Transcriptome‐based identification and functional characterization of iridoid synthase involved in monotropein biosynthesis in blueberry

Abstract

Blueberries (Vaccinium spp.) are well known for their nutritional quality, and recent work has shown that Vaccinium spp. also produce iridoids, which are specialized metabolites with potent health‐promoting benefits. The iridoid glycoside monotropein, which has anti‐inflammatory and antinociceptive activities, has been detected in several wild blueberry species but in only a few cultivated highbush blueberry cultivars. How monotropein is produced in blueberry and the genes involved in its biosynthesis remain to be elucidated. Using a monotropein‐positive (M+) and monotropein‐negative (M−) cultivar of blueberry, we employed transcriptomics and comparative genomics to identify candidate genes in the blueberry iridoid biosynthetic pathway. Orthology analysis was completed using de novo transcript assemblies for both the M+ and M− blueberry cultivars along with the known iridoid‐producing plant species Catharanthus roseus to identify putative genes involved in key steps in the early iridoid biosynthetic pathway. From the identified orthologs, we functionally characterized iridoid synthase (ISY), a key enzyme involved in formation of the iridoid scaffold, from both the M+ and M− cultivars. Detection of nepetalactol suggests that ISY from both the M+ and M− cultivars produce functional enzymes that catalyze the formation of iridoids. Transcript accumulation of the putative ISY gene did not correlate with monotropein production, suggesting other genes in the monotropein biosynthetic pathway may be more directly responsible for differential accumulation of the metabolite in blueberry. Mutual rank analysis revealed that ISY is co‐expressed with UDP‐glucuronosyltransferase, which encodes an enzyme downstream of the ISY step. Results from this study contribute new knowledge in our understanding of iridoid biosynthesis in blueberry and could lead to development of new cultivars with increased human health benefits.

1. INTRODUCTION

Blueberry, a species in the genus Vaccinium which is native to North America, is grown globally and has seen an increase in both harvested area and yield over the last decades (Food and Agriculture Organization [FAO], 2022). This can be attributed to its economic importance and well‐known nutritional quality. Blueberry fruits contain health‐promoting compounds such as anthocyanins and flavonoids (Moyer et al., 2002; Rossi et al., 2022; Skrovankova et al., 2015), which have antioxidant properties that help prevent chronic diseases such as cancer, diabetes, cardiovascular, and neurological disorders (Grace et al., 2009; Seeram et al., 2006; Zafra‐Stone et al., 2007). Additionally, blueberries and other Vaccinium species contain iridoid compounds (Heffels et al., 2017; Jensen et al., 2002; Leisner et al., 2017; Ma et al., 2013; Swiatek & Komorowski, 1972), a class of plant natural products with known human health benefits (Dinda et al., 2011; Tundis et al., 2008; Viljoen et al., 2012; Wang et al., 2020). Recent work has detected the iridoid glycoside monotropein in blueberry (Leisner et al., 2017). In a survey of 13 wild and 71 cultivated blueberries, it was shown that all wild Vaccinium species contained monotropein, yet this compound was only present in select cultivated blueberries (Leisner et al., 2017). The highest abundance of monotropein was found in floral buds and stems but was also present in all tissues examined in cultivars that were monotropein‐positive (M+). On the other hand, monotropein‐negative (M−) cultivars did not produce the compound in any tissues surveyed (Leisner et al., 2017) with a cutoff for quantitation (10 ng/mg) for classifying cultivars as M+ or M−. Most of the M+ cultivars were of the southern highbush ecotype, while the majority of M− cultivars surveyed were northern highbush. Monotropein is also found in other plant species, including Indian mulberry (Morinda officinalis), where it has been shown to have antinociceptive, anti‐inflammatory, anti‐osteoporosis, and wound healing properties (Chen et al., 2020; Choi et al., 2005; Karna et al., 2019; Shen et al., 2018; Wang et al., 2018; Zhang et al., 2016). Due to its potential positive human health benefits and differential production across blueberry cultivars, it is of interest to understand more about the biosynthesis of this metabolite at the molecular level.

To identify key genes in iridoid biosynthesis in blueberry, transcriptomics, comparative genomics, and biochemistry were employed. The use of multiple “omic” tools have been used in elucidation of the monoterpene indole alkaloid (MIA) pathway in the model medicinal plant Catharanthus roseus (Kellner et al., 2015; Miettinen et al., 2014; van Moerkercke et al., 2013), as well as identification of a key cyclization enzyme (iridoid synthase, ISY) in iridoid biosynthesis in olive and Nepeta species (Alagna et al., 2016; Sherden et al., 2018). Outside of the iridoid biosynthetic pathway, comparative genomics has been critical in identification of genes in the terpene biosynthetic pathway in tomato (Matsuba et al., 2013) and the glucosinolate pathway in Arabidopsis (Bekaert et al., 2012). Furthermore, it has been observed that specialized metabolite biosynthetic pathway genes have the proclivity to be physically clustered in plant genomes (DellaPena and O'Connor, 2012). Taken together, the use of comparative genomics integrated with transcriptomics and metabolite profile analysis is a powerful tool to identify genes involved in the biosynthesis of specialized metabolites.

Among plants, the most complete elucidation of the iridoid biosynthetic pathway is in C. roseus (Collu et al., 2001; Geu‐Flores et al., 2012; Irmler et al., 2000; Miettinen et al., 2014; Murata et al., 2008; Simkin et al., 2013). The iridoid pathway stems from the methylerythritol phosphate (MEP) pathway through the conversion of geranyl pyrophosphate (GPP) to geraniol in the early iridoid pathway. The early portion of the iridoid pathway is then linked to the late iridoid pathway by the activity of ISY (Mint Evolutionary Genomics Consortium, 2018). Beyond this first committed step in the formation of the iridoid skeleton by ISY, tailoring enzymes account for the biosynthesis of specific iridoid compounds.

ISY is part of the larger progesterone 5β‐reductase (P5βR)/iridoid synthase (PRISE) family (Nguyen & O'Connor, 2020; Petersen et al., 2016) and performs a key transition step by conducting reductive cyclization that generates the iridoid ring scaffold, converting 8‐oxogeranial to nepetalactol (Geu‐Flores et al., 2012). Due to its role in this key step in iridoid biosynthesis, we hypothesize that either the presence or absence of the ISY gene or its gene expression in blueberry may account for differential monotropein production among cultivars. Validation of the putative ISY gene identified from targeted orthology analysis was carried out using heterologous expression in Escherichia coli and in vitro end‐point enzyme assays, as this has proven effective to validate ISY in other plant species (Alagna et al., 2016; Geu‐Flores et al., 2012; Lichman et al., 2020), as well as monoterpene synthase in Camelina sativa (Borghi & Xie, 2018). We also propose here a possible pathway for monotropein biosynthesis (Figure 1). Overall, elucidation of the iridoid biosynthetic pathway in blueberry would increase our basic knowledge of natural product biosynthesis in an economically important fruit crop, shed light on the mechanisms leading to iridoid production in specific germplasm, and could aid in developing cultivars with increased iridoid/monotropein content for human health.

FIGURE 1.

Proposed monotropein biosynthetic pathway in Vaccinium corymbosum . GPP—geranyl diphosphate; GES—geraniol synthase; G8H—geraniol 8‐hydroxylase; GOR—8‐hydroxygeraniol oxidoreductase; ISY—iridoid synthase; IO—iridoid oxidase; UGT—UDP‐glucuronosyltransferase.

2. RESULTS AND DISCUSSION

2.1. De novo transcriptome assembly of differential monotropein cultivars and transcriptome‐enabled gene discovery

A diverse set of tissues from two highbush blueberry cultivars with contrasting monotropein content (Table S1) were subjected to RNA‐Sequencing (RNA‐Seq) and when aligned to the V. corymbosum cv. Draper reference genome (Colle et al., 2019), had an overall alignment rate of 89.1%–92.3% (Table S2). As the reference genome (cv. Draper) is M−, independent transcriptome assemblies were completed for both Ornablue (M+) and Concord (M−) (Table S3) using young leaf, mature leaf, floral bud, open flower (Ornablue only), stem, unripe fruit, and ripe fruit tissues (Table S1) to ensure the difference in monotropein biosynthesis was not due to a presence/absence variant. Benchmarking Universal Single‐Copy Orthologue (BUSCO) analysis revealed 98.8% (420/425) and 98.4% (418/425) of the Viridiplantae BUSCO genes for Ornablue (M+) and Concord (M−), respectively (Table S3), suggesting of a highly representative transcriptome for gene discovery.

Translated peptide sequences generated from representative transcripts for both Ornablue (M+) and Concord (M−) were used to identify orthologs of C. roseus iridoid biosynthetic genes using OrthoFinder (Figure 2). OrthoFinder assigned 158,620 genes (50.5% of total) to 56,571 orthogroups; 50% of all genes were in orthogroups with two or more genes (G50 = 2). There were 6,432 orthogroups with all species present and 2,158 of these consisted entirely of single‐copy genes. Gene occupancy analysis revealed orthologs in both Ornablue (M+) and Concord (M−) for all genes involved in the early portion of the iridoid biosynthetic pathway, except for geraniol synthase (GES) (Table S4). An additional BLAST analysis using C. roseus GES as a query against custom blueberry peptide databases resulted in the identification of four potential orthologs of GES in blueberry, all of which were present in Ornablue (M+) (Table S5). Alignment of these potential orthologs to the V. corymbosum genome (cv. Draper) identified six possible candidate GES genes (Table S5). The orthogroup containing the C. roseus ISY gene contained only a single ortholog of ISY for both Ornablue (M+) and Concord (M−), respectively (Figure 2). Orthologs of the late iridoid pathway genes including 7‐deoxyloganetic acid glucosyltransferase (7‐DLGT), the UDP‐glucuronosyltransferase (UGT) in C. roseus, were also identified (Figure 2), although the specific UGT for iridoid/monotropein production in blueberry is yet to be determined (Figure 1).

FIGURE 2.

Number of genes from C. roseus and blueberry (Ornablue [M+] and Concord [M−]) in each orthogroup identified using OrthoFinder. Additional possible orthologs identified by BLAST are presented in Table S5. GES—geraniol synthase; G8H—geraniol 8‐hydroxylase; GOR—8‐hydroxygeraniol oxidoreductase; ISY—iridoid synthase; IO—iridoid oxidase; 7‐DLGT—7‐deoxyloganetic acid glucosyltransferase (the likely UDP‐glucuronosyltransferase involved in monotropein biosynthesis).

2.2. Multiple sequence alignment and analysis

To characterize the candidate ISY gene in blueberry (VcISY), multiple sequence alignment of the amino acid sequences of the Ornablue (M+) and Concord (M−) VcISY was completed and revealed 98% identity to each other and 63% identity to the C. roseus ISY (CrISY) (Figure S1). Further comparison of other ISY amino acid sequences demonstrated that VcISY contained the amino acid residues previously identified to be critical for ISY function (Table 1) (Kries et al., 2016, 2017). The VcISY sequence was also compared with other known ISY sequences that are structurally different from CrISY (Table 1). Analysis of critical residues revealed that except for the amino acid at residue 346, VcISY had the same amino acid residue with CrISY and Olea europaea ISY (OeISY) compared with the ISY residues for Antirrhinum majus (AmISY), which catalyzes formation of an alternative iridoid stereoisomer. This also suggests the specificity of VcISY to 8‐oxogeranial as substrate since the Ala343‐Ser354 loop has been shown to determine substrate specificity of CrISY, particularly in substrate recognition (Kries et al., 2016).

TABLE 1.

Comparison of amino acid residues at the binding pocket of characterized iridoid synthase (ISY) in different species. Column headers indicate amino acid position. Cr— C. roseus ; Oe— Olea europaea ; Vc— Vaccinium corymbosum ; Am— Antirrhinum majus .

	149	246	342	345	346	349	352
CrISY	F	A	F	I	A	S	L
OeISY	I	V	F	I	A	L	V
VcISY	F	A	F	I	V	S	L
AmISY	W	W	L	V	V	N	T

Phylogenetic analysis of the Ornablue VcISY sequence using the maximum likelihood method revealed clustering with functionally validated ISY genes from C. roseus (Madagascar periwinkle), O. europaea (olive), Digitalis purpurea (foxglove), A. majus (snapdragon), and Sesamum indicum (sesame) and not with ISY genes associated with the VEP1 clade, other progesterone‐5‐β‐reductase genes (including the CrP5βR6 clade), or Nepetoideae‐specific VEP1 clade (Figure 3). Although P5βRs have intrinsic iridoid synthase activities (Munkert et al., 2015), VcISY did not cluster with ISY genes associated with the VEP1 clade, other P5βR genes, or Nepetoideae‐specific VEP1 clade, as these ISYs have unique iridoid synthase structures compared with the majority of characterized iridoid synthases (Lichman et al., 2020). This shows that VcISY has a higher sequence similarity with the other functionally validated ISYs.

FIGURE 3.

Phylogenetic tree of ISY and related P5βR‐like enzymes. Relationships are based on coding sequences of characterized and putative ISY and P5βR/ISY‐like enzymes (PRISE) analyzed by maximum likelihood method. Clades with bootstrap values ≥90 are labeled with black circles. The VEP1 clade is shown in blue; CrP5βR4 clade in green; ISY clade in purple including blueberry ISY (VcISY) in orange; and CrP5βR6 clade in brown. Species acronyms: Antirrhinum majus (Am), Catharanthus roseus (Cr), Digitalis purpurea (Dp), Erythranthe guttatus (Eg), Nepeta cataria (Nc), Nepeta mussinii (Nm), Olea europaea (Oe), Plantago major (Pm), Sesamum indicum (Si), Vaccinium corymbosum (Vc). VcISY sequence is based on the monotropein‐producing cultivar Ornablue.

2.3. Biochemical assay of blueberry ISY

The VcISY gene from both Ornablue (M+) and Concord (M−) were expressed in E. coli and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) (Figure S2) showed that the purified protein corresponds to the expected size of 42 kDa and that the His‐tagged protein was successfully isolated from other expressed proteins. The purified protein was used for functional enzyme analysis. End‐point enzyme assays and analysis of the end product using gas chromatography–mass spectrometry (GC‐MS) revealed that both the Ornablue (M+) and Concord (M−) VcISY catalyzed the reaction converting 8‐oxogeranial (8‐OG) to cis,trans‐nepetalactol (Figure 4), with the substrate fully consumed in both reactions. Analysis of the enzyme reaction products also showed the presence of other peaks in the GC‐MS chromatograms (Figures 4 and S3), which correspond to the partially cyclized iridodials that are typically found in the ISY reaction (Nguyen & O'Connor, 2020; Sherden et al., 2018).

FIGURE 4.

Functional characterization of blueberry ISY by gas chromatography–mass spectrometry analysis of in vitro enzyme assay reaction products. (a) Total ion chromatograms of negative control reaction (substrate [8‐oxo‐geranial, 8‐OG] and NADPH only; no enzyme), c,t‐nepetalactol (reference standard), and reaction products with ISY from Ornablue (M+) and Concord (M−) in the presence of NADPH are compared. ISY converted 8‐OG to c,t‐nepetalactol (peak 3) and iridodials (peaks 1 and 2). (b) Mass spectra of c,t‐nepetalactol (peak 3) labeled in panel a. Mass spectra of peaks 1 and 2 are shown in Figure S3. Chromatograms and mass spectra were generated using the Agilent Enhanced ChemStation software.

2.4. Expression analysis of iridoid biosynthetic genes

Gene expression analysis of orthologs identified using OrthoFinder and BLAST was performed to determine if transcript abundance mirrored metabolite levels across tissues and cultivars (Figure 5; Table S6). To minimize differences in transcript assembly between the two de novo transcriptomes, we aligned our Ornablue (M+) and Concord (M−) transcriptomes to the Draper reference genome (Colle et al., 2019), and annotated genes located in the aligned regions of the Draper genome were then analyzed for their gene expression using the RNA‐Seq data. Some orthologs mapped to more than one annotated gene in the Draper genome, while others mapped to regions without any annotated genes (Table S4). The highest transcript expression, represented as fragments per kilobase of exon per million mapped fragments (FPKM), across all samples and genes was in Ornablue floral buds for the majority of the orthologs in the iridoid biosynthetic pathway (Figure 5; Table S6). Transcript expression of ISY in Ornablue (M+) was highest in unripe fruits followed by floral buds (Figure 5), which demonstrate a different trend in comparison with metabolite production across tissues analyzed (Figure S4) (Spearman Rank coefficient R = 0.45, p = 0.14) (Leisner et al., 2017). Transcript levels of ISY in Concord (M−) had a different pattern compared with the metabolite data. Notably, the relative expression levels in mature leaves of Concord are comparable with Ornablue floral buds, but metabolite levels are lower in Concord (Figures 5 and S4). Since both Ornablue (M+) and Concord (M−) show expression of the ISY ortholog, but Concord does not produce monotropein, it is possible that the monoterpene cyclase in blueberry is structurally different than ISY in C. roseus. There is a precedent for this hypothesis, as the monoterpene cyclase in C. roseus uses an alternative biochemical mechanism to create cyclic terpenes (Geu‐Flores et al., 2012).

FIGURE 5.

Transcript abundance of iridoid biosynthetic genes in Ornablue (M+) and Concord (M−). Transcript data is shown as Z‐scores of FPKM values of transcripts across tissue samples in Ornablue (M+) and Concord (M−) with hierarchical clustering across samples. FPKM values were based on alignment to the V. corymbosum cv. Draper genome (Colle et al., 2019). Alignment of orthologs to the V. corymbosum genome was done using GMAP (v.20130509), and transcripts located in the aligned regions were then analyzed for their gene expression (FPKM). Expression values of orthologs are indicated in Table S6. Cells shown in gray depict identified transcripts that are not expressed (FPKM = 0) in the analyzed samples. Transcript abbreviations: GES—geraniol synthase; G8H—geraniol 8‐hydroxylase; GOR—8‐hydroxygeraniol oxidoreductase; ISY—iridoid synthase; IO—iridoid oxidase; 7‐DLGT—7‐deoxyloganetic acid glucosyltransferase. Sample acronyms: O—Ornablue; C—Concord; FB—floral buds; St—stems; UF—unripe fruits; RF—ripe fruits; YL—young leaves; ML—mature leaves.

Due to the discordant metabolite and transcript expression levels, we hypothesize that VcISY is expressed in both cultivars, yet the expression of genes encoding enzymes catalyzing the other steps of the biosynthetic pathway may affect monotropein levels. For example, in Lamiaceae, GES and ISY were significantly associated with gene expression and iridoid production across multiple Lamiaceae species (Mint Evolutionary Genomics Consortium, 2018). Low or no detectable expression of GES was observed in non‐iridoid producing species, while expression of GES was highly correlated with iridoid production. Regardless of iridoid production, ISY had some expression in all species, although iridoid‐producing species had higher gene expression levels compared with non‐producing species (Mint Evolutionary Genomics Consortium, 2018). In blueberry, we observed a similar pattern with iridoid (monotropein) production and ISY, where it is expressed in both M+ and M− cultivars (Figure 5). In our current dataset, we observed that both Ornablue (M+) and Concord (M−) have detectable levels of potential GES orthologs, with high expression (FPKM >50) of two GES orthologs (GES_2 and GES_4) and moderate expression of one GES ortholog (GES_3) in Ornablue floral buds (Figure 5; Table S6). Another GES ortholog (GES_1) had the highest expression in leaves from Concord (Figure 5), although expression levels are generally low (FPKM <5) (Table S6). This follows a similar pattern as that found in Lamiaceae species (Mint Evolutionary Genomics Consortium, 2018), but further analysis and experimentation is needed to determine functionality of these orthologs.

Additionally, post‐transcriptional modifications and/or post‐translational modifications could lead to the absence of monotropein. This discordant pattern between metabolite and transcript expression levels has been observed in other plant species, including metabolites in the central metabolic pathways in Brassica napus (Schwender et al., 2014). It is also possible that formation of other iridoid compounds that have the same stereochemistry as monotropein, such as gardenoside which has been previously identified in neotropical blueberry (Ma et al., 2013), is favored in M− cultivars such as Concord as opposed to production of monotropein. Additionally, low expression of iridoid biosynthetic genes could lead to low levels of monotropein that is below the limit of quantitation (10 ng/mg monotropein), which was the basis of classifying cultivars as M+ or M− (Leisner et al., 2017).

Other steps in the iridoid biosynthetic pathway may also limit monotropein production as genes downstream of ISY are required for the complete biosynthesis of monotropein (Figure 1). To identify additional candidate genes in blueberry, mutual rank‐based co‐expression analysis using expression data across the transcriptome (Table S7) was also performed (Table S8). The 10 best co‐expressed transcripts with VcISY from the mutual rank analysis are shown in Table 2, which includes UDP‐glucuronosyltransferase (UGT). This gene has higher expression in Ornablue (M+) tissues than in Concord (M−), except for leaves (Figure S5). In the proposed monotropein biosynthetic pathway (Figure 1) UGT is downstream of ISY, following enzyme action of iridoid oxidase (IO). Based on analysis in C. roseus, the likely UGT is 7‐DLGT, which we identified potential candidate genes of from our orthology analysis (Figure 2; Tables S4 and S6). The UGT identified from mutual rank analysis, however, is not one of the 7‐DLGT candidate genes identified from orthology analysis (Tables 2 and S6). Analysis of the 7‐DLGT candidate genes demonstrated these orthologs have the lowest expression in fruits across all tissues in both Ornablue (M+) and Concord (M−) (Figure 5; Table S6). Altogether, we have identified candidates for three biosynthetic genes (ISY, GES, and UGT/7‐DLGT) in the blueberry monotropein biosynthetic pathway using transcriptomics, orthology, and co‐expression analysis that will allow us to investigate the mechanism behind differential monotropein production between M+ and M− blueberry cultivars.

TABLE 2.

Top 10 co‐expressed genes with iridoid synthase in blueberry. Co‐expressed genes were identified by mutual rank analysis. Functional annotation was based on Wisecaver and Edger (2019). The top 200 co‐expressed genes are listed in Table S8.

Gene ID	Annotation
maker‐VaccDscaff17‐snap‐gene‐35.46	Adenylate kinase/UMP‐CMP kinase
snap_masked‐VaccDscaff48‐processed‐gene‐44.18	UDP‐glycosyltransferase family
maker‐VaccDscaff2‐snap‐gene‐409.60	UDP‐glucuronosyl/UDP‐glucosyltransferase
maker‐VaccDscaff31‐snap‐gene‐15.28	Armadillo‐type fold
maker‐VaccDscaff34‐augustus‐gene‐236.23	3,4‐dihydroxy‐2‐butanone 4‐phosphate synthase, RibB
maker‐VaccDscaff28‐augustus‐gene‐338.25	ABC transporter type 1, transmembrane domain
maker‐VaccDscaff2‐augustus‐gene‐25.22	LOG family (possible lysine decarboxylase)
augustus_masked‐VaccDscaff34‐processed‐gene‐61.1	Stress up‐regulated Nod 19
maker‐VaccDscaff24‐augustus‐gene‐156.30	Vacuolar protein sorting‐associated protein 13, N‐terminal domain
maker‐VaccDscaff20‐snap‐gene‐300.25	Protein kinase domain

3. CONCLUSION

Overall, we have shown that multiple “omic” (transcriptomics, co‐expression analysis, and comparative genomics) approaches identified candidate genes for the iridoid biosynthetic pathway in blueberry. Among the candidate genes identified, we functionally characterized ISY using in vitro enzyme assays and showed that the putative VcISYs from both Ornablue (M+) and Concord (M−) produce functional enzymes that catalyze the formation of the iridoid c,t‐nepetalactol. With the success of the methodology pipeline, other genes in the pathway will also be validated using the same approach. Among the suite of genes involved in iridoid/monotropein production, GES in the early iridoid pathway as well as UGT and oxidases in the tailoring steps leading to monotropein synthesis are of special interest for the next analysis based on orthology and co‐expression analyses. Findings from this study expand our knowledge on iridoid production in blueberry that could be used in developing new cultivars with increased nutritional quality.

4. MATERIALS AND METHODS

4.1. Sample collection

Sampling was performed on blueberry plants grown by the Michigan Blueberry Growers Association in Grand Junction, MI, USA (42°24′09.4″N, 86°04′20.9″W) in 2016. Tissue samples were collected from Ornablue, a monotropein‐positive cultivar, and Concord, a monotropein‐negative cultivar (Leisner et al., 2017). Unripe and ripe fruits, young and mature leaves (at fruit ripening), floral buds, and stems were harvested from both Ornablue and Concord, with additional open flowers from Ornablue. Tissues were sampled from one plant per cultivar. Samples were placed on ice and transported to East Lansing, MI, where they were subsequently flash frozen with liquid nitrogen and stored at −80°C until use. Monotropein content of the same set of samples (except open flowers) was previously quantified by liquid chromatography‐mass spectrometry (Leisner et al., 2017).

4.2. RNA‐sequencing (RNA‐Seq) and de novo transcriptome assembly

Total RNA was extracted from each of the tissue samples for both Ornablue and Concord. Frozen tissue was homogenized into a fine powder using a mortar and pestle. RNA from young leaves, mature leaves, floral buds, open flowers (Ornablue only), and stems was extracted using the Spectrum Plant Total RNA Kit (Sigma‐Aldrich, St. Louis, MO, USA) according to manufacturer's protocol. RNA from unripe and ripe fruits was extracted using a modified protocol from Vashisth et al. (2011). Genomic DNA contamination was removed from RNA samples using Turbo DNase treatment (Applied Biosystems by Life Technologies, Austin, TX, USA) according to the manufacturer's protocol. RNA quantity was determined with a spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific, Waltham, MA, USA), while quality was assessed by running samples on a 1.5% nondenaturing agarose gel and on a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). All cDNA libraries were constructed by Novogene (Novogene Corporation, Chula Vista, CA, USA). RNA‐Seq was also completed by Novogene generating 150‐nt paired‐end reads for all tissues of both cultivars using an Illumina HiSeq 4000 (Illumina Inc, San Diego, CA, USA). FASTQ files from all sequencing runs have been deposited in the Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gove/sra) under BioProject PRJNA477303.

Paired‐end reads were assessed for quality using FASTQC (v.0.11.5) and cleaned using Cutadapt (v.2.10; Martin, 2011) with a minimum quality score of 20 (‐q 20), minimum length of 36 (‐m 36) and custom adapter sequences. Libraries were checked for contamination by comparison to the NCBI non‐redundant nucleotide database using BLAST (v.2.9.0+; Camacho et al., 2009). Separate transcriptome assemblies were made for Ornablue and Concord utilizing all tissue libraries for each cultivar using Trinity (v. 2.10.0; Haas et al., 2013) with a minimum contig length of 200 bp (‐‐min_contig_length 200) and ‐‐full_cleanup mode with remaining default parameters. Completeness of the assembled transcriptomes based on expected gene content was assessed by BUSCO (v.5.3.2; Manni et al., 2021) using the dataset viridiplantae_odb10 (2022‐05‐19).

4.3. Gene expression and co‐expression analysis

Gene expression analysis was performed by aligning each RNA‐Seq library to the V. corymbosum genome (cv. Draper; Colle et al., 2019) using HISAT2 (v.2.2.1; Kim et al., 2019) with default parameters, followed by determination of FPKM values using StringTie (v.2.2.0; Pertea et al., 2016) (Table S7). Transcript abundances of potential orthologs of iridoid biosynthetic genes were converted to Z‐scores, which were plotted on an expression heatmap with hierarchical clustering across samples using the pheatmap package (v.1.0.12; Kolde, 2019) in RStudio (v.4.1.1; RStudio Team, 2021). Spearman correlation test between monotropein levels and transcript expression (FPKM) of ISY in all tissues was performed using the cor.test function in RStudio (v.4.1.1; RStudio Team, 2021).

Co‐expression analysis by mutual rank was also performed. First, the Pearson correlation coefficient was calculated using the filtered FPKM matrix (FPKM >1). A mutual rank list was then generated following Geu‐Flores et al. (2012). The top 200 genes co‐expressed with ISY were picked (Table S8). Annotation of the top 10 co‐expressed genes was determined based on Wisecaver and Edger (2019).

4.4. Orthology analysis

Orthology analysis using OrthoFinder (v.0.7.1; Emms & Kelly, 2015) was performed with representative peptide sequences from C. roseus and Transdecoder‐derived peptides (Haas et al., 2013) from the Ornablue and Concord de novo transcriptomes. Potential orthologs were analyzed for location in the genome by aligning transcript sequences to the V. corymbosum reference genome (cv. Draper; Colle et al., 2019) using GMAP (v.20130509; Wu & Watanabe, 2005). Annotated genes located in the aligned regions of the Draper genome were then analyzed for their gene expression as stated above. In the case of absence of an ortholog identified using Orthofinder (e.g., GES), BLAST (v.2.2.31+; Camacho et al., 2009) was also used to identify potential orthologs. BLAST was performed using a custom database built using either the Ornablue or Concord peptide sequences derived from Transdecoder. The C. roseus peptide sequence for GES was used as the query to search the custom databases. Functional annotation of the top BLAST hits was determined by BLAST analysis using The Arabidopsis Information Resource (TAIR) database (TAIR10; https://www.arabidopsis.org/Blast/). Transcript abundance of the GES orthologs determined by BLAST was also determined by aligning transcript sequences to the V. corymbosum reference genome (cv. Draper; Colle et al., 2019) using GMAP (v.20130509) and then annotated genes located in the aligned regions of the Draper genome were analyzed for their gene expression as stated above.

4.5. Analysis and sequence alignment of ISY

To determine sequence similarity, the putative blueberry ISY (VcISY) was compared with C. roseus ISY (CrISY) at the amino acid level using MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/). Additionally, the amino acids at the enzyme binding pocket site of VcISY and CrISY were compared based on the structure of CrISY (Kries et al., 2016).

The coding sequences of VcISY (Ornablue [M+], DN135366_c0_g1_i1) and CrISY/P5βR5 (JX974564) from the ISY orthogroup were used for alignment using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). A phylogenetic tree of known ISYs and P5ßR enzymes was constructed using additional sequences from GenBank from C. roseus: CrP5βR6 (KJ873887.1); A. majus (Am): AmISY (MF281392.1), Am18685 (MF281393.1), Am26155 (MF281394.1), Am29566 (MF281395.1); O. europaea (Oe): OeISY (KT954038.1), Oe1,4‐R1.1a (KT954039.1), Oe1,4‐R2 (KT954043.1), Oe1,4‐R3 (KT954044.1); D. purpurea (Dp): DpP5βR1 (AJ310673.1), DpP5βR2 (GU062787.1); Rosmarinus officinalis: ROOF‐P5βR (JQ894868.1); S. indicum (Si): SiP5βR1 (XM 011071474.2), SiP5βR2/ISY (XM 011071475.2), SiP5βR3 (XM 011089869.2), SiP5βR4 (XM 011094221.2), SiP5βR5 (XM 020695852.1); Erythranthe guttatus (Eg): EgP5βR1 (XM 012981417.1), EgP5βR2 (XM 012981418.1); Plantago major (Pm): PmP5βR1 (GU354233.1); Nepeta cataria (Nc): NcISYlike1A (KY882233.1), NcISY2 (KY882234.1); and Nepeta mussinii (Nm): NmISYlike1 (KY882235.1), NmISY2 (38) (KY882236.1). Using the inferred guide tree, sequences were realigned using PRANK (v.170427; Löytynoja & Goldman, 2008). Maximum likelihood (ML) tree inference was performed using IQ‐TREE (v.1.6.12; Nguyen et al., 2015) with the best‐fit substitution model (TIM3 + F + I + G4 based on Bayesian Information Criterion) determined by ModelFinder (Kalyaanamoorthy et al., 2017). The ML tree was generated with 200 iterations and ultrafast bootstrap approximation was used to obtain branch supports (1000 replicates; Hoang et al., 2018). The tree was visualized using FigTree (v1.4.4; http://tree.bio.ed.ac.uk/software/figtree/).

4.6. Iridoid synthase protein expression and purification

Both the full‐length Ornablue and Concord VcISY could not be amplified; hence, genes were synthesized (Twist Bioscience, San Francisco, CA, USA) and cloned in the pET‐28a(+) expression vector with N‐terminal 6× His‐tag and codon‐optimized for E. coli (Table S9). Enzyme expression and purification was done following Lichman et al. (2020) with modifications. The plasmids were subcloned into E. coli (BL21 DE3) which were grown overnight in LB medium with kanamycin at 37°C with shaking at 200 rpm. The overnight starter culture was used to inoculate 1 L of LB medium with kanamycin and grown at 37°C at 200 rpm until OD₆₀₀ = 0.7. The cultures were then cooled to 18°C with shaking before adding 0.25 mM IPTG to induce protein expression. The bacterial cultures were incubated at 18°C for 17 h. Cells were harvested by centrifugation (4000g, 15 min, 4°C), resuspended in lysis buffer, and incubated on ice for 15 min. Cells were then lysed using a sonicator (Qsonica, Newtown, CT, USA; amplitude, 50% pulses) for two 5‐min cycles of 20 s on, 40 s off for a total of 10 min. The lysate was centrifuged (35,000g, 30 min, 4°C) and the resulting supernatant was added with 500 μL Ni‐nitrilotriacetic acid (Ni‐NTA) agarose (QIAGEN, Hilden, Germany) washed in binding buffer. The mixture was incubated in a rocking incubator at 4°C for 1 h (30 rpm) and then centrifuged (2000g, 3 min, 4°C) to collect the Ni‐NTA pellet. The pellet was washed three times with 10 mL binding buffer, then eluted with 600 μL elution buffer. Following centrifugation (2000g, 5 min, 4°C), the supernatant was collected and filtered using a centrifugal filter (0.1 μm, Ultrafree®‐MC, Merck Millipore, Tullagreen Carrigtwohill Co. Cork, Ireland). The elution buffer was exchanged with sample buffer using a Ultracel‐10 regenerated cellulose membrane (Amicon Ultra‐0.5, Merck Millipore, Tullagreen Carrigtwohill Co. Cork, Ireland). Protein concentration was measured using NanoDrop. The purified proteins were divided into 20 μL aliquots and flash‐frozen in liquid nitrogen before storage at −80°C. The purity of the samples was checked by running samples (≤5 μg) on an SDS‐PAGE gel and stained with Coomassie blue.

4.7. End‐point enzyme assay and GC‐MS

Purified proteins were used in an end‐point enzyme assay with 8‐oxogeranial (8‐OG) as substrate. The reactions were based on Lichman et al. (2020). Briefly, buffer (HEPES [50 mM, pH 8.0]), NADPH (1 mM), ISY (0.5 mM), and 8‐OG (0.5 mM) were mixed and incubated at 30°C with shaking at 200 rpm for 3 h. The reactions were quenched with 100 μL ethyl acetate mixed with D‐camphor (1 mM, 10 μL) as internal standard, and the organic phase was analyzed by GC‐MS. An Agilent 6890N GC and 5975 MS with a Restek Rxi‐5Sil MS column with Integra‐Guard column (15 m, 0.25 mm ID, 0.25 μm df) was used to analyze the samples. The temperature of the injection port was 220°C and 1 μL of sample was injected in split mode with a ratio of 5:1. The pressure was constant at 2.2 psi and flow was set to 1.2 mL/min with Helium gas. The GC oven temperature was programmed with an initial temperature of 40°C for 3 min, ramp to 110°C at 5°C/min, and held for 2 min and then ramp to 280°C at 120°C/min with a 4 min hold, thus requiring a total run time of 24.4 min. The MS transfer line was set at 250°C. The MS source was set to 230°C and the EI was set to 70 eV emission with a scan range of 45 to 300 mz. Selected ion monitoring was used to monitor D‐camphor at 80.8 and 108.8 mz and c,t‐nepetalactol as reference standard at 54.6 and 120.7 mz beginning at 14 min. The raw data were processed and analyzed using Agilent Enhanced ChemStation software.

AUTHOR CONTRIBUTIONS

CPL, CRB, and SEO conceived the project and designed the research. CPL and LMFL performed the experiments and analyzed the data. LMFL, MOK, and CPL interpreted and validated the data. LMFL and CPL wrote the paper. All authors reviewed and edited the manuscript.

CONFLICT OF INTEREST STATEMENT

The authors did not report any conflict of interest.

Supporting information

Data S1. Supporting information.

Table S1. Blueberry samples used in the current study. List includes biological information, sequencing library information, and SRA Run number.

Table S2. Alignment of RNA‐Seq reads to V. corymbosum cv. Draper reference genome. Number of trimmed reads, total concordant alignment rate, and overall alignment rate are shown for each sequencing library. Reads were aligned to the blueberry tetraploid reference genome (Colle et al., 2019) using HISAT2 (v.2.2.1).

Table S3. De novo transcriptome assembly statistics. Listed are the number of genes and transcripts and contiguity statistics (N50) of Ornablue (M+) and Concord (M‐) transcriptome assemblies. Assemblies were completed with Trinity (v. 2.10.0) and completeness of the assemblies were assessed with BUSCO (v.5.3.2).

Table S4. Gene occupancy of iridoid biosynthetic genes in C. roseus and blueberry (Ornablue [M+] and Concord [M‐]). Orthology analysis was performed using OrthoFinder (v.0.7.1). The genes in the V. corymbosum genome associated with the identified orthologs were determined by aligning Ornablue or Concord transcript sequences to the V. corymbosum reference genome (cv. Draper; Colle et al., 2019) using GMAP (v.20130509). Some orthologs aligned to more than one gene model in the Draper genome, while some aligned to regions with no annotated genes; in these cases the number of annotated genes located in the aligned regions are indicated in parenthesis. Gene abbreviations are in the Figure 2 legend.

Table S5. Potential orthologs of C. roseus geraniol synthase (GES) in blueberry. The peptide sequence of C. roseus GES was used as query for a BLAST search against custom blueberry peptide databases. Potential orthologs were identified in Ornablue (M+) but not in Concord (M‐). Ornablue transcripts were aligned to the V. corymbosum cv. Draper reference genome (Colle et al., 2019) and the corresponding Draper gene ID is provided. Two of the candidate GES genes aligned to a region in the Draper genome that contained two gene models.

Table S6. Transcript expression of iridoid biosynthetic gene orthologs in blueberry, expressed as FPKM, in different tissues of Ornablue (M+) and Concord (M‐). To identify the genes in the Draper genome associated with the orthologs identified in Ornablue and Concord, the Ornablue or Concord transcripts were aligned to the Draper genome using GMAP (v.20130509), and annotated genes located in the aligned regions of the Draper genome were then analyzed for their gene expression (FPKM). FPKM values were based on alignment of the RNA‐Seq reads to the V. corymbosum cv. Draper genome (Colle et al., 2019) using HISAT2 (v.2.2.1) and quantified using StringTie (v.2.2.0). The Cultivar column indicates which genotype the ortholog was identified in. For GOR and ISY the ortholog mapped to the same gene in the Draper genome. Transcript abbreviations: GES—geraniol synthase; G8H—geraniol 8‐hydroxylase; GOR – 8‐hydroxygeraniol oxidoreductase; ISY – iridoid synthase; IO – iridoid oxidase; 7‐DLGT – 7‐deoxyloganetic acid glucosyltransferase. Sample acronyms: O – Ornablue; C – Concord; FB – floral buds; St – stems; UF – unripe fruits; RF – ripe fruits; YL – young leaves; ML – mature leaves.

Table S7. Transcript expression, expressed as FPKM, of different tissues of Ornablue (M+) and Concord (M‐). RNA‐Seq reads were mapped to the V. corymbosum cv. Draper genome (Colle et al., 2019) using HISAT2 (v.2.2.1) and transcript quantification was performed using StringTie (v.2.2.0). Sample names are the same as in Table S1.

Table S8. Top 200 co‐expressed genes with iridoid synthase in blueberry. Co‐expressed genes were identified by mutual rank analysis.

Table S9. Codon‐optimized DNA sequences of blueberry iridoid synthase. Sequences from the cultivars Ornablue (M+) and Concord (M‐) were codon optimized for E. coli.

Figure S1. Alignment of the amino acid sequences of C. roseus iridoid synthase (CrISY) and blueberry iridoid synthase (VcISY) from the monotropein‐positive cultivar Ornablue and monotropein‐negative Concord. Alignment was done using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).

Figure S2. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) analysis of purified iridoid synthase (ISY) from heterologous expression in E. coli . (A) Ornablue ISY; (B) Concord ISY. Lanes: 1 – lysate after sonication of cells; 2 – pellet after centrifugation of lysed cells; 3 – supernatant after centrifugation of lysed cells; 4 – proteins unbound to Ni‐nitrilotriacetic acid agarose; 5 – His‐tagged ISY; M – marker (PageRuler™ Prestained Protein Ladder). Protein samples loaded in lane 5 are 4 μg and 5 μg for A and B, respectively.

Figure S3. Gas chromatography–mass spectrometry analysis (GC‐MS) of in vitro iridoid synthase (ISY) assay reaction products showing the formation of c,t‐nepetalactol and iridodials. (A) Total ion chromatograms of c,t‐nepetalactol (reference standard) and reaction products with ISY from Ornablue (M+) and Concord (M‐) in the presence of NADPH. (B) Mass spectra of peaks 1 and 2 labeled in panel A. Chromatograms and mass spectra were generated using the Agilent Enhanced ChemStation software.

Figure S4. Monotropein content in Ornablue (M+) and Concord (M‐). Metabolite data is shown as concentration (ng/mg dry weight) in different tissues. The monotropein concentration in Concord tissues is below 10 ng/mg. Redrawn from Leisner et al., 2017.

Figure S5. Transcript expression of UDP‐glucuronosyl/UDP‐glucosyltransferase (UGT) in different tissues of Ornablue (M+) and Concord (M‐). UGT (gene ID: maker‐VaccDscaff2‐snap‐gene‐409.60) was identified to be co‐expressed with iridoid synthase using mutual rank analysis (Table 2). The expression data, expressed as FPKM, was taken from Table S7.

Lawas, L. M. F. , Kamileen, M. O. , Buell, C. R. , O'Connor, S. E. , & Leisner, C. P. (2023). Transcriptome‐based identification and functional characterization of iridoid synthase involved in monotropein biosynthesis in blueberry. Plant Direct, 7(7), e512. 10.1002/pld3.512

DATA AVAILABILITY STATEMENT

The data set supporting the results of this manuscript is available in the Sequence Read Archive (http://www.ncbi.nlm.nih.gove/sra) under BioProject PRJNA477303.