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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.

A micropore-filtration method was used to reduce the proportion of plant DNA in microbial DNA samples isolated from roots prior to sequencing. We tested the impact of this pre-sequencing filtration methodology and used it to characterize the root microbiome of maize grown on two soils with different fertility levels. The micropore filtration reduced plant DNA contamination and unveiled potential in the N-poor soil for N fixation in roots and phosphate uptake by roots in the phosphate-poor soil. Our methodology and findings allude to the potential capability of plants to initiate plant-microbe interactions under sub-optimal soil fertility.

Most land plants entertain a mutualistic symbiosis known as arbuscular mycorrhiza with fungi (Glomeromycota) that provide them with essential mineral nutrients, in particular phosphate (Pi), and protect them from biotic and abiotic stress. Arbuscular mycorrhizal (AM) symbiosis increases plant productivity and biodiversity and is therefore relevant for both natural plant communities and crop production. However, AM fungal populations suffer from intense farming practices in agricultural soils, in particular Pi fertilization. The dilemma between natural fertilization from AM symbiosis and chemical fertilization has raised major concern and emphasizes the need to better understand the mechanisms by which Pi suppresses AM symbiosis. Here, we test the hypothesis that Pi may interfere with AM symbiosis via the phytohormone gibberellic acid (GA) in the Solanaceous model systems Petunia hybrida and Nicotiana tabacum. Indeed, we find that GA is inhibitory to AM symbiosis and that Pi may cause GA levels to increase in mycorrhizal roots. Consistent with a role of endogenous GA as an inhibitor of AM development, GA-defective N. tabacum lines expressing a GA-metabolizing enzyme (GA methyltransferase—GAMT) are colonized more quickly by the AM fungus Rhizoglomus irregulare, and exogenous Pi is less effective in inhibiting AM colonization in these lines. Systematic gene expression analysis of GA-related genes reveals a complex picture, in which GA degradation by GA2 oxidase plays a prominent role. These findings reveal potential targets for crop breeding that could reduce Pi suppression of AM symbiosis, thereby reconciling the advantages of Pi fertilization with the diverse benefits of AM symbiosis.

Most plants associate with beneficial arbuscular mycorrhizal (AM) fungi that facilitate soil nutrient acquisition. Prior to contact, partner recognition triggers reciprocal genetic remodelling to enable colonisation. The plant Dwarf14-Like (D14L) receptor conditions pre-symbiotic perception of AM fungi, and also detects the smoke constituent karrikin. D14L-dependent signalling mechanisms, underpinning AM symbiosis are unknown. Here, we present the identification of a negative regulator from rice, which operates downstream of the D14L receptor, corresponding to the homologue of the Arabidopsis thaliana Suppressor of MAX2-1 (AtSMAX1) that functions in karrikin signalling. We demonstrate that rice SMAX1 is a suppressor of AM symbiosis, negatively regulating fungal colonisation and transcription of crucial signalling components and conserved symbiosis genes. Similarly, rice SMAX1 negatively controls strigolactone biosynthesis, demonstrating an unexpected crosstalk between the strigolactone and karrikin signalling pathways. We conclude that removal of SMAX1, resulting from D14L signalling activation, de-represses essential symbiotic programmes and increases strigolactone hormone production.

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. Supplementary Information The online version contains supplementary material available at 10.1186/s13071-021-04953-6.

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).

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.

During millions of years of evolution, land plants and microorganisms have established elaborate partnerships. Microbes play essential roles in plant fitness and help plants cope with environmental challenges. Vice versa, plants provide the microbes with a niche and food. In the soil, a complex network of interactions mediated by metabolic signals drives the relationship between plants and microbes. Here, we review the roles of metabolic signaling in the plant-microbiome interaction. We discuss how plant-produced small molecules are involved in the recruitment of the microbiome. Also the microbial partners in this relationship use small molecules, such as quorum sensing molecules and volatiles for intra-species and inter-species communication. We give an overview of the regulation of the biosynthesis, secretion and perception of both plant and microbial small molecules and discuss the examples of biotechnological approaches to engineer the plant-microbiome interaction by targeting these metabolic dialogues. Ultimately, an improved understanding of the plant-microbiome interaction and engineering possibilities will pave the way to a more sustainable agriculture.

During arbuscular mycorrhizal symbiosis, colonization of the root is modulated in response to the physiological status of the plant, with regulation occurring locally and systemically. Here, we identify differentially expressed genes encoding CLAVATA3/ESR-related (CLE) peptides that negatively regulate colonization levels by modulating root strigolactone content. CLE function requires a receptor-like kinase, SUNN; thus, a CLE-SUNN-strigolactone feedback loop is one avenue through which the plant modulates colonization levels.

Almost 80 years after the discovery of the first plant hormone, auxin, a few years ago a new class of plant hormones, the strigolactones, was discovered. These molecules have unprecedented biological activity in a number of highly important biological processes in plants but also outside the plant in the rhizosphere, the layer of soil surrounding the roots of plants and teeming with life. The exploitation of this amazing biological activity is not without challenges: the synthesis of strigolactones is complicated and designing the desired activity a difficult task. This minireview describes the current state of knowledge about the strigolactones and how synthetic analogs can be developed that can potentially contribute to the development of a sustainable agriculture.

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.

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.

Main conclusionThe sunflower sesquiterpene lactones 8-epixanthatin and tomentosin can bind to the hydrophobic pocket of sunflower KAI2 with an affinity much higher than for the exogenous ligand KAR.AbstractSesquiterpene lactones (STLs) are secondary plant metabolites with a wide range of biological, such as anti-microbial, activities. Intriguingly, the STLs have also been implicated in plant development: in several Asteraceae, STL levels correlate with the photo-inhibition of hypocotyl elongation. Although this effect was suggested to be due to auxin transport inhibition, there is no structural–functional evidence for this claim. Intriguingly, the light-induced inhibition of hypocotyl elongation in Arabidopsis has been ascribed to HYPOSENSITIVE TO LIGHT/KARRIKIN-INSENSITIVE2 (HTL/KAI2) signaling. KAI2 was discovered because of its affinity to the smoke-derived karrikin (KAR), though it is generally assumed that KAI2 has another, endogenous but so far elusive, ligand rather than the exogenous KARs. Here, we postulate that the effect of STLs on hypocotyl elongation is mediated through KAI2 signaling. To support this hypothesis, we have generated homology models of the sunflower KAI2s (HaKAI2s) and used them for molecular docking studies with STLs. Our results show that particularly two sunflower STLs, 8-epixanthatin and tomentosin, can bind to the hydrophobic pockets of HaKAI2s with high affinity. Our results are in line with a recent study, showing that these two STLs accumulate in the light-exposed hypocotyls of sunflower. This finding sheds light on the effect of STLs in hypocotyl elongation that has been reported for many decades but without conclusive insight in the elusive mechanism underlying this effect.

Main conclusions Sugar-mediated osmotic acclimation and a strong antioxidative response reduce drought-induced biomass loss at the vegetative stage in rice. Abstract 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.

Guaianolides are an important class of sesquiterpene lactones with unique biological and pharmaceutical properties. They have been postulated to be derived from germacranolides, but for years no progress has been made in the elucidation of their biosynthesis that requires an unknown cyclization mechanism. Here we demonstrate the isolation and characterization of a cytochrome P450 from feverfew (Tanacetum parthenium), kauniolide synthase. Kauniolide synthase catalyses the formation of the guaianolide kauniolide from the germacranolide substrate costunolide. Unlike most cytochrome P450s, kauniolide synthase combines stereoselective hydroxylation of costunolide at the C3 position, with water elimination, cyclization and regioselective deprotonation. This unique mechanism of action is supported by in silico modelling and docking experiments. The full kauniolide biosynthesis pathway is reconstructed in the heterologous hosts Nicotiana benthamiana and yeast, paving the way for biotechnological production of guaianolide-type sesquiterpene lactones., Q.L. was funded as a part of Terpmed (Plant Terpenoids for Human Health: a chemical and genomic approach to identify and produce bioactive compounds) (Project ID 227448) and A.B.K. was funded by Iranian Ministry of Science Research and Technology.

Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different terpenes. The relationship between TPS sequence and resulting terpene product is not completely understood. In this work we describe two TPSs from the Camphor tree Cinnamomum camphora (L.) Presl. One of these, CiCaMS, acts as a monoterpene synthase (monoTPS), and mediates the production of myrcene, while the other, CiCaSSy, acts as a sesquiterpene synthase (sesquiTPS), and catalyses the production of α-santalene, β-santalene and trans-α-bergamotene. Interestingly, these enzymes share 97% DNA sequence identity and differ only in 22 amino acid residues out of 553. To understand which residues are essential for the catalysis of monoterpenes resp. sesquiterpenes, a number of hybrid synthases were prepared, and supplemented by a set of single-residue variants. These were tested for their ability to produce monoterpenes and sesquiterpenes by in vivo production of sesquiterpenes in E. coli, and by in vitro enzyme assays. This analysis pinpointed three residues in the sequence which could mediate the change in product specificity from a monoterpene synthase to a sesquiterpene synthase. Another set of three residues defined the sesquiterpene product profile, including the ratios between sesquiterpene products.

Parasitic plants have a unique heterotrophic lifestyle based on the extraction of water and nutrients from host plants. Some parasitic plant species, particularly those of the family Orobanchaceae, attack crops and cause substantial yield losses. The breeding of resistant crop varieties is an inexpensive way to control parasitic weeds, but often does not provide a long-lasting solution because the parasites rapidly evolve to overcome resistance. Understanding mechanisms underlying naturally occurring parasitic plant resistance is of great interest and could help to develop methods to control parasitic plants. In this review, we describe the virulence mechanisms of parasitic plants and resistance mechanisms in their hosts, focusing on obligate root parasites of the genera Orobanche and Striga. We noticed that the resistance (R) genes in the host genome often encode proteins with nucleotide-binding and leucine-rich repeat domains (NLR proteins), hence we proposed a mechanism by which host plants use NLR proteins to activate downstream resistance gene expression. We speculated how parasitic plants and their hosts co-evolved and discussed what drives the evolution of virulence effectors in parasitic plants by considering concepts from similar studies of plant–microbe interaction. Most previous studies have focused on the host rather than the parasite, so we also provided an updated summary of genomic resources for parasitic plants and parasitic genes for further research to test our hypotheses. Finally, we discussed new approaches such as CRISPR/Cas9-mediated genome editing and RNAi silencing that can provide deeper insight into the intriguing life cycle of parasitic plants and could potentially contribute to the development of novel strategies for controlling parasitic weeds, thereby enhancing crop productivity and food security globally.

Evolution of lipid transfer from plants to fungi allowed plants to colonize land

To gain insight into the regulatory networks that underlie the induced defense in cucumber against spider mites, genes encoding transcription factors (TFs) were identified in the cucumber (Cucumissativus) genome and their regulation by two-spotted spider mite (Tetranychusurticae) herbivory was analyzed using RNA-seq. Of the total 1212 annotated TF genes in the cucumber genome, 119 were differentially regulated upon spider-mite herbivory during a period of 3 days. These TF genes belong to different categories but the MYB, bHLH, AP2/ERF and WRKY families had the highest relative numbers of differentially expressed genes. Correlation analysis of the expression of TF genes with defense-associated genes during herbivory and pathogen infestation, and in different organs resulted in the putative identification of regulators of herbivore-induced terpenoid and green-leaf-volatile biosynthesis. Analysis of the cis-acting regulatory elements (CAREs) present in the promoter regions of the genes responsive to spider-mite feeding revealed potential TF regulators. This study describes the TF genes in cucumber that are potentially involved in the regulation of induced defense against herbivory by spider mites.

Monoterpene methylperillate can be used efficiently as a convenient starting material for polyethylene terephthalate plastic. We describe a biosynthetic pathway towards methylperillate from Salvia and report enzymes for key reactions., Plants produce a large variety of highly functionalized terpenoids. Functional groups such as partially unsaturated rings and carboxyl groups provide handles to use these compounds as feedstock for biobased commodity chemicals. For instance, methylperillate, a monoterpenoid found in Salvia dorisiana, may be used for this purpose, as it carries both an unsaturated ring and a methylated carboxyl group. The biosynthetic pathway of methylperillate in plants is still unclear. In this work, we identified glandular trichomes from S. dorisiana as the location of biosynthesis and storage of methylperillate. mRNA from purified trichomes was used to identify four genes that can encode the pathway from geranyl diphosphate towards methylperillate. This pathway includes a (–)-limonene synthase (SdLS), a limonene 7-hydroxylase (SdL7H, CYP71A76), and a perillyl alcohol dehydrogenase (SdPOHDH). We also identified a terpene acid methyltransferase, perillic acid O-methyltransferase (SdPAOMT), with homology to salicylic acid OMTs. Transient expression in Nicotiana benthamiana of these four genes, in combination with a geranyl diphosphate synthase to boost precursor formation, resulted in production of methylperillate. This demonstrates the potential of these enzymes for metabolic engineering of a feedstock for biobased commodity chemicals.

Flower sepals are critical for flower development and vary greatly in lifespan depending on their function postpollination. However, very little is known on what controls sepal longevity. Using a sepal senescence mutant screen, we directly connected strigolactones (SL) with sepal longevity. We identified two Arabidopsis mutants that harbour novel mutations in the SL biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) and receptor DWARF14 (AtD14). The mutation in AtD14 caused a substitution of the catalytic Ser-97 to Phe in the enzyme active site. The lesion in MAX1 changed a highly conserved Gly-469 to Arg in the haem-iron ligand signature of the cytochrome P450 protein, which caused loss-of-function of MAX1. nCounter-based transcriptional analysis suggested an interaction between SL and sugar signalling in controlling dark-induced inflorescence senescence. The results uncover an important function for SL in regulating floral organ senescence in addition to its other diverse functions in plant development and stress response.One-sentence summaryTwo novel mutants in the strigolactone pathway demonstrate a role for the hormone in sepal senescence, and transcriptional analysis highlights interaction between strigolactones and sugar signalling.

Plants regulate responses towards herbivory through fine-tuning of defence-related hormone production, expression of defence genes, and production of secondary metabolites. Jasmonic acid (JA) plays a key role in plant–herbivorous arthropod interactions. To understand how pepper (Capsicum annuum) responds to herbivory, leaf transcriptomes and metabolomes of two genotypes different in their susceptibility to spider mites were studied. Mites induced both JA and salicylic acid (SA) signalling. However, mite infestation and exogenous JA resulted in distinct transcriptome profiles. Compared with JA, mites induced fewer differentially expressed genes involved in metabolic processes (except for genes involved in the phenylpropanoid pathway) and lipid metabolic processes. Furthermore, pathogen-related defence responses including WRKY transcription factors were more strongly induced upon mite infestation, probably as a result of induced SA signalling. Untargeted analysis of secondary metabolites confirmed that JA treatment induced larger changes in metabolism than spider mite infestation, resulting in higher terpenoid and flavonoid production. The more resistant genotype exhibited a larger increase in endogenous JA and volatile and non-volatile secondary metabolites upon infestation, which could explain its stronger defence. Reasoning that in JA–SA antagonizing crosstalk, SA defences are prioritized over JA defences, we hypothesize that lack of SA-mediated repression of JA-induced defences could result in gain of resistance towards spider mites in pepper., Lack of SA-mediated repression of JA-induced specialized metabolites results in gain of resistance towards spider mites in pepper

In the above mentioned publication, part of Fig. 1b was distorted (48 h after TSSM Infestation). The original article has been corrected and the proper version of Fig. 1 is also published here. (Figure presented.).

Background Recently, putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene pre-mRNA and maturation of the miRNAs from the intron. Here, we demonstrated that such dual functionality is indeed possible, using as host gene the firefly luciferase gene with intron (ffgLUC), and different artificial intronic miRNAs (aimiRNA) placed within the intron of ffgLUC. Results The miRNAs were based on the structure of the natural miR319a. Luciferase (LUC) activity in planta was used to evaluate a correct splicing of the ffgLUC mRNA. Different target sequences were inserted into the aimiRNA to monitor efficiency of silencing of different target mRNAs. After adjusting the insertion cloning strategy, the ffgLUCaimiR-319a gene showed dual functionality with correct splicing of ffgLUC and efficient silencing of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 transcription factor genes targeted in-trans by aimiR-319a or targeting the transgene ffLUC in-cis by an aimiR-LUC. Silencing of endogenous target genes by aimiRNA or amiRNA is efficient both in transient assays and stable transformants. A behave as strong phenotype the PHYTOCHROME B (PHYB) gene was also targeted by ffgLUCaimiR-PHYB. The lack of silencing of the PHYB target was most likely due to an insensitive target site within the PHYB mRNA which can potentially form a double stranded stem structure. Conclusion The combination of an overexpression construct with an artificial intronic microRNA allows for a simultaneous dual function in plants. The concept therefore adds new options to engineering of plant traits that require multiple gene manipulations.

Strigolactones (SLs) represent a class of plant hormones that regulate developmental processes and play a role in the response of plants to various biotic and abiotic stresses. Both in planta hormonal roles and ex planta signalling effects of SLs are potentially interesting agricultural targets. In this review, we explore various aspects of SL function and highlight distinct areas of agriculture that may benefit from the use of synthetic SL analogues, and we identify possible bottlenecks. Our objective is to identify where the contribution of science and stakeholders are still needed to achieve harnessing the benefits of SLs for a sustainable agriculture of the near future.

Phorodon humuli (Damson-hop aphid) is one of the major pests of hops in the northern hemisphere. It causes significant yield losses and reduces hop quality and economic value. Damson-hop aphid is currently controlled with insecticides, but the number of approved pesticides is steadily decreasing. In addition, the use of insecticides almost inevitably results in the development of resistant aphid genotypes. An integrated approach to pest management in hop cultivation is therefore badly needed in order to break this cycle and to prevent the selection of strains resistant to the few remaining registered insecticides. The backbone of such an integrated strategy is the breeding of hop cultivars that are resistant to Damson-hop aphid. However, up to date mechanisms of hops resistance towards Damson-hop aphids have not yet been unraveled. In the experiments presented here, we used metabolite profiling followed by multivariate analysis and show that metabolites responsible for hop aroma and flavor (sesquiterpenes) in the cones can also be found in the leaves, long before the hop cones develop, and may play a role in resistance against aphids. In addition, aphid feeding induced a change in the metabolome of all hop genotypes particularly an increase in a number of oxidized compounds, which suggests this may be part of a resistance mechanism. Electronic supplementary material The online version of this article (10.1007/s10886-018-0980-y) contains supplementary material, which is available to authorized users.

Strigolactones (SLs) are a class of signalling molecules secreted by the roots of plants into the rhizosphere. On the one hand, they serve as the signal for recruiting arbuscular mycorrhizal fungi which have a symbiotic relationship with plants. On the other hand, they are also host detection signals for the non-symbiotic, pathogenic, root parasitic plants, which use the SLs as germination stimulants. Finally, recently the SLs were discovered to be a new class of plant hormones that regulate processes such as branching/tillering and root architecture. Intriguingly, >25 different SLs have already been discovered that all have the so-called D-ring but otherwise display many differences in structure and functional groups. In this review, we will critically discuss the structural diversity in the SLs. How are they synthesized in plants; how has this structural diversity possibly evolved; what is the biological relevance of this diversity; and what does this imply for the perception of the SLs by receptors in the plant itself and in other organisms? Finally, we conclude that only little is known about the biological significance of this structural diversity, and we will give an outlook on how to elucidate their importance further.

Both strigolactones (SLs) and abscisic acid (ABA) biosynthetically originate from carotenoids. Considering their common origin, the interaction of these two hormones at the biosynthetic and/or regulatory level may be anticipated. Here we show that, in rice, drought simultaneously induces SL production in the root, and ABA production and the expression of SL biosynthetic genes in the shoot. Under control conditions, the ABA concentration was higher in shoots of the SL biosynthetic rice mutants dwarf10 (d10) and d17 than in wild-type plants, while a similar trend was observed for the SL perception mutant d3. These differences were enhanced under drought. However, drought did not result in an increase in leaf ABA content in the rice mutant line d27, carrying a mutation in the gene encoding the first committed enzyme in SL biosynthesis, to the same extent as in the other SL mutants and the wild type. Accordingly, d10, d17, and d3 lines were more drought tolerant than wild-type plants, whereas d27 displayed decreased tolerance. Finally, overexpression of OsD27 in rice resulted in increased levels of ABA when compared with wild-type plants. We conclude that the SL and ABA pathways are connected with each other through D27, which plays a crucial role in determining ABA and SL content in rice.

Genome-scale models capture the remarkable specialization of epithelial cells for oleoresin biosynthesis in needles of loblolly pine (Pinus taeda L.)., The shoot system of pines contains abundant resin ducts, which harbor oleoresins that play important roles in constitutive and inducible defenses. In a pilot study, we assessed the chemical diversity of oleoresins obtained from mature tissues of loblolly pine trees (Pinus taeda L.). Building on these data sets, we designed experiments to assess oleoresin biosynthesis in needles of 2-year-old saplings. Comparative transcriptome analyses of single cell types indicated that genes involved in the biosynthesis of oleoresins are significantly enriched in isolated epithelial cells of resin ducts, compared with those expressed in mesophyll cells. Simulations using newly developed genome-scale models of epithelial and mesophyll cells, which incorporate our data on oleoresin yield and composition as well as gene expression patterns, predicted that heterotrophic metabolism in epithelial cells involves enhanced levels of oxidative phosphorylation and fermentation (providing redox and energy equivalents). Furthermore, flux was predicted to be more evenly distributed across the metabolic network of mesophyll cells, which, in contrast to epithelial cells, do not synthesize high levels of specialized metabolites. Our findings provide novel insights into the remarkable specialization of metabolism in epithelial cells.

[EN] Production of citrus, the main fruit tree crop worldwide, is severely threatened by Huanglongbing (HLB), for which as yet a cure is not available. Spread of this bacterial disease in America and Asia is intimately connected with dispersal and feeding of the insect vector Diaphorina citri, oligophagous on rutaceous host plants. Effective control of this psyllid is an important component in successful HLB management programs. Volatiles released from the non-host guava have been shown to be repellent to the psyllid and to inhibit its response to citrus odour. By analysing VOC emission from guava we identified one volatile compound, (E)-ß-caryophyllene, which at certain doses exerts a repellent effect on D. citri. Non-host plant rejection mediated by (E)-ß-caryophyllene is demonstrated here by using Arabidopsis over-expression and knock-out lines. For the first time, results indicate that genetically engineered Arabidopsis plants with modified emission of VOCs can alter the behaviour of D. citri. This study shows that transgenic plants with an inherent ability to release (E)-ß-caryophyllene can potentially be used in new protection strategies of citrus trees against HLB., We thank Dr. Pedro Serra (IBMCP, Valencia, Spain) for his help with statistical analysis, and Prof. Dr. Luiz A.B. de Moraes (Chemistry Department, FFCLRP, USP, Riberao Preto, Brazil) and Prof. Dr. Edson Rodrigues Filho (LaBioMMi, Chemistry Department, UFSCar, Sao Carlos, Brazil) for the use of GC-MS equipment. Use of the Citrus Germplasm Bank (IVIA, Valencia, Spain) is gratefully acknowledged. This work was funded by the Fundo de Defesa da Citricultura (Fundecitrus) and FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, 2015/0711-3). In memoriam of Prof. J.M.Bove.

The new phytologist 213(3), 1346-1362 (2017). doi:10.1111/nph.14220, Published by Wiley-Blackwell, Oxford [u.a.]

In the root exudate and root extracts of maize hybrid cv NK Falkone seven putative strigolactones were detected using UPLC-TQ-MS-MS. All seven compounds displayed MS-MS-fragmentation common for strigolactones and particularly the presence of a fragment of m/z 97 Da, which may indicate the presence of the so-called D-ring, suggests they are strigolactones. The levels of all these putative strigolactones increased upon phosphate starvation and decreased upon fluridone (carotenoid biosynthesis inhibitor) treatment, both of which are a common response for strigolactones. All seven compounds were subsequently isolated with prep-HPLC-MS. They all exhibited Striga hermonthica seed germination inducing activity just as the synthetic strigolactone analog GR24. The structure of two of the seven compounds was elucidated by NMR spectroscopy as: methyl (2E,3E)-4-(3,3-dimethyl-5-oxo-2-(prop-1-en-2-yl)tetrahydrofuran-2-yl)-2-(((4-methyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)methylene)but-3-enoate (two diastereomers 1a and 1b). Strigolactones (1a/b) are closely related to the methyl ester of carlactonoic acid (MeCLA) and heliolactone. However, they contain a unique 4,4-dimethyltetrahydrofuran-2-one motif as the “A-ring” instead of the classical (di)methylcyclohexene. Because these compounds were isolated from maize (Zea mays) we called them “zealactone 1a and 1b”. The implications of this discovery for our view on strigolactones and their biosynthesis are discussed.

The role of phloem proteins in plant resistance to aphids is still largely elusive. By genome-wide association mapping of aphid behavior on 350 natural Arabidopsis thaliana accessions, we identified the small heat shock-like SIEVE ELEMENT-LINING CHAPERONE1 (SLI1). Detailed behavioral studies on near-isogenic and knockout lines showed that SLI1 impairs phloem feeding. Depending on the haplotype, aphids displayed a different duration of salivation in the phloem. On sli1 mutants, aphids prolonged their feeding sessions and ingested phloem at a higher rate than on wild-type plants. The largest phenotypic effects were observed at 26°C, when SLI1 expression is upregulated. At this moderately high temperature, sli1 mutants suffered from retarded elongation of the inflorescence and impaired silique development. Fluorescent reporter fusions showed that SLI1 is confined to the margins of sieve elements where it lines the parietal layer and colocalizes in spherical bodies around mitochondria. This localization pattern is reminiscent of the clamp-like structures observed in previous ultrastructural studies of the phloem and shows that the parietal phloem layer plays an important role in plant resistance to aphids and heat stress.

Our lack of full understanding of transport and sequestration of the heterologous products currently limit metabolic engineering in plants for the production of high value terpenes. For instance, although all genes of the artemisinin/arteannuin B (AN/AB) biosynthesis pathway (AN-PW) from Artemisia annua have been identified, ectopic expression of these genes in Nicotiana benthamiana yielded mostly glycosylated pathway intermediates and only very little free (dihydro)artemisinic acid [(DH)AA]. Here we demonstrate that Lipid Transfer Protein 3 (AaLTP3) and the transporter Pleiotropic Drug Resistance 2 (AaPDR2) from A. annua enhance accumulation of (DH)AA in the apoplast of N. benthamiana leaves. Analysis of apoplast and cell content and apoplast exclusion assays show that AaLTP3 and AaPDR2 prevent reflux of (DH)AA from the apoplast back into the cells and enhances overall flux through the pathway. Moreover, AaLTP3 is stabilized in the presence of AN-PW activity and co-expression of AN-PW+AaLTP3+AaPDR2 genes yielded AN and AB in necrotic N. benthamiana leaves at 13 days post-agroinfiltration. This newly discovered function of LTPs opens up new possibilities for the engineering of biosynthesis pathways of high value terpenes in heterologous expression systems.

Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua, was integrated into the moss Physcomitrella patens. Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS, into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens. Introduction of three pathway genes, ADS-CYP71AV1-ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens.

The therapeutic properties of complex terpenes often depend on the stereochemistry of their functional groups. However, stereospecific chemical synthesis of terpenes is challenging. To overcome this challenge, metabolic engineering can be employed using enzymes with suitable stereospecific catalytic activity. Here we used a combinatorial metabolic engineering approach to explore the stereospecific modification activity of the Artemisia annua artemisinic aldehyde ∆11(13) double bond reductase2 (AaDBR2) on products of the feverfew sesquiterpene biosynthesis pathway (GAS, GAO, COS and PTS). This allowed us to produce dihydrocostunolide and dihydroparthenolide. For dihydroparthenolide we demonstrate that the preferred order of biosynthesis of dihydroparthenolide is by reduction of the exocyclic methylene of parthenolide, rather than through C4-C5 epoxidation of dihydrocostunolide. Moreover, we demonstrate a promiscuous activity of feverfew CYP71CB1 on dihydrocostunolide and dihydroparthenolide for the production of 3β-hydroxy-dihydrocostunolide and 3β-hydroxy-dihydroparthenolide, respectively. Combined, these results offer new opportunities for engineering novel sesquiterpene lactones with potentially improved medicinal value.

Chicory (Cichorium intybus L.) is a medicinal and industrial plant from the Asteraceae family that produces a variety of sesquiterpene lactones (STLs), most importantly bitter guaianolides: lactucin, lactucopicrin and 8-deoxylactucin as well as their modified forms such as oxalates. These compounds have medicinal properties; however, they also hamper the extraction of inulin – a very important food industry product from chicory roots. The first step in guaianolide biosynthesis is catalyzed by germacrene A synthase (GAS) which in chicory exists in two isoforms – GAS long (encoded by CiGASlo) and GAS short (encoded by CiGASsh). AmiRNA silencing was used to obtain plants with reduced GAS gene expression and level of downstream metabolites, guaianolide-15-oxalates, as the major STLs in chicory. This approach could be beneficial for engineering new chicory varieties with varying STL content, and especially varieties with reduced bitter compounds more suitable for inulin production.

Plants exhibit a vast array of sesquiterpenes, C15 hydrocarbons which often function as herbivore-repellents or pollinator-attractants. These in turn are produced by a diverse range of sesquiterpene synthases. A comprehensive analysis of these enzymes in terms of product specificity has been hampered by the lack of a centralized resource of sufficient functionally annotated sequence data. To address this, we have gathered 262 plant sesquiterpene synthase sequences with experimentally characterized products. The annotated enzyme sequences allowed for an analysis of terpene synthase motifs, leading to the extension of one motif and recognition of a variant of another. In addition, putative terpene synthase sequences were obtained from various resources and compared with the annotated sesquiterpene synthases. This analysis indicated regions of terpene synthase sequence space which so far are unexplored experimentally. Finally, we present a case describing mutational studies on residues altering product specificity, for which we analyzed conservation in our database. This demonstrates an application of our database in choosing likely-functional residues for mutagenesis studies aimed at understanding or changing sesquiterpene synthase product specificity.

International audience; Chicory (Cichorium intybus L.) produces bitter sesquiterpene lactones (STLs). Some enzymes in the biosynthetic pathway towards these compounds have been characterized. However, the genomic organization and tissue specificity of their biosynthesis is largely unknown. Concentrations of two sesquiterpene lactones and expression of genes involved in the first dedicated biosynthetic step were measured in different chicory tissues. BAC clones containing different genes encoding germacrene A synthase were sequenced, and revealed several tightly linked paralogs. Promoters of genes encoding two germacrene A synthases were fused to GFP and expressed in plants regenerated from transformed chicory hairy root cultures. Highest expression was observed in the epidermis of leaves and external root tissue. This work opens the possibility to select for chicory germplasm diversified in STL content, and to study their role in chicory in defence and physiology.

Strigolactones (SLs) are a group of carotenoid derivatives that act as a hormone regulating plant development and response to environmental stimuli. SLs are also released into soil as a signal indicating the presence of a host for symbiotic arbuscular mycorrhizal fungi and root parasitic weeds. In this chapter, we provide an overview on the enormous progress that has been recently made in elucidating SL biosynthesis and signal transduction. We describe the tailoring pathway from the carotenoid precursor to the central intermediate carlactone, highlighting the stereospecificity of the involved enzymes, the all-trans/9-cis-β-carotene isomerase (D27), the 9-cis-specific CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), as well as CCD8 and its unusual catalytic activity. We then outline the oxidation of carlactone by cytochrome P450 enzymes, such as the Arabidopsis MORE AXILLARY GROWTH 1 (MAX1), into different SLs and the role of other enzymes in generating this diversity, and discuss why plants produce many different SLs. This is followed by depicting hormonal and nutritional factors that regulate SL biosynthesis and release, and by a description of transport mechanisms. In the second part of our chapter, we focus on SL perception and signal transduction, describing the SL receptor DECREASED APICAL DOMINANCE 2 (DAD2)/DWARF14 (D14) and its unique features, the central function of protein degradation mediated by the F-box protein MAX2 and its homologs. We also discuss the latest advances in understanding how SLs regulate the transcription of target genes and the role of SMXL/D53 transcription inhibitors.

Summary Volatiles mediate the interaction of plants with pollinators, herbivores and their natural enemies, other plants and micro‐organisms. With increasing knowledge about these interactions the underlying mechanisms turn out to be increasingly complex. The mechanisms of biosynthesis and perception of volatiles are slowly being uncovered. The increasing scientific knowledge can be used to design and apply volatile‐based agricultural strategies., Significance Statement Volatiles mediate the interaction of plants with pollinators, micro‐organisms, other plants and herbivores and their natural enemies. The increasing knowledge about the role of volatiles in these interactions, the underlying mechanisms, the biosynthesis and perception of volatiles are reviewed and the perspective for volatile‐based agricultural control strategies discussed.

A parasitic plant is a flowering plant that attaches itself morphologically and physiologically to a host (another plant) by a modified root (the haustorium). Only about 25 out of the 270 genera of parasitic plants have a negative impact in agriculture and forestry, and thus can be considered weeds. Among them, the most damaging root-parasitic weeds belong to the genera Orobanche and Phelipanche (commonly named broomrapes) and Striga (witchweeds) (all belonging to the Orobanchaceae family). Considering the aims of the book, this chapter will focus only on this group of parasitic weeds, as in these plants strigolactones have a key role both in their life cycle, and in management strategies to control them. Distribution, agricultural importance and life cycle of these parasitic weeds are briefly introduced, after which we focus on the role of strigolactones in seed germination, parasite development, host specificity, plant nutrition, and microbiome composition. Furthermore, some weed control approaches involving strigolactones are discussed.

A potent stimulant induces parasitic plant germination that causes it to die

A striking feature of the angiosperms that use animals as pollen carriers to sexually reproduce is the great diversity of their flowers with regard to morphology and traits such as color, odor, and nectar. These traits are underpinned by the synthesis of secondary metabolites such as pigments and volatiles, as well as carbohydrates and amino acids, which are used by plants to lure and reward animal pollinators. We review here the knowledge of the metabolic network that supports the biosynthesis of these compounds and the behavioral responses that these molecules elicit in the animal pollinators. Such knowledge provides us with a deeper insight into the ecology and evolution of plant-pollinator interactions, and should help us to better manage these ecologically essential interactions in agricultural ecosystems. Novel routes for the biosynthesis of floral volatiles in unusual subcellular compartments are being identified and new theories on the emission of scent are proposed.Key genes in betalain biosynthesis have been identified and new theories on the evolutionary origin of the pathway have been suggested.Master regulators that coordinately control the production of pigments and scent in flowers are emerging.Loci have been identified that contribute to reproductive isolation by pollination preferences for visual and olfactory cues and ultimately lead to speciation.

Flowers of Nicotiana species emit a characteristic blend including the cineole cassette monoterpenes. This set of terpenes is synthesized by multiproduct enzymes, with either 1,8-cineole or α-terpineol contributing most to the volatile spectrum, thus referring to cineole or terpineol synthase, respectively. To understand the molecular and structural requirements of the enzymes that favor the biochemical formation of α-terpineol and 1,8-cineole, site-directed mutagenesis, in silico modeling, and semiempiric calculations were performed. Our results indicate the formation of α-terpineol by a nucleophilic attack of water. During this attack, the α-terpinyl cation is stabilized by π-stacking with a tryptophan side chain (tryptophan-253). The hypothesized catalytic mechanism of α-terpineol-to-1,8-cineole conversion is initiated by a catalytic dyad (histidine-502 and glutamate-249), acting as a base, and a threonine (threonine-278) providing the subsequent rearrangement from terpineol to cineol by catalyzing the autoprotonation of (S)-(-)-α-terpineol, which is the favored enantiomer product of the recombinant enzymes. Furthermore, by site-directed mutagenesis, we were able to identify amino acids at positions 147, 148, and 266 that determine the different terpineol-cineole ratios in Nicotiana suaveolens cineole synthase and Nicotiana langsdorffii terpineol synthase. Since amino acid 266 is more than 10 A away from the active site, an indirect effect of this amino acid exchange on the catalysis is discussed.

BACKGROUND Significant losses in sorghum biomass and grain yield occur in sub-Saharan Africa owing to infection by the root-parasitic weed Striga hermonthica (Del.) Benth. One strategy to avoid these losses is to adopt resistant crop varieties. For further delineation of the role of germination stimulants in resistance, we conducted a field experiment employing six sorghum genotypes, in eastern Sudan, and in parallel analysed the strigolactone levels in the root exudates of these genotypes under controlled conditions in Wageningen. RESULTS The root exudates of these genotypes displayed large differences in strigolactone composition and Striga-germination-inducing activity. Korokollow, Fakimustahi and Wadfahel exuded the highest amounts of 5-deoxystrigol. Fakimustahi was by far the highest sorgomol producer, and Wadbaco and SRN39 produced the highest amount of orobanchol. The concentration of 5-deoxystrigol in the root exudate showed a significant positive correlation with in vitro Striga germination and was positively associated with Striga infection in the field experiments, whereas orobanchol was negatively associated with Striga infection in the field experiments. CONCLUSION For the first time a close association is reported between strigolactone levels analysed under laboratory conditions and Striga infection in the field in sorghum genotypes. These genotypes may be used for further study of this resistance mechanism and for the introgression of the low germination trait in other sorghum varieties to breed for a strigolactone composition with low stimulant activity. The use of such improved varieties in combination with other Striga management tools could possibly alleviate the current Striga problem on the African continent. © 2016 Society of Chemical Industry

Highlight The transcription factor WRKY22 increases susceptibility to aphids in Arabidopsis via the suppression of salicylic acid signalling., Aphids induce many transcriptional perturbations in their host plants, but the signalling cascades responsible and the effects on plant resistance are largely unknown. Through a genome-wide association (GWA) mapping study in Arabidopsis thaliana, we identified WRKY22 as a candidate gene associated with feeding behaviour of the green peach aphid, Myzus persicae. The transcription factor WRKY22 is known to be involved in pathogen-triggered immunity, and WRKY22 gene expression has been shown to be induced by aphids. Assessment of aphid population development and feeding behaviour on knockout mutants and overexpression lines showed that WRKY22 increases susceptibility to M. persicae via a mesophyll-located mechanism. mRNA sequencing analysis of aphid-infested wrky22 knockout plants revealed the up-regulation of genes involved in salicylic acid (SA) signalling and down-regulation of genes involved in plant growth and cell-wall loosening. In addition, mechanostimulation of knockout plants by clip cages up-regulated jasmonic acid (JA)-responsive genes, resulting in substantial negative JA–SA crosstalk. Based on this and previous studies, WRKY22 is considered to modulate the interplay between the SA and JA pathways in response to a wide range of biotic and abiotic stimuli. Its induction by aphids and its role in suppressing SA and JA signalling make WRKY22 a potential target for aphids to manipulate host plant defences.