BACKGROUND
the female inflorescences  of humulus lupulus l.  contain prenylated acylphloroglucinols and prenylchalcones  that are important for the brewing industry and have potential medicinal uses  <cit> .
the acylphloroglucinols, referred to as bitter acids because they contribute the characteristic bitter flavour of beer, include the α-acid humulone and its acyl-side chain variants cohumulone and adhumulone .
antimicrobial β-acids , which differ from α-acids by the presence of an additional dimethylallyl prenyl group, are also abundant.
hop cultivars vary widely in their content and composition of bitter acids with some “super-alpha” cultivars containing greater than 20% humulone by dry weight in cones  <cit> .
the bitter acids and other prenylated polyketides occur mainly in lupulin glands, which are glandular trichomes present on hop cones and leaves  <cit> .
glandular trichomes typically have little or no photosynthetic activity and often contain only a few highly active biosynthetic pathways, which has made transcriptome analysis of isolated trichome secretory cells useful for the identification of the genes involved in the production of specialized metabolites  <cit> .
enzymes involved in the biosynthesis of xanthohumol and terpenoids in hop lupulin glands  <cit>  and cannabinoids in glandular trichomes of the closely related species cannabis sativa   <cit>  have been identified using a combination of trichome-targeted est analysis and biochemical assays.
the precursors for bitter acid biosynthesis are branched-chain acyl-coas thioesters, which function as primers for polyketide synthesis, malonyl-coa, which is the polyketide extender and dimethylallyl diphosphate , which serves as a prenyl donor.
the acyl-coas, isovaleryl-coa, isobutyryl-coa and 2-methylbutyryl-coa, are obtained by the degradation of the branched-chain amino acids  leucine, valine and isoleucine, respectively .
the study of bcaa metabolism is of particular interest in hop since it is one of the few plants that produces large amounts of bcaa-derived natural products in specialized secretory structures.
some solanaceae species utilize bcaas in the trichome-localized production of acyl sugars  <cit> .
bcaa biosynthesis in plants consists of eight enzymatic steps to produce leucine and valine from pyruvate, or isoleucine from threonine and pyruvate  .
branched-chain aminotransferase  enzymes catalyze both the final step in bcaa biosynthesis and the first step in bcaa degradation.
these two phases are physically separated, with biosynthesis occurring in plastids and catabolism in mitochondria.
bcats have been studied in both arabidopsis and tomato with each containing six and seven isoforms, respectively  <cit> .
bcaa derived coa-esters are generated by the branched-chain ketoacid dehydrogenase complex  which is made up of four subunits  <cit> .
three of these subunits, ketoacid dehydrogenase e1α and e1β, and dihydrolipoyl acyltransferase , are specific to the bckdh complex while the dihydrolipoyl dehydrogenase  subunit also functions in other metabolic pathways  <cit> .
labeling studies have shown that the dmapp required for the prenylation of bitter acids is provided by the methyl-d-erythritol 4-phosphate  pathway localized in plastids  <cit> .
mep-derived isoprenoids are also used in the biosynthesis of mono- and sesquiterpenes, which co-occur with bitter acids in lupulin glands  <cit> .
the mep pathway is initiated with glyceraldehyde-3-phosphate and pyruvate; the latter is also used in bcaa biosynthesis .
previous investigations have shown that transcripts for the seven mep pathway enzymes and dmapp/ipp isomerase are abundant in lupulin glands but their expression was not compared with leaves or other organs  <cit> .
the final steps in bitter acid biosynthesis are unique to hop.
the type iii polyketide synthase enzyme valerophenone synthase  condenses a bcaa-derived acyl-coa starter molecule with three molecules of malonyl-coa to form the polyketide core   <cit>  .
one or more aromatic prenyltransferase enzymes perform two c <dig> prenylations to yield diprenyl intermediates   <cit> .
at this point the α- and β-acid pathways diverge with a third prenylation forming the β-acids while an oxygenation reaction, catalyzed by an as yet unknown humulone synthase enzyme, yields the α-acids  <cit> .
here we used rna-seq to assemble and compare the transcriptomes of lupulin glands, cones  and leaves.
we analyzed the expression of genes involved in precursor biosynthesis, including bcaas, mep-derived isoprenoids and malonyl-coa, and the final steps in bitter acid formation.
we focused on the bcat enzymes which play key roles in the formation of branched-chain acyl-coas.
two bcat-encoding genes were present, hlbcat <dig> and hlbcat <dig>  with transcripts for both more abundant in lupulin glands compared with leaves and cones.
hlbcat <dig> and hlbcat <dig> were experimentally-localized using gfp fusion proteins to the mitochondrion and plastid, respectively.
assays of recombinant hlbcat <dig> and hlbcat <dig> showed they catalyze biosynthetic and catabolic reactions.
these findings provide evidence for lupulin gland-specific bcaa and isoprenoid metabolism to produce precursors for bitter acid biosynthesis.
RESULTS
transcriptome sequencing and assembly
lupulin glands were isolated from the mature hop cones of four commercial cultivars grown for their high α-acid content: ‘taurus’, ‘nugget’, ‘magnum’ and ‘apollo’.
leaves and cones were also collected from ‘taurus’ and ‘apollo’ cultivars.
the cone samples consisted of the photosynthetic bracts and bracteoles that remained after the removal of lupulin glands.
rna purified from eleven samples  was reverse-transcribed and the cdnas sequenced on a single lane of an illumina hiseq  <dig> instrument as a multiplexed sample.
each cdna produced  <dig> - <dig>  million reads .
the reads obtained from all samples were assembled using trinity  <cit>  and refined using cdhit-est  <cit>  to produce a transcriptome of ~ <dig>  <dig> contigs with an average read length of  <dig> bp.
this transcriptome was used as the reference for rna-seq analysis of the individual tissues using clc bio.
at least 87% of the reads from each sample were mapped to the reference transcriptome for each tissue .
comparison of metabolic transcripts in lupulin glands, cones and leaves
we compared the expression of mrnas encoding primary metabolic enzymes  cycle and glycolysis) between the three tissues .
as expected, calvin cycle genes were predominantly represented in the photosynthetic tissue  but not in the lupulin glands.
notable exceptions were the calvin cycle enzymes involved in glycolysis, which are important for the flow of carbon from sucrose to pyruvate in the glands.
an isoform of the rubisco small subunit was one of the most abundant genes in the leaf transcriptome, while the rubisco large subunit was not highly expressed.
tca cycle genes showed only minor differences in expression levels between the three tissues.
transcripts encoding glycolytic enzymes were also represented at comparable levels in leaves and lupulin glands, however, some genes within this pathway showed specificity to one tissue over the other.
several genes encoding enzymes required for sucrose utilization  were more highly expressed in the gland versus leaf tissue.
mep pathway genes were more highly expressed in lupulin glands compared with green tissues.
as expected from the need for malonyl-coa for polyketide synthesis, transcripts encoding acetyl-coa carboxylase  <cit>  and atp-citrate lyase  <cit>  were abundant in the glands .
most genes in the bcaa biosynthetic pathway showed higher expression in lupulin glands compared to cones and leaves , with the transcript for ketol-acid reductoisomerase showing very high abundance.
exceptions were the low expression of threonine deaminase, which is only required for isoleucine biosynthesis, and the regulatory subunit of acetohydroxyacid synthase, which had minimal expression in all three tissues .
four putative bcat-encoding genes were identified in the transcriptome.
two of these genes, denoted humulus lupulus bcat <dig>  and hlbcat <dig>  were abundant in the lupulin glands and hlbcat <dig> showed gland-specific expression .
formation of the bcaa derived coa-esters is carried out by the bckdh complex .
two isoforms of the e1α subunit were identified in the dataset and the expression of both isoforms along with the e1β and e <dig> subunits were increased in the lupulin glands .
the expression of the e <dig> subunit was similar in all tissues.
vps was one of the most abundant mrnas in the lupulin gland dataset, dwarfing all other genes involved in secondary metabolism examined here .
two aromatic prenyltransferase genes, the previously characterized hlpt <dig> <cit>  and hlpt <dig>  showed similar gland-specific expression.
lupulin gland-specific transcription factors
we also analyzed the rna-seq data for transcription factors that may be involved in regulating bitter acid biosynthesis or other lupulin gland-specific metabolic activities.
forty-eight transcription factors were significantly more abundant in lupulin glands compared to the leaves .
of these, a subset of  <dig> was also higher in glands compared to cones.
eleven transcription factors were expressed at more than ten-fold higher levels in the lupulin glands compared to cones and leaves .
fold change represents the difference between mean rpkm values for gland compared to leaf and cone.
a n= <dig>
b n= <dig>
c marks et al.
<cit> .
validation of gene expression by quantitative rt-pcr
the rna-seq analysis of the bckdh complex, lupulin gland-specific bcats, vps and hlpt <dig> was confirmed using qrt-pcr.
hlbcat <dig> was lupulin gland specific while hlbcat <dig> was expressed in both lupulin glands and leaves .
e1α, e1β and e <dig> of the bckdh complex were more highly expressed in lupulin glands while the e <dig> subunit was represented equally in all tissues.
the genes encoding vps and hlpt <dig> were highly expressed in the lupulin glands while essentially non-existent in the leaf tissue.
all of these findings were in agreement with the rna-seq data .
metabolite profiling of bcaas, acyl-coas and bitter acids
we quantified bcaa levels in lupulin glands, cones and leaves using hplc with fluorescence detection of their derivatized adducts.
leucine, valine and isoleucine levels were similar in lupulin glands and leaves, with valine the most prominent .
bcaa levels in the cone tissue were highly variable and, in the case of leucine and isoleucine, were also elevated compared to glands and leaves.
using a sensitive lc-ms method with multiple reaction monitoring, we measured the abundance of the acyl-coas that are used as substrates in bitter acid biosynthesis .
although acetyl-coa is not used by vps directly, it is converted to malonyl-coa by acetyl-coa carboxylase and therefore we included it in the analysis.
both acetyl-coa and malonyl-coa exhibit elevated levels in lupulin glands .
the starter substrates for the vps reaction, isovaleryl-coa, isobutyryl-coa and 2-methylbutyryl-coa, accumulated in glands while remaining largely absent in other tissues.
isovaleryl-coa was five times more abundant than the other branched-chain acyl-coas.
hplc analysis of the major bitter acids and their immediate precursors showed, as expected, that the α-acids accumulated to higher levels than the β-acids and that humulone was the most abundant end-product .
these results correlated well with the levels of acyl-coa precursors .
we also quantified the diprenylated precursors  of the bitter acids.
small amounts of deoxyhumulone and deoxycohumulone were present while deoxyadhumulone could not be detected .
the absence of deoxyadhumulone may reflect the overall lower levels of isoleucine-derived bitter acids.
although we attempted to identify the products from the vps reaction such as phlorisovalerophenone, as well as the monoprenylated forms, these metabolites were undetectable.
hlbcat <dig> and hlbcat <dig> catalyze aminotransferase reactions with bcaas
to further investigate bcaa metabolism in lupulin glands, we characterized the biochemical properties of the two gland-specific bcat enzymes.
hlbcat <dig> is  <dig> amino acids long with calculated molecular weight of  <dig>  kda; hlbcat <dig> is a  <dig>  kda  protein.
nucleotide sequences for both enzymes are available in genbank hlbcat <dig>  jq063073; hlbcat <dig>  jq063074].
the mature forms  of hlbcat <dig> and hlbcat <dig> were expressed as his-tagged recombinant proteins in e. coli and purified.
enzyme assays were performed in both the forward  and reverse  directions to test catalytic activities and to determine kinetic parameters .
both enzymes functioned as bcats and showed reversibility, as would be expected based on their ping pong reaction mechanism  <cit> .
unlike previous work in tomato, where the bcats exhibited kinetic preferences corresponding to anabolic or catabolic functions  <cit> , the hop bcats had similar km values in either direction.
amean ± standard error .
subcellular localization of hop bcats
hlbcat <dig>  hlbcat <dig> and hlbcat <dig> contain putative n-terminal signal peptides while hlbcat <dig> does not.
targetp  <cit>  analysis predicted that hlbcat <dig> and hlbcat <dig> are localized to the mitochondria, and hlbcat <dig> to the plastids.
we tested the subcellular localization of the two bcats with gland-specific expression, hlbcat <dig> and hlbcat <dig>  using transient expression in nicotiana benthamiana leaves.
hop leaves were not used for this experiment because they proved difficult to infiltrate with agrobacterium solution.
hlbcat1-gfp constructs with signal peptide  or signal peptide with mature protein  co-localized with the mitochondrial mtrk marker  <cit> .
the full-length hlbcat <dig> failed to localize to a recognizable compartment, however its signal peptide fused to gfp colocalized with the plastid ptrk marker .
hlbcat <dig> and hlbcat <dig> constructs without signal peptide accumulated in the cytosol .
phylogenetic analysis of hop bcats
a phylogenetic tree was constructed using bcat protein sequences from hop, arabidopsis, tomato, cannabis sativa and other plants obtained using the phytozome database  <cit> .
we observed a separation of dicot bcats into two clades, which could be classified as biosynthetic/plastidial and catabolic/mitochondrial based on the presence of experimentally-localized proteins in each group .
hlbcat <dig> and  <dig> grouped into the mitochondrial bcat clade and hlbcat <dig> grouped into the plastidial clade, which agrees with their predicted localizations by targetp and our analysis of intracellular localization .
hlbcat <dig> was grouped with the other bcats which do not appear to localize to either the plastid or mitochondrion.
although the monocot bcats clustered into two distinct groups that were positioned between the dicot clades, there is no experimental localization data for monocot bcats at this time.
discussion
illumina sequencing provides new genomic resources for hop
although the transcriptome of lupulin glands has been extensively studied using ests obtained through sanger sequencing  <cit>  and several  <dig> pyrosequencing datasets are available in ncbi, an in-depth comparison of the lupulin gland transcriptome with green tissues  has not been reported.
schilmiller et al.
<cit>  previously used  <dig> pyrosequencing to compare gene expression in tomato trichomes, leaves and stems, and rna-seq analysis was used to identify terpene synthases in tomato trichomes  <cit> .
microarrays have also been used to compare the trichome and stem transcriptome of alfalfa  <cit> .
the illumina sequences reported here significantly expand the genomic resources available for hop, in particular leaves and cones for which limited sequences are available.
the high number of contigs in the de novo transcriptome assembly probably relates to the presence of “broken contigs”, as evidenced by the relatively short contig length .
this may have resulted from our use of combined rna-seq data from several hop cultivars, the lack of sufficient sequencing depth and the presence of introns from alternative splicing events in some transcripts.
in addition to the further development of transcriptome resources for hop, an important next step is to link the transcriptome with a whole genome sequence and genetic map.
however, the estimated  <dig>  gbp size of the humulus lupulus genome  <cit>  and the highly heterozygous nature of this dioecious species present obvious challenges for genome assembly.
lupulin glands synthesize precursor metabolites for bitter acid production
the expression pattern for genes related to photosynthesis , and the absence of bitter acids from the cone minus gland samples , indicates that separation of the non-photosynthetic glands from photosynthetic cones was largely complete.
analysis of gene expression for primary metabolic pathways  showed the reliance of lupulin glands on surrounding green tissues for the supply of sugars.
the genes involved in the calvin cycle were more abundant in the leaves and cones compared to lupulin glands .
the only exceptions to this pattern were genes encoding enzymes that function in both the calvin cycle and glycolysis.
the tca cycle had similar expression levels across the leaf, cone and lupulin gland samples which can be attributed to its role in supplying intermediates for many pathways.
among the glycolysis transcripts three genes  stood out for their high expression in the lupulin glands compared to leaves.
all three are involved in sucrose mobilization, underscoring the exogenous supply of sugars to the lupulin gland.
this is in agreement with the previous suggestion that sucrose would be an ideal energy source and precursor for secondary metabolism in glandular trichomes  <cit> .
the high amounts of bitter acids produced by elite hop cultivars represents a large carbon commitment within the lupulin glands.
given that sugars are imported, we analyzed the rna-seq dataset to determine if other precursors  are imported or synthesized in situ.
both the mep and bcaa pathways were more abundantly represented in the lupulin glands , a finding that provides evidence for the de novo biosynthesis of bcaas and dmapp in these cells.
bcaa biosynthesis in lupulin glands is logical given the demand for the acyl side-chains of the bcaas but not the amino group, and the need to prevent the accumulation of excess nitrogen.
this finding is in agreement with schilmiller et al.
<cit>  who noted that while phenylpropanoid-derived metabolites are synthesized in trichomes  the import of phenylalanine into glandular trichomes was minimal.
transcripts corresponding to the enzymes of the polyketide portion of the bitter acid pathway  were among the most abundant in the glandular trichome dataset .
the presence of two highly expressed aromatic prenyltransferases is notable since hlpt <dig> has only been shown to catalyze the first prenylation step in bitter acid biosynthesis  <cit> .
the α- and β-acids require two and three prenyl groups, respectively, and the second prenyltransferase enzyme  may catalyze these steps.
lupulin gland-specific expression of transcription factors
rna-seq analysis also revealed a number of lupulin gland-specific transcription factors which may be involved in regulating bitter acid biosynthesis or other metabolic or developmental processes in glandular trichomes .
matouček and co-workers have identified several transcription factors that may control flavonoid biosynthesis in hop through the regulation of chalcone synthase  .
although all of these transcription factors could be identified in the hop transcriptome reported here, only transcripts of hlmyb <dig> and hlbhlh <dig> were more abundant in lupulin glands compared to leaves .
this expression pattern makes sense since chs, which plays a role in both the gland-specific production of xanthohumol and in the formation of flavonoids, is expressed throughout the plant.
indeed our rna-seq analysis shows that chs_h <dig> is expressed in lupulin glands and leaves , with the three-fold higher expression levels in glands likely reflecting the production of xanthohumol.
two myb transcription factors, can <dig> and can <dig>  have been shown to be highly expressed in cannabis glandular trichomes and implicated in regulating trichome-specific metabolic processes  <cit> .
both have close homologs to hop transcription factors that are over ten-fold more abundant in the lupulin glands .
can <dig>  which has 70% amino acid identity to the hop myb protein encoded by comp63432_c0_seq <dig>  was proposed to be involved in regulating cannabinoid formation.
since cannabis and humulus are closely related genera with glandular trichome-localized polyketide pathways, comp63432_c0_seq <dig> may be a candidate for regulating the bitter acid pathway.
lupulin glands contain both mitochondrial and plastidial bcats
the biosynthesis and subsequent degradation of the bcaas requires both metabolic and catabolic bcats.
the lupulin gland datasets contained two bcats, hlbcat <dig> and hlbcat <dig>  which were abundant in the glandular trichomes.
based on rna-seq and qrt-pcr analysis  hlbcat <dig> is exclusively expressed in the lupulin glands.
transient expression of the hlbcat1-gfp fusion construct confirmed its mitochondrial targeting .
hlbcat <dig> with n-terminal signal peptide failed to localize to the plastid and appeared to form aggregates in the n. benthamiana epidermal cells , however, the predicted hlbcat <dig> signal peptide fused to gfp was targeted to the plastid .
these localizations were further supported through the construction of a phylogenetic tree comparing bcats from a variety of plant species .
problems with the experimental localization of hop plastid proteins have been previously reported for a gpp synthase small subunit  identified in lupulin glands  <cit> .
kinetic analysis suggests lack of specialization for hop bcats
kinetic analysis has provided equivocal evidence for the catalytic specialization of bcats for bcaa biosynthesis or catabolism.
in tomato, biosynthetic bcats  had km values with the keto acids that were an order-of-magnitude less than the catabolic enzymes , indicating specialization of the former for forward  reactions  <cit> .
in contrast, arabidopsis atbcat <dig>  which has been shown to be a catabolic enzyme, had km values for the keto acids  that were much lower than for bcaa substrates , suggesting a preference for the biosynthetic reaction.
indeed, the km values for atbcat <dig> with the keto acids were lower than those reported with the plastidial, biosynthetic enzyme atbcat <dig>  <cit> .
the kinetic properties of the hop bcats appear to be more similar to the arabidopsis enzymes.
the catabolic enzyme hlbcat <dig> exhibited lower km values for keto acids, and therefore an apparent preference for the forward  reaction, compared with than hlbcat <dig> .
it is noteworthy that hlbcat <dig> showed a lower km for leucine compared with valine and isoleucine which may reflect this enzyme’s preferred role in synthesizing isovaleryl-coa for humulone biosynthesis.
the lower vmax values for the hlbcat <dig> may also explain why it is more highly expressed compared to hlbcat <dig> in the lupulin gland tissue .
phylogenetic analysis indicates separation of biosynthetic and catabolic bcats
to further analyze the function of the hop bcats, we constructed a phylogenetic tree of diverse bcats including well characterized enzymes from arabidopsis and tomato.
this analysis was successful in separating biosynthetic  and catabolic  bcats into separate clades.
bcats are also reported to accumulate in alternative cellular compartments such as the cytosol and vacuole for an as of yet unknown function  <cit>  and these genes likely make up the third group that includes several cytosolic enzymes and hlbcat <dig> .
an anomalous grouping was observed for atbcat <dig>  which has been shown to be plastid localized  <cit>  but which was placed in the mitochondrial clade.
a previous study found atbcat <dig> to be the only arabidopsis gene to be co-expressed with other genes involved in bcaa catabolism  <cit> , a role one would expect to be occupied by a mitochondrial bcat.
in addition, targetp analysis of atbcat <dig> predicts mitochondrial rather than plastidial localization.
CONCLUSIONS
this study provides an in-depth analysis of the transcriptional activity of primary and specialized metabolism leading to hop bitter acids.
rna-seq analysis showed that both the mep and bcaa pathways were highly expressed in lupulin glands together with genes required for remobilization of carbon from sucrose.
branched-chain acyl-coas and bitter acids are present in higher levels in isolated lupulin glands compared with cones  and leaves.
a plastidial bcat enzyme involved in bcaa biosynthesis and a mitochondrial bcat catalyzing bcaa degradation both show lupulin gland-specific expression.
together these findings indicate that the glandular trichomes are the site of biosynthesis for the precursors for bitter acid biosynthesis.
the deep transcriptome sequencing reported here significantly expands the genomic resources available for hop.
this new information, and our analysis of bcaa metabolism, will be useful for the further elucidation of enzymes and regulatory proteins involved in bitter acid production and for breeding hop cultivars with increased or modified bitter acid content.

 the female inflorescences of humulus lupulus l. contain prenylated acylphloroglucinols and prenylchalcones that are important for the brewing industry and have potential medicinal uses. the acylphloroglucinols, referred to as bitter acids because they contribute the characteristic bitter flavour of beer, include the α-acid humulone and its acyl-side chain variants cohumulone and adhumulone. antimicrobial β-acids, which differ from α-acids by the presence of an additional dimethylallyl prenyl group, are also abundant. hop cultivars vary widely in their content and composition of bitter acids with some “super-alpha” cultivars containing greater than 20% humulone by dry weight in cones. the bitter acids and other prenylated polyketides occur mainly in lupulin glands, which are glandular trichomes present on hop cones and leaves. glandular trichomes typically have little or no photosynthetic activity and often contain only a few highly active biosynthetic pathways, which has made transcriptome analysis of isolated trichome secretory cells useful for the identification of the genes involved in the production of specialized metabolites. enzymes involved in the biosynthesis of xanthohumol and terpenoids in hop lupulin glands and cannabinoids in glandular trichomes of the closely related species cannabis sativa have been identified using a combination of trichome-targeted est analysis and biochemical assays. the precursors for bitter acid biosynthesis are branched-chain acyl-coas thioesters, which function as primers for polyketide synthesis, malonyl-coa, which is the polyketide extender and dimethylallyl diphosphate, which serves as a prenyl donor. the acyl-coas, isovaleryl-coa, isobutyryl-coa and 2-methylbutyryl-coa, are obtained by the degradation of the branched-chain amino acids leucine, valine and isoleucine, respectively. the study of bcaa metabolism is of particular interest in hop since it is one of the few plants that produces large amounts of bcaa-derived natural products in specialized secretory structures. some solanaceae species utilize bcaas in the trichome-localized production of acyl sugars. bcaa biosynthesis in plants consists of eight enzymatic steps to produce leucine and valine from pyruvate, or isoleucine from threonine and pyruvate. branched-chain aminotransferase enzymes catalyze both the final step in bcaa biosynthesis and the first step in bcaa degradation. these two phases are physically separated, with biosynthesis occurring in plastids and catabolism in mitochondria. bcats have been studied in both arabidopsis and tomato with each containing six and seven isoforms, respectively. bcaa derived coa-esters are generated by the branched-chain ketoacid dehydrogenase complex which is made up of four subunits. three of these subunits, ketoacid dehydrogenase e1α and e1β, and dihydrolipoyl acyltransferase, are specific to the bckdh complex while the dihydrolipoyl dehydrogenase subunit also functions in other metabolic pathways. labeling studies have shown that the dmapp required for the prenylation of bitter acids is provided by the methyl-d-erythritol 4-phosphate pathway localized in plastids. mep-derived isoprenoids are also used in the biosynthesis of mono- and sesquiterpenes, which co-occur with bitter acids in lupulin glands. the mep pathway is initiated with glyceraldehyde-3-phosphate and pyruvate; the latter is also used in bcaa biosynthesis. previous investigations have shown that transcripts for the seven mep pathway enzymes and dmapp/ipp isomerase are abundant in lupulin glands but their expression was not compared with leaves or other organs. the final steps in bitter acid biosynthesis are unique to hop. the type iii polyketide synthase enzyme valerophenone synthase condenses a bcaa-derived acyl-coa starter molecule with three molecules of malonyl-coa to form the polyketide core. one or more aromatic prenyltransferase enzymes perform two c prenylations to yield diprenyl intermediates. at this point the α- and β-acid pathways diverge with a third prenylation forming the β-acids while an oxygenation reaction, catalyzed by an as yet unknown humulone synthase enzyme, yields the α-acids. here we used rna-seq to assemble and compare the transcriptomes of lupulin glands, cones and leaves. we analyzed the expression of genes involved in precursor biosynthesis, including bcaas, mep-derived isoprenoids and malonyl-coa, and the final steps in bitter acid formation. we focused on the bcat enzymes which play key roles in the formation of branched-chain acyl-coas. two bcat-encoding genes were present, hlbcat and hlbcat with transcripts for both more abundant in lupulin glands compared with leaves and cones. hlbcat and hlbcat were experimentally-localized using gfp fusion proteins to the mitochondrion and plastid, respectively. assays of recombinant hlbcat and hlbcat showed they catalyze biosynthetic and catabolic reactions. these findings provide evidence for lupulin gland-specific bcaa and isoprenoid metabolism to produce precursors for bitter acid biosynthesis.  transcriptome sequencing and assembly lupulin glands were isolated from the mature hop cones of four commercial cultivars grown for their high α-acid content: ‘taurus’, ‘nugget’, ‘magnum’ and ‘apollo’. leaves and cones were also collected from ‘taurus’ and ‘apollo’ cultivars. the cone samples consisted of the photosynthetic bracts and bracteoles that remained after the removal of lupulin glands. rna purified from eleven samples was reverse-transcribed and the cdnas sequenced on a single lane of an illumina hiseq instrument as a multiplexed sample. each cdna produced - million reads. the reads obtained from all samples were assembled using trinity and refined using cdhit-est to produce a transcriptome of ~ contigs with an average read length of bp. this transcriptome was used as the reference for rna-seq analysis of the individual tissues using clc bio. at least 87% of the reads from each sample were mapped to the reference transcriptome for each tissue. comparison of metabolic transcripts in lupulin glands, cones and leaves we compared the expression of mrnas encoding primary metabolic enzymes cycle and glycolysis) between the three tissues. as expected, calvin cycle genes were predominantly represented in the photosynthetic tissue but not in the lupulin glands. notable exceptions were the calvin cycle enzymes involved in glycolysis, which are important for the flow of carbon from sucrose to pyruvate in the glands. an isoform of the rubisco small subunit was one of the most abundant genes in the leaf transcriptome, while the rubisco large subunit was not highly expressed. tca cycle genes showed only minor differences in expression levels between the three tissues. transcripts encoding glycolytic enzymes were also represented at comparable levels in leaves and lupulin glands, however, some genes within this pathway showed specificity to one tissue over the other. several genes encoding enzymes required for sucrose utilization were more highly expressed in the gland versus leaf tissue. mep pathway genes were more highly expressed in lupulin glands compared with green tissues. as expected from the need for malonyl-coa for polyketide synthesis, transcripts encoding acetyl-coa carboxylase and atp-citrate lyase were abundant in the glands. most genes in the bcaa biosynthetic pathway showed higher expression in lupulin glands compared to cones and leaves, with the transcript for ketol-acid reductoisomerase showing very high abundance. exceptions were the low expression of threonine deaminase, which is only required for isoleucine biosynthesis, and the regulatory subunit of acetohydroxyacid synthase, which had minimal expression in all three tissues. four putative bcat-encoding genes were identified in the transcriptome. two of these genes, denoted humulus lupulus bcat and hlbcat were abundant in the lupulin glands and hlbcat showed gland-specific expression. formation of the bcaa derived coa-esters is carried out by the bckdh complex. two isoforms of the e1α subunit were identified in the dataset and the expression of both isoforms along with the e1β and e subunits were increased in the lupulin glands. the expression of the e subunit was similar in all tissues. vps was one of the most abundant mrnas in the lupulin gland dataset, dwarfing all other genes involved in secondary metabolism examined here. two aromatic prenyltransferase genes, the previously characterized hlpt and hlpt showed similar gland-specific expression. lupulin gland-specific transcription factors we also analyzed the rna-seq data for transcription factors that may be involved in regulating bitter acid biosynthesis or other lupulin gland-specific metabolic activities. forty-eight transcription factors were significantly more abundant in lupulin glands compared to the leaves. of these, a subset of was also higher in glands compared to cones. eleven transcription factors were expressed at more than ten-fold higher levels in the lupulin glands compared to cones and leaves. fold change represents the difference between mean rpkm values for gland compared to leaf and cone. a n= b n= c marks et al.. validation of gene expression by quantitative rt-pcr the rna-seq analysis of the bckdh complex, lupulin gland-specific bcats, vps and hlpt was confirmed using qrt-pcr. hlbcat was lupulin gland specific while hlbcat was expressed in both lupulin glands and leaves. e1α, e1β and e of the bckdh complex were more highly expressed in lupulin glands while the e subunit was represented equally in all tissues. the genes encoding vps and hlpt were highly expressed in the lupulin glands while essentially non-existent in the leaf tissue. all of these findings were in agreement with the rna-seq data. metabolite profiling of bcaas, acyl-coas and bitter acids we quantified bcaa levels in lupulin glands, cones and leaves using hplc with fluorescence detection of their derivatized adducts. leucine, valine and isoleucine levels were similar in lupulin glands and leaves, with valine the most prominent. bcaa levels in the cone tissue were highly variable and, in the case of leucine and isoleucine, were also elevated compared to glands and leaves. using a sensitive lc-ms method with multiple reaction monitoring, we measured the abundance of the acyl-coas that are used as substrates in bitter acid biosynthesis. although acetyl-coa is not used by vps directly, it is converted to malonyl-coa by acetyl-coa carboxylase and therefore we included it in the analysis. both acetyl-coa and malonyl-coa exhibit elevated levels in lupulin glands. the starter substrates for the vps reaction, isovaleryl-coa, isobutyryl-coa and 2-methylbutyryl-coa, accumulated in glands while remaining largely absent in other tissues. isovaleryl-coa was five times more abundant than the other branched-chain acyl-coas. hplc analysis of the major bitter acids and their immediate precursors showed, as expected, that the α-acids accumulated to higher levels than the β-acids and that humulone was the most abundant end-product. these results correlated well with the levels of acyl-coa precursors. we also quantified the diprenylated precursors of the bitter acids. small amounts of deoxyhumulone and deoxycohumulone were present while deoxyadhumulone could not be detected. the absence of deoxyadhumulone may reflect the overall lower levels of isoleucine-derived bitter acids. although we attempted to identify the products from the vps reaction such as phlorisovalerophenone, as well as the monoprenylated forms, these metabolites were undetectable. hlbcat and hlbcat catalyze aminotransferase reactions with bcaas to further investigate bcaa metabolism in lupulin glands, we characterized the biochemical properties of the two gland-specific bcat enzymes. hlbcat is amino acids long with calculated molecular weight of kda; hlbcat is a kda protein. nucleotide sequences for both enzymes are available in genbank hlbcat jq063073; hlbcat jq063074]. the mature forms of hlbcat and hlbcat were expressed as his-tagged recombinant proteins in e. coli and purified. enzyme assays were performed in both the forward and reverse directions to test catalytic activities and to determine kinetic parameters. both enzymes functioned as bcats and showed reversibility, as would be expected based on their ping pong reaction mechanism. unlike previous work in tomato, where the bcats exhibited kinetic preferences corresponding to anabolic or catabolic functions, the hop bcats had similar km values in either direction. amean ± standard error. subcellular localization of hop bcats hlbcat hlbcat and hlbcat contain putative n-terminal signal peptides while hlbcat does not. targetp analysis predicted that hlbcat and hlbcat are localized to the mitochondria, and hlbcat to the plastids. we tested the subcellular localization of the two bcats with gland-specific expression, hlbcat and hlbcat using transient expression in nicotiana benthamiana leaves. hop leaves were not used for this experiment because they proved difficult to infiltrate with agrobacterium solution. hlbcat1-gfp constructs with signal peptide or signal peptide with mature protein co-localized with the mitochondrial mtrk marker. the full-length hlbcat failed to localize to a recognizable compartment, however its signal peptide fused to gfp colocalized with the plastid ptrk marker. hlbcat and hlbcat constructs without signal peptide accumulated in the cytosol. phylogenetic analysis of hop bcats a phylogenetic tree was constructed using bcat protein sequences from hop, arabidopsis, tomato, cannabis sativa and other plants obtained using the phytozome database. we observed a separation of dicot bcats into two clades, which could be classified as biosynthetic/plastidial and catabolic/mitochondrial based on the presence of experimentally-localized proteins in each group. hlbcat and grouped into the mitochondrial bcat clade and hlbcat grouped into the plastidial clade, which agrees with their predicted localizations by targetp and our analysis of intracellular localization. hlbcat was grouped with the other bcats which do not appear to localize to either the plastid or mitochondrion. although the monocot bcats clustered into two distinct groups that were positioned between the dicot clades, there is no experimental localization data for monocot bcats at this time. discussion illumina sequencing provides new genomic resources for hop although the transcriptome of lupulin glands has been extensively studied using ests obtained through sanger sequencing and several pyrosequencing datasets are available in ncbi, an in-depth comparison of the lupulin gland transcriptome with green tissues has not been reported. schilmiller et al. previously used pyrosequencing to compare gene expression in tomato trichomes, leaves and stems, and rna-seq analysis was used to identify terpene synthases in tomato trichomes. microarrays have also been used to compare the trichome and stem transcriptome of alfalfa. the illumina sequences reported here significantly expand the genomic resources available for hop, in particular leaves and cones for which limited sequences are available. the high number of contigs in the de novo transcriptome assembly probably relates to the presence of “broken contigs”, as evidenced by the relatively short contig length. this may have resulted from our use of combined rna-seq data from several hop cultivars, the lack of sufficient sequencing depth and the presence of introns from alternative splicing events in some transcripts. in addition to the further development of transcriptome resources for hop, an important next step is to link the transcriptome with a whole genome sequence and genetic map. however, the estimated gbp size of the humulus lupulus genome and the highly heterozygous nature of this dioecious species present obvious challenges for genome assembly. lupulin glands synthesize precursor metabolites for bitter acid production the expression pattern for genes related to photosynthesis, and the absence of bitter acids from the cone minus gland samples, indicates that separation of the non-photosynthetic glands from photosynthetic cones was largely complete. analysis of gene expression for primary metabolic pathways showed the reliance of lupulin glands on surrounding green tissues for the supply of sugars. the genes involved in the calvin cycle were more abundant in the leaves and cones compared to lupulin glands. the only exceptions to this pattern were genes encoding enzymes that function in both the calvin cycle and glycolysis. the tca cycle had similar expression levels across the leaf, cone and lupulin gland samples which can be attributed to its role in supplying intermediates for many pathways. among the glycolysis transcripts three genes stood out for their high expression in the lupulin glands compared to leaves. all three are involved in sucrose mobilization, underscoring the exogenous supply of sugars to the lupulin gland. this is in agreement with the previous suggestion that sucrose would be an ideal energy source and precursor for secondary metabolism in glandular trichomes. the high amounts of bitter acids produced by elite hop cultivars represents a large carbon commitment within the lupulin glands. given that sugars are imported, we analyzed the rna-seq dataset to determine if other precursors are imported or synthesized in situ. both the mep and bcaa pathways were more abundantly represented in the lupulin glands, a finding that provides evidence for the de novo biosynthesis of bcaas and dmapp in these cells. bcaa biosynthesis in lupulin glands is logical given the demand for the acyl side-chains of the bcaas but not the amino group, and the need to prevent the accumulation of excess nitrogen. this finding is in agreement with schilmiller et al. who noted that while phenylpropanoid-derived metabolites are synthesized in trichomes the import of phenylalanine into glandular trichomes was minimal. transcripts corresponding to the enzymes of the polyketide portion of the bitter acid pathway were among the most abundant in the glandular trichome dataset. the presence of two highly expressed aromatic prenyltransferases is notable since hlpt has only been shown to catalyze the first prenylation step in bitter acid biosynthesis. the α- and β-acids require two and three prenyl groups, respectively, and the second prenyltransferase enzyme may catalyze these steps. lupulin gland-specific expression of transcription factors rna-seq analysis also revealed a number of lupulin gland-specific transcription factors which may be involved in regulating bitter acid biosynthesis or other metabolic or developmental processes in glandular trichomes. matouček and co-workers have identified several transcription factors that may control flavonoid biosynthesis in hop through the regulation of chalcone synthase. although all of these transcription factors could be identified in the hop transcriptome reported here, only transcripts of hlmyb and hlbhlh were more abundant in lupulin glands compared to leaves. this expression pattern makes sense since chs, which plays a role in both the gland-specific production of xanthohumol and in the formation of flavonoids, is expressed throughout the plant. indeed our rna-seq analysis shows that chs_h is expressed in lupulin glands and leaves, with the three-fold higher expression levels in glands likely reflecting the production of xanthohumol. two myb transcription factors, can and can have been shown to be highly expressed in cannabis glandular trichomes and implicated in regulating trichome-specific metabolic processes. both have close homologs to hop transcription factors that are over ten-fold more abundant in the lupulin glands. can which has 70% amino acid identity to the hop myb protein encoded by comp63432_c0_seq was proposed to be involved in regulating cannabinoid formation. since cannabis and humulus are closely related genera with glandular trichome-localized polyketide pathways, comp63432_c0_seq may be a candidate for regulating the bitter acid pathway. lupulin glands contain both mitochondrial and plastidial bcats the biosynthesis and subsequent degradation of the bcaas requires both metabolic and catabolic bcats. the lupulin gland datasets contained two bcats, hlbcat and hlbcat which were abundant in the glandular trichomes. based on rna-seq and qrt-pcr analysis hlbcat is exclusively expressed in the lupulin glands. transient expression of the hlbcat1-gfp fusion construct confirmed its mitochondrial targeting. hlbcat with n-terminal signal peptide failed to localize to the plastid and appeared to form aggregates in the n. benthamiana epidermal cells, however, the predicted hlbcat signal peptide fused to gfp was targeted to the plastid. these localizations were further supported through the construction of a phylogenetic tree comparing bcats from a variety of plant species. problems with the experimental localization of hop plastid proteins have been previously reported for a gpp synthase small subunit identified in lupulin glands. kinetic analysis suggests lack of specialization for hop bcats kinetic analysis has provided equivocal evidence for the catalytic specialization of bcats for bcaa biosynthesis or catabolism. in tomato, biosynthetic bcats had km values with the keto acids that were an order-of-magnitude less than the catabolic enzymes, indicating specialization of the former for forward reactions. in contrast, arabidopsis atbcat which has been shown to be a catabolic enzyme, had km values for the keto acids that were much lower than for bcaa substrates, suggesting a preference for the biosynthetic reaction. indeed, the km values for atbcat with the keto acids were lower than those reported with the plastidial, biosynthetic enzyme atbcat. the kinetic properties of the hop bcats appear to be more similar to the arabidopsis enzymes. the catabolic enzyme hlbcat exhibited lower km values for keto acids, and therefore an apparent preference for the forward reaction, compared with than hlbcat. it is noteworthy that hlbcat showed a lower km for leucine compared with valine and isoleucine which may reflect this enzyme’s preferred role in synthesizing isovaleryl-coa for humulone biosynthesis. the lower vmax values for the hlbcat may also explain why it is more highly expressed compared to hlbcat in the lupulin gland tissue. phylogenetic analysis indicates separation of biosynthetic and catabolic bcats to further analyze the function of the hop bcats, we constructed a phylogenetic tree of diverse bcats including well characterized enzymes from arabidopsis and tomato. this analysis was successful in separating biosynthetic and catabolic bcats into separate clades. bcats are also reported to accumulate in alternative cellular compartments such as the cytosol and vacuole for an as of yet unknown function and these genes likely make up the third group that includes several cytosolic enzymes and hlbcat. an anomalous grouping was observed for atbcat which has been shown to be plastid localized but which was placed in the mitochondrial clade. a previous study found atbcat to be the only arabidopsis gene to be co-expressed with other genes involved in bcaa catabolism, a role one would expect to be occupied by a mitochondrial bcat. in addition, targetp analysis of atbcat predicts mitochondrial rather than plastidial localization.  this study provides an in-depth analysis of the transcriptional activity of primary and specialized metabolism leading to hop bitter acids. rna-seq analysis showed that both the mep and bcaa pathways were highly expressed in lupulin glands together with genes required for remobilization of carbon from sucrose. branched-chain acyl-coas and bitter acids are present in higher levels in isolated lupulin glands compared with cones and leaves. a plastidial bcat enzyme involved in bcaa biosynthesis and a mitochondrial bcat catalyzing bcaa degradation both show lupulin gland-specific expression. together these findings indicate that the glandular trichomes are the site of biosynthesis for the precursors for bitter acid biosynthesis. the deep transcriptome sequencing reported here significantly expands the genomic resources available for hop. this new information, and our analysis of bcaa metabolism, will be useful for the further elucidation of enzymes and regulatory proteins involved in bitter acid production and for breeding hop cultivars with increased or modified bitter acid content. 
bitter acids are prenylated polyketides synthesized in lupulin glands of the hop plant which are important contributors to the bitter flavour and stability of beer. we used rna sequencing to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. transcripts encoding all the enzymes of bcaa metabolism were significantly more abundant in lupulin glands, indicating that bcaa biosynthesis and subsequent degradation occurs in these specialized cells. the biosynthetic pathway leading to bcaas in lupulin glands involves the plastidial enzyme, hlbcat the mitochondrial enzyme hlbcat degrades bcaas as the first step in the catabolic pathway leading to branched chain-acyl-coas. rna-seq analysis showed the gland-specific expression of the mep pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. hlbcat is a plastidial enzyme likely involved in bcaa biosynthesis. our analysis of the hop transcriptome significantly expands the genomic resources available for this agriculturally-important crop. bitter acids are formed from acyl-coa precursors derived from branched-chain amino acid degradation and c prenyl diphosphates from the methyl-d-erythritol 4-phosphate pathway. we also measured the levels of bcaas, acyl-coa intermediates, and bitter acids in glands, cones and leaves. branched-chain acyl-coas and bitter acids were present at higher levels in glands compared with leaves and cones. this study provides evidence for the lupulin gland-specific biosynthesis of bcaas and prenyl diphosphates to provide precursors for the production of bitter acids. phylogenetic analysis of the hop bcats and those from other plants showed that they group into distinct biosynthetic and catabolic clades.
 bitter acids are prenylated polyketides synthesized in lupulin glands of the hop plant which are important contributors to the bitter flavour and stability of beer. bitter acids are formed from acyl-coa precursors derived from branched-chain amino acid degradation and c prenyl diphosphates from the methyl-d-erythritol 4-phosphate pathway. we used rna sequencing to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. we also measured the levels of bcaas, acyl-coa intermediates, and bitter acids in glands, cones and leaves.  transcripts encoding all the enzymes of bcaa metabolism were significantly more abundant in lupulin glands, indicating that bcaa biosynthesis and subsequent degradation occurs in these specialized cells. branched-chain acyl-coas and bitter acids were present at higher levels in glands compared with leaves and cones. rna-seq analysis showed the gland-specific expression of the mep pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. two branched-chain aminotransferase enzymes, hlbcat and hlbcat were abundant, with gene expression quantification by rna-seq and qrt-pcr indicating that hlbcat was specific to glands while hlbcat was present in glands, cones and leaves. recombinant hlbcat and hlbcat catalyzed forward and reverse reactions with similar kinetic parameters. hlbcat is targeted to mitochondria where it likely plays a role in bcaa catabolism. hlbcat is a plastidial enzyme likely involved in bcaa biosynthesis. phylogenetic analysis of the hop bcats and those from other plants showed that they group into distinct biosynthetic and catabolic clades.  our analysis of the hop transcriptome significantly expands the genomic resources available for this agriculturally-important crop. this study provides evidence for the lupulin gland-specific biosynthesis of bcaas and prenyl diphosphates to provide precursors for the production of bitter acids. the biosynthetic pathway leading to bcaas in lupulin glands involves the plastidial enzyme, hlbcat the mitochondrial enzyme hlbcat degrades bcaas as the first step in the catabolic pathway leading to branched chain-acyl-coas. 