteins. However, this comes at a cost, as NLRs can also induce detrimental autoimmunity in genetic interactions with foreign alleles. This may occur when independently evolved genomes are combined in inter- or intraspecific crosses, or when foreign alleles are introduced by mutagenesis or transgenesis. Most autoimmunity-inducing NLRs are encoded within highly variable NLR gene clusters with no known immune functions, which were termed autoimmune risk loci. Whether risk NLRs differ from sensor NLRs operating in natural pathogen resistance and how risk NLRs are activated in autoimmunity is unknown. Here, we analyzed the DANGEROUS MIX2 risk locus, a major autoimmunity hotspot in Arabidopsis thaliana. By gene editing and heterologous expression, we show that a single gene, DM2h, is necessary and sufficient for autoimmune induction in three independent cases of autoimmunity in accession Landsberg erecta. We focus on autoimmunity provoked by an EDS1-yellow fluorescent protein (YFP)NLS fusion protein to characterize DM2h functionally and determine features of EDS1-YFPNLS activating the immune receptor. Our data suggest that risk NLRs function in a manner reminiscent of sensor NLRs, while autoimmunity-inducing properties of EDS1-YFPNLS in this context are unrelated to the protein\'s functions as an immune regulator. We propose that autoimmunity, at least in some cases, may be caused by spurious, stochastic interactions of foreign alleles with coincidentally matching risk NLRs.

Protein glycation is usually referred to as an array of non-enzymatic post-translational modifications formed by reducing sugars and carbonyl products of their degradation. The resulting advanced glycation end products (AGEs) represent a heterogeneous group of covalent adducts, known for their pro-inflammatory effects in mammals, and impacting on pathogenesis of metabolic diseases and ageing. In plants, AGEs are the markers of tissue ageing and response to environmental stressors, the most prominent of which is drought. Although water deficit enhances protein glycation in leaves, its effect on seed glycation profiles is still unknown. Moreover, the effect of drought on biological activities of seed protein in mammalian systems is still unstudied with respect to glycation. Therefore, here we address the effects of a short-term drought on the patterns of seed protein-bound AGEs and accompanying alterations in pro-inflammatory properties of seed protein in the context of seed metabolome dynamics. A short-term drought, simulated as polyethylene glycol-induced osmotic stress and applied at the stage of seed filling, resulted in the dramatic suppression of primary seed metabolism, although the secondary metabolome was minimally affected. This was accompanied with significant suppression of NF-kB activation in human SH-SY5Y neuroblastoma cells after a treatment with protein hydrolyzates, isolated from the mature seeds of drought-treated plants. This effect could not be attributed to formation of known AGEs. Most likely, the prospective anti-inflammatory effect of short-term drought is related to antioxidant effect of unknown secondary metabolite protein adducts, or down-regulation of unknown plant-specific AGEs due to suppression of energy metabolism during seed filling.

Background: The plant phyllosphere is a well-studied habitat characterized by low nutrient availability and high community dynamics. In contrast, plant trichomes, known for their production of a large number of metabolites, are a yet unexplored habitat for microbes. We analyzed the phyllosphere as well as trichomes of two tomato genotypes (Solanum lycopersicum LA4024, S. habrochaites LA1777) by targeting bacterial 16S rRNA gene fragments. Results: Leaves, leaves without trichomes, and trichomes alone harbored similar abundances of bacteria (108–109 16S rRNA gene copy numbers per gram of sample). In contrast, bacterial diversity was found significantly increased in trichome samples (Shannon index: 4.4 vs. 2.5). Moreover, the community composition was significantly different when assessed with beta diversity analysis and corresponding statistical tests. At the bacterial class level, Alphaproteobacteria (23.6%) were significantly increased, whereas Bacilli (8.6%) were decreased in trichomes. The bacterial family Sphingomonadacea (8.4%) was identified as the most prominent, trichome-specific feature; Burkholderiaceae and Actinobacteriaceae showed similar patterns. Moreover, Sphingomonas was identified as a central element in the core microbiome of trichome samples, while distinct low-abundant bacterial families including Hymenobacteraceae and Alicyclobacillaceae were exclusively found in trichome samples. Niche preferences were statistically significant for both genotypes and genotype-specific enrichments were further observed. Conclusion: Our results provide first evidence of a highly specific trichome microbiome in tomato and show the importance of micro-niches for the structure of bacterial communities on leaves. These findings provide further clues for breeding, plant pathology and protection as well as so far unexplored natural pathogen defense strategies.

The exchange of small RNAs (sRNAs) between hosts and pathogens can lead to gene silencing in the recipient organism, a mechanism termed cross-kingdom RNAi (ck-RNAi). While fungal sRNAs promoting virulence are established, the significance of ck-RNAi in distinct plant pathogens is not clear. Here, we describe that sRNAs of the pathogen Hyaloperonospora arabidopsidis, which represents the kingdom of oomycetes and is phylogenetically distant from fungi, employ the host plant's Argonaute (AGO)/RNA-induced silencing complex for virulence. To demonstrate H. arabidopsidis sRNA (HpasRNA) functionality in ck-RNAi, we designed a novel CRISPR endoribonuclease Csy4/GUS reporter that enabled in situ visualization of HpasRNA-induced target suppression in Arabidopsis. The significant role of HpasRNAs together with AtAGO1 in virulence was revealed in plant atago1 mutants and by transgenic Arabidopsis expressing a short-tandem-target-mimic to block HpasRNAs, that both exhibited enhanced resistance. HpasRNA-targeted plant genes contributed to host immunity, as Arabidopsis gene knockout mutants displayed quantitative enhanced susceptibility.

Mitogen‐activated protein kinase (MAPK) cascades are key signalling modules of plant defence responses to pathogen‐associated molecular patterns (PAMPs, e.g. bacterial flg22 peptide). The Tandem Zinc Finger Protein 9 (TZF9) is an RNA‐binding protein that is phosphorylated by two PAMP‐responsive MAPKs, MPK3 and MPK6. We mapped the major phosphosites in TZF9 and showed their importance for controlling in vitro RNA‐binding activity, in vivo flg22‐induced rapid disappearance of TZF9‐labelled processing body‐like structures and TZF9 protein turnover. Microarray analysis showed a strong discordance between transcriptome (total mRNA) and translatome (polysome‐associated mRNA) in the tzf9 mutant, with more mRNAs associated to ribosomes in the absence of TZF9. This suggests that TZF9 may sequester and inhibit translation of subsets of mRNAs. Fittingly, TZF9 physically interacts with poly(A)‐binding protein 2 (PAB2), a hallmark constituent of stress granules – a site for stress‐induced translational stalling/arrest. TZF9 even promotes stress granule assembly in the absence of stress. Hence, MAPKs may control defence gene expression post‐transcriptionally through release from translation arrest within TZF9‐PAB2‐containing RNA granules or perturbing PAB2 functions in translation control (e.g. in the mRNA closed‐loop model of translation).

Black pepper (Piper nigrum L.) is known for the high content of piperine, a cinnamoyl amide derivative regarded as largely responsible for the pungent taste of this widely used spice. Despite its long history and worldwide use, the biosynthesis of piperine and related amides has been enigmatic up to now. In this report we describe a specific piperic acid CoA ligase from immature green fruits of P. nigrum. The corresponding enzyme was cloned and functionally expressed in E. coli. The recombinant enzyme displays a high specificity for piperic acid and does not accept the structurally related feruperic acid characterized by a similar C‐2 extension of the general C6‐C3 phenylpropanoid structure. The enzyme is also inactive with the standard set of hydroxycinnamic acids tested including caffeic acid, 4‐coumaric acid, ferulic acid, and sinapic acid. Substrate specificity is corroborated by in silico modeling which suggests a perfect fit of the substrate piperic acid to the active site of the piperic acid CoA ligase. The CoA ligase gene shows highest expression levels in immature green fruits, is also expressed in leaves and flowers, but not in roots. Virus‐induced gene silencing provided some preliminary indications that the production of piperoyl‐CoA is required for the biosynthesis of piperine in black pepper fruits.

Glandular trichomes are epidermal outgrowths that are the site of biosynthesis and storage of large quantities of specialized metabolites. Besides their role in the protection of plants against biotic and abiotic stresses, they have attracted interest owing to the importance of the compounds they produce for human use; for example, as pharmaceuticals, flavor and fragrance ingredients, or pesticides. Here, we review what novel concepts investigations on glandular trichomes have brought to the field of specialized metabolism, particularly with respect to chemical and enzymatic diversity. Furthermore, the next challenges in the field are understanding the metabolic network underlying the high productivity of glandular trichomes and the transport and storage of metabolites. Another emerging area is the development of glandular trichomes. Studies in some model species, essentially tomato, tobacco, and Artemisia, are now providing the first molecular clues, but many open questions remain: How is the distribution and density of different trichome types on the leaf surface controlled? When is the decision for an epidermal cell to differentiate into one type of trichome or another taken? Recent advances in gene editing make it now possible to address these questions and promise exciting discoveries in the near future.

Secretions from glandular trichomes potentially protect the plant against a variety of aggressors. In the tomato genus, wild species constitute a rich source of chemical diversity produced at the leaf surface by glandular trichomes. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we identify two derivatives of 7-epi-zingiberene from S. habrochaites that had not been reported as yet. We identified them as 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene. Using a combination of genetics and transcriptomics we identified a single cytochrome P450 oxygenase, ShCYP71D184 that carries out two successive oxidations to generate the two sesquiterpenoids. Bioactivity assays showed that only 9-hydroxy-10,11-epoxyzingiberene exhibits substantial toxicity against B. tabaci. In addition, both 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene display substantial growth inhibitory activities against a range of microorganisms, including Bacillus subtilis, Phytophtora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.