Genetic and Phytochemical Characterization of Lettuce Flavonoid Biosynthesis Mutants

We previously developed red lettuce (Lactuca sativa L.) cultivars with high flavonoid and phenolic acid content and demonstrated their anti-diabetic effect. Here we report on developing three fertile and true-breeding lettuce lines enriched with flavonoids with reported beneficial health effects. These lines were identified in a segregating population of EMS-mutagenized red lettuce and characterized biochemically and genetically. Change in red coloration was used as a visual indicator of a mutation in a flavonoid pathway gene, leading to accumulation of flavonoid precursors of red anthocyanins. Pink-green kaempferol overproducing kfoA and kfoB mutants accumulated kaempferol to 0.6–1% of their dry weight, higher than in any vegetable reported. The yellow-green naringenin chalcone overproducing mutant (nco) accumulated naringenin chalcone, not previously reported in lettuce, to 1% dry weight, a level only observed in tomato peel. Kfo plants carried a mutation in the FLAVONOID-3′ HYDROXYLASE (F3′H) gene, nco in CHALCONE ISOMERASE (CHI). This work demonstrates how non-GMO approaches can transform a common crop plant into a functional food with possible health benefits.


Isolation of flavonoid biosynthesis mutants. An ethyl methanesulfonate (EMS)-mutagenized cv.
Firecracker red leaf lettuce segregating population derived from seeds of self-pollinated mutagenized plants was screened for anthocyanin (cyanidin 3-O-malonylglucoside) loss manifested by changes in color. 1522 mutagenized (M1) plants were grown from seed mutagenized by 0.10 or 0.15% EMS, selfed, and the mature dry inflorescences collected to obtain the M2 segregating population. 136 M1 lines were sterile. Seed from the remaining 1386 M1 lines were planted (12 seeds per plant, if available) in growth chambers equipped with cool fluorescent lights emitting high levels of both photosynthetically active radiation and ultraviolet (UV); lighting conditions known to induce strong anthocyanin accumulation, and, thus, red color. Forty-three lines harboring color variants were identified visually. Methanolic extracts of the twenty most prominent color mutants were biochemically profiled using an Ultra Performance Liquid Chromatography -Tandem Mass Spectrometer (UPLC-MS/MS) system. Three mutants were selected for further studies.
Pink-green kaempferol overproducer kfoA had high levels of kaempferol glycosides (mostly kaempferol 3-O-malonylglucoside, low amounts of kaempferol 3-O-glucoside and kaempferol 3-O-glucuronide) but lacked quercetin or cyanidin derivatives. Another kaempferol overproducer, kfoB, accumulated the same kaempferol derivatives as kfoA, and had low but detectable cyanidin and quercetin glycoside content. The yellow-green naringenin chalcone overproducer nco line had high levels of glycosylated compounds (hexosides and malonylhexoside) ( Supplementary Fig. S1g,h,i), with a shared aglycone ion of m/z 273 [M + H] ( Supplementary Fig. S1m,n), which corresponds to the isomers naringenin chalcone (Supplementary Fig. S1l) and naringenin ( Supplementary  Fig. S1j). However, naringenin and naringenin chalcone have characteristically different UV spectra, naringenin chalcone having its absorption maximum at 365 nm ( Supplementary Fig. S1q), and naringenin at 289 nm ( Supplementary Fig. S1o). Additionally, the UV absorbance spectra of naringenin glycosides and naringenin chalcone glycosides are similar to the spectra of their aglycones 27 and Supplementary Fig. S1p. Both glycosides in nco lettuce had the characteristic UV absorbance spectra of naringenin chalcone ( Supplementary Fig. S1r,s). Based on these data and on genetic data below, we concluded that nco lettuce accumulated naringenin chalcone glycosides. Nco lacked detectable kaempferol or cyanidin derivatives and had greatly reduced quercetin level compared to cv. Firecracker. Supplementary Fig. S2 shows major peaks of cv. Firecracker, kfo and nco extract chromatograms. Accumulation of high levels of kaempferol or naringenin chalcone is a novel trait in lettuce 28 , therefore, kfoA, kfoB and nco were further characterized. Figure 2 shows representative photos of 15-week old cv. Firecracker, kfoA, kfoB and nco plants grown under UV-emitting, cool fluorescent lights. Under these conditions, Firecracker plants were deep red (Fig. 2a,b), kfoA (Fig. 2c,d) and kfoB (Fig. 2e,f) were pink-green, and nco (Fig. 2g,h) were yellow-green color. All mutants grew slower than wild type cv. Firecracker plants under fluorescent lights (UV light intensity 0.4 ± 0.1 mol/m 2 d), a trait previously described in A. thaliana flavonoid biosynthesis mutants 29,30 . KfoA and nco accumulate high amounts of flavonoid compounds missing from parental line cv. Firecracker. KfoA, kfoB and nco mutants and wild type cv. Firecracker were grown under identical conditions illuminated by cool fluorescent lights and subjected to further UPLC-MS/MS analysis. Leaves were harvested from 18-week old plants, lyophilized, mixed with HCl-acidified methanol, and subjected to acid hydrolysis, based on the method of Hertog et al. 31 . This treatment results in the removal of glycosylation from all flavonoids and chalcones, allowing for the quantification of aglycones, or, in case of nco, their derivatives using UPLC-MS/MS (Table 1). Supplementary Fig. S3 shows representative chromatograms of cv. Firecracker, kfoA, kfoB and nco acid hydrolyzed extracts.
The anthocyanin cyanidin and the flavonol quercetin were detected in cv. Firecracker extracts, as expected in red leaf lettuce 14,15,17 . Additionally, low levels of pelargonidin were observed (Table 1). In kfoA plants cyanidin and quercetin were not detectable. Instead, accumulation of the flavonol kaempferol and the anthocyanidin pelargonidin was observed. While kaempferol has been reported in lettuce 28,[32][33][34] , kfoA plants accumulated >10 mg kaempferol/ g dry weight, or ~103 mg kaempferol/100 g fresh weight, two orders of magnitude higher than previously reported. Additionally, kfoA and kfoB plants contained more pelargonidin (0.33 and 0.60 mg pelargonidin/g dry weight), the predominant anthocyanin in strawberries 35 , than cv. Firecracker (<0.2 mg pelargonidin/g dry weight). KfoB plants accumulated >6 mg kaempferol/g dry weight, lower than kfoA. However, they accumulated more pelargonidin than kfoA, and contained quantifiable cyanidin and quercetin. To our best knowledge, this is the first report on the accumulation of pelargonidin in lettuce leaves.
Nco acid hydrolyzed extracts lacked cyanidin, kaempferol or pelargonidin, but contained >10 mg naringenin/g dry weight, and, on average, 0.6 mg quercetin/g dry weight. As naringenin chalcone glycosides, but not naringenin glycosides were observed in non-hydrolyzed nco extracts (see previous section), we tested the effect of acid hydrolysis on pure naringenin chalcone and observed full conversion to naringenin. Therefore, the levels of naringenin in hydrolyzed extracts of nco correspond to the levels of naringenin chalcone glycosides in the plant. Small amounts of quercetin observed in nco were also likely derived from naringenin formed spontaneously in planta from naringenin chalcone, as naringenin chalcone can spontaneously isomerize by C ring closure to naringenin 36 . To our best knowledge, naringenin chalcone has not been described in lettuce before.
Total polyphenol levels were measured in ten plants per line, using a modified Folin-Ciocalteu assay 17 . Wild type cv. Firecracker and kfoB both had 45 mg gallic acid equivalent/g dry weight. KfoA and nco plants had somewhat lower total polyphenol levels: 23 and 36 mg gallic acid equivalent/g dry weight, respectively (Table 1).
Nco is a chalcone isomerase mutant. The nco flavonoid profile (Table 1) resembled A. thaliana tt5 null mutants, which have nonfunctional CHALCONE ISOMERASE (CHI), an enzyme that converts naringenin chalcone to naringenin 29,37 . Therefore, primers designed based on lettuce Expressed Sequence Tags (ESTs) homologous to the A. thaliana CHI gene (TAIR AT3G55120) were used to amplify the full coding sequence (CDS) of the putative lettuce CHI from cDNA in cv. Firecracker and nco. The wild type cv. Firecracker CHI (CHI+, NCBI MG981123) was predicted to code for a 235-amino acid protein, and the CDS was identical to XM_023891334, a predicted CHALCONE ISOMERASE from green crisphead lettuce cv. Salinas. Additionally, it was identical to LG9_805610, identified as the only CHI expressed (of two putative CHI genes) in the lettuce genome 11 . Nco plants were homozygous for an allele (chi1, NCBI MG981124) that harbors a premature stop codon caused by a G to A mutation in codon 120, truncating the CHI enzyme. The CHI1 truncated protein lacks two conserved residues of the naringenin binding cleft, as well as a residue of the active site hydrogen bond network 38 ; therefore, it is expected to be nonfunctional.
Of the M2 population, one nco mutant and 4 wild type siblings were genotyped. The mutant was homozygous for the chi1 allele, whereas wild type plants were heterozygous or homozygous for CHI+ allele. The M2 mutant www.nature.com/scientificreports www.nature.com/scientificreports/ and its wild type red siblings were selfed, and segregation ratios in M3 individuals were observed. In addition, selfed seed from two M3 mutants were planted. (Table 2; Supplementary Table S1 for segregation ratios of individual parents). Homozygous chi1 mutants always produced yellow-green offspring, heterozygotes produced yellow-green and red offspring, and homozygous CHI+ plants always produced red offspring, indicating that the mutant allele is recessive and responsible for the observed phenotype.
We then genotyped 5 mutants and 14 wild-type siblings from self-pollinated offspring of nco M2 plants (M3 generation) and found that only yellow-green mutants were homozygous for chi1 (Fig. 3a). Additionally, we www.nature.com/scientificreports www.nature.com/scientificreports/ amplified the full CDS of the putative lettuce F3H gene in cv. Firecracker and nco from cDNA using primers designed based on lettuce ESTs homologous to the A. thaliana FLAVANONE-3-HYDROXYLASE (F3H) gene (TAIR AT3G51240). F3H converts naringenin to dihydrokaempferol (Fig. 1), and, in A. thaliana, f3h mutants accumulate a mix of chalcones, flavonols and anthocyanins 39 . As in Arabidopsis, F3H in lettuce is a single-copy gene 11 . The F3H coding sequence of nco was found to be identical to that of cv. Firecracker. Our data suggest that though two putative CHI copies exist in the lettuce genome 11 , losing both functional copies of the LG9_805610 gene leads to low anthocyanin (yellow-green) phenotype, and that nco is a chi mutant.
KfoA and kfoB are flavonoid 3′-hydroxylase mutants. The kfoA flavonoid profile (Table 1) resembled Arabidopsis thaliana tt7 null mutants, which have a nonfunctional FLAVONOID-3′ HYDROXYLASE (F3′H) enzyme 40,41 . Therefore, primers designed based on lettuce ESTs homologous to the A. thaliana F3′H gene (TAIR AT5G07990) were used to amplify the full CDS of the putative lettuce F3′H in cv. Firecracker, kfoA and kfoB. The wild type cv. Firecracker F3′H (F3′H+, NCBI MG981125) was a gene containing three exons, predicted to code for a 512-amino acid protein, and was identical to XM_023887166, a predicted FLAVONOID 3′-MONOOXYGENASE-LIKE gene from green crisphead lettuce cv. Salinas. Additionally, it was identical to LG5_471950, one of five putative F3′H genes in the lettuce genome identified by Zhang et al. 11 .
KfoA plants were homozygous for an allele (f3′h1, NCBI MG981126) harboring a G to A mutation in the splice acceptor site of intron 2, while kfoB plants were homozygous for an allele (f3′h2, NCBI MG981127) harboring a premature stop codon caused by a C to T mutation in codon 233. Translated kfo F3′H proteins harbor the CR1 active site (amino acids 171-186) responsible for the hydroxylating activity, but lack three substrate recognition sites as well as the EXXR motif necessary for core stabilization 42 , thus, it is expected that both kfoA and kfoB mutant F3′H proteins are nonfunctional. Of the M2 population, one kfoA mutant and seven wild type siblings were genotyped, as well as one kfoB and five wild type siblings. Mutants were homozygous for f3′h1 or f3′h2, while wild type plants were heterozygous or homozygous for the wild type F3′H+. Mutant plants and wild type red siblings were selfed, and segregation ratios in M3 and M4 individuals were observed (Table 2; Supplementary Table S1 for segregation ratios of individual parents). Homozygous f3′h mutants always produced pink-green offspring, heterozygotes produced pink-green and red offspring, and homozygous wild type plants always produced red offspring, indicating that the mutant alleles are recessive and responsible for the observed phenotype. Genotyping 15 mutants and 5 wild-type siblings from self-pollinated offspring of kfoA M2 plants (M3 generation), we found that only pink-green mutants were homozygous for f3′h1 (Fig. 3b). Our data suggest that kfoA and kfoB are mutants for one of the five F3′H gene copies in lettuce (LG5_471950 in 11 ), and that this gene is predominantly responsible for the synthesis of dihydroquercetin, a precursor in anthocyanin biosynthesis in wild type cv. Firecracker.  Table 1. Flavonoid aglycones in 18-week old red cv. Firecracker, kfoA, kfoB and nco lettuce grown under cool fluorescent lights. Acid hydrolysis was used to convert compounds to aglycones. Mean mg compound/ g dry leaf weight, and, in parenthesis, as mg compound/ 100 g fresh leaf weight ± standard deviation is shown for cv. Firecracker (n = 6), kfoA, kfoB and nco (n = 10). Pelargonidin was quantified in cyanidin equivalents. Total polyphenol content is calculated as gallic acid (GA) equivalent in mg GA/ g dry leaf weight. BQ, below quantification limits. Naringenin chalcone glycosides were converted to naringenin during acid hydrolysis. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Nco lettuce plants have a characteristic yellow-green leaf color due to the accumulation of yellow-colored naringenin chalcone glycosides and the lack of red anthocyanins. Naringenin chalcone has not been reported in lettuce before. Tomato (Solanum lycopersicum) skin is the best-known food source of this compound, where it accumulates up to 1% dry weight 43 , a level similar to that of nco lettuce. Naringenin chalcone is anti-inflammatory, anti-allergic (e.g. 44,45 ) and anti-obesity 46 in vitro and in vivo, and was found to improve symptoms of perennial allergic rhinitis in a clinical trial 47 . Therefore, nco lettuce could be a useful dietary source for naringenin chalcone, with one US leaf lettuce serving of 85 g containing ~79 mg of the compound.
The color phenotype in nco lettuce is caused by a nonsense mutation in the CHI gene. To our knowledge, nco is the first chi mutant in lettuce. While CHI is ubiquitous in higher plants, chi mutants have been characterized from just a handful of species, with individual flavonoids from these mutants not quantified. Chi mutants www.nature.com/scientificreports www.nature.com/scientificreports/ have been described in ornamental flowers such as Petunia hybrida 48 , Callistephus chinensis 49 and Dianthus caryophillus 50 , in crops such as barley (Hordeum vulgare) 51,52 , rice (Oryza sativa) 53 and onion (Allium cepa) 54 and in A. thaliana 29,37,55 . In all species, the chi mutant phenotype results in yellowish tissues: hull and internodes in rice 53 , bulb color in onion 54 , petals in C. chinensis 49 and D. caryophillus 50 , seed coat in A. thaliana 56 and pollen in P. hybrida 48 . P. hybrida chi mutants accumulate naringenin chalcone as aglycone 48 , while C. chinensis 49 , D. caryophillus 50 and barley (Hordeum vulgare) 51 chi mutants accumulate naringenin chalcone 2′-glucoside (isosalipurposide). Detailed metabolome analysis of A. thaliana chi mutants revealed the presence of multiple naringenin chalcone glycosides 37 , while in chi onion 54 and rice 53 the compound responsible for the yellowish or golden color was not identified. We found that nco lettuces accumulate naringenin chalcone hexoside and malonylhexoside, but not the aglycone, similarly to most chi mutants.
Like nco lettuce, chi mutants of A. thaliana 30,37 , P. hybrida 48 , C. chinensis 49 and D. caryophyllus 50 had low but detectable levels of flavonols. In Arabidopsis, Peer et al. 39 hypothesized that spontaneous isomerisation of naringenin chalcone in planta to naringenin, the substrate of the next enzyme in the anthocyanidin biosynthesis pathway, F3H, was responsible for the presence of flavonols.
Kfo phenotypes were caused by mutations in the F3′H gene: a mutation of the intron 2 splice acceptor site in kfoA, and a nonsense mutation in kfoB. In lettuce, no f3′h mutant has been described, but in a study of 240 lettuce accessions five genes were identified as F3′H, three of which were expressed and two of which (including the F3′H gene mutant in kfoA and kfoB) carried expressed Qualitative Trait Loci (eQTL) for flavonoid composition 11 . The lettuce f3′h phenotype is very similar to A. thaliana f3′h mutants (called tt7), which accumulate kaempferol, and the anthocyanin pelargonidin 40,41 that differs from cyanidin by the lack of 3′-hydroxylation. Kfo f3′h mutant lettuces also accumulate pelargonidin, although pelargonidin levels in kfo are much lower than cyanidin levels in red parent line cv. Firecracker (Table 1). This difference suggests reduced substrate specificity of the DFR enzyme for its substrate in f3′h mutants, dihydrokaempferol, compared to its substrate in wild type lettuce, dihydroquercetin (Fig. 1). Interestingly, f3′h mutants in morning glory (Ipomoea ssp.) accumulate pelargonidin derivatives producing magenta, pink or fuschia flowers 63 . In carnation (D. caryophillus), f3′h mutants have pink petals, accumulating a pelargonidin glycoside, while plants with functional F3'H have purple petals accumulating a cyanidin glycoside 64 .
In plants, many environmental stresses trigger the accumulation of antioxidants including flavonoids and other phenolics 65 . Flavonoids are hypothesized to act as UV absorbers and reduce the levels of damaging reactive oxygen species 66 . In lettuce, exposure to UV or blue light increases flavonoid levels, but reduces yield (e.g. [67][68][69][70][71][72][73] ). This effect was observed during different months in the field growth season 71 , and in field 67,69,70 and greenhouse 68 experiments, where levels of UV exposure were controlled using UV-blocking cover foils, as well as in controlled growth chambers supplemented by UV or blue light emitting LED diodes 72,73 . UV-induced increase in flavonoid and total phenolic content was observed across different green and red cultivars 74 , indicating that it is a universal phenomenon in lettuce. Armas Gutierrez 75 reported that continuous exposure to cool fluorescent lights resulted in high accumulation of total phenolics, total antioxidants and total anthocyanins. Therefore, in our experiment, we replicated the growth conditions optimal for high phenolic content determined by Armas Gutierrez 75 .
A. thaliana flavonoid biosynthesis mutants are more sensitive to high UV 29,30,76 and visible light stress 77 than wild type plants. As in lettuce, wild type A. thaliana plants have a decreased rate of biomass accumulation under high UV stress compared to low UV conditions, but the effects are more severe in flavonoid biosynthesis mutants 29,30 . This sensitivity has been attributed to enhanced photoinhibition 77 and increased lipid and protein peroxidation 76,77 in mutants lacking flavonoids that absorb UV and scavenge reactive oxygen species. However, the UV sensitivity of the different A. thaliana mutants is not equal. The kaempferol-accumulating f3′h mutant is less UV sensitive than chs, chi and f3h mutants, which accumulate low levels of flavonols 30 . We found that nco and kfo lettuces grew somewhat slower than wild type cv. Firecracker plants under UV-emitting cool fluorescent lights, though we did not observe visible growth retardation under greenhouse conditions (natural light plus supplemental white light). Under cool fluorescent lights, kfo (f3′h) lettuce grew faster than nco (chi) but not as fast as wild type cv. Firecracker, similarly to A. thaliana f3′h and chi mutants 30 . Potentially, desirable high biomass and flavonoid levels could be obtained by growing nco and kfo under low UV conditions, and subjecting them to higher levels UV or blue light before harvest. 3-day supplemental UV treatment for 16 h/day has significantly increased total anthocyanin and antioxidant levels in red leaf lettuce, with no effect on the total leaf biomass 73 . Similarly, 6-day pre-harvest exposure to UV resulted in a 4.6x increase in total anthocyanin content and 2.3x www.nature.com/scientificreports www.nature.com/scientificreports/ increase in total phenolic content in red leaf lettuce field grown under UV-blocking foil 70 , while total biomass of these plants was not significantly different from those not exposed to UV.
In conclusion, we created and characterized flavonoid biosynthetic mutants in lettuce with potential health benefits. The modified flavonoid profile characterized by record high accumulation of kaempferol and naringenin chalcone may transform lettuce into a food with health benefits. However, animal studies and human clinical trials will be needed to confirm the health benefits of the high flavonoid lettuce varieties described here. Innovative mutagenizing and selection strategies producing higher levels of beneficial phytochemicals could be an important strategy for adding value added output traits to common crops. Methods eMs mutagenesis of cv. Firecracker lettuce seeds. Lettuce cv. Firecracker (Johnny's Selected Seeds) seeds were mutagenized with 0.10 or 0.15% EMS, using the protocol in 75 . In short, seeds (M0) were soaked in distilled water containing 0.1% or 0.15% (v/v) EMS and incubated for 12 h at room temperature in a rotary shaker. Thereafter, the EMS solution was decanted, the seeds were washed five times with 50 ml of distilled water and dried. Mutagenized M1 seeds were planted and grown under standard greenhouse conditions at the Rutgers New Jersey Agricultural Experiment Station (NJAES) glass research greenhouse under the following settings: 25 °C/19 °C day/night temperature, 16 h light/8 h dark photoperiod with natural light supplemented with 400 W high pressure sodium lamps. Inflorescences were individually collected from 1,522 mature M1 plants, and the M2 seed was threshed, dried out in the greenhouse and placed in paper coin envelopes. The envelopes were placed in re-sealable plastic storage bags with desiccant and stored at 4 °C. To determine the flavonoid composition of putative color mutants, extracts were prepared from lyophilized and ground leaf tissue, using the method described in 18 . In short, leaves were kept at −80 °C prior to lyophilization. Freeze-dried leaves were ground to a fine powder with a mortar and pestle. 50 or 100 mg lyophilized leaf powder was placed in a 15 ml plastic tube, protected from light, then, respectively, 1.5 ml or 3 ml solvent (methanol/water/ acetic acid; 85:14.5:0.5 v/v), was added. The leaf powder was vortexed with the solvent for 30 sec, sonicated for 5 min, vortexed for another 30 sec, kept for 10 min at room temperature, and centrifuged at 1700 rcf for 5 min. The supernatant was decanted, then the extraction was repeated twice, and the decanted extracts pooled. The decanted solution was centrifuged at 1700 rcf for 8 min and filtered through 0.45 µm polytetrafluoroethylene (PTFE) filters (Fisher Scientific) for UPLC-MS/MS analysis.
Extracts were separated and analyzed by a UPLC-MS/MS system using the protocol described in 78 . Since this protocol results in the co-elution of chlorogenic acid and cyanidin 3-O-malonylglucoside, we used a modified gradient elution to separate these two compounds. For this protocol, the mobile phase consisted of two components: Solvent A (0.5% ACS grade acetic acid in double distilled de-ionized water, pH 3-3.5), and Solvent B (100% acetonitrile). The initial conditions of the gradient were 95% A and 5% B; for 20 minutes the proportion reached linearly 80% A and 20% B. Within the next 3 minutes the proportion was 5% A and 95% B, which was maintained for 4 minutes. Within the following 3 minutes the gradient was adjusted to initial conditions, and 5 additional minutes were included for equilibration before subsequent injections.
Acid hydrolysis, UPLC-MS/MS analysis and quantification of flavonoid aglycones and total polyphenol content. Lyophilized lettuce leaves from 18-week old cv. Firecracker, kfoA, kfoB and nco plants were ground using a mortar and pestle. Fifty mg leaf powder was placed in a plastic tube and subjected to acid hydrolysis, based on the method of Hertog et al. 31 . In short, 4 ml solvent (methanol/water; 62.5:37.5 v/v, 2 g/l tert-butylhydroquinone, Sigma Aldrich) was added, then the mix was acidified with 1.0 ml 6 M HCl, vortexed www.nature.com/scientificreports www.nature.com/scientificreports/ for a few seconds, and placed in a 90 °C water bath for 2 h. Afterwards, 100% methanol was used to make up the volume of the extract to 10 ml. The extract was then sonicated for 5 min, centrifuged at 2500 rpm for 8 min, and filtered through 0.45 μm PTFE filters (Fisher Scientific) for UPLC-MS/MS analysis. Total polyphenol content of the extracts was measured by a modified Folin-Ciocalteu assay 17 based on 79 and 80 .
Extracts were separated and analyzed by a UPLC-MS/MS consisting of the Dionex ® UltiMate 3000 RSLC ultra-high-pressure liquid chromatography system, consisting of a workstation with ThermoFisher Scientific's Xcalibur v. 4.0 software package combined with Dionex ® 's SII LC control software, solvent rack/degasser SRD-3400, pulseless chromatography pump HPG-3400RS, autosampler WPS-3000RS, column compartment TCC-3000RS, and photodiode array detector DAD-3000RS. After passing through the photodiode array detector, the eluent flow was guided to a Q Exactive Plus Orbitrap high-resolution high-mass-accuracy mass spectrometer (MS). Mass detection was full MS scan with low energy collision induced dissociation (CID) from 100 to 1000 m/z in either positive, or negative ionization mode with electrospray (ESI) interface. Sheath gas flow rate was 30 arbitrary units, auxiliary gas flow rate was 7, and sweep gas flow rate was 1. The spray voltage was 3500 volts (−3500 for negative ESI) with a capillary temperature of 275 °C. The mass resolution was 140,000. Column and run conditions were identical to 78 apart from that the average pump pressure was 3900 psi for the initial conditions.
Putative formulas of flavonoids and other compounds were determined by isotope abundance analysis on the high-resolution mass spectral data with Xcalibur v.4.0 software and reporting the best fitting empirical formula. Database searches were performed using reaxys.com (Elsevier RELX Intellectual Properties SA) and SciFinder (American Chemical Society).
Nucleic acid isolation, and genotyping kfo and nco lettuces. Total cellular DNA was isolated from leaves of lettuces grown in growth chambers, using a modified cetyltrimethylammonium bromide (CTAB) method 81 . Total RNA was isolated using the QIAGEN RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer's instructions. Nucleic acids were quantified using a NanoDrop UV-Vis spectrophotometer (Thermo Fisher Scientific). cDNA synthesis was performed from total RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. Primers (Supplementary Table S2) were designed based on lettuce ESTs or genomic DNA homologs of A. thaliana CHI (TAIR AT3G55120), F3′H (TAIR AT5G07990) and F3H (TAIR AT3G51240). PCR-amplification of the full CDS of F3H and CHI was performed on nco cDNA, and of F3′H was performed on kfoA and kfoB genomic DNA, with the following PCR program: 5 min at 94 °C; 34 cycles of 30 sec at 94 °C, 30 sec at 60 °C, 90 sec at 72 °C; 10 min at 72 °C. PCR products were treated with ExoSAP-IT (Affymetrix), and Sanger sequenced. Raw sequence reads were assembled using SeqMan Pro (DNASTAR). Of the M2 generation, one kfoA mutant and seven wild type siblings, as well as one kfoB and five wild type siblings were genotyped for their F3′H alleles. Fifteen kfoA mutants and five wild-type siblings were genotyped for their F3′H alleles in the M3 generation. One M2 generation nco mutant and four wild type siblings were genotyped for their CHI alleles. Five nco mutants and fourteen wild-type siblings were genotyped for their CHI alleles in the M3 generation.