Recruitment of distinct UDP-glycosyltransferase families demonstrates dynamic evolution of chemical defense within Eucalyptus L'Hér.

The economic and ecologically important genus Eucalyptus is rich in structurally diverse specialized metabolites. While some specialized metabolite classes are highly prevalent across the genus, the cyanogenic glucoside prunasin is only produced by c. 3% of species. To investigate the evolutionary mechanisms behind prunasin biosynthesis in Eucalyptus, we compared de novo assembled transcriptomes, together with online resources between cyanogenic and acyanogenic species. Identified genes were characterized in vivo and in vitro. Pathway characterization of cyanogenic Eucalyptus camphora and Eucalyptus yarraensis showed for the first time that the final glucosylation step from mandelonitrile to prunasin is catalyzed by a novel UDP-glucosyltransferase UGT87. This step is typically catalyzed by a member of the UGT85 family, including in Eucalyptus cladocalyx. The upstream conversion of phenylalanine to mandelonitrile is catalyzed by three cytochrome P450 (CYP) enzymes from the CYP79, CYP706, and CYP71 families, as previously shown. Analysis of acyanogenic Eucalyptus species revealed the loss of different ortholog prunasin biosynthetic genes. The recruitment of UGTs from different families for prunasin biosynthesis in Eucalyptus demonstrates important pathway heterogeneities and unprecedented dynamic pathway evolution of chemical defense within a single genus. Overall, this study provides relevant insights into the tremendous adaptability of these long-lived trees.

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.

Glutathione transferases catalyze recycling of auto-toxic cyanogenic glucosides in sorghum.

Cyanogenic glucosides are nitrogen-containing specialized metabolites that provide chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide. It has been suggested that cyanogenic glucosides are also a store of nitrogen that can be remobilized for general metabolism via a previously unknown pathway. Here we reveal a recycling pathway for the cyanogenic glucoside dhurrin in sorghum (Sorghum bicolor) that avoids hydrogen cyanide formation. As demonstrated in vitro, the pathway proceeds via spontaneous formation of a dhurrin-derived glutathione conjugate, which undergoes reductive cleavage by glutathione transferases of the plant-specific lambda class (GSTLs) to produce p-hydroxyphenyl acetonitrile. This is further metabolized to p-hydroxyphenylacetic acid and free ammonia by nitrilases, and then glucosylated to form p-glucosyloxyphenylacetic acid. Two of the four GSTLs in sorghum exhibited high stereospecific catalytic activity towards the glutathione conjugate, and form a subclade in a phylogenetic tree of GSTLs in higher plants. The expression of the corresponding two GSTLs co-localized with expression of the genes encoding the p-hydroxyphenyl acetonitrile-metabolizing nitrilases at the cellular level. The elucidation of this pathway places GSTs as key players in a remarkable scheme for metabolic plasticity allowing plants to reverse the resource flow between general and specialized metabolism in actively growing tissue.

The cytochrome P450 CYP72A552 is key to production of hederagenin-based saponins that mediate plant defense against herbivores.

Plants continuously evolve new defense compounds. One class of such compounds is triterpenoid saponins. A few species in the Barbarea genus produce saponins as the only ones in the large crucifer family. However, the molecular mechanism behind saponin biosynthesis and their role in plant defense remains unclear. We used pathway reconstitution in planta, enzymatic production of saponins in vitro, insect feeding assays, and bioinformatics to identify a missing gene involved in saponin biosynthesis and saponin-based herbivore defense. A tandem repeat of eight CYP72A cytochromes P450 colocalise with a quantitative trait locus (QTL) for saponin accumulation and flea beetle resistance in Barbarea vulgaris. We found that CYP72A552 oxidises oleanolic acid at position C-23 to hederagenin. In vitro-produced hederagenin monoglucosides reduced larval feeding by up to 90% and caused 75% larval mortality of the major crucifer pest diamondback moth and the tobacco hornworm. Sequence analysis indicated that CYP72A552 evolved through gene duplication and has been under strong selection pressure. In conclusion, CYP72A552 has evolved to catalyse the formation of hederagenin-based saponins that mediate plant defense against herbivores. Our study highlights the evolution of chemical novelties by gene duplication and selection for enzyme innovations, and the importance of chemical modification in plant defense evolution.

Reconfigured Cyanogenic Glucoside Biosynthesis in Eucalyptus cladocalyx Involves a Cytochrome P450 CYP706C55.

Cyanogenic glucosides are a class of specialized metabolites widespread in the plant kingdom. Cyanogenic glucosides are α-hydroxynitriles, and their hydrolysis releases toxic hydrogen cyanide, providing an effective chemical defense against herbivores. Eucalyptus cladocalyx is a cyanogenic tree, allocating up to 20% of leaf nitrogen to the biosynthesis of the cyanogenic monoglucoside, prunasin. Here, mass spectrometry analyses of E. cladocalyx tissues revealed spatial and ontogenetic variations in prunasin content, as well as the presence of the cyanogenic diglucoside amygdalin in flower buds and flowers. The identification and biochemical characterization of the prunasin biosynthetic enzymes revealed a unique enzyme configuration for prunasin production in E. cladocalyx This result indicates that a multifunctional cytochrome P450 (CYP), CYP79A125, catalyzes the initial conversion of l-phenylalanine into its corresponding aldoxime, phenylacetaldoxime; a function consistent with other members of the CYP79 family. In contrast to the single multifunctional CYP known from other plant species, the conversion of phenylacetaldoxime to the α-hydroxynitrile, mandelonitrile, is catalyzed by two distinct CYPs. CYP706C55 catalyzes the dehydration of phenylacetaldoxime, an unusual CYP reaction. The resulting phenylacetonitrile is subsequently hydroxylatedby CYP71B103 to form mandelonitrile. The final glucosylation step to yield prunasin is catalyzed by a UDP-glucosyltransferase, UGT85A59. Members of the CYP706 family have not been reported previously to participate in the biosynthesis of cyanogenic glucosides, and the pathway structure in E. cladocalyx represents an example of convergent evolution in the biosynthesis of cyanogenic glucosides in plants.

NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds.

In plants, transport processes are important for the reallocation of defence compounds to protect tissues of high value, as demonstrated in the plant model Arabidopsis, in which the major defence compounds, glucosinolates, are translocated to seeds on maturation. The molecular basis for long-distance transport of glucosinolates and other defence compounds, however, remains unknown. Here we identify and characterize two members of the nitrate/peptide transporter family, GTR1 and GTR2, as high-affinity, proton-dependent glucosinolate-specific transporters. The gtr1 gtr2 double mutant did not accumulate glucosinolates in seeds and had more than tenfold over-accumulation in source tissues such as leaves and silique walls, indicating that both plasma membrane-localized transporters are essential for long-distance transport of glucosinolates. We propose that GTR1 and GTR2 control the loading of glucosinolates from the apoplasm into the phloem. Identification of the glucosinolate transporters has agricultural potential as a means to control allocation of defence compounds in a tissue-specific manner.

Biosynthesis of the leucine derived α-, ß- and γ-hydroxynitrile glucosides in barley (Hordeum vulgare L.).

Barley (Hordeum vulgare L.) produces five leucine-derived hydroxynitrile glucosides (HNGs), of which only epiheterodendrin is a cyanogenic glucoside. The four non-cyanogenic HNGs are the ß-HNG epidermin and the γ-HNGs osmaronin, dihydroosmaronin and sutherlandin. By analyzing 247 spring barley lines including landraces and old and modern cultivars, we demonstrated that the HNG level varies notably between lines whereas the overall ratio between the compounds is constant. Based on sequence similarity to the sorghum (Sorghum bicolor) genes involved in dhurrin biosynthesis, we identified a gene cluster on barley chromosome 1 putatively harboring genes that encode enzymes in HNG biosynthesis. Candidate genes were functionally characterized by transient expression in Nicotiana benthamiana. Five multifunctional P450s, including two CYP79 family enzymes and three CYP71 family enzymes, and a single UDP-glucosyltransferase were found to catalyze the reactions required for biosynthesis of all five barley HNGs. Two of the CYP71 enzymes needed to be co-expressed for the last hydroxylation step in sutherlandin synthesis to proceed. This observation, together with the constant ratio between the different HNGs, suggested that HNG synthesis in barley is organized within a single multi-enzyme complex.

Methyl Transfer in Glucosinolate Biosynthesis Mediated by Indole Glucosinolate O-Methyltransferase 5.

Indole glucosinolates (IGs) are plant secondary metabolites that are derived from the amino acid tryptophan. The product of Arabidopsis (Arabidopsis thaliana) IG core biosynthesis, indol-3-ylmethyl glucosinolate (I3M), can be modified by hydroxylation and subsequent methoxylation of the indole ring in position 1 (1-IG modification) or 4 (4-IG modification). Products of the 4-IG modification pathway mediate plant-enemy interactions and are particularly important for Arabidopsis innate immunity. While CYP81Fs encoding cytochrome P450 monooxygenases and IGMTs encoding indole glucosinolate O-methyltransferases have been identified as key genes for IG modification, our knowledge about the IG modification pathways is not complete. In particular, it is unknown which enzyme is responsible for methyl transfer in the 1-IG modification pathway and whether this pathway plays a role in defense, similar to 4-IG modification. Here, we analyze two Arabidopsis transfer DNA insertion lines with targeted metabolomics. We show that biosynthesis of 1-methoxyindol-3-ylmethyl glucosinolate (1MOI3M) from I3M involves the predicted unstable intermediate 1-hydroxyindol-3-ylmethyl glucosinolate (1OHI3M) and that IGMT5, a gene with moderate similarity to previously characterized IGMTs, encodes the methyltransferase that is responsible for the conversion of 1OHI3M to 1MOI3M. Disruption of IGMT5 function increases resistance against the root-knot nematode Meloidogyne javanica and suggests a potential role for the 1-IG modification pathway in Arabidopsis belowground defense.

Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications.

Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM(1) characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.

Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana.

The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces ß-amyrin and α-amyrin, and is preferentially expressed in resistant plants; ß-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs).

Dhurrin metabolism in the developing grain of Sorghum bicolor (L.) Moench investigated by metabolite profiling and novel clustering analyses of time-resolved transcriptomic data.

BACKGROUND: The important cereal crop Sorghum bicolor (L.) Moench biosynthesize and accumulate the defensive compound dhurrin during development. Previous work has suggested multiple roles for the compound including a function as nitrogen storage/buffer. Crucial for this function is the endogenous turnover of dhurrin for which putative pathways have been suggested but not confirmed. RESULTS: In this study, the biosynthesis and endogenous turnover of dhurrin in the developing sorghum grain was studied by metabolite profiling and time-resolved transcriptome analyses. Dhurrin was found to accumulate in the early phase of grain development reaching maximum amounts 25 days after pollination. During the subsequent maturation period, the dhurrin content was turned over, resulting in only negligible residual dhurrin amounts in the mature grain. Dhurrin accumulation correlated with the transcript abundance of the three genes involved in biosynthesis. Despite the accumulation of dhurrin, the grains were acyanogenic as demonstrated by the lack of hydrogen cyanide release from macerated grain tissue and by the absence of transcripts encoding dhurrinases. With the missing activity of dhurrinases, the decrease in dhurrin content in the course of grain maturation represents the operation of hitherto uncharacterized endogenous dhurrin turnover pathways. Evidence for the operation of two such pathways was obtained by metabolite profiling and time-resolved transcriptome analysis. By combining cluster- and phylogenetic analyses with the metabolite profiling, potential gene candidates of glutathione S-transferases, nitrilases and glycosyl transferases involved in these pathways were identified. The absence of dhurrin in the mature grain was replaced by a high content of proanthocyanidins. Cluster- and phylogenetic analyses coupled with metabolite profiling, identified gene candidates involved in proanthocyanidin biosynthesis in sorghum. CONCLUSIONS: The results presented in this article reveal the existence of two endogenous dhurrin turnover pathways in sorghum, identify genes putatively involved in these transformations and show that dhurrin in addition to its insect deterrent properties may serve as a storage form of reduced nitrogen. In the course of sorghum grain maturation, proanthocyanidins replace dhurrin as a defense compound. The lack of cyanogenesis in the developing sorghum grain renders this a unique experimental system to study CNglc synthesis as well as endogenous turnover.

Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803.

Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo. The engineered strains produced up to 66mg/L of p-hydroxyphenylacetaldoxime and 5mg/L of dhurrin in lab-scale cultures after 3 days of cultivation and 3mg/L of dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation.

Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis.

Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons.

Integration of biosynthesis and long-distance transport establish organ-specific glucosinolate profiles in vegetative Arabidopsis.

Although it is essential for plant survival to synthesize and transport defense compounds, little is known about the coordination of these processes. Here, we investigate the above- and belowground source-sink relationship of the defense compounds glucosinolates in vegetative Arabidopsis thaliana. In vivo feeding experiments demonstrate that the glucosinolate transporters1 and 2 (GTR1 and GTR2), which are essential for accumulation of glucosinolates in seeds, are likely to also be involved in bidirectional distribution of glucosinolates between the roots and rosettes, indicating phloem and xylem as their transport pathways. Grafting of wild-type, biosynthetic, and transport mutants show that both the rosette and roots are able to synthesize aliphatic and indole glucosinolates. While rosettes constitute the major source and storage site for short-chained aliphatic glucosinolates, long-chained aliphatic glucosinolates are synthesized both in roots and rosettes with roots as the major storage site. Our grafting experiments thus indicate that in vegetative Arabidopsis, GTR1 and GTR2 are involved in bidirectional long-distance transport of aliphatic but not indole glucosinolates. Our data further suggest that the distinct rosette and root glucosinolate profiles in Arabidopsis are shaped by long-distance transport and spatially separated biosynthesis, suggesting that integration of these processes is critical for plant fitness in complex natural environments.

A recycling pathway for cyanogenic glycosides evidenced by the comparative metabolic profiling in three cyanogenic plant species.

Cyanogenic glycosides are phytoanticipins involved in plant defence against herbivores by virtue of their ability to release toxic hydrogen cyanide (HCN) upon tissue disruption. In addition, endogenous turnover of cyanogenic glycosides without the liberation of HCN may offer plants an important source of reduced nitrogen at specific developmental stages. To investigate the presence of putative turnover products of cyanogenic glycosides, comparative metabolic profiling using LC-MS/MS and high resolution MS (HR-MS) complemented by ion-mobility MS was carried out in three cyanogenic plant species: cassava, almond and sorghum. In total, the endogenous formation of 36 different chemical structures related to the cyanogenic glucosides linamarin, lotaustralin, prunasin, amygdalin and dhurrin was discovered, including di- and tri-glycosides derived from these compounds. The relative abundance of the compounds was assessed in different tissues and developmental stages. Based on results common to the three phylogenetically unrelated species, a potential recycling endogenous turnover pathway for cyanogenic glycosides is described in which reduced nitrogen and carbon are recovered for primary metabolism without the liberation of free HCN. Glycosides of amides, carboxylic acids and 'anitriles' derived from cyanogenic glycosides appear as common intermediates in this pathway and may also have individual functions in the plant. The recycling of cyanogenic glycosides and the biological significance of the presence of the turnover products in cyanogenic plants open entirely new insights into the multiplicity of biological roles cyanogenic glycosides may play in plants.

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants.

Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, ß-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate ß-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13ß-epoxy-3ß,16ß-dihydroxy-oleanane (12,13ß-epoxy-16ß-hydroxy-ß-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family--as a multifunctional stereo- and regio-specific hydroxylase in plant specialized metabolism.

The evolutionary appearance of non-cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous ß-glucosidases resulting from a crucial amino acid substitution.

Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic α-hydroxynitrile glucosides lotaustralin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific ß-glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non-cyanogenic γ- and ß-hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only ß-glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site-directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non-flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic ß-glucosidase, resembling BGD2, and required no more than a single amino acid substitution.

Lotus japonicus flowers are defended by a cyanogenic ß-glucosidase with highly restricted expression to essential reproductive organs.

Flowers and leaves of Lotus japonicus contain α-, ß-, and γ-hydroxynitrile glucoside (HNG) defense compounds, which are bioactivated by ß-glucosidase enzymes (BGDs). The α-HNGs are referred to as cyanogenic glucosides because their hydrolysis upon tissue disruption leads to release of toxic hydrogen cyanide gas, which can deter herbivore feeding. BGD2 and BGD4 are HNG metabolizing BGD enzymes expressed in leaves. Only BGD2 is able to hydrolyse the α-HNGs. Loss of function mutants of BGD2 are acyanogenic in leaves but fully retain cyanogenesis in flowers pointing to the existence of an alternative cyanogenic BGD in flowers. This enzyme, named BGD3, is identified and characterized in this study. Whereas all floral tissues contain α-HNGs, only those tissues in which BGD3 is expressed, the keel and the enclosed reproductive organs, are cyanogenic. Biochemical analysis, active site architecture molecular modelling, and the observation that L. japonicus accessions lacking cyanogenic flowers contain a non-functional BGD3 gene, all support the key role of BGD3 in floral cyanogenesis. The nectar of L. japonicus flowers was also found to contain HNGs and additionally their diglycosides. The observed specialisation in HNG based defence in L. japonicus flowers is discussed in the context of balancing the attraction of pollinators with the protection of reproductive structures against herbivores.

A Functional EXXEK Motif is Essential for Proton Coupling and Active Glucosinolate Transport by NPF2.11.

The proton-dependent oligopeptide transporter (POT/PTR) family shares a highly conserved E1X1X2E2RFXYY (E1X1X2E2R) motif across all kingdoms of life. This motif is suggested to have a role in proton coupling and active transport in bacterial homologs. For the plant POT/PTR family, also known as the NRT1/PTR family (NPF), little is known about the role of the E1X1X2E2R motif. Moreover, nothing is known about the role of the X1 and X2 residues within the E1X1X2E2R motif. We used NPF2.11-a proton-coupled glucosinolate (GLS) symporter from Arabidopsis thaliana-to investigate the role of the E1X1X2E2K motif variant in a plant NPF transporter. Using liquid chromatography-mass spectrometry (LC-MS)-based uptake assays and two-electrode voltage clamp (TEVC) electrophysiology, we demonstrate an essential role for the E1X1X2E2K motif for accumulation of substrate by NPF2.11. Our data suggest that the highly conserved E1, E2 and K residues are involved in translocation of protons, as has been proposed for the E1X1X2E2R motif in bacteria. Furthermore, we show that the two residues X1 and X2 in the E1X1X2E2[K/R] motif are conserved as uncharged amino acids in POT/PTRs from bacteria to mammals and that introducing a positive or negative charge in either position hampers the ability to overaccumulate substrate relative to the assay medium. We hypothesize that introducing a charge at X1 and X2 interferes with the function of the conserved glutamate and lysine residues of the E1X1X2E2K motif and affects the mechanism behind proton coupling.

Elucidating the role of transport processes in leaf glucosinolate distribution.

In Arabidopsis (Arabidopsis thaliana), a strategy to defend its leaves against herbivores is to accumulate glucosinolates along the midrib and at the margin. Although it is generally assumed that glucosinolates are synthesized along the vasculature in an Arabidopsis leaf, thereby suggesting that the margin accumulation is established through transport, little is known about these transport processes. Here, we show through leaf apoplastic fluid analysis and glucosinolate feeding experiments that two glucosinolate transporters, GTR1 and GTR2, essential for long-distance transport of glucosinolates in Arabidopsis, also play key roles in glucosinolate allocation within a mature leaf by effectively importing apoplastically localized glucosinolates into appropriate cells. Detection of glucosinolates in root xylem sap unambiguously shows that this transport route is involved in root-to-shoot glucosinolate allocation. Detailed leaf dissections show that in the absence of GTR1 and GTR2 transport activity, glucosinolates accumulate predominantly in leaf margins and leaf tips. Furthermore, we show that glucosinolates accumulate in the leaf abaxial epidermis in a GTR-independent manner. Based on our results, we propose a model for how glucosinolates accumulate in the leaf margin and epidermis, which includes symplasmic movement through plasmodesmata, coupled with the activity of putative vacuolar glucosinolate importers in these peripheral cell layers.