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.

A carlactonoic acid methyltransferase that contributes to the inhibition of shoot branching in Arabidopsis.

SignificanceStrigolactones (SLs) are a group of apocarotenoid hormones, which regulates shoot branching and other diverse developmental processes in plants. The major bioactive form(s) of SLs as endogenous hormones has not yet been clarified. Here, we identify an Arabidopsis methyltransferase, CLAMT, responsible for the conversion of an inactive precursor to a biologically active SL that can interact with the SL receptor in vitro. Reverse genetic analysis showed that this enzyme plays an essential role in inhibiting shoot branching. This mutant also contributed to specifying the SL-related metabolites that could move from root to shoot in grafting experiments. Our work has identified a key enzyme necessary for the production of the bioactive form(s) of SLs.

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.

An Independent Evolutionary Origin for Insect Deterrent Cucurbitacins in Iberis amara.

Pieris rapae and Phyllotreta nemorum are Brassicaceae specialists, but do not feed on Iberis amara spp. that contain cucurbitacins. The cucurbitacins are highly oxygenated triterpenoid, occurring widespread in cucurbitaceous species and in a few other plant families. Using de novo assembled transcriptomics from I. amara, gene co-expression analysis and comparative genomics, we unraveled the evolutionary origin of the insect deterrent cucurbitacins in I. amara. Phylogenetic analysis of five oxidosqualene cyclases and heterologous expression allowed us to identify the first committed enzyme in cucurbitacin biosynthesis in I. amara, cucurbitadienol synthase (IaCPQ). In addition, two species-specific cytochrome P450s (CYP708A16 and CYP708A15) were identified that catalyze the unique C16 and C22 hydroxylation of the cucurbitadienol backbone, enzymatic steps that have not been reported before. Furthermore, the draft genome assembly of I. amara showed that the IaCPQ was localized to the same scaffold together with CYP708A15 but spanning over 100 kb, this contrasts with the highly organized cucurbitacin gene cluster in the cucurbits. These results reveal that cucurbitacin biosynthesis has evolved convergently via different biosynthetic routes in different families rather than through divergence from an ancestral pathway. This study thus provides new insight into the mechanism of recurrent evolution and diversification of a plant defensive chemical.

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.

Adaptation of the parasitic plant lifecycle: germination is controlled by essential host signaling molecules.

Parasitic plants are plants that connect with a haustorium to the vasculature of another, host, plant from which they absorb water, assimilates, and nutrients. Because of this parasitic lifestyle, parasitic plants need to coordinate their lifecycle with that of their host. Parasitic plants have evolved a number of host detection/host response mechanisms of which the germination in response to chemical host signals in one of the major families of parasitic plants, the Orobanchaceae, is a striking example. In this update review, we discuss these germination stimulants. We review the different compound classes that function as germination stimulants, how they are produced, and in which host plants. We discuss why they are reliable signals, how parasitic plants have evolved mechanisms that detect and respond to them, and whether they play a role in host specificity. The advances in the knowledge underlying this signaling relationship between host and parasitic plant have greatly improved our understanding of the evolution of plant parasitism and are facilitating the development of more effective control measures in cases where these parasitic plants have developed into weeds.

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).

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.

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.

Plant triterpenoids with bond-missing skeletons: biogenesis, distribution and bioactivity.

Covering: up to December 2018 The polycyclic ABCD(E) framework of triterpenoids can miss a single endocyclic C-C bond as a result of a modification of the cyclization cascade that triggers their formation (interrupted- or diverted cascades), or can be the result of post-cyclization ring cleavage by late-stage oxidative modifications (seco-triterpenoids). Because of mechanistic and biogenetic differences, ring opening associated with loss of a skeletal fragment, as in nor-seco-triterpenoids (limonoids, quassinoids), will not be covered, nor will compounds where ring opening is part of a fragmentation cascade or of a multiple diversion from it. Even with these limitations, 342 bond-missing triterpenoids could be retrieved from the literature, with transversal distribution in the plant kingdom. Their structural diversity translates into a variety of biological targets, with dominance of potential applications in the realm of cancer, neuroprotection, and anti-infective therapy. In addition to the bioactivity and chemotaxonomic relevance of bond-missing triterpenoids, current knowledge on the genetic basis of interrupted- and diverted oxidosqualene cyclases will be summarized. This untapped source of enzymes could be useful to selectively modify triterpenoids by metabolic engineering, circumventing the bottlenecks of their isolation (poor yield or inadequate supply chain) to explore new areas of their chemical space.

A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid α-Onocerin.

8,14-seco-Triterpenoids are characterized by their unusual open C-ring. Their distribution in nature is rare and scattered in taxonomically unrelated plants. The 8,14-seco-triterpenoid α-onocerin is only known from the evolutionarily distant clubmoss genus Lycopodium and the leguminous genus Ononis, which makes the biosynthesis of this seco-triterpenoid intriguing from an evolutionary standpoint. In our experiments with Ononis spinosa, α-onocerin was detected only in the roots. Through transcriptome analysis of the roots, an oxidosqualene cyclase, OsONS1, was identified that produces α-onocerin from squalene-2,3;22,23-dioxide when transiently expressed in Nicotiana bethamiana In contrast, in Lycopodium clavatum, two sequential cyclases, LcLCC and LcLCD, are required to produce α-onocerin in the N. benthamiana transient expression system. Expression of OsONS1 in the lanosterol synthase knockout yeast strain GIL77, which accumulates squalene-2,3;22,23-dioxide, verified the α-onocerin production. A phylogenetic analysis predicts that OsONS1 branches off from specific lupeol synthases and does not group with the known L. clavatum α-onocerin cyclases. Both the biochemical and phylogenetic analyses of OsONS1 suggest convergent evolution of the α-onocerin pathways. When OsONS1 was coexpressed in N. benthamiana leaves with either of the two O. spinosa squalene epoxidases, OsSQE1 or OsSQE2, α-onocerin production was boosted, most likely because the epoxidases produce higher amounts of squalene-2,3;22,23-dioxide. Fluorescence lifetime imaging microscopy analysis demonstrated specific protein-protein interactions between OsONS1 and both O. spinosa squalene epoxidases. Coexpression of OsONS1 with the two OsSQEs suggests that OsSQE2 is the preferred partner of OsONS1 in planta. Our results provide an example of the convergent evolution of plant specialized metabolism.

Co-expression of squalene epoxidases with triterpene cyclases boosts production of triterpenoids in plants and yeast.

Triterpene cyclases catalyze the first committed step in triterpene biosynthesis, by forming mono- to pentacyclic backbone structures from oxygenated C30 isoprenoid precursors. Squalene epoxidase precedes this cyclization by providing the oxygenated and activated substrate for triterpene biosynthesis. Three squalene epoxidases from Cucurbita pepo (CpSEs) were isolated and shown to have evolved under purifying selection with signs of sites under positive selection in their N- and C-termini. They all localize to the Endoplasmic Reticulum (ER) and produce 2,3-oxidosqualene and 2,3:22,23-dioxidosqualene when expressed in a yeast erg1 (squalene epoxidase) erg7 (lanosterol synthase) double mutant. Co-expression of the CpSEs with four different triterpene cyclases, either transiently in Nicotiana benthamiana or constitutively in yeast, showed that CpSEs boost triterpene production. CpSE2 was the best performing in this regard, which could reflect either increased substrate production or superior channeling of the substrate to the triterpene cyclases. Fluorescence Lifetime Imaging Microscopy (FLIM) analysis with C. pepo cucurbitadienol synthase (CpCPQ) revealed a specific interaction with CpSE2 but not with the other CpSEs. When CpSE2 was transformed into C. pepo hairy root lines, cucurbitacin E production was increased two folds compared to empty vector control lines. This study provides new insight into the importance of SEs in triterpene biosynthesis, suggesting that they may facilitate substrate channeling, and demonstrates that SE overexpression is a new tool for increasing triterpene production in plants and yeast.

Monoterpene biosynthesis potential of plant subcellular compartments.

Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol.

A strategy for differential abundance analysis of sparse microbiome data with group-wise structured zeros.

Comparing the abundance of microbial communities between different groups or obtained under different experimental conditions using count sequence data is a challenging task due to various issues such as inflated zero counts, overdispersion, and non-normality. Several methods and procedures based on counts, their transformation and compositionality have been proposed in the literature to detect differentially abundant species in datasets containing hundreds to thousands of microbial species. Despite efforts to address the large numbers of zeros present in microbiome datasets, even after careful data preprocessing, the performance of existing methods is impaired by the presence of inflated zero counts and group-wise structured zeros (i.e. all zero counts in a group). We propose and validate using extensive simulations an approach combining two differential abundance testing methods, namely DESeq2-ZINBWaVE and DESeq2, to address the issues of zero-inflation and group-wise structured zeros, respectively. This combined approach was subsequently successfully applied to two plant microbiome datasets that revealed a number of taxa as interesting candidates for further experimental validation.

The potential of strigolactones to shift competitive dynamics among two Rhizophagus irregularis strains.

Strigolactones are phytohormones that influence arbuscular mycorrhizal fungal (AMF) spore germination, pre-symbiotic hyphal branching, and metabolic rates. Historically, strigolactone effects have been tested on single AMF strains. An open question is whether intraspecific variation in strigolactone effects and intraspecific interactions can influence AMF competition. Using the Rhizophagus irregularis strains A5 and C2, we tested for intraspecific variation in the response of germination and pre-symbiotic growth (i.e., hyphal length and branching) to the strigolactones GR24 and 5-deoxystrigol. We also tested if interactions between these strains modified their germination rates and pre-symbiotic growth. Spore germination rates were consistently high (> 90%) for C2 spores, regardless of treatment and the presence of the other strain. For A5 spores, germination was increased by strigolactone presence from approximately 30 to 70% but reduced when grown in mixed culture. When growing together, branching increased for both strains compared to monocultures. In mixed cultures, strigolactones increased the branching for both strains but led to an increase in hyphal length only for the strain A5. These strain-specific responses suggest that strigolactones may have the potential to shift competitive dynamics among AMF species with direct implications for the establishment of the AMF community.

OsCYP706C2 diverts rice strigolactone biosynthesis to a noncanonical pathway branch.

Strigolactones exhibit dual functionality as regulators of plant architecture and signaling molecules in the rhizosphere. The important model crop rice exudes a blend of different strigolactones from its roots. Here, we identify the inaugural noncanonical strigolactone, 4-oxo-methyl carlactonoate (4-oxo-MeCLA), in rice root exudate. Comprehensive, cross-species coexpression analysis allowed us to identify a cytochrome P450, OsCYP706C2, and two methyl transferases as candidate enzymes for this noncanonical rice strigolactone biosynthetic pathway. Heterologous expression in yeast and Nicotiana benthamiana indeed demonstrated the role of these enzymes in the biosynthesis of 4-oxo-MeCLA, which, expectedly, is derived from carlactone as substrate. The oscyp706c2 mutants do not exhibit a tillering phenotype but do have delayed mycorrhizal colonization and altered root phenotype. This work sheds light onto the intricate complexity of strigolactone biosynthesis in rice and delineates its role in symbiosis and development.

Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed.

Geraniol hydroxylase and hydroxygeraniol oxidase activities of the CYP76 family of cytochrome P450 enzymes and potential for engineering the early steps of the (seco)iridoid pathway.

The geraniol-derived (seco)iridoid skeleton is a precursor for a large group of bioactive compounds with diverse therapeutic applications, including the widely used anticancer molecule vinblastine. Despite of this economic prospect, the pathway leading to iridoid biosynthesis from geraniol is still unclear. The first geraniol hydroxylation step has been reported to be catalyzed by cytochrome P450 enzymes such as CYP76B6 from Catharanthus roseus and CYP76C1 from Arabidopsis thaliana. In the present study, an extended functional analysis of CYP76 family members was carried-out to identify the most effective enzyme to be used for pathway reconstruction. This disproved CYP76C1 activity and led to the characterization of CYP76C4 from A. thaliana as a geraniol 9- or 8-hydroxylase. CYP76B6 emerged as a highly specialized multifunctional enzyme catalyzing two sequential oxidation steps leading to the formation of 8-oxogeraniol from geraniol. This dual function was confirmed in planta using a leaf-disc assay. The first step, geraniol hydroxylation, was very efficient and fast enough to outcompete geraniol conjugation in plant tissues. When the enzyme was expressed in leaf tissues, 8-oxogeraniol was converted into further oxidized and/or reduced compounds in the absence of the next enzyme of the iridoid pathway.

Lessons learned from metabolic engineering in hairy roots: Transcriptome and metabolic profile changes caused by Rhizobium-mediated plant transformation in Cucurbitaceae species.

Cucurbitaceae species are used in traditional medicine around the world. Cucurbitacins are highly oxygenated triterpenoids found in Cucurbitaceae species and exhibit potent anticancer activity alone and in combination with other existing chemotherapeutic drugs. Therefore, increasing production of these specialized metabolites is of great relevance. We recently showed that hairy roots of Cucurbita pepo can be used as a platform for metabolic engineering of cucurbitacins to modify their structure and increase their production. To study the changes in cucurbitacin accumulation upon formation of hairy roots, an empty vector (EV) control and Cucurbitacin inducing bHLH transcription factor 1 (CpCUCbH1)-overexpressing hairy roots of C. pepo were compared to untransformed (WT) roots. Whilst CpCUCbH1-overexpression increased production of cucurbitacins I and B by 5-fold, and cucurbitacin E by 3-fold when compared to EV lines, this increase was not significantly different when compared to WT roots. This indicated that Rhizobium rhizogenes transformation lowered the cucurbitacins levels in hairy roots, but that increasing expression of cucurbitacin biosynthetic genes by CpCUCbH1-overexpression restored cucurbitacin production to WT levels. Subsequent metabolomic and RNA-seq analysis indicated that the metabolic profile and transcriptome of hairy roots was significantly changed when compared to WT roots. Interestingly, it was observed that 11% of the differentially expressed genes were transcription factors. It was noteworthy that the majority of transcripts showing highest Pearson correlation coefficients to the Rhizobium rhizogenes genes rolB, rolC and ORF13a, were predicted to be transcription factors. In summary, hairy roots are an excellent platform for metabolic engineering of plant specialized metabolites, but these extensive transcriptome and metabolic profile changes should be considered in subsequent studies.

A Combination of Metabolomics and Machine Learning Results in the Identification of a New Cyst Nematode Hatching Factor.

Potato Cyst Nematodes (PCNs) are an economically important pest for potato growers. A crucial event in the life cycle of the nematode is hatching, after which the juvenile will move toward the host root and infect it. The hatching of PCNs is induced by known and unknown compounds in the root exudates of host plant species, called hatching factors (HFs, induce hatching independently), such as solanoeclepin A (solA), or hatching stimulants (HSs, enhance hatching activity of HFs). Unraveling the identity of unknown HSs and HFs and their natural variation is important for the selection of cultivars that produce low amounts of HFs and HSs, thus contributing to more sustainable agriculture. In this study, we used a new approach aimed at the identification of new HFs and HSs for PCNs in potato. Hereto, root exudates of a series of different potato cultivars were analyzed for their PCN hatch-inducing activity and their solA content. The exudates were also analyzed using untargeted metabolomics, and subsequently the data were integrated using machine learning, specifically random forest feature selection, and Pearson's correlation testing. As expected, solA highly correlates with hatching. Furthermore, this resulted in the discovery of a number of metabolite features present in the root exudate that correlate with hatching and solA content, and one of these is a compound of m/z 526.18 that predicts hatching even better than solA with both data methods. This compound's involvement in hatch stimulation was confirmed by the fractionation of three representative root exudates and hatching assays with the resulting fractions. Moreover, the compound shares mass fragmentation similarity with solA, and we therefore assume it has a similar structure. With this work, we show that potato likely produces a solA analogue, and we contribute to unraveling the hatch-inducing cocktail exuded by plant roots.