Scots Pines With Tolerance to Melampsora pinitorqua and Diplodia sapinea Show Distinct Metabolic Profiles.

Diplodia sapinea causes Diplodia tip blight (DTB) and is recognised as an opportunistic necrotrophic pathogen affecting conifers. While DTB is associated with abiotic stress, the impact of biotic stress in the host on D. sapinea's lifestyle shift is unknown. Observed co-occurrences of D. sapinea and Melampsora pinitorqua, causing pine twisting rust on Scots pine (Pinus sylvestris), instigated an investigation into their interaction with and influence on the defence mechanisms of the host. We hypothesised that M. pinitorqua infections predispose the trees to D. sapinea by stressing the host and altering the shoot metabolites. Pines in a plantation were sampled over time to study pathogen biomass and host metabolites. Symptoms of both pathogens were consistent over years, and the preceding season's symptoms affected the metabolic profiles pre-infection and M. pinitorqua's proliferation. Symptoms of M. pinitorqua altered shoot metabolites more than fungal biomass, with co-symptomatic trees exhibiting elevated M. pinitorqua biomass. Specific phenolic compounds had a strong positive association with the shoot symptom × D. sapinea interaction. D. sapinea's biomass presymptoms was independent of previous disease symptoms and infection by M. pinitorqua. Some trees showed disease tolerance, with delayed rust infections and minimal DTB symptoms. Further investigations on this trait are needed.

Glucosinolate structural diversity shapes recruitment of a metabolic network of leaf-associated bacteria.

Host defenses can have broader ecological roles, but how they shape natural microbiome recruitment is poorly understood. Aliphatic glucosinolates (GLSs) are secondary defense metabolites in Brassicaceae plant leaves. Their genetically defined structure shapes interactions with pests in Arabidopsis thaliana leaves, and here we find that it also shapes bacterial recruitment. In model genotype Col-0, GLSs (mostly 4-methylsulfinylbutyl-GLS) have no clear effect on natural leaf bacterial recruitment. In a genotype from a wild population, however, GLSs (mostly allyl-GLS) enrich specific taxa, mostly Comamonadaceae and Oxalobacteraceae. Consistently, Comamonadaceae are also enriched in wild A. thaliana, and Oxalobacteraceae are enriched from wild plants on allyl-GLS as carbon source, but not on 4-methylsulfinylbutyl-GLS. Recruitment differences between GLS structures most likely arise from bacterial myrosinase specificity. Community recruitment is then defined by metabolic cross-feeding among bacteria. The link of genetically defined metabolites to recruitment could lead to new strategies to shape plant microbiome balance.

Insights into the molecular basis of beet curly top resistance in sugar beet through a transcriptomic approach at the early stage of symptom development.

Curly top disease caused by Beet curly top virus (BCTV) is a limiting factor for sugar beet production. The most economical and sustainable control of BCTV in sugar beet would be via the growth of resistant cultivars, although most commercial cultivars possess only low-to-moderate quantitative resistance. A double haploid line (KDH13) showed a high level of resistance to BCTV infection. However, the mechanism of resistance and response of this line to BCTV infection is unknown. Here, we tested the response of this line to both local and systemic BCTV infections. The virus replicated at a high level in locally infected tissue but lower than in susceptible KDH19 plants. Resistant KDH13 plants systemically infected with BCTV showed only mild enation without leaf curling after 30 days. In contrast, severe leaf curling appeared after 12 days in susceptible plants with higher virus accumulation. Transcriptome analysis of the BCTV-infected KDH13 plants at the early stage of symptom development showed only 132 genes that were exclusively deregulated compared to the regulation of a large number of genes (1018 genes) in KDH19 plants. Pathway enrichment analysis showed that differentially expressed genes were predominantly involved in hormone metabolism, DNA methylation, immune response, cell cycle, biotic stress and oxidative stress. The auxin level in both resistant and susceptible plants increased in response to BCTV infection. Remarkably, exogenous application of auxin caused leaf curling phenotype in the absence of the virus. This study demonstrates the response of resistant and susceptible plants to BCTV infection at both local and systemic infections and highlights the defence-related genes and metabolic pathways including auxin for their contribution towards BCTV symptom development and resistance in sugar beet.

Glutamine as sole nitrogen source prevents induction of nitrate transporter gene NRT2.4 and affects amino acid metabolism in Arabidopsis.

Plants assimilate inorganic nitrogen (N) to glutamine. Glutamine is the most abundant amino acid in most plant species, the N-supplying precursor of all N-containing compounds in the cell and the first organic nitrogen molecule formed from inorganic nitrogen taken up by the roots. In addition to its role in plant nutrition, glutamine most likely also has a function as a signaling molecule in the regulation of nitrogen metabolism. We investigated whether glutamine influences the high-affinity transporter system for nitrate uptake. Therefore, we analyzed the expression of the nitrate transporter NRT2.4, which is inducible by N deficiency, in Arabidopsis thaliana grown under different nitrogen starvation scenarios, comparing nitrate or glutamine as the sole nitrogen source. Using the reporter line ProNRT2.4:GFP and two independent knockout lines, nrt2.4-1 and nrt2.4-2, we analyzed gene expression and amino acid profiles. We showed that the regulation of NRT2.4 expression depends on available nitrogen in general, for example on glutamine as a nitrogen source, and not specifically on nitrate. In contrast to high nitrate concentrations, amino acid profiles changed to an accumulation of amino acids containing more than one nitrogen during growth in high glutamine concentrations, indicating a switch to nitrogen storage metabolism. Furthermore, we demonstrated that the nrt2.4-2 line shows unexpected effects on NRT2.5 gene expression and the amino acids profile in shoots under high glutamine supply conditions compared to Arabidopsis wild type and nrt2.4-1, suggesting non-NRT2.4-related metabolic consequences in this knockout line.

Cellooligomer/CELLOOLIGOMER RECEPTOR KINASE1 Signaling Exhibits Crosstalk with PAMP-Triggered Immune Responses and Sugar Metabolism in Arabidopsis Roots.

The degradation of cellulose generates cellooligomers, which function as damage-associated molecular patterns and activate immune and cell wall repair responses via the CELLOOLIGOMER RECEPTOR KINASE1 (CORK1). The most active cellooligomer for the induction of downstream responses is cellotriose, while cellobiose is around 100 times less effective. These short-chain cellooligomers are also metabolized after uptake into the cells. In this study, we demonstrate that CORK1 is mainly expressed in the vascular tissue of the upper, fully developed part of the roots. Cellooligomer/CORK1-induced responses interfere with chitin-triggered immune responses and are influenced by BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE1 and the receptor kinase FERONIA. The pathway also controls sugar transporter and metabolism genes and the phosphorylation state of these proteins. Furthermore, cellotriose-induced ROS production and WRKY30/40 expression are controlled by the sugar transporters SUCROSE-PROTON SYMPORTER1, SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER11 (SWEET11), and SWEET12. Our data demonstrate that cellooligomer/CORK1 signaling is integrated into the pattern recognition receptor network and coupled to the primary sugar metabolism in Arabidopsis roots.

Strigolactone deficiency induces jasmonate, sugar and flavonoid phytoalexin accumulation enhancing rice defense against the blast fungus Pyricularia oryzae.

Strigolactones (SLs) are carotenoid-derived phytohormones that regulate plant growth and development. While root-secreted SLs are well-known to facilitate plant symbiosis with beneficial microbes, the role of SLs in plant interactions with pathogenic microbes remains largely unexplored. Using genetic and biochemical approaches, we demonstrate a negative role of SLs in rice (Oryza sativa) defense against the blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae). We found that SL biosynthesis and perception mutants, and wild-type (WT) plants after chemical inhibition of SLs, were less susceptible to P. oryzae. Strigolactone deficiency also resulted in a higher accumulation of jasmonates, soluble sugars and flavonoid phytoalexins in rice leaves. Likewise, in response to P. oryzae infection, SL signaling was downregulated, while jasmonate and sugar content increased markedly. The jar1 mutant unable to synthesize jasmonoyl-l-isoleucine, and the coi1-18 RNAi line perturbed in jasmonate signaling, both accumulated lower levels of sugars. However, when WT seedlings were sprayed with glucose or sucrose, jasmonate accumulation increased, suggesting a reciprocal positive interplay between jasmonates and sugars. Finally, we showed that functional jasmonate signaling is necessary for SL deficiency to induce rice defense against P. oryzae. We conclude that a reduction in rice SL content reduces P. oryzae susceptibility by activating jasmonate and sugar signaling pathways, and flavonoid phytoalexin accumulation.

Integrative multi-omics analyses of date palm (Phoenix dactylifera) roots and leaves reveal how the halophyte land plant copes with sea water.

Date palm (Phoenix dactylifera L.) is able to grow and complete its life cycle while being rooted in highly saline soils. Which of the many well-known salt-tolerance strategies are combined to fine-tune this remarkable resilience is unknown. The precise location, whether in the shoot or the root, where these strategies are employed remains uncertain, leaving us unaware of how the various known salt-tolerance mechanisms are integrated to fine-tune this remarkable resilience. To address this shortcoming, we exposed date palm to a salt stress dose equivalent to seawater for up to 4 weeks and applied integrative multi-omics analyses followed by targeted metabolomics, hormone, and ion analyses. Integration of proteomic into transcriptomic data allowed a view beyond simple correlation, revealing a remarkably high degree of convergence between gene expression and protein abundance. This sheds a clear light on the acclimatization mechanisms employed, which depend on reprogramming of protein biosynthesis. For growth in highly saline habitats, date palm effectively combines various salt-tolerance mechanisms found in both halophytes and glycophytes: "avoidance" by efficient sodium and chloride exclusion at the roots, and "acclimation" by osmotic adjustment, reactive oxygen species scavenging in leaves, and remodeling of the ribosome-associated proteome in salt-exposed root cells. Combined efficiently as in P. dactylifera L., these sets of mechanisms seem to explain the palm's excellent salt stress tolerance.

The Beneficial Fungus Mortierella hyalina Modulates Amino Acid Homeostasis in Arabidopsis under Nitrogen Starvation.

Non-mycorrhizal but beneficial fungi often mitigate (a)biotic stress-related traits in host plants. The underlying molecular mechanisms are mostly still unknown, as in the interaction between the endophytic growth-promoting soil fungus Mortierella hyalina and Arabidopsis thaliana. Here, abiotic stress in the form of nitrogen (N) deficiency was used to investigate the effects of the fungus on colonized plants. In particular, the hypothesis was investigated that fungal infection could influence N deficiency via an interaction with the high-affinity nitrate transporter NRT2.4, which is induced by N deficiency. For this purpose, Arabidopsis wild-type nrt2.4 knock-out and NRT2.4 reporter lines were grown on media with different nitrate concentrations with or without M. hyalina colonization. We used chemical analysis methods to determine the amino acids and phytohormones. Experimental evidence suggests that the fungus does not modulate NRT2.4 expression under N starvation. Instead, M. hyalina alleviates N starvation in other ways: The fungus supplies nitrogen (15N) to the N-starved plant. The presence of the fungus restores the plants' amino acid homeostasis, which was out of balance due to N deficiency, and causes a strong accumulation of branched-chain amino acids. We conclude that the plant does not need to invest in defense and resources for growth are maintained, which in turn benefits the fungus, suggesting that this interaction should be considered a mutualistic symbiosis.

Acclimation of circadian rhythms in woodland strawberries (Fragaria vesca L.) to Arctic and mid-latitude photoperiods.

BACKGROUND: Though many abiotic factors are constantly changing, the photoperiod is a predictable factor that enables plants to time many physiological responses. This timing is regulated by the circadian clock, yet little is known about how the clock adapts to the differences in photoperiod between mid-latitudes and high latitudes. The primary objective of this study was to compare how clock gene expression is modified in four woodland strawberry (Fragaria vesca L.) accessions originating from two different populations in Italy (IT1: Tenno, Italy, 45°N, IT4: Salorno, Italy, 46°N) and two in Northern Norway (NOR2: Alta, Norway, 69°N, NOR13: Indre Nordnes, Norway 69°N) when grown under simulated daylength conditions of an Arctic or mid-latitude photoperiod. The second objective was to investigate whether population origin or the difference in photoperiod influenced phytohormone accumulation. RESULTS: The Arctic photoperiod induced lower expression in IT4 and NOR13 for six clock genes (FvLHY, FvRVE8, FvPRR9, FvPRR7, FvPRR5, and FvLUX), in IT1 for three genes (FvLHY, FvPRR9, and FvPRR5) and in NOR2 for one gene (FvPRR9). Free-running rhythms for FvLHY in IT1 and IT4 were higher after the Arctic photoperiod, while the free-running rhythm for FvLUX in IT4 was higher after the mid-latitude photoperiod. IT1 showed significantly higher expression of FvLHY and FvPRR9 than all other accessions, as well as significantly higher expression of the circadian regulated phytohormone, abscisic acid (ABA), but low levels of salicylic acid (SA). NOR13 had significantly higher expression of FvRVE8, FvTOC1, and FvLUX than all other accessions. NOR2 had extremely low levels of auxin (IAA) and high levels of the jasmonate catabolite, hydroxyjasmonic acid (OH-JA). CONCLUSIONS: Our study shows that circadian rhythms in Fragaria vesca are driven by both the experienced photoperiod and genetic factors, while phytohormone levels are primarily determined by specific accessions' genetic factors rather than the experienced photoperiod.

Biosynthesis, herbivore induction, and defensive role of phenylacetaldoxime glucoside.

Aldoximes are well-known metabolic precursors for plant defense compounds such as cyanogenic glycosides, glucosinolates, and volatile nitriles. They are also defenses themselves produced in response to herbivory; however, it is unclear whether aldoximes can be stored over a longer term as defense compounds and how plants protect themselves against the potential autotoxic effects of aldoximes. Here, we show that the Neotropical myrmecophyte tococa (Tococa quadrialata, recently renamed Miconia microphysca) accumulates phenylacetaldoxime glucoside (PAOx-Glc) in response to leaf herbivory. Sequence comparison, transcriptomic analysis, and heterologous expression revealed that 2 cytochrome P450 enzymes, CYP79A206 and CYP79A207, and the UDP-glucosyltransferase UGT85A123 are involved in the formation of PAOx-Glc in tococa. Another P450, CYP71E76, was shown to convert PAOx to the volatile defense compound benzyl cyanide. The formation of PAOx-Glc and PAOx in leaves is a very local response to herbivory but does not appear to be regulated by jasmonic acid signaling. In contrast to PAOx, which was only detectable during herbivory, PAOx-Glc levels remained high for at least 3 d after insect feeding. This, together with the fact that gut protein extracts of 3 insect herbivore species exhibited hydrolytic activity toward PAOx-Glc, suggests that the glucoside is a stable storage form of a defense compound that may provide rapid protection against future herbivory. Moreover, the finding that herbivory or pathogen elicitor treatment also led to the accumulation of PAOx-Glc in 3 other phylogenetically distant plant species suggests that the formation and storage of aldoxime glucosides may represent a widespread plant defense response.

Occurrence and conversion of progestogens and androgens are conserved in land plants.

Progestogens and androgens have been found in many plants, but little is known about their biosynthesis and the evolution of steroidogenesis in these organisms. Here, we show that the occurrence and biosynthesis of progestogens and androgens are conserved across the viridiplantae lineage. An UHPLC-ESI-MS/MS method allowed high-throughput analysis of the occurrence and chemical conversion of progestogens and androgens in 41 species across the green plant lineage. Dehydroepiandrosterone, testosterone, and 5α-dihydrotestosterone are plants' most abundant mammalian-like steroids. Progestogens are converted into 17α-hydroxyprogesterone and 5α-pregnane-3,20-dione. Androgens are converted into testosterone and 5α-dihydrotestosterone. 17,20-Lyases, essential for converting progestogens to androgens, seem to be most effective in monocot species. Our data suggest that the occurrence of progestogens and androgens is highly conserved in plants, and their biosynthesis might favor a route using the Δ4 pathway.

Metabolism of plant-derived toxins from its insect host increases the success of the entomopathogenic fungus Beauveria bassiana.

Beauveria bassiana is a soil fungus that parasitizes a large number of arthropod species, including numerous crop pests, causing white muscardine disease and is therefore used as a biological insecticide. However, some insects, such as the cabbage aphid (Brevicoryne brassicae), defend themselves chemically by sequestering dietary pro-toxins (glucosinolates) from their Brassicales host plants. Glucosinolates are accumulated by cabbage aphids and activated to form toxic isothiocyanates when under attack. While isothiocyanate formation protects aphids against most attackers, B. bassiana is still able to infect the cabbage aphid under natural conditions. We therefore investigated how this fungus is able to circumvent the chemical defense system of the cabbage aphid. Here, we describe how B. bassiana infection activates the cabbage aphid defense system, but the resulting toxins are metabolized by B. bassiana via the mercapturic acid pathway, of which the first step is catalyzed by glutathione-S-transferases of low substrate specificity. This detoxification pathway enhances B. bassiana growth when isothiocyanates are present in natural concentrations, and so appears to be an important factor in fungal parasitization of these chemically defended aphids.

Combination of bacterial N-acyl homoserine lactones primes Arabidopsis defenses via jasmonate metabolism.

N-acyl homoserine lactones (AHLs) are important players in plant-bacteria interactions. Different AHL-producing bacteria can improve plant growth and resistance against plant pathogens. In nature, plants may host a variety of AHL-producing bacteria and frequently experience numerous AHLs at the same time. Therefore, a coordinated response to combined AHL molecules is necessary. The purpose of this study was to explore the mechanism of AHL-priming using combined AHL molecules including N-(3-oxo-hexanoyl)-L-homoserine lactone, N-3-oxo-octanoyl-L-homoserine lactone, N-3-oxo-dodecanoyl-L-homoserine lactone, and N-3-oxo-tetradecanoyl-L-homoserine lactone and AHL-producing bacteria including Serratia plymuthica HRO-C48, Rhizobium etli CFN42, Burkholderia graminis DSM17151, and Ensifer meliloti (Sinorhizobium meliloti) Rm2011. We used transcriptome analysis, phytohormone measurements, as well as genetic and microbiological approaches to assess how the combination of structurally diverse AHL molecules influence Arabidopsis (Arabidopsis thaliana). Our findings revealed a particular response to a mixture of AHL molecules (AHL mix). Different expression patterns indicated that the reaction of plants exposed to AHL mix differs from that of plants exposed to single AHL molecules. In addition, different content of jasmonic acid (JA) and derivatives revealed that jasmonates play an important role in AHL mix-induced priming. The fast and stable decreased concentration of COOH-JA-Ile after challenge with the flagellin-derived peptide flg22 indicated that AHL mix modifies the metabolism of jasmonates. Study of various JA- and salicylic acid-related Arabidopsis mutants strengthened the notion that JA homeostasis is involved in AHL-priming. Understanding how the combination of AHLs primes plants for enhanced resistance has the potential to broaden our approaches in sustainable agriculture and will help to effectively protect plants against pathogens.

Testing hypotheses of a coevolutionary key innovation reveals a complex suite of traits involved in defusing the mustard oil bomb.

Coevolutionary interactions are responsible for much of the Earth's biodiversity, with key innovations driving speciation bursts on both sides of the interaction. One persistent question is whether macroevolutionary traits identified as key innovations accurately predict functional performance and selection dynamics within species, as this necessitates characterizing their function, investigating their fitness consequences, and exploring the selection dynamics acting upon them. Here, we used CRISPR-Cas9 mediating nonhomologous end joining (NHEJ) in the butterfly species Pieris brassicae to knock out and directly assess the function and fitness impacts of nitrile specifier protein (NSP) and major allergen (MA). These are two closely related genes that facilitate glucosinolate (GSL) detoxification capacity, which is a key innovation in mustard feeding Pierinae butterflies. We find NSP and MA are both required for survival on plants containing GSLs, with expression differences arising in response to variable GSL profiles, concordant with detoxification performance. Importantly, this concordance was only observed when using natural host plants, likely reflecting the complexity of how these enzymes interact with natural plant variation in GSLs and myrosinases. Finally, signatures of positive selection for NSP and MA were detected across Pieris species, consistent with these genes' importance in recent coevolutionary interactions. Thus, the war between these butterflies and their host plants involves more than the mere presence of chemical defenses and detoxification mechanisms, as their regulation and activation represent key components of complex interactions. We find that inclusion of these dynamics, in ecologically relevant assays, is necessary for coevolutionary insights in this system and likely others.

Rhizobia Contribute to Salinity Tolerance in Common Beans (Phaseolus vulgaris L.).

Rhizobia are soil bacteria that induce nodule formation on leguminous plants. In the nodules, they reduce dinitrogen to ammonium that can be utilized by plants. Besides nitrogen fixation, rhizobia have other symbiotic functions in plants including phosphorus and iron mobilization and protection of the plants against various abiotic stresses including salinity. Worldwide, about 20% of cultivable and 33% of irrigation land is saline, and it is estimated that around 50% of the arable land will be saline by 2050. Salinity inhibits plant growth and development, results in senescence, and ultimately plant death. The purpose of this study was to investigate how rhizobia, isolated from Kenyan soils, relieve common beans from salinity stress. The yield loss of common bean plants, which were either not inoculated or inoculated with the commercial R. tropici rhizobia CIAT899 was reduced by 73% when the plants were exposed to 300 mM NaCl, while only 60% yield loss was observed after inoculation with a novel indigenous isolate from Kenyan soil, named S3. Expression profiles showed that genes involved in the transport of mineral ions (such as K+, Ca2+, Fe3+, PO43-, and NO3-) to the host plant, and for the synthesis and transport of osmotolerance molecules (soluble carbohydrates, amino acids, and nucleotides) are highly expressed in S3 bacteroids during salt stress than in the controls. Furthermore, genes for the synthesis and transport of glutathione and γ-aminobutyric acid were upregulated in salt-stressed and S3-inocculated common bean plants. We conclude that microbial osmolytes, mineral ions, and antioxidant molecules from rhizobia enhance salt tolerance in common beans.

Combined -omics framework reveals how ant symbionts benefit the Neotropical ant-plant Tococa quadrialata at different levels.

Ant-plant defensive mutualism is a widely studied phenomenon, where ants protect their host plants (myrmecophytes) against herbivores in return for the provision of nesting sites and food. However, few studies addressed the influence of ant colonization and herbivory on the plant's metabolism. We chose the Amazonian plant Tococa quadrialata, living in association with Azteca cf. tonduzi ants for an ant-exclusion study to reveal the chemistry behind this symbiosis. We found that colonized plants did not only benefit from protection but also from increased amino acid and nitrogen content, enabling better performance even in an herbivore-free environment. In contrast, ant-deprived T. quadrialata plants accumulated more ellagitannins, a major class of constitutive defense compounds. Moreover, herbivory-induced jasmonate-mediated defense responses, including the upregulation of signaling and defense genes and the emission of volatiles irrespective of colonization status. Altogether, we show how ant-colonization can influence the general and defense-related metabolism and performance of myrmecophytes.

Dynamics of alkannin/shikonin biosynthesis in response to jasmonate and salicylic acid in Lithospermum officinale.

Alkannin/shikonin and their derivatives are specialised metabolites of high pharmaceutical and ecological importance exclusively produced in the periderm of members of the plant family Boraginaceae. Previous studies have shown that their biosynthesis is induced in response to methyl jasmonate but not salicylic acid, two phytohormones that play important roles in plant defence. However, mechanistic understanding of induction and non-induction remains largely unknown. In the present study, we generated the first comprehensive transcriptomic dataset and metabolite profiles of Lithospermum officinale plants treated with methyl jasmonate and salicylic acid to shed light on the underlying mechanisms. Our results highlight the diverse biological processes activated by both phytohormones and reveal the important regulatory role of the mevalonate pathway in alkannin/shikonin biosynthesis in L. officinale. Furthermore, by modelling a coexpression network, we uncovered structural and novel regulatory candidate genes connected to alkannin/shikonin biosynthesis. Besides providing new mechanistic insights into alkannin/shikonin biosynthesis, the generated methyl jasmonate and salicylic acid elicited expression profiles together with the coexpression networks serve as important functional genomic resources for the scientific community aiming at deepening the understanding of alkannin/shikonin biosynthesis.

An LC-MS/MS assay for enzymatic characterization of methylthioalkylmalate synthase (MAMS) involved in glucosinolate biosynthesis.

Brassicaceae are blessed with specialized metabolites called glucosinolates (GSLs), which along with their degradation products, are beneficial in agriculture and human health. To date, more than 130 GSL structures have been identified, mostly derived from the amino acid methionine. The biosynthesis of methionine-derived aliphatic GSLs starts with a side-chain elongation step involving a recursive three-step cyclic process that incorporates a new methylene group into the 2-oxo acid to form a series of elongated 2-oxo acids. Methylthioalkylmalate synthase (MAMS) catalyzes the first committed step in the side-chain elongation of methionine-derived GSLs. The substrate specificity of MAMS with different 2-oxo acids determines whether reaction products of a given cycle enter for an additional round of chain elongation or enter into core GSLs structure formation. Multiple MAMS encoding genes are present in the Brassicaceae species and are known to play a central role in shaping the diverse profile of aliphatic GSLs. We recently established a highly sensitive LC-MS/MS-based methodology that quantifies the MAMS activity by estimating the amount of the next intermediate of the pathway, the 2-malate derivatives. Overall, this chapter describes the protocol for the expression, purification, and steady-state kinetic analysis of the recombinant MAMS protein.

Alternative transcript splicing regulates UDP-glucosyltransferase-catalyzed detoxification of DIMBOA in the fall armyworm (Spodoptera frugiperda).

Herbivorous insects often possess the ability to detoxify chemical defenses from their host plants. The fall armyworm (Spodoptera frugiperda), which feeds principally on maize, detoxifies the maize benzoxazinoid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by stereoselective re-glucosylation using a UDP-glucosyltransferase, SfUGT33F28. SfUGT33F28 activity is induced by feeding on a DIMBOA-containing diet, but how this induction is regulated is unknown. In the present work, we describe the alternative splicing of the SfUGT33F28 transcript. Variant transcripts are differentially expressed in response to DIMBOA, and this transcriptional response is mediated by an insect aryl hydrocarbon receptor. These variants have large deletions leading to the production of truncated proteins that have no intrinsic UGT activity with DIMBOA but interact with the full-length enzyme to raise or lower its activity. Therefore, the formation of SfUGT33F28 splice variants induces DIMBOA-conjugating UGT activity when DIMBOA is present in the insect diet and represses activity in the absence of this plant defense compound.