The Capsicum terpenoid biosynthetic module is affected by spider-mite herbivory.

In response to herbivory, Capsicum annuum leaves adapt their specialized metabolome that may protect the plant against herbivore feeding either directly or indirectly through volatile metabolites acting as cues for natural enemies of the herbivore. The volatile blend of spider-mite infested leaves differs from non-challenged leaves predominantly by a higher contribution of mono- and sesquiterpenes. In addition to these terpenoids released into the headspace, the terpenoid composition of the leaves alters upon herbivory. All this suggests an important role for terpenoids and their biosynthetic machinery in the defence against herbivory. Here, we show that the C. annuum genome contains a terpene synthase (TPS) gene family of 103 putative members of which structural analysis revealed that 27 encode functional enzymes. Transcriptome analysis showed that several TPS loci were differentially expressed upon herbivory in leaves of two C. annuum genotypes, that differ in susceptibility towards spider mites. The relative expression of upstream biosynthetic genes from the mevalonate and the methylerythritol phosphate pathway also altered upon herbivory, revealing a shift in the metabolic flux through the terpene biosynthetic module. The expression of multiple genes potentially acting downstream of the TPSs, including cytochrome P450 monooxygenases, UDP-glucosyl transferases, and transcription factors strongly correlated with the herbivory-induced TPS genes. A selection of herbivory-induced TPS genes was functionally characterized through heterologous expression and the products that these enzymes catalysed matched with the volatile and non-volatile terpenoids induced in response to herbivory.

Tomato geranylgeranyl diphosphate synthase isoform 1 is involved in the stress-triggered production of diterpenes in leaves and strigolactones in roots.

Carotenoids are photoprotectant pigments and precursors of hormones such as strigolactones (SL). Carotenoids are produced in plastids from geranylgeranyl diphosphate (GGPP), which is diverted to the carotenoid pathway by phytoene synthase (PSY). In tomato (Solanum lycopersicum), three genes encode plastid-targeted GGPP synthases (SlG1 to SlG3) and three genes encode PSY isoforms (PSY1 to PSY3). Here, we investigated the function of SlG1 by generating loss-of-function lines and combining their metabolic and physiological phenotyping with gene co-expression and co-immunoprecipitation analyses. Leaves and fruits of slg1 lines showed a wild-type phenotype in terms of carotenoid accumulation, photosynthesis, and development under normal growth conditions. In response to bacterial infection, however, slg1 leaves produced lower levels of defensive GGPP-derived diterpenoids. In roots, SlG1 was co-expressed with PSY3 and other genes involved in SL production, and slg1 lines grown under phosphate starvation exuded less SLs. However, slg1 plants did not display the branched shoot phenotype observed in other SL-defective mutants. At the protein level, SlG1 physically interacted with the root-specific PSY3 isoform but not with PSY1 and PSY2. Our results confirm specific roles for SlG1 in producing GGPP for defensive diterpenoids in leaves and carotenoid-derived SLs (in combination with PSY3) in roots.

Role of Strigolactones in the Host Specificity of Broomrapes and Witchweeds.

Root parasitic plants of the Orobanchaceae, broomrapes and witchweeds, pose a severe problem to agriculture in Europe, Asia and especially Africa. These parasites are totally dependent on their host for survival, and therefore, their germination is tightly regulated by host presence. Indeed, their seeds remain dormant in the soil until a host root is detected through compounds called germination stimulants. Strigolactones (SLs) are the most important class of germination stimulants. They play an important role in planta as a phytohormone and, upon exudation from the root, function in the recruitment of symbiotic arbuscular mycorrhizal fungi. Plants exude mixtures of various different SLs, possibly to evade detection by these parasites and still recruit symbionts. Vice versa, parasitic plants must only respond to the SL composition that is exuded by their host, or else risk germination in the presence of non-hosts. Therefore, parasitic plants have evolved an entire clade of SL receptors, called HTL/KAI2s, to perceive the SL cues. It has been demonstrated that these receptors each have a distinct sensitivity and specificity to the different known SLs, which possibly allows them to recognize the SL-blend characteristic of their host. In this review, we will discuss the molecular basis of SL sensitivity and specificity in these parasitic plants through HTL/KAI2s and review the evidence that these receptors contribute to host specificity of parasitic plants.

Genetic Mapping of the Root Mycobiota in Rice and its Role in Drought Tolerance.

BACKGROUND: Rice is the second most produced crop worldwide, but is highly susceptible to drought. Micro-organisms can potentially alleviate the effects of drought. The aim of the present study was to unravel the genetic factors involved in the rice-microbe interaction, and whether genetics play a role in rice drought tolerance. For this purpose, the composition of the root mycobiota was characterized in 296 rice accessions (Oryza sativa L. subsp. indica) under control and drought conditions. Genome wide association mapping (GWAS) resulted in the identification of ten significant (LOD > 4) single nucleotide polymorphisms (SNPs) associated with six root-associated fungi: Ceratosphaeria spp., Cladosporium spp., Boudiera spp., Chaetomium spp., and with a few fungi from the Rhizophydiales order. Four SNPs associated with fungi-mediated drought tolerance were also found. Genes located around those SNPs, such as a DEFENSIN-LIKE (DEFL) protein, EXOCYST TETHERING COMPLEX (EXO70), RAPID ALKALINIZATION FACTOR-LIKE (RALFL) protein, peroxidase and xylosyltransferase, have been shown to be involved in pathogen defense, abiotic stress responses and cell wall remodeling processes. Our study shows that rice genetics affects the recruitment of fungi, and that some fungi affect yield under drought. We identified candidate target genes for breeding to improve rice-fungal interactions and hence drought tolerance.

Transcriptome analysis of the phosphate starvation response sheds light on strigolactone biosynthesis in rice.

Phosphorus (P) is a major element required for plant growth and development. To cope with P shortage, plants activate local and long-distance signaling pathways, such as an increase in the production and exudation of strigolactones (SLs). The role of the latter in mitigating P deficiency is, however, still largely unknown. To shed light on this, we studied the transcriptional response to P starvation and replenishment in wild-type rice and a SL mutant, dwarf10 (d10), and upon exogenous application of the synthetic SL GR24. P starvation resulted in major transcriptional alterations, such as the upregulation of P TRANSPORTER, SYG1/PHO81/XPR1 (SPX) and VACUOLAR PHOSPHATE EFFLUX TRANSPORTER. Gene Ontology (GO) analysis of the genes induced by P starvation showed enrichment in phospholipid catabolic process and phosphatase activity. In d10, P deficiency induced upregulation of genes enriched for sesquiterpenoid production, secondary shoot formation and metabolic processes, including lactone biosynthesis. Furthermore, several genes induced by GR24 treatment shared the same GO terms with P starvation-induced genes, such as oxidation reduction, heme binding and oxidoreductase activity, hinting at the role that SLs play in the transcriptional reprogramming upon P starvation. Gene co-expression network analysis uncovered a METHYL TRANSFERASE that displayed co-regulation with known rice SL biosynthetic genes. Functional characterization showed that this gene encodes an enzyme catalyzing the conversion of carlactonoic acid to methyl carlactonoate. Our work provides a valuable resource to further studies on the response of crops to P deficiency and reveals a tool for the discovery of SL biosynthetic genes.

Maize domestication phenotypes reveal strigolactone networks coordinating grain size evolution with kernel-bearing cupule architecture.

The maize (Zea mays) ear represents one of the most striking domestication phenotypes in any crop species, with the cob conferring an exceptional yield advantage over the ancestral form of teosinte. Remodeling of the grain-bearing surface required profound developmental changes. However, the underlying mechanisms remain unclear and can only be partly attributed to the known domestication gene Teosinte glume architecture 1 (Tga1). Here we show that a more complete conversion involves strigolactones (SLs), and that these are prominent players not only in the Tga1 phenotype but also other domestication features of the ear and kernel. Genetic combinations of a teosinte tga1 allele with three SL-related mutants progressively enhanced ancestral morphologies. The SL mutants, in addition to modulating the tga1 phenotype, also reshaped kernel-bearing pedicels and cupules in a teosinte-like manner. Genetic and molecular evidence are consistent with SL regulation of TGA1, including direct interaction of TGA1 with components of the SL-signaling system shown here to mediate TGA1 availability by sequestration. Roles of the SL network extend to enhancing maize seed size and, importantly, coordinating increased kernel growth with remodeling of protective maternal tissues. Collectively, our data show that SLs have central roles in releasing kernels from restrictive maternal encasement and coordinating other factors that increase kernel size, physical support, and their exposure on the grain-bearing surface.

Coumarin biosynthesis genes are required after foliar pathogen infection for the creation of a microbial soil-borne legacy that primes plants for SA-dependent defenses.

Plants deposit photosynthetically-fixed carbon in the rhizosphere, the thin soil layer directly around the root, thereby creating a hospitable environment for microbes. To manage the inhabitants of this nutrient-rich environment, plant roots exude and dynamically adjust microbe-attracting and -repelling compounds to stimulate specific members of the microbiome. Previously, we demonstrated that foliar infection of Arabidopsis thaliana by the biotrophic downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) leads to a disease-induced modification of the rhizosphere microbiome. Soil conditioned with Hpa-infected plants provided enhanced protection against foliar downy mildew infection in a subsequent population of plants, a phenomenon dubbed the soil-borne legacy (SBL). Here, we show that for the creation of the SBL, plant-produced coumarins play a prominent role as coumarin-deficient myb72 and f6'h1 mutants were defective in creating a Hpa-induced SBL. Root exudation profiles changed significantly in Col-0 upon foliar Hpa infection, and this was accompanied by a compositional shift in the root microbiome that was significantly different from microbial shifts occurring on roots of Hpa-infected coumarin-deficient mutants. Our data further show that the Hpa-induced SBL primes Col-0 plants growing in SBL-conditioned soil for salicylic acid (SA)-dependent defenses. The SA-signaling mutants sid2 and npr1 were unresponsive to the Hpa-induced SBL, suggesting that the protective effect of the Hpa-induced shift in the root microbiome results from an induced systemic resistance that requires SA-signaling in the plant.

Probing strigolactone perception mechanisms with rationally designed small-molecule agonists stimulating germination of root parasitic weeds.

The development of potent strigolactone (SL) agonists as suicidal germination inducers could be a useful strategy for controlling root parasitic weeds, but uncertainty about the SL perception mechanism impedes real progress. Here we describe small-molecule agonists that efficiently stimulate Phelipanchce aegyptiaca, and Striga hermonthica, germination in concentrations as low as 10-8 to 10-17 M. We show that full efficiency of synthetic SL agonists in triggering signaling through the Striga SL receptor, ShHTL7, depends on the receptor-catalyzed hydrolytic reaction of the agonists. Additionally, we reveal that the stereochemistry of synthetic SL analogs affects the hydrolytic ability of ShHTL7 by influencing the probability of the privileged conformations of ShHTL7. Importantly, an alternative ShHTL7-mediated hydrolysis mechanism, proceeding via nucleophilic attack of the NE2 atom of H246 to the 2'C of the D-ring, is reported. Together, our findings provide insight into SL hydrolysis and structure-perception mechanisms, and potent suicide germination stimulants, which would contribute to the elimination of the noxious parasitic weeds.

The tomato cytochrome P450 CYP712G1 catalyses the double oxidation of orobanchol en route to the rhizosphere signalling strigolactone, solanacol.

Strigolactones (SLs) are rhizosphere signalling molecules and phytohormones. The biosynthetic pathway of SLs in tomato has been partially elucidated, but the structural diversity in tomato SLs predicts that additional biosynthetic steps are required. Here, root RNA-seq data and co-expression analysis were used for SL biosynthetic gene discovery. This strategy resulted in a candidate gene list containing several cytochrome P450s. Heterologous expression in Nicotiana benthamiana and yeast showed that one of these, CYP712G1, can catalyse the double oxidation of orobanchol, resulting in the formation of three didehydro-orobanchol (DDH) isomers. Virus-induced gene silencing and heterologous expression in yeast showed that one of these DDH isomers is converted to solanacol, one of the most abundant SLs in tomato root exudate. Protein modelling and substrate docking analysis suggest that hydroxy-orbanchol is the likely intermediate in the conversion from orobanchol to the DDH isomers. Phylogenetic analysis demonstrated the occurrence of CYP712G1 homologues in the Eudicots only, which fits with the reports on DDH isomers in that clade. Protein modelling and orobanchol docking of the putative tobacco CYP712G1 homologue suggest that it can convert orobanchol to similar DDH isomers as tomato.

High-energy-level metabolism and transport occur at the transition from closed to open flowers.

During the maturation phase of flower development, the onset of anthesis visibly marks the transition from buds to open flowers, during which petals stretch out, nectar secretion commences, and pollination occurs. Analysis of the metabolic changes occurring during this developmental transition has primarily focused on specific classes of metabolites, such as pigments and scent emission, and far less on the whole network of primary and secondary metabolites. To investigate the metabolic changes occurring at anthesis, we performed multi-platform metabolomics alongside RNA sequencing in individual florets harvested from the main inflorescence of Arabidopsis (Arabidopsis thaliana) ecotype Col-0. To trace metabolic fluxes at the level of the whole inflorescence and individual florets, we further integrated these studies with radiolabeled experiments. These extensive analyses revealed high-energy-level metabolism and transport of carbohydrates and amino acids, supporting intense metabolic rearrangements occurring at the time of this floral transition. These comprehensive data are discussed in the context of our current understanding of the metabolic shifts underlying flower opening. We envision that this analysis will facilitate the introgression of floral metabolic traits promoting pollination in crop species for which a comprehensive knowledge of flower metabolism is still limited.

Effect of strigolactones on recruitment of the rice root-associated microbiome.

Strigolactones are endogenous plant hormones regulating plant development and are exuded into the rhizosphere when plants experience nutrient deficiency. There, they promote the mutualistic association of plants with arbuscular mycorrhizal fungi that help the plant with the uptake of nutrients from the soil. This shows that plants actively establish-through the exudation of strigolactones-mutualistic interactions with microbes to overcome inadequate nutrition. The signaling function of strigolactones could possibly extend to other microbial partners, but the effect of strigolactones on the global root and rhizosphere microbiome remains poorly understood. Therefore, we analyzed the bacterial and fungal microbial communities of 16 rice genotypes differing in their root strigolactone exudation. Using multivariate analyses, distinctive differences in the microbiome composition were uncovered depending on strigolactone exudation. Moreover, the results of regression modeling showed that structural differences in the exuded strigolactones affected different sets of microbes. In particular, orobanchol was linked to the relative abundance of Burkholderia-Caballeronia-Paraburkholderia and Acidobacteria that potentially solubilize phosphate, while 4-deoxyorobanchol was associated with the genera Dyella and Umbelopsis. With this research, we provide new insight into the role of strigolactones in the interplay between plants and microbes in the rhizosphere.

Terpene synthases in cucumber (Cucumis sativus) and their contribution to herbivore-induced volatile terpenoid emission.

Terpenoids play important roles in flavour, pollinator attraction and defence of plants. In cucumber (Cucumis sativus) they are important components of the herbivore-induced plant volatile blend that attracts natural enemies of herbivores. We annotated the cucumber TERPENE SYNTHASE gene (CsTPS) family and characterized their involvement in the response towards herbivores with different feeding guilds using a combined molecular and biochemical approach. Transcripts of multiple CsTPS genes were upregulated in leaves upon herbivory and the products generated by the expressed proteins match the terpenoids recorded in the volatile blend released by herbivore-damaged leaves. Spatial and temporal analysis of the promoter activity of CsTPS genes showed that cell content-feeding spider mites (Tetranychus urticae) and thrips (Frankliniella occidentalis) induced promoter activity of CsTPS9 and CsTPS19 within hours after initiation of infestation, while phloem-feeding aphids (Myzus persicae) induced CsTPS2 promoter activity. Our findings offer detailed insights into the involvement of the TPS gene family in the dynamics and fine-tuning of the emission of herbivore-induced plant volatiles in cucumber, and open a new avenue to understand molecular mechanisms that affect plant-herbivore interactions.

UPLC-MS/MS analysis and biological activity of the potato cyst nematode hatching stimulant, solanoeclepin A, in the root exudate of Solanum spp.

MAIN CONCLUSION: Solanoeclepin A is a hatching stimulant for potato cyst nematode in very low (pM) concentrations. We report a highly sensitive method for the analysis of SolA in plant root exudates using UHPLC-MS/MS and show that there is considerable natural variation in SolA production in Solanum spp. corresponding with their hatching inducing activity. Potato cyst nematode (PCN) is a plant root sedentary endoparasite, specialized in the infection of solanaceous species such as potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Earlier reports (Mulder et al. in Hatching agent for the potato cyst nematode, Patent application No. PCT/NL92/00126, 1996; Schenk et al. in Croat Chem Acta 72:593-606, 1999) showed that solanoeclepin A (SolA), a triterpenoid metabolite that was isolated from the root exudate of potato, induces the hatching of PCN. Its low concentration in potato root exudate has hindered progress in fully understanding its hatching inducing activity and exploitation in the control of PCN. To further investigate the role of SolA in hatching of PCN, the establishment of a highly sensitive analytical method is a prerequisite. Here we present the efficient single-step extraction and UHPLC-MS/MS based analysis for rapid determination of SolA in sub-nanomolar concentrations in tomato root exudate. This method was used to analyze SolA production in different tomato cultivars and related solanaceous species, including the trap crop Solanum sisymbriifolium. Hatching assays with PCN, Globodera pallida, with root exudates of tomato genotypes revealed a significant positive correlation between SolA concentration and hatching activity. Our results demonstrate that there is natural variation in SolA production within solanaceous species and that this has an effect on PCN hatching. The analytical method we have developed can potentially be used to support breeding for crop genotypes that induce less hatching and may therefore display reduced infection by PCN.

On the role of dauer in the adaptation of nematodes to a parasitic lifestyle.

Nematodes are presumably the most abundant Metazoa on Earth, and can even be found in some of the most hostile environments of our planet. Various types of hypobiosis evolved to adapt their life cycles to such harsh environmental conditions. The five most distal major clades of the phylum Nematoda (Clades 8-12), formerly referred to as the Secernentea, contain many economically relevant parasitic nematodes. In this group, a special type of hypobiosis, dauer, has evolved. The dauer signalling pathway, which culminates in the biosynthesis of dafachronic acid (DA), is intensively studied in the free-living nematode Caenorhabditis elegans, and it has been hypothesized that the dauer stage may have been a prerequisite for the evolution of a wide range of parasitic lifestyles among other nematode species. Biosynthesis of DA is not specific for hypobiosis, but if it results in exit of the hypobiotic state, it is one of the main criteria to define certain behaviour as dauer. Within Clades 9 and 10, the involvement of DA has been validated experimentally, and dauer is therefore generally accepted to occur in those clades. However, for other clades, such as Clade 12, this has hardly been explored. In this review, we provide clarity on the nomenclature associated with hypobiosis and dauer across different nematological subfields. We discuss evidence for dauer-like stages in Clades 8 to 12 and support this with a meta-analysis of available genomic data. Furthermore, we discuss indications for a simplified dauer signalling pathway in parasitic nematodes. Finally, we zoom in on the host cues that induce exit from the hypobiotic stage and introduce two hypotheses on how these signals might feed into the dauer signalling pathway for plant-parasitic nematodes. With this work, we contribute to the deeper understanding of the molecular mechanisms underlying hypobiosis in parasitic nematodes. Based on this, novel strategies for the control of parasitic nematodes can be developed.

The role of strigolactones in P deficiency induced transcriptional changes in tomato roots.

BACKGROUND: Phosphorus (P) is an essential macronutrient for plant growth and development. Upon P shortage, plant responds with massive reprogramming of transcription, the Phosphate Starvation Response (PSR). In parallel, the production of strigolactones (SLs)-a class of plant hormones that regulates plant development and rhizosphere signaling molecules-increases. It is unclear, however, what the functional link is between these two processes. In this study, using tomato as a model, RNAseq was used to evaluate the time-resolved changes in gene expression in the roots upon P starvation and, using a tomato CAROTENOID CLEAVAGE DIOXYGENASES 8 (CCD8) RNAi line, what the role of SLs is in this. RESULTS: Gene ontology (GO)-term enrichment and KEGG analysis of the genes regulated by P starvation and P replenishment revealed that metabolism is an important component of the P starvation response that is aimed at P homeostasis, with large changes occurring in glyco-and galactolipid and carbohydrate metabolism, biosynthesis of secondary metabolites, including terpenoids and polyketides, glycan biosynthesis and metabolism, and amino acid metabolism. In the CCD8 RNAi line about 96% of the PSR genes was less affected than in wild-type (WT) tomato. For example, phospholipid biosynthesis was suppressed by P starvation, while the degradation of phospholipids and biosynthesis of substitute lipids such as sulfolipids and galactolipids were induced by P starvation. Around two thirds of the corresponding transcriptional changes depend on the presence of SLs. Other biosynthesis pathways are also reprogrammed under P starvation, such as phenylpropanoid and carotenoid biosynthesis, pantothenate and CoA, lysine and alkaloids, and this also partially depends on SLs. Additionally, some plant hormone biosynthetic pathways were affected by P starvation and also here, SLs are required for many of the changes (more than two thirds for Gibberellins and around one third for Abscisic acid) in the gene expression. CONCLUSIONS: Our analysis shows that SLs are not just the end product of the PSR in plants (the signals secreted by plants into the rhizosphere), but also play a major role in the regulation of the PSR (as plant hormone).

Integration of omics data to unravel root microbiome recruitment.

The plant microbiome plays an essential role in supporting plant growth and health, but plant molecular mechanisms underlying its recruitment are still unclear. Multi-omics data integration methods can be used to unravel new signalling relationships. Here, we review the effects of plant genetics and root exudates on root microbiome recruitment, and discuss methodological advances in data integration approaches that can help us to better understand and optimise the crop-microbiome interaction for a more sustainable agriculture.

Drought tolerance in selected aerobic and upland rice varieties is driven by different metabolic and antioxidative responses.

MAIN CONCLUSIONS: Sugar-mediated osmotic acclimation and a strong antioxidative response reduce drought-induced biomass loss at the vegetative stage in rice. A clear understanding of the physiological and biochemical adaptations to water limitation in upland and aerobic rice can help to identify the mechanisms underlying their tolerance to low water availability. In this study, three indica rice varieties-IR64 (lowland), Apo (aerobic), and UPL Ri-7 (upland)-, that are characterized by contrasting levels of drought tolerance, were exposed to drought at the vegetative stage. Drought-induced changes in biomass, leaf metabolites and oxidative stress markers/enzyme activities were analyzed in each variety at multiple time points. The two drought-tolerant varieties, Apo and UPL Ri-7 displayed a reduced water use in contrast to the susceptible variety IR64 that displayed high water consumption and consequent strong leaf dehydration upon drought treatment. A sugar-mediated osmotic acclimation in UPL Ri-7 and a strong antioxidative response in Apo were both effective in limiting the drought-induced biomass loss in these two varieties, while biomass loss was high in IR64, also after recovery. A qualitative comparison of these results with the ones of a similar experiment conducted in the field at the reproductive stage showed that only Apo, which also in this stage showed the highest antioxidant power, was able to maintain a stable grain yield under stress. Our results show that different metabolic and antioxidant adaptations confer drought tolerance to aerobic and upland rice varieties in the vegetative stage. The effectiveness of these adaptations differs between developmental stages. Unraveling the genetic control of these mechanisms might be exploited in breeding for new rice varieties adapted to water-limited environments.

Strigolactones regulate sepal senescence in Arabidopsis.

Flower sepals are critical for flower development and vary greatly in life span depending on their function post-pollination. Very little is known about what controls sepal longevity. Using a sepal senescence mutant screen, we identified two Arabidopsis mutants with delayed senescence directly connecting strigolactones with senescence regulation in a novel floral context that hitherto has not been explored. The mutations were in the strigolactone biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) and in the strigolactone receptor gene DWARF14 (AtD14). The mutation in AtD14 changed the catalytic Ser97 to Phe in the enzyme active site, which is the first mutation of its kind in planta. The lesion in MAX1 was in the haem-iron ligand signature of the cytochrome P450 protein, converting the highly conserved Gly469 to Arg, which was shown in a transient expression assay to substantially inhibit the activity of MAX1. The two mutations highlighted the importance of strigolactone activity for driving to completion senescence initiated both developmentally and in response to carbon-limiting stress, as has been found for the more well-known senescence-associated regulators ethylene and abscisic acid. Analysis of transcript abundance in excised inflorescences during an extended night suggested an intricate relationship among sugar starvation, senescence, and strigolactone biosynthesis and signalling.