ELEVATION OF CONDENSED TANNINS IN THE LEAVES OF Ta

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Elevation of Condensed Tannins in the Leaves of Ta-MYB14‑1 White Clover (Trifolium repens L.) Outcrossed with High Anthocyanin Lines Marissa B. Roldan,*,† Greig Cousins,‡ Karl Fraser,†,§ Kerry R. Hancock,∥ Vern Collette,⊥ Jerome Demmer,# Derek R. Woodfield,‡ John R. Caradus,⊗ Chris Jones,× and Christine R. Voisey*,† †

AgResearch Limited, Palmerston North 4442, New Zealand PGG Wrightson Seeds Ltd., Palmerston North 4442, New Zealand § Riddet Institute, Massey University, Palmerston North 4442, New Zealand ∥ University of Southern Queensland, Toowoomba, Queensland 4350, Australia ⊥ Plant and Food Research, Palmerston North 4442, New Zealand # Halcyon Bioconsulting Ltd., Auckland 0571, New Zealand ⊗ Grasslanz Technology Ltd., Palmerston North 4442, New Zealand × International Livestock Research Institute, Nairobi 00100, Kenya

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S Supporting Information *

ABSTRACT: Condensed tannins (CT) are highly desirable in forage as they sequester dietary protein and reduce bloat and methane emissions in ruminants. However, the widely used forage legume white clover (Trifolium repens) only produces CTs in flowers and trichomes and at levels too low to achieve therapeutic effects. Genetic transformation with transcription factor TaMYB14-1 from Trifolium arvense was effective in inducing CTs to 0.6% of leaf dry matter. CT synthesis has been elevated further by crossing the primary white clover transgenic line with wild type genotypes producing the related phenylpropanoids, anthocyanins. CT levels in leaves were highest under the anthocyanin leaf marks associated with the “red midrib” trait; however, there was no evidence for CT accumulation in leaf sections with the “red V” anthocyanin marking. Ta-MYB14-1 was stably inherited in two generations of crosses, and T2 progeny produced up to 3.6-fold higher CTs than the T0 parent. The profile of small CT oligomers such as dimers and trimers was consistent in T0, T1, T2, and BC2 progeny and consisted predominantly of prodelphinidins (PD), with lesser amounts of procyanidins (PC) and mixed PC:PD oligomers. KEYWORDS: white clover, condensed tannins, anthocyanins, pasture bloat, R2R3MYB transcription factor

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nematodes15−19 and deliver an antiherbivore effect to plants.20−22 Importantly, modest concentrations of CTs also reduce greenhouse gas (GHGs) emissions by ruminants.23−25 In 2016, methane (CH4) and nitrous oxide (N2O) emissions from enteric fermentation and nitrogen fertilizer, respectively, accounted for 93.9% of total emissions from the New Zealand agricultural sector, approximately 49% of New Zealand’s total GHG emissions.26 This is an important consideration since CH4 and N2O have higher global warming potential than carbon dioxide (CO2).27 Reduction in rumen CH4 emissions associated with CT-rich forages has been attributed to both direct and indirect effects on rumen methanogens. For example, certain CT building blocks are directly toxic to rumen methanogens28 such as Methanobrevibacter ruminantium,23 and indirect effects occur through their reduction of

INTRODUCTION

Condensed tannins (CTs), also known as proanthocyanidins (PAs), are widely distributed polyphenolic plant secondary metabolites that are oligomers or polymers of two or more flavan-3-ol units. The building blocks of CTs are monomeric flavan-3-ols in the cis or trans configuration, such as (epi)afzelechin, (epi)catechin, and (epi)gallocatechin, respectively (Figure 1). Depending on their composition, these metabolites may provide benefits to human health, particularly therapeutic effects against cancer1−3 and cardiovascular dysfunction.4 CTs are also beneficial in forage-based agricultural systems when incorporated into animal feed in moderate amounts (2−4% of dry weight (DW)). They bind to protein, forming insoluble complexes5,6 that render proteins resistant to degradation by rumen microbes. The CT−protein complexes later disassociate as they pass through the acidic abomasum,5 and the released proteins are then absorbed in the small intestine. The reduction in protein digestion by rumen microbes reduces nitrogen (N) loss through urine, increases N utilization by animals, and improves production of meat and milk as well as animal reproductive performance7−9 and the symptoms of pasture bloat.10−14 Further, CTs have anthelmintic properties and are active against gastrointestinal © XXXX American Chemical Society

Special Issue: Advances in Polyphenol Chemistry: Implications for Nutrition, Health, and the Environment Received: Revised: Accepted: Published: A

February 19, 2019 May 24, 2019 June 26, 2019 June 26, 2019 DOI: 10.1021/acs.jafc.9b01185 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Structures of condensed tannin flavan-3-ol building blocks in the trans (above) and cis (below) configurations.

ruminal crude protein digestibility29,30 and metabolic H2, which is used by methanogens to reduce CO2 to CH4.24 White clover (Trifolium repens L.) is a valuable forage legume in temperate regions due to its high nutritive value31,32 and ability to fix atmospheric nitrogen in symbiosis with Rhizobia bacteria. However, while white clover forage is high in soluble protein,33 it has extremely low levels of CTs in leaves. Consequently, rumen bacteria rapidly degrade white clover protein into ammonia34 (that is voided in urine), resulting in nitrogen loss to the soil. Digestion of soluble white clover protein by rumen bacteria can also cause a stable foam that traps gases in the rumen and may result in bloat10,33 which prevents animal feeding and can be fatal. The development of white clover with 2−4% (of dry matter (DM)) CTs in the leaves8,31 would therefore be of significant benefit to animal productivity and welfare and may also reduce GHG emissions from livestock. White clover CTs accumulate almost exclusively in floral tissues in a developmentally regulated32 manner. Previous research suggests they consist predominantly of oligomers of (epi)gallocatechin ((E)GC) units with a mean degree of polymerization of 5.8.33 In vegetative organs, CTs are restricted only to trichomes on the underside of leaves and are therefore at levels far below that required for therapeutic effects. Conventional methods to elevate CTs in white clover through mutagenesis or interspecific hybridization have not been successful.34 Additionally, utilization of forages such as sainfoin (Onobrychis viciifolia), sulla (Hedysarum coronarium L.), and Lotus corniculatus, which accumulate 2.5−12% DM CTs in their leaves,35 and are considered bloat-safe,5,36−39 have failed due to poor persistence under grazing. Given that white clover is able to produce CTs in leaf trichomes and flowers, genetic modification approaches offer a solution to produce white clover plants that synthesize CTs in the leaves. CTs are end products of the acetate/malonate and phenylpropanoid pathways.40 These pathways are tightly regulated by a family of R2R3-MYB transcription factors (TF) that, together with specific members of the basic-helix− loop−helix (bHLH) and WD-repeat (WDR) families, coordinate the activity of genes involved in the synthesis of several different classes of phenypropanoids, including CTs

and the red pigmented UV protectants, anthocyanins.41,42 Heterologous expression of an R2R3-MYB TF, Ta-MYB14-1, originally cloned from hare’s-foot clover (Trifolium arvense L.), has been shown to induce CT synthesis to levels of 1.8% of DM in the leaves of white clover and alfalfa.43 This was considered a breakthrough toward achieving bloat-safe leguminous forages with the potential to reduce GHG emissions. One transgenic white clover plant produced, line 3320, was also the subject of transcriptomic analysis which confirmed that white clover genes that encode enzymes putatively required for CT synthesis, such as anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR), and the multidrug and toxin extrusion 1 transporter (MATE1), were also upregulated by insertion of Ta-MYB14-1.44 Since the long-term goal is to produce white clover plants that express an average of 2−4% CT of DM in the leaves, the objective now is to elevate CTs in primary transformants by backcrossing to wild type (WT) genotypes that express the appropriate transcription regulators for high CT production. White clover leaves produce a variety of genetically inherited readily distinguishable anthocyanin markings that are regulated by different R2R3-MYB transcription factors.45,46,42 The R2R3-MYB gene Tr-RED LEAF, at the so-called white clover “R” locus, regulates anthocyanin leaf patterns such as “red leaf”, “red midrib”, and “red fleck”.42 Tr-RED V, an R2R3-MYB gene at the “V” locus, regulates the “red V” (Vh2) leaf pattern, an inverted “V” anthocyanin marking near the base of each leaflet.42,46,47 White clover anthocyanin leaf markings correlate closely with the presence or absence of the MYB TF in the corresponding genomes. Since R2R3-MYB TFs are known to activate different branches of the phenylpropanoid pathway, and some also operate as negative regulators, we reasoned that the cocktail of compounds expressed by each progeny plant is expected to be dependent on the TF genes inherited from each of its parents and is likely to vary between parents and their progeny and between siblings. The objective of this study was to investigate whether wild type white clover pollen donors, which differ with respect to their expression of anthocyanin leaf markings, alter the quantity and quality of soluble CTs in the progeny and whether successive cycles of selection for high CTs in leaves B

DOI: 10.1021/acs.jafc.9b01185 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Plant materials and breeding scheme used in this study. (A) Representative examples of trifoliate leaves from T0(3320) (left), WT-LAC (middle), and WT-HAC (right). (B) Crossing scheme. The white and gray boxes show the names and categories, respectively, of the plants and their progenies. T0(3320) was first crossed with two high (HAC) and two low anthocyanin (LAC) genotypes to produce T1 progeny. The plant from each T1-HAC progeny with the highest CT ranking (T1-HAC1a and 2a) was backcrossed to a third wild type (WT) genotype (WT-HAC3) to create BC2 progenies (pBC2-HAC1a and pBC2-HAC2a). T2 progenies were also produced by intercrossing the highest (a) and second highest (b) CT producers from each T1-HAC progeny. The hatched lines show the origin of genotypes used to make the BC2 and T2 progenies. accession JN049641.1 and was identical to it except for a single base (T663G) which results in an amino acid change (N221 K; Supplemental Figure S1A,B). The open reading frame was fused 5′ to the CaMV35S promoter and 3′ to the octopine synthase (OCS) terminator in the binary vector pHZBar_35STaMYB14-1.43 The selectable marker gene, bar, was similarly bordered 5′ with the CaMV35S promoter and 3′ with the OCS terminator (Supplemental Figure S2A). Transgenic shoots were selected on media containing phosphinothricin (Basta) using the previously published method.48 The wild type nontransgenic (WT) white clover genotypes used for crossing experiments had either low (WT-LAC) or high anthocyanin (WT-HAC) accumulation in the leaves. The WT-LAC genotypes had a weak diffuse anthocyanin pigmentation pattern at the base of each leaflet between the “white V” marking and the petiole (Figure 2A, middle panel). The WT-LAC1 and WT-LAC2, genotypes 213001/ 310 and 213001/168, respectively, were obtained from a cross between the white clover cultivar Kopu II and the self-compatible line Crau S8. The WT-HAC pollen donors (WT-HAC1 and 2), used to produce the pT1-HAC1 and pT1-HAC2 progeny, were more intensely pigmented with anthocyanin localization consistent with the expression of two distinct leaf markings within each leaflet, the “red V” and “red midrib” (also called the feathermark)49 (Figure 2A,

elevated average levels in each generation. We also aimed to confirm that the segregation of Ta-MYB14-1 in white clover progeny was consistent with Mendelian inheritance theory and to assess the effect of homozygosity on CT accumulation. Leaves at different developmental stages were quantified for CTs, and CT localization in leaflets was correlated with specific anthocyanin leaf marks such as the “red midrib”, “red fleck”, or “red V”. Finally, we used LC-MS to identify and compare the profiles of monomeric CT building blocks, dimers, and trimers in progeny from different generations. Together these data confirm that the development of white clover primary transgenic plants is an appropriate strategy to breed novel cultivars with efficacious CT characteristics.

2. MATERIALS AND METHODS 2.1. Plant Materials. White clover primary transgenic line T0(3320) (Figure 2A, left) was produced previously by Agrobacterium-mediated T-DNA transfer of the R2R3MYB transcription factor Ta-MYB14-1, originally from T. arvense, into cotyledonary explants.43 The Ta-MYB14-1 gene used was an allelic variant of NCBI C


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leaves using the protocol described previously,51 digested with restriction enzyme HindIII and hybridized with a digoxigenin (DIG)-labeled probe which was synthesized using a plasmid DNA template pHZBAR_35STa-MYB14-1 with the primers given above. Blotting, probe synthesis, hybridization, and detection were carried out as described in the DIG application manual for filter hybridization (Roche Diagnostics, GmbH, Mannheim, Germany). 2.7. Quantitation of Condensed Tannins in White Clover Leaves. Fully expanded white clover leaves were harvested for CT quantitation in two independent samplings (approximately 8 and 12 weeks after the seeds were sown). Leaves were frozen in liquid nitrogen, freeze-dried, and finely ground using a bead mill homogenizer (OMNI Bead Ruptor, VWR OMNI International Inc. CA). Phenolic extraction was carried out as previously described.50,52 Briefly, approximately 10 mg of each powdered sample was extracted in 0.5 mL of aqueous acetone (70% (v/v) acetone, 0.5% (v/v) acetic acid), vortexed, sonicated (Ultrasonic Bath, VWR model SM2200AHT, Ultrasonic PTY Ltd., NSW, Australia) at room temperature for 1 h and centrifuged at 2 500g for 5 min. The pellet was re-extracted as described above, and the supernatants from both extractions were pooled and extracted with a 1.25 volume of chloroform, followed by centrifugation at 2 500g for 5 min. The aqueous solution, which is the crude soluble CT extract, was carefully transferred to a fresh tube. An aliquot of 2.5 μL of CT extract was pipetted in duplicate into a 96-well plate, and 197.5 μL of 0.2% (w/v) DMACA reagent in methanol/hydrochloric acid (1:1 [v/v]) was added and mixed well by pipetting. After incubation for 5 min, the absorbance was read at 640 nm on a VersaMax microplate reader equipped with Softmax Pro software (Molecular Devices LLC) for data acquisition. The CT in the sample was quantified against a standard curve of epigallocatechin (Indofine Chemical Company, Hillsborough, NJ). 2.8. Liquid Chromatography-Mass Spectrometry (LC-MS). To determine CT composition, liquid chromatography-mass spectrometry (LC-MS) was conducted on leaves of the founder plant T0(3320), plus 2 independent genotypes of the T1 (T1-HAC1a and T1-HAC2a), T2 (highest and second highest CT producers in pT2HACa), and BC2 (highest and second highest CT producers in pBC2-HAC1a) generations. The CT levels of the samples used for LC-MS analyses are shown in Supplemental Table S3. The negative control was a null segregant from pT1-HAC2. Freeze-dried leaf material was extracted and analyzed using the previously described method.53 Briefly, leaf tissue (100 mg DM) was extracted with 2 mL of aqueous acetic acid (0.1% [v/v])/methanol (2:8 [v/v]) for 1 h at 4 °C. The debris was pelleted in a microcentrifuge at 15 000g for 10 min. The supernatant was removed and evaporated to dryness under a stream of nitrogen and reconstituted in 500 μL of 10% (v/v) methanol to yield the crude CT extract. The reference sample, a commercial preparation of grape seed extract (Vitis, Good Health Limited, Auckland, New Zealand), was extracted as described above and was included at the start and end of every run to monitor the quality and reproducibility of the mass spectrometry ionization and chromatography. LC-MS analyses were conducted using a Thermo Accela 1250 UHPLC system coupled to a linear ion trap mass spectrometer (Thermo LTQ-XL, both San Jose, CA) with the same column and detection parameters as described previously.54 Briefly, a 5 μL aliquot of sample was injected onto a 150 mm × 2.1 mm Luna C18(2) column (Phenomenex, Torrance, CA) held at a constant 25 °C. The flow rate was 200 μL min−1, and gradient elution was performed with solvent A = 0.1% (v/v) formic acid in water; solvent B = 0.1% (v/v) formic acid in acetonitrile. The mass spectrometer was set for electrospray ionization in the positive mode, with a spray voltage of 4.5 kV and a capillary temperature of 275 °C. Flow rates of nitrogen sheath gas, auxiliary gas, and sweep gas were set (in arbitrary units/ min) to 20, 10, and 5, respectively. The MS was programmed to scan from 150 to 2000 m/z (MS1 scan), then to sequentially perform product ion scans for selected m/z [M + H]+ ions of propelargonidin (PP), procyanidin (PC), and prodelphinidin (PD) monomers and dimer masses and then remeasured with a second method to measure

right panel). The WT-HAC1 and WT-HAC2 were obtained from crosses between the cultivars Kopu II and MFGC accession no. C24328 [4(“red midrib” + “red V” × Kopu II)-F2], and Tribute and MFGC accession no. C24328 [4(“red midrib” + “red V” × Tribute)BC3], respectively. 2.2. Crossing of Transgenic and Nontransgenic Clovers and Seed Planting. All crosses were performed manually under PC2 containment glasshouse conditions. The crossing scheme used in the study is illustrated in Figure 2B. The primary transgenic T0(3320) was crossed with two WT genotypes with high and low anthocyanin markings, WT-HAC1 and 2 and WT-LAC1 and 2, respectively. The first generation (T1) progenies from these crosses were named pT1HAC1 and 2 and pT1-LAC1 and 2. The plants in each T1 progeny were ranked according to CT accumulation from highest to lowest (a to z). The plant from each T1-HAC progeny with the highest CT ranking (T1-HAC1a and 2a) was backcrossed to a third wild type (WT) genotype (WT-HAC3) to create the second generation backcross (BC2) progenies (pBC2-HAC1a and pBC2-HAC2a). The WT-HAC3 is an elite parent plant (HS99/1 R2) of the cultivar Mainstay, which expresses the anthocyanin leaf mark “red fleck”. T2 progeny were made by intercrossing T1-HAC1a and T1-HAC2a (the highest CT producers in each T2 progeny) and T1-HAC1b and T1HAC2b (the second highest CT producers in each T2 progeny). A month after crossing, mature seeds were collected, cleaned and stored at 4 °C until sowing. Prior to sowing, seeds were scarified with sand paper (P120 Grit, Bear Master Painters), placed on moist filter paper in a Petri dish, and incubated at 4 °C overnight to break any seed dormancy. After a further 24−48 h incubation in the dark at 25 °C, pregerminated seeds were sown into peat plugs, transferred to potting mix when true trifoliate leaves emerged, and maintained in the glasshouse as described below. 2.3. Growing Conditions. Plants were maintained and crossed under PC2 containment glasshouse conditions throughout the duration of the study. During the crossing period, in addition to ambient light, plants were also illuminated by artificial light that supplied ∼450 μmol s−1 m−1 of photosynthetically active radiation (PAR) for 18 h per day. The temperature range in the glasshouse was between 15 and 20 °C. 2.4. Reagents. The 4-(dimethylamino) cinnamaldehyde (DMACA) was purchased from Sigma-Aldrich (St. Louis, MO), and (−)-epigallocatechin was from Indofine Chemical Company (Hillsborough, NJ). The buffers, reagents, and detection solutions used for Southern blot hybridization were purchased from Roche Diagnostics (Mannheim, Germany). 2.5. Histochemical CT Assay and Segregation Analysis. A histochemical assay using the chromogenic reagent DMACA was used for qualitative assessments of CT accumulation and localization in plant tissues.50 The assay was routinely carried out in a 48-well plate whereby a leaflet was harvested into each well, soaked immediately in a solution containing ethanol/glacial acetic acid (3:1 [v/v]), and allowed to decolorize for at least 2 h or overnight. Decolorized leaves were then stained in 0.3% (w/v) DMACA in methanol/hydrochloric acid (1:1 [v/v]) for 20 min followed by destaining with several washes of 70% (v/v) ethanol. Leaves that exhibited blue coloration, irrespective of the intensity, were considered positive, and those that remained opaque were considered negative. This assay was also used as a preliminary assessment of Ta-MYB14-1 segregation in progeny plants. Qualitative DMACA assays for Ta-MYB14-1 segregation were confirmed by PCR as described below. 2.6. Molecular Characterization of Plants. DNA for PCR was isolated from young folded white clover leaves using the Geneaid Genomic DNA Mini Kit (Geneaid International, Taiwan) according to the manufacturer’s protocol. For segregation analysis, routine PCR reactions were performed using forward (MR183) and reverse (MR184) primers (Supplemental Table S1) homologous to the CaMV35S promoter upstream of Ta-MYB14-1. An amplification product of 627 bp was obtained using the following cycling conditions: 1× 94 °C for 2 min, 30× [94 °C for 15 s, 55 °C for 30 s; 72 °C for 1 min], and 1× [72 °C for 5 min], 10 °C hold. For Southern blot hybridization, genomic DNA was isolated from young D


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Figure 3. Segregation of Ta-MYB14-1 in T1 progeny and corresponding changes in CT levels. (A) The percentage of T1 segregants in crosses between T0(3320) and WT-LAC1 and 2 (pT1-LAC1 and 2) or WT-HAC1 and 2 (pT1-HAC1 and 2). (B) Fold increase in CT accumulation in leaves of individual plants (purple circles, averaged across two harvest dates). There were two technical replicates per extraction. The population mean is shown as a green circle. Progeny with different letters are significantly different at the 5% level. T0(3320) and the null segregants (−ve) were excluded from the statistical analysis. The individual from pT1-HAC1 with the highest CT fold change was detected as an outlier. combinations of trimers. Isolation windows for each selected m/z value of 2.0 mu and a collision energy of 35% were used, and each sample was measured twice by LC-MS/MS to detect monomers, dimers, and trimers. 2.9. Statistical Analyses. The data on CT quantity in individual plants were routinely averaged across two harvest dates with two technical replicates per extraction. The differences in CT accumulation among progeny groups were determined using Minitab 18 Statistical software (Pennsylvania State University) and Genstat for Windows (18th edition). The data, which were normally distributed, were subjected to one-way analysis of variance, and where differences were significant at P < 0.05, the means were compared using Fisher’s Least Significant Difference, P < 0.05.

integrated into the genome and that it contained a copy of the transgene at a single locus (Supplemental Figure S2B). PCR was also used to amplify the sequences of the CaMV35S promoter, Ta-MYB14-1 gene, OCS terminator, and T-DNA flanking the right (RB) and left borders (LB). Sequencing of the PCR amplicons confirmed that the inserted T-DNA was intact and free of vector contamination and that the TaMYB14-1 gene was an allelic variant of the published sequence (NCBI accession JN049641.1) with a single base pair (T663G) difference which results in an amino acid change (N221 K; Supplemental Figure S1). The primers used are given in Supplemental Table S1. On the basis of the single gene integration event, the progeny from any crosses with T0(3320) were predicted to segregate according to Mendelian principles, assuming no effects of the transgene on seed production, gemination, or plant viability. 3.2. Segregation of Ta-MYB14-1 in T1 Progeny. First generation (T1) progeny seed (pT1-LAC1 and 2 and pT1-

3. RESULTS AND DISCUSSION 3.1. Verification of the Ta-MYB14-1 Integration Locus. The primary transgenic genotype T0(3320), genetically transformed with the R2R3-MYB TF Ta-MYB14-1 from T. arvense, was produced previously as described.43,44 Southern blot hybridization was used to confirm that Ta-MYB14-1 had E


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Figure 4. Quantitation of condensed tannins in leaf sections. (A) Representative images of the anthocyanin marks in leaflets from a pT1-HAC2 genotype (left) and the corresponding DMACA-staining of the same leaflet (right). A null segregant is shown above a Ta-MYB14-1-positive segregant. (B) Trifoliate leaf showing leaf sections (S1−S4) taken for independent CT quantitation. The “red midrib” (S1) was dissected from the tip of each leaflet to the junction with the “red V”. The leaf lamina on either side of the red midrib contained “red fleck” markings (S2). The “red V” was taken from either side of the “red midrib” (S3), and S4 was the green lamina between the petiole and the “red V”. (C) Boxplot of soluble CT accumulation in leaf sections indicated in part B. Each purple circle represents the mean of two technical replicates from three independent genotypes from pT1-HAC2. Pairwise comparison of means was by the Fisher LSD method. Means that do not share a letter are significantly different at P < 0.05. Null segregant controls (negative) were excluded from the statistical analysis.

of Ta-MYB14-1 occurred according to Mendelian principles. Together these results confirmed the stable inheritance of TaMYB14-1 in the T1 generation. 3.3. Quantity of Condensed Tannins in T1 Progeny. Anthocyanins and condensed tannins are metabolites derived from the complex phenylpropanoid pathway,55 and their biosynthesis is dependent on several shared metabolites. We therefore reasoned that white clover genotypes with extensive anthocyanin leaf markings would have a more active phenylpropanoid pathway than genotypes with low anthocyanin marks, and their progeny with T0(3320) may therefore be able to synthesize CTs at higher concentrations. We tested this hypothesis by crossing T0(3320) with two WT genotypes with high and low anthocyanin markings, WT-HAC1 and 2 and

HAC1 and 2) were sown and analyzed for segregation of TaMYB14-1 by DMACA staining and PCR. There were 21 and 18 seed from independent crosses with WT-LAC1 and 2, respectively, and 45 seed each from the crosses with WTHAC1 and 2 (Figure 3A). Positive scores for Ta-MYB14-1 and CT synthesis in leaflets, by PCR and DMACA, respectively, were 100% concordant; however, there was considerable variation with respect to the intensity of staining (data not shown). DMACA staining was therefore a cheap and robust method for scoring progeny for inheritance of Ta-MYB14-1. The data were subjected to a Pearson’s chi-square test and the results (χob2 = 1.72; χtab2 = 9.35, df = 3) confirmed that TaMYB14-1 segregated at a ratio consistent with 1:1 for negative vs positive individuals (Figure 3A), indicating that inheritance F


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phenotype) activated the Tr-DFR promoter, whereas Tr-RED V failed to induce any activity.42 The reported activation of the Tr-DFR promoter by Tr-RED LEAFRm implies that it enhances metabolic flux through the phenylpropanoid pathway, leading to the anthocyanin marks characteristic of the “red midrib”. Furthermore, since Ta-MYB14-1 induces expression of proanthocyanidin-specific genes such as leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS), and anthocyanidin reductase (ANR),43 this MYB may enhance the proportion of metabolic intermediates that are synthesized into flavan-3-ols, the monomeric precursors of CTs. The combined activity of these two MYB proteins in the same host may therefore underlie the increase in CTs observed in the HAC progeny. In contrast, the progeny of LAC parents produced significantly lower CTs compared with HAC progeny (Figure 3B). It is possible that, since LAC parents only express the “red V”, which is a phenotype of Tr-RED V activity, the Tr-DFR promoter may be less active,42 resulting in lower metabolic flux for CT synthesis, even in the presence of TaMYB14-1. The effects of other MYB genes at the V locus on phenylpropanoid pathway genes are untested. Further studies need to be conducted to confirm that Ta-MYB14-1 is being expressed at similar levels in LAC and HAC progenies and that the Tr-RED LEAF and phenylpropanoid pathway genes are more highly expressed in HAC vs LAC progenies. 3.4. Quantitation of Condensed Tannins in Leaf Dissections with Different Anthocyanin Marks. White clover leaves expressing anthocyanin markings consistent with both the R and V loci in the same leaflets (such as in the HAC progeny) offered the opportunity to determine whether the distribution of CTs overlapped with the “red midrib”, “red fleck” (R locus), or “red V” (V locus) marks. Leaves of TaMYB14-1-positive pT1-HAC2 progeny were stained with DMACA to show the distribution of CTs. CT distribution was concentrated along the length of the midrib from the petiole to the leaf tip, consistent with the distribution of the anthocyanin “red midrib” (Figure 4A). CTs were also present in a more diffuse pattern over the leaf lamina (where the “red fleck” was expressed) but, apart from the occasional discrete blue fleck, did not colocalize with the “red V” of the V locus or within the lamina enclosed by it (Figure 4A). Therefore, CT expression was confined to regions of the leaflets expressing the “red midrib” or “red fleck” marks associated with Tr-RED LEAF regulation at the white clover R locus. To confirm these observations, the anthocyanin leaf markings were then dissected (as shown in Figure 4B) and soluble CTs quantified (Figure 4C). The sections taken included the “red midrib” from the junction of the “red V” to the leaf tip (S1), the lamina containing the “red fleck” on either side of the “red midrib” (S2), the “red V” on either side of the “red midrib” (S3), and the green lamina beneath the “red V” to the base of the petiole (S4) (Figure 4B). The same sections were collected from the leaflets of Ta-MYB14-1 null segregants for comparison. The “red midrib” (S1) produced the highest CTs with a mean of 1.39% DW (p < 0.01) followed by the “red fleck” of the leaf lamina (S2) with 0.21% DW, and the lowest sections were the “red V” and the green lamina below, S3 and S4, respectively. These results confirmed the DMACA-staining observations and indicated that the anthocyanin markings associated with the expression of the R locus, such as the “red midrib” and “red fleck”, are required for elevated condensed tannin synthesis in leaves.

Figure 5. Accumulation of CTs in leaves of different ages. (A) Leaf position (L1−L6) along a white clover stolon (a creeping stem). The youngest leaf is at the tip and they become progressively older in the retrograde direction. (B) Quantitation of soluble CTs in leaves from the youngest (L1) to the oldest (L6). Each data point (purple circles) represents the mean of two independent extractions from a pooled sample of milled leaves of the same age from a single genotype. Each genotype was represented by two clonal replicates, and leaves at each position from at least 20 stolons were bulked for the analysis. Means were not significantly different at P < 0.05.

WT-LAC1 and 2, respectively (Figure 2B). The progenies from high anthocyanin parents, pT1-HAC1 and pT1-HAC2, produced mean CTs of 0.78% and 0.74% of DM, respectively (Supplemental Table S2A) while T0(3320) produced 0.61% CTs. In contrast, progenies from the low anthocyanin plants, pT1-LAC1 and pT1-LAC2 produced lower CTs compared to T0(3320) and the HAC progenies, with 0.50% and 0.43% CT of DM, respectively. Mean CT accumulation in pT1-HAC1 and pT1-HAC2 progeny was 1.6−1.7-fold higher than pT1LAC1 and pT1-LAC2, and only pT1-HAC2 was not significantly different from pT1-LAC1 at the 5% level. The highest T1 CT producer (T1-HAC1a) accumulated 1.71% CT of DM (Supplemental Table S2A), 2.5-fold higher than T0(3320) (Figure 3B). In contrast, the highest T1 CT producer in the LAC plants (from pT1-LAC1) accumulated 0.91% CT of DM (Supplemental Table S2A), 1.5-fold relative to T0(3320) (Figure 3B). CTs were not detected in the leaves of null segregants (which did not inherit TaMYB14-1) (Figure 3B). These results show that white clover parents expressing both the “red V” and “red midrib” anthocyanin leaf markings increased the synthesis of CTs in their progeny relative to plants with low anthocyanin markings. Dihydroflavonol reductase (DFR) catalyzes the reduction of dihydroflavonol to leucoanthocyanidin and is the first committed step in the synthesis of anthocyanin and proanthocyanidins.56,57 Functional characterization of white clover R2R3MYB genes linked to the R and V loci has shown that Tr-RED LEAFRm (which conditions the “red midrib” G


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Figure 6. Ta-MYB14-1 segregation and CT accumulation in T2 and BC2 progenies (A) Percentage of BC2 segregants in crosses between the highest (T1-HAC1a and T1-HAC2a × WT-HAC3) CT producers (pBC2-HAC1 and HAC2, respectively) and in T2 segregants in crosses between the highest (T1-HAC1a and T1-HAC2a; pT2-HACa) and second highest (T1-HAC1b and T1-HAC2b; pT2-HACb) CT producers (see Figure 2 for the plant crossing scheme). (B) CT accumulation in leaves of individual plants (purple circles), averaged across two harvest dates. There were two technical replicates per extraction. The population mean is shown as a green circle. Progeny with different letters are significantly different at the P < 0.05 level. The T0, T1 parent, and null segregants (−ve) were excluded from the analysis of variance.

Our results, together with previous findings that proanthocyanin-specific genes are upregulated in white clover by TaMYB14-1,43 suggest that the Tr-RED LEAF MYB, which activates the Tr-DFR promoter, may have contributed to the enhanced CT production in leaf sections with “red midrib” and “red flecks”. In contrast, the “red V” anthocyanin mark (associated with the Tr-RED V MYB at the V locus) does not activate the Tr-DFR promoter.42 This may reduce the flow of substrate in the specific leaf section containing the “red V”. Anthocyanins and condensed tannins share regulatory genes in the early biosynthetic pathway, and the availability of the intermediates may redirect the metabolic flux toward the synthesis of either anthocyanins or the flavan-3-ols which are oligomerized to CTs. Loss of function of ANR in the Arabidopsis banyuls mutant resulted in loss of proanthocyanidins and accumulation of anthocyanins in the seed coat.58,59 Conversely, in a model legume Medicago truncatula, proanthocyanidin production increased by almost 3-fold with

ectopic expression of ANR but resulted in a 50% reduction in anthocyanins.60 A similar result was observed in tobacco,61 suggesting that ANR is specific to proanthocyanidin synthesis. In this study we did not measure anthocyanin concentrations to determine whether expression of Ta-MYB14-1 diverted metabolites from the anthocyanins to CT biosynthesis, but this remains a key objective in the future. 3.5. CT Accumulation in Leaves of Different Ages. To investigate whether the accumulation of CTs is developmentally regulated in white clover, we collected trifoliate leaves of different ages starting with the youngest (unopened) at the stolon tip (L1) to the oldest at the sixth node (L6), which was mature but not yet starting to senesce (Figure 5A). Four independent genotypes were used as biological replicates, and there were two clonal replicates for each genotype. Leaves (at least 20) from the same position of all the stolons on both clonal replicates of each genotype were pooled. Soluble CTs were quantified in two independent extractions from each H


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Figure 7. Extracted ion chromatograms of summed MS2 product ions (in bold below) from the [M + H] parent ions for propelargonidin (PP) building blocks afzelechin and epiafzelechin (275.3 → 139 m/z), procyanidin building blocks catechin and epicatechin (291.3 → 123 + 139 m/z), prodelphinidin (PD) building blocks (307.3 → 139 + 151 m/z), PP dimers (547.3 → 275 + 411 m/z), PP:PC dimers (563.3 → 273 + 275 + 411 m/z), PC dimers (579.3 → 291 + 409 + 427 + 453 m/z), PC:PD dimers (595.3 → 291 + 425 + 443 m/z), and PD dimers (611.3 → 305 + 307 + 443 m/z) measured in leaves of a representative pT2-HACa plant (see Supplemental Table S3 for details).

produce 1.7% (T1-HAC1a) and 1.4% (T1-HAC2a) of CTs, we next pairwise-crossed the two highest and second highest (T1-HAC1b, 1.52% and T1-HAC2b, 1.26%) CT-producing genotypes to create the T2 progenies pT2-HACa and pT2HACb (Figure 2B). The first objective of these crosses was to confirm that the gene had segregated according to expectations in the next generation of progeny. Control crosses (BC2), where the same T1 plants were backcrossed to a high anthocyanin wild type genotype (WT-HAC3), were also produced (Figure 2B). Ta-MYB14-1 segregated approximately 3:1 (positive/negative plants) in the T2 progeny and 1:1 in the BC2 progeny (Figure 6A) as determined by PCR. We analyzed the data using the Pearson’s chi-squared test. The results (χob2 = 0.8008; χtab2 = 9.35, df = 3) confirmed there was no difference between expected and observed values, indicating that the segregation of the gene was consistent with Mendelian patterns of inheritance. All plants that inherited Ta-MYB14-1

pool. There was a trend for increasing CT accumulation from L1 to L6 in two of the four genotypes (Figure 5B). However, mean CTs in leaves at different positions were not significantly different (P < 0.05) when the four genotypes were considered together. These data indicate that leaf age is not a major factor in CT variability in these genotypes. Accumulation of CTs in transgenic white clover leaves and flowers therefore differ in this regard, as CTs in flowers are developmentally regulated, with higher CTs in the older tissues.32 The difference between these tissues with respect to developmental regulation of CTs may be due to the CaMV35S promoter which regulates TaMYB14-1 expression in the transgenic plants, as this promoter may be expressed in a relatively stable way in white clover leaves of different ages, but this remains to be confirmed. 3.6. Segregation of Ta-MYB14−1 in T2 and BC2 progeny of HAC breeding lines. Since the highest T1 plants (containing a single copy of Ta-MYB14-1) were able to I


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Figure 8. Extracted ion chromatograms of summed MS2 product ions (in bold below) from the [M + H] parent ions for (i) 611.3 → 305 + 307 + 443 m/z transition of prodelphinidin (PD) dimer peaks demonstrating the presence of PD dimers in (A) the T0(3320) plant and (B) a null segregant from pT1-HAC2 (negative control), plus representatives of (C) a pT1-HAC1, (D) a pT2-HACa, and (E) a pBC2-HAC1a plant (see Supplemental Table S3 for plant details). The Y-axis is fixed at the same level for all leaf extracts and (ii) 579.3 → 291 + 409 + 427 + 453 m/z transition of procyanidin (PC) demonstrating the presence of PC dimers across the same samples (A−E).

expressed CTs in the leaves to a greater or lesser extent, as was observed in the T1 progeny, as determined by DMACA staining (data not shown). We expected that 33% of the CT positive plants in the T2 progenies would be homozygous (two gene copies) for Ta-MYB-14-1; however, we were not able to test all the plants for the number of gene copies. 3.7. Quantitation of CTs in T2 and BC2 Progenies. To determine whether there was a gene dosage effect on CT accumulation in the white clover leaves, we quantified CTs in leaves from all Ta-MYB14-1 plants in the T2 progenies which segregated 3:1 (we presumed this was made up of 25% homozygous, 50% hemizygous, 25% null segregants) and compared them with plants from BC2 progenies with approximately 50% hemizygous and 50% null segregants (Figure 6A). The pT2-HACa progeny, obtained by crossing the highest T1 CT producers, accumulated significantly more CTs compared to the BC2 progenies and was not significantly different from the T2 progeny pT2-HACb. The progeny from pT2-HACb, a cross between the second highest T1 CT producers, accumulated intermediate CT levels and was not significantly different to the BC2 or pT2-HACa crosses. This suggests that both gene dosage and the amount of CT in the parents contributed to average CT accumulation in the progeny; however, this needs to be confirmed with more crossing experiments. The highest CT-producing genotype (from pT2-HACa) had 3.6-fold higher CT levels than the T0(3320) parent (Figure

6B) and 1.2-fold higher relative to the mean of T1 parents. This plant, plus the highest producer from the pT2-HACb cross, were zygosity tested by crossing them to a nontransgenic wild type plant, and 100% of both progenies tested positive for Ta-MYB14-1 by PCR, indicating both were homozygous for this gene. 3.8. Qualitative Analysis of CTs in Plants from Different Generations. LC-MS analysis revealed that the monomers, dimers, and trimers detected in all plants containing CTs were predominantly composed of prodelphinidin units, along with amounts of procyanidin (catechin and epicatechin). PC and mixed PC:PD oligomers were also detected (Figure 7). There were trace levels of propelargonidin (PP) building blocks (afzelechin and epiafzelechin), a PP dimer and mixed PP:PC dimers were also observed (Figure 7); however, no PP or mixed PP trimers were detected in any samples (data not shown). Similar profiles of monomeric CT building blocks, dimer (Figure 8), and trimers (Supplemental Figure S4) were detected across all generational extracts. LC-MS analysis also provided conclusive evidence of oligomer formation in the intergenerational plants. As the profiles were consistent across all plants containing CTs, and across all intergenerational species, representative examples are shown in Figure 8. This highlights that the relative levels and profile of PD dimer peaks from the original T0(3320) plant extracts (Figure 8A) through to the T2 generation (Figure 8D) are consistent with similar or slightly greater levels in the J



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generational plants when compared to T0(3320). These data show that no CT was detected in the null segregant (Figure 8B), highlighting Ta-MYB14-1 transformation as the key to CT production. In conclusion, our results show that the progeny of transgenic plant T0(3320) when crossed with HAC WT parents accumulated significantly higher condensed tannins in the leaves than progeny from crosses with LAC WT parents, indicating that the anthocyanin marks which condition the “red midrib” at the R locus may be of use in breeding efforts to elevate CT levels in the progeny. When the highest and second highest progeny were pairwise crossed to produce the T2 generation, the full siblings varied greatly with respect to the amount of CTs produced; however, on average, T2 progeny from the highest expressers produced significantly more CTs than pairwise crosses of the second highest expressers and also elevated the levels attained by the most productive genotypes. Furthermore, progeny exhibited Mendelian segregation over two generations. CT levels in the leaves were highest under the anthocyanin leaf marks associated with the “red midrib” traits, but there was no evidence for CTs enhancement under the “red V” which was consistent with observations that the V locus MYBs are not positive regulators of leaf condensed tannins in these white clover genotypes. Similar profiles of monomers and CT dimers and trimers were detected across all generational extracts. We have not yet investigated whether other white clover MYB transcription factors are able to inhibit CT production. Further studies on the role played by the cDNA variants of the Tr-RED V as well as the Tr-RED LEAF and other MYB transcription factors are essential to further understand the role of the MYB proteins in the regulation of CT synthesis in white clover leaves. Taken together, this indicates that the CT trait in Ta-MYB14-1 transformed white clover can be enhanced by breeding with parental lines with the appropriate anthocyanin traits. Further, it should be possible to produce a white clover cultivar with stable elevated foliar CTs that will improve animal health and productivity.

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Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Marissa B. Roldan: 0000-0002-4902-9248 Author Contributions

All authors contributed to experimental design. M.B.R., G.C., K.R.H., and K.F. conducted the experiments. The manuscript was written by M.B.R., K.F., and C.R.V., with editing by all authors. Data analysis was conducted by M.B.R. in consultation with Statistical Scientists, C.M.K. and D.L. (see Acknowledgments). Funding

This work was supported by the Primary Growth Partnership (PGP) Program of the Ministry for Primary Industries, New Zealand, with additional support from PGG Wrightson Seeds Limited, New Zealand, and Grasslanz Technology Limited, New Zealand. Notes

The authors declare the following competing financial interest(s): John Caradus is employed by Grasslanz Technology Ltd., the owner of the patent protecting the TaMYB14 transcription factor. All other authors declare no conflict of interests.

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ACKNOWLEDGMENTS We are grateful to Dr. Zulfi Jahufer for kindly sharing his insights on breeding strategy and for critically reviewing this manuscript. We are also thankful to AgResearch Statistical Scientists Catherine McKenzie and Dr. Donqwen Luo for statistical advice and to Rupinder Kaur for technical assistance.

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ABBREVIATIONS USED CT, condensed tannins; PD, prodelphinidin; PC, procyanidin; T0, primary transgenic plant; T1, first generation progeny; T2, second generation progeny from a pairwise cross (T1 × T1); BC, backcross; DM, dry matter; TF, transcription factor; WT, wild type; LAC, low anthocyanin; HAC, high anthocyanin

ASSOCIATED CONTENT

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01185.

REFERENCES

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Supplemental Figure S1, alignment of nucleotide and translated amino acid sequences of Ta-MYB14-1 previously submitted to NCBI (GenBank JN049641.1) and the Ta-MYB14-1 sequence in the transgenic white clover plant T0(3320); Supplemental Figure S2, TaMYB14-1 transformation vector and Southern blot hybridization of T0(3320); Supplemental Figure S3, representative PCR analysis of the T1 progeny of a cross between T0(3320) and WT-LAC1 showing approximately 50% positive for the transgene; Supplemental Figure S4, extracted ion chromatograms of summed MS2 product ions demonstrating the presence of trimers; Supplemental Table S1, oligonucleotide sequences and corresponding binding sites; Supplemental Table S2A, mean and highest soluble CTs in T1 progeny; and Supplemental Table S3, mean and highest quantity of soluble CTs produced in the leaves of progeny (PDF) K


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