Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus)

Abstract

Background: Bitter acids (e.g. humulone) are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus) 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 (BCAA) degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP) pathway. We used RNA sequencing (RNA-seq) 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.

Results: 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 (BCAT) enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic) and reverse (catabolic) reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 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 (plastidial) and catabolic (mitochondrial) clades.

Conclusions: 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, HlBCAT2. The mitochondrial enzyme HlBCAT1 degrades BCAAs as the first step in the catabolic pathway leading to branched chain-acyl-CoAs.

Figure 1

Biosynthesis of hop bitter acids from branched-chain amino acid (BCAA) precursors.a) The acyl side-chains of humulone/lupulone are derived from leucine, cohumulone/colupulone from valine and adhumulone/adlupulone from isoleucine via the corresponding acyl-CoA thioesters. The carbon chain of the BCAAs is shown in red to indicate their incorporation into the α- and β-acids. b) Schematic depiction of the major pathways contributing to the biosynthesis of the major α-acid product, humulone, in lupulin glands. Pathways are coloured to match Figure 2b: BCAA biosynthesis, purple; BCKDH complex, brown; MEP pathway, orange; malonyl-CoA biosynthesis, blue; bitter acid biosynthesis, green. The movement of metabolic intermediates between cellular compartments is indicated by dashed line. Note: the subcellular localization of the putative humulone synthase enzyme is not known.

Figure 2

Scatter plot comparison of gene expression of metabolic pathways between lupulin glands, leaves and cones. Data is presented as reads per kb exon model per million mapped reads (RPKM). Corresponding gene names and expression values can be found in Table S2. a) Expression of genes encoding enzymes of primary metabolic pathways (Calvin cycle, TCA cycle and glycolysis). b) Expression of genes encoding enzymes of BCAA metabolism (BCAA, BCKDH complex, HlBCAT1 and HlBCAT2), MEP pathway, malonyl-CoA synthesis and bitter acid biosynthesis.

Figure 3

Metabolite analysis of lupulin glands, leaves and cones.a) Amounts of BCAAs (leucine, valine and isoleucine). b) Amounts of acyl-CoA precursors for bitter acid biosynthesis. c) Amounts of major bitter acid products. Inset shows amounts of diprenylated acylphloroglucinol intermediates. For all analyses, values represent mean ± standard error (n=5 for glands, n=3 for cone and leaf).

Figure 4

Subcellular co-localization of BCAT signal peptides fused to GFP with organelle specific markers using transient expression in Nicotiana benthamiana leaves. The HlBCAT1 signal peptide fused to GFP colocalized with the mitochondrial MTRK marker (upper panel) while the HlBCAT2 signal peptide-GFP fusion colocalized with the plastidial PTRK marker (lower panel). Plasmid constructs in Agrobacterium were agro-infiltrated into N. benthamiana leaves and fluorescent protein expression visualized using confocal microscopy. Scale bars represent 20 μm.

Figure 5

A phylogenetic tree of BCAT proteins in plants constructed using the neighbour-joining method. BCATs group into plastidial (orange line) and mitochondrial (blue line) clades, with apparent division into dicot and monocot subclades. A third clade (grey line) includes BCATs that fall into neither group. BCATs with experimentally-confirmed organelle localization are labelled: P, plastid; M, mitochondrion; C, cytosol. Species abbreviations are found in Methods (At, Arabidopsis thaliana; Hl, Humulus lupulus; Sl, Solanum lycopersicon). *As we note in the Discussion, the plastid localization of AtBCAT2 requires further analysis.