Limited Tyrosine Utilization Explains Lower Betalain Contents in

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Limited Tyrosine Utilization Explains Lower Betalain Contents in Yellow than Red Table Beet Genotypes Minmin Wang, Samuel Lopez-Nieves, Irwin L. Goldman, and Hiroshi A. Maeda J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 09 May 2017 Downloaded from http://pubs.acs.org on May 10, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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RESEARCH ARTICLES

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Title:

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Limited Tyrosine Utilization Explains Lower Betalain Contents in Yellow than Red Table

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Beet Genotypes

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Authors: Minmin Wang†, Samuel Lopez-Nieves†, Irwin L. Goldman§, Hiroshi A. Maeda†,*

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Affiliations:

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†

Department of Botany, University of Wisconsin-Madison, 430 Lincoln Dr. Madison, WI,

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53706, USA

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§

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WI 53706, USA

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*Corresponding author, Hiroshi A. Maeda ([email protected])

Department of Horticulture, University of Wisconsin-Madison, 1575 Linden Drive, Madison,

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ABSTRACT

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Betalains are tyrosine-derived pigments that consist of red-violet betacyanins and yellow

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betaxanthins. These pigments are major sources of natural food dyes in the U.S. Decades of table

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beet breeding efforts have increased betalain pigmentation, but yellow betaxanthin accumulation

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has been lower than red betacyanins. To identify possible bottlenecks in betalain production,

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here we conducted comparative analyses of betalains and their precursor, tyrosine, in various

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beet genotypes. Consistent with previous studies, red beets had much higher betalain

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concentrations than yellow beets. Conversely, tyrosine levels were higher in yellow than red

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genotypes in both table beet and Swiss chard. Interestingly, increased tyrosine levels were

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positively correlated with elevated betalain accumulations among red but not yellow genotypes

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especially at a later developmental stage, suggesting that yellow beets are not efficiently

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converting tyrosine into betalain pigments. Based on these results, we hypothesize that further

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increase in tyrosine production will likely enhance betacyanin accumulation in red beets,

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whereas better utilization of the accumulated tyrosine will be required to further improve

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betaxanthin production in yellow beets.

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KEYWORDS:

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Betalain pigments, tyrosine, betacyanins, betaxanthins, natural pigments, table beets, Beta

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vulgaris

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INTRODUCTION

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Plant natural products, also known as plant secondary metabolites, provide essential components

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of human nutrients and pharmaceuticals. For example, β-carotene and tocopherols are essential

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micronutrients (vitamin A and E, respectively),1–3 while isoquinoline alkaloids provide various

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human medicines (e.g. analgesic morphine, cancer drug noscapine).4–7 Extensive breeding efforts

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have increased the levels of important plant natural products.8–10 Molecular studies further

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identified genes and enzymes directly involved in the production of these secondary metabolites

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which enabled marker assisted molecular breeding and genetic engineering.11–14 However,

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secondary metabolites are produced from primary metabolite precursors (e.g. amino acids), and

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little is known about the roles that upstream primary metabolism plays in the production of plant

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natural products.15,16 This fundamental knowledge gap may limit further improvement of

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secondary metabolite production in plants.

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Betalains are classified as the red-violet betacyanins and the yellow betaxanthins (Figure

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1). These pigments are used to attract pollinators and seed dispersers in plant species in the order

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Caryophyllales, including the genus Beta.17–19 Betalains likely have similar physiological roles to

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anthocyanins in B. vulgaris, though intense betalain coloration in some tissues of crops (e.g. beet

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roots) is due to domestication. In addition, alkaloid pigments, betalains, likely play a role in

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nitrogen storage.20 Recent studies showed that betalains have antioxidant activities21–23 and a

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number of beneficial health-related properties,24 such as induction of Phase II enzymes that

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offers protection against certain cancers,25 inhibition of the expression of Intercellular Adhesion

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Molecule-1, a response factor in the inflammatory reaction, to protect endothelium,26 and

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cytotoxic effects on various types of cancer cells.27 These pigments are currently used as major

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sources of natural red dyes in food industry in the U.S., where heat stability is not a primary

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limitation.28 The use of natural food colorants is becoming popular in recent years due to

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concerns over the safety of synthetic dyes.29 In addition, betalain-based food dyes do not stain

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and thus are sought after for processed foods, as well as frozen and refrigerated dairy products.30

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Among the most well-known betalain pigment producers are table beet (B. vulgaris), cactus (i.e.

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Opuntia ficus-indica), bougainvillea (i.e. Bougainvillea glabra), and four o’clock (Mirabilis

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jalapa). Of these, only table beet and cactus have production acreage devoted to pigment

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production for food and fabric dyes.10

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The group of cultivated types associated with Beta vulgaris, which includes sugar beet,

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table beet, Swiss chard, and mangel, is of Mediterranean origin. Leafy Beta crops, such as Swiss

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chard, were documented in Roman times.31,32 It appears that movement of these crops out of the

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Mediterranean basin into northern Europe was accompanied by selection for the biennial habit

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and swollen roots that could be stored during cold periods. Selection for visual appearance (i.e.,

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root pigmentation) also likely took place during this time, resulting in the red table beet. In more

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recent times, selections for animal feed (e.g., fodder beet) and sugar extraction (sugar beet) have

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focused on size and sucrose content and against color, which could interfere with sugar

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extraction, resulting in unpigmented forms.33,34 Thus, humans selected beet for various colors

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during the breeding history of B. vulgaris.

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Beginning in the 1970s, researchers at the University of Wisconsin-Madison began

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experimenting with approaches to increase native levels of betalain pigments in table beet for use

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of these pigments as natural food dyes.10,35 The concentration process of betalains in beet root

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juice is time and energy intensive, and can contribute to the degradation of color.36 These

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limitations fueled efforts to breed for elevated concentrations of betalains, which have been

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realized in table beet.35 Today, table beet populations have been created that contain betalain

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pigment concentrations at 5-fold higher than standard table beet cultivars.35 Gains were

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substantial during the first sixteen cycles of selection; however, a plateau in pigment

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concentration in later cycles has been noted.35 This observation suggests that alternative

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approaches may be necessary to further increase the concentration of betalain pigments in table

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beet. Beginning in the 1990s, yellow table beet populations were selected for elevated

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betaxanthin concentrations. Although betaxanthin pigment concentrations were increased in

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these populations, the rates of gain were far less than those for betacyanins, and subsequent

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efforts have resulted in only moderate increases in betaxanthins.35 The biochemical mechanism

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underlying this observation is currently unknown.

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Betalains are produced from an aromatic amino acid precursor, L-tyrosine, which is first

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converted to L-3,4-dihydroxyphenylalanine (L-DOPA) via hydroxylation (Figure 1).37,38

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Subsequently, L-DOPA is either converted to betalamic acid via the extradiol ring cleavage

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reaction catalyzed by L-DOPA 4,5-dioxygenase (DODA)39,40 or oxidized to L-dopaquinone,41

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which is further spontaneously cyclized to cyclo-DOPA. Betalamic acid is spontaneously

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conjugated with cyclo-DOPA or various amines to produce red betacyanins and yellow

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betaxanthins, respectively.17,18 Prior studies revealed the presence of dominant alleles at two

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linked loci (R and Y, 7.4 ± 1.7 cM)42 that condition the qualitative production of betalain

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pigments in B. vulgaris. Red-pigmented roots are observed only in the presence of dominant

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alleles at both the R and Y loci, while white roots are conditioned by recessive alleles at the Y

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locus, and yellow roots by the genotype rrY- (homozygous recessive at the R locus while

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carrying at least one dominant Y allele). Thus, the R locus controls the red vs. yellow

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pigmentation, while the dominant Y allele is required for overall betalain production in table

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beet. Recently, the R locus was identified as a novel cytochrome P450, CYP76AD1,41 which

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catalyzes both tyrosine hydroxylation and L-DOPA oxidation to provide the cyclo-DOPA moiety

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required for all betacyanin synthesis, although CYP76AD5 and CYP76AD6 also catalyze the

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first tyrosine hydroxylation step redundantly with CYP76AD1.37,38 The Y locus encodes MYB1

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transcription factor controlling the expression of DODA and CYP76AD143 (Figure 1). An

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additional locus, ‘blotchy’ (bl), conditions a blotchy or irregular pigment patterning in either red

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or yellow roots,44 though its molecular identity is currently unknown.

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Although the functions of the betalain pathway loci (e.g. R and Y) have been studied

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extensively, the role of the upstream tyrosine biosynthesis on quantitative production of betalain

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pigments has not been investigated. Tyrosine is required for protein biosynthesis in all living

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cells, and also serves as a precursor of a diverse array of plant natural products, including

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plastoquinone, tocopherols, rosmarinic acid, isoquinoline alkaloids, catecholamines, and

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betalains.45–50 Tyrosine is synthesized from prephenate, the product of the shikimate pathway

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(Figure 1).51–54 Most plants synthesize tyrosine mainly via arogenate intermediate, in which

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prephenate is first transaminated to arogenate55–57 followed by oxidative decarboxylation to

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tyrosine catalyzed by arogenate dehydrogenase (ADH).

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To better understand how tyrosine is produced and utilized for betalain synthesis, this

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study analyzed and compared the levels of betalains and tyrosine using various B. vulgaris

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genotypes accumulating different levels and types of betalain pigments, including table beet

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germplasm that was developed through a process of half-sib family recurrent selection over the

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last two decades.10,35 The results revealed that red and yellow genotypes have distinct bottlenecks

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in betalain production, limited by supply and utilization of tyrosine, respectively.

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MATERIALS AND METHODS

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Plant Materials

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Two accessions of red (the population UWHiRed and the inbred line W357B) and yellow (the

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populations UWHiYellow1 and UWHiYellow2) beets were the most recent breeding cycle

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developed via half-sib family recurrent selections over a period of sixteen cycles.35 Seeds were

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also saved at each breeding cycle of the red and yellow recurrent selection programs and used for

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this study. The inbred line W357B was also developed at the University of Wisconsin-Madison,

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and is a standard inbred line used to make F1 hybrid table beet cultivars. The sugar beet analyzed

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in this study is a standard inbred line with green hypocotyls obtained from Michigan State

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University. ‘Badger Torch’ is a new cultivar released from the University of Wisconsin-Madison

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breeding program, which was bred using the germplasm that contained higher betaxanthin levels

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derived from a red table beet breeding material. This cultivar was included because it possesses a

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unique internal coloring pattern, with concentric circles of orange and yellow pigmentation.

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‘Rhubarb Chard’ and ‘Bright Lights’ Swiss chard, the latter containing white, pink, and yellow

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chards, were used for this study.

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Growth Conditions

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For analyzing developmental changes in metabolite levels (Figure 2), seeds of accessions

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‘W357B’, ‘UWHiRed’, ‘UWHiYellow1’ and ‘UWHiYellow2’ were sown on 2 June, 2015 in 0.7

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L plastic pots with a 3:1 mixture of field soil (Madison, WI) and Metromix (2.67 kg m-3 dolomite

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1:1 by volume) in the greenhouse. Lights and temperature were maintained at 16/8 hour

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light/dark cycle and approximately 22°C, respectively. Plants were watered daily and fertilized

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monthly with a modified half-strength Hoagland solution (6 ppm NH4+, 96 ppm NO3, 26 ppm P,

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124 ppm K, 90 ppm Ca, 24 ppm Mg, 16 ppm S, 1.6 ppm Fe, 0.27 ppm Mn, 0.25 ppm B, 0.16 Wang%et%al.%


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ppm Cu, 0.12 ppm Zn, 0.016 ppm Mo). Seeds germinated on 15 June, which was designated as

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Week 0. Tissues were harvested at 4, 8, 10, 12 and 14 weeks after germination. For the analysis

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of different beet varieties (Figure 3), all accessions were sown in pots in the greenhouse as

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described above on 1 June, 2016 and harvested at 4 and 12 weeks after germination. The

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germination rate of red beet germplasm from recurrent selection cycles in years 1987 and 1993

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(R87 and R93, Figure 3) was low, and we were not able to collect 4-weeks-old root/hypocotyl

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tissue for these two accessions. On 31 August, 2016, Swiss chard cultivars of different colors

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were planted, grown in the same conditions as described above for beets, individual plants with

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white, pink, red, and yellow petioles and hypocotyls were selected at the seedling stage (~1

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week-old), and harvested at 12 weeks after germination on 30 November, 2016.

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For harvesting samples for all metabolite analyses, approximately 70 mg fresh weight

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(FW) samples were collected, the exact FW was recorded, and snap-frozen in liquid nitrogen

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(Supporting Information Figure S1). For leaf tissues, the middle of a newly formed leaf was

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horizontally excised to include both vein and intervein regions. For root/hypocotyl tissues, a

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transverse section containing all tissue types was collected (Supporting Information Figure S1).%

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For developmental metabolite changes, the frozen tissues were lyophilized in a benchtop freeze

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dry system (Labconco, Kansas City, MO), and the lyophilized weight was recorded. For the

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remaining analysis, frozen tissues were used directly for metabolite extraction as described

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below.

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Extraction of Metabolites from Beet Roots/Hypocotyls and Leaves

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For metabolite extractions, ~7 mg lyophilized or ~70 mg fresh tissues were first submerged

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overnight (16 hrs) in a 1.5 mL centrifuge tube containing 400 μL extraction solution [2:1 ratio

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(v/v) of methanol:chloroform, 100 μM norvaline (recovery standard for GC-MS), 100 μM 4-

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chlorobenzoic acid (recovery standard for HPLC)]. Betalain pigments and tyrosine were then

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extracted by adding 300 μL water and 125 μL chloroform, followed by vigorous vortex mixing

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for 5 min and centrifugation at 20,000g for 5 min for phase separation. The upper polar phase

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(~400 μL) was transferred to a new centrifuge tube and dried down in a benchtop speed vacuum

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(Labconco). The dried polar phase was resuspended in 100 μL water for HPLC for the

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confirmation of pigments or GCMS analysis for the analysis of tyrosine (see below). The sample

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was further diluted for pigment quantification on the spectrophotometer (see below).

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Betalain Analysis

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Betacyanin and betaxanthin contents were measured from the resuspended polar extract using a

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spectrophotometer (Beckman Coulter, Brea, CA) at 480 and 538 nm.58 Red and yellow beets

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extractions were diluted by 100 to 500 times to reach OD reading between 0.1 to 1, where a

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linear increase in spectrometric readings was observed. Full wavelength scans were performed

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on Nanodrop spectrophotometer (ThermoScientific, Waltham, MA) to determine if the samples

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had peaks at 480 or 538 nm for quantification. To confirm the identity of the pigments, 5 μL of

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the same diluted pigment extracts were injected into HPLC (Infinity 1260, Agilent, Santa Clara,

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CA), equipped with Atlantis T3 C-18 column (3 μm, 2.1x150 mm, Waters,% Milford, MA), and

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separated by the following gradient of acetonitrile (B) in 1% formic acid (A): 0% B for 5 min,

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followed by a linear increase to 76% B at 25 min, an isocratic elution at 76% B until 31 min, a

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linear decrease to 0% B at 32 min, and an equibiliration at 0% for another 10 min. Betacyanins

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and betaxanthins were monitored at 534 nm and 474 nm, respectively, by the photodiode array

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detector (Agilent) and their identity was confirmed by their absorption spectra (Supporting

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Information Figure S2). In addition to the absorption spectra, betaxanthin peaks were monitored

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by the fluorescence detector (Agilent) at 460 and 510 nm for excitation and emission

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wavelengths, respectively.

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HPLC Quantification of Tyrosine Content

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For quantification of tyrosine contents, 5 μL of the same soluble metabolite extracts (without

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dilution) were analyzed by HPLC with the same setting as described above for betalain analysis.

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The fluorescence detector was set at 274 and 303 nm for excitation and emission wavelengths,

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respectively, to detect tyrosine. Data were normalized by the peak area of recovery standard 4-

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chlorobenzoic acid, which was detected by UV absorbance at 270 nm. Quantification was based

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on the standard curves generated by injecting 5 μL of serial dilutions of authentic tyrosine

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(Thermo Fisher Scientific-Acros Organics, Geel, Belgium) from 10 μM to 1 mM.

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GCMS Quantification of Tyrosine Level

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Tyrosine contents were also analyzed by GC-MS as tert-butyldimethylsilyl derivative to further

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confirm the HPLC quantification data.59 The same soluble extracts (10 μL out of 100 μL) were

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dried down in a 200 μL volume glass insert, and 40 μL pyridine (Acros Organics) was added

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before sonication for 10 min. For derivatization, 40 μL N-tert-butyldimethylsilyl-N-

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methyltrifluoroacetamide with 1% tert-butyldimethylchlorosilane (Cerilliant, Round Rock, TX)

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was added to the pyridine resuspension and incubated at 80°C for 1 hour. One microliter of

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derivatized sample was injected into GC-MS (ISQ-LT, ThermoScientific, MA) equipped with 5%

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phenyl phase column (TG-5MS, ThermoScientific) by 1:10 split mode with a helium flow rate of

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1.2 mL/min. The inlet temperature was set at 260°C. The oven program began at 70°C and was

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held for 2 min, before being ramped up to 300°C at 5°C per min, and then was held at 300°C for

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10 min. The ion source temperature and MS transfer line temperature were both set to 300°C.

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The electron energy was 70 eV. Both data of selected-ion monitoring (SIM) and total ion Wang%et%al.%


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chromatogram were collected between the retention time of 9.3 and 50 min. SIM ions for

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derivatized tyrosine and norvaline (recovery standard) were set at 186 and 260 m/z and 302 and

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466 m/z, respectively. Tyrosine and norvaline peaks were identified based on the retention time

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and MS spectra of their respective authentic standards. Quantification was based on the standard

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curves generated by derivatizing and injecting different concentrations of authentic tyrosine from

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10 μM to 1 mM as described above for samples.

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RESULTS

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Betalain Concentrations in Roots/Hypocotyl and Leaves under Different Developmental

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Stages

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Betalain pigment coloration usually becomes visible at the seedling stage in most beet cultivars.10

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However, quantitative changes in these pigments are not well understood during beet

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development. Using the betalain extraction, separation, and quantification methods described

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above, red and yellow pigments were analyzed from red and yellow beets, respectively (Figure

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2A). The chemical natures of the red and yellow pigments were further confirmed by high-

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performance liquid chromatography (HPLC) to be betacyanins and betaxanthins based on their

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maximum absorption and, for betaxanthins, their unique fluorescence emission60,61 (Supporting

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Information Figure S2). Root/hypocotyl tissues accumulated much higher levels of betalain

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pigments than leaf tissues (up to 1.7 vs. 0.4 mg/gFW, respectively, Figure 2A). Although leaf

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betalain contents increased during plant development, root/hypocotyl-derived betalains remained

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relatively constant throughout plant development, consistent with visual observation. In

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root/hypocotyl tissues, yellow beet genotypes accumulated much less betalain (five to eight

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times) than red genotypes (Figure 2A) in agreement with previous studies.35

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Tyrosine Contents under Different Developmental Stages

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Next, we analyzed the levels of tyrosine, the precursor of betalains, from the same polar phase

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using HPLC fluorescence detection and GCMS as tert-butyldimethylsilyl derivative as described

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above. In root/hypocotyl tissues, red genotypes (or accessions) had relatively low levels of

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tyrosine (10 to 50 nmol/gFW), whereas yellow genotypes (or accessions) accumulated much

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higher levels of tyrosine, which reached up to 370 nmol/gFW (Figure 2B). Tyrosine levels were

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relatively constant throughout the development of yellow hypocotyl/roots except for slightly low

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level at 4 weeks (Figure 2B). In leaves, similar levels of tyrosine were detected among different

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genotypes, except for somewhat high tyrosine contents in HiRed and HiYellow2 at 4 weeks

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(Figure 2B). These results showed that tyrosine levels were consistently higher in yellow than

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red beets in root/hypocotyl tissues, where we focused on the remaining analysis.

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Betalain and Tyrosine Concentrations in Different Beet and Swiss Chard Genotypes

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To further examine the relationship between betalain and tyrosine accumulations in different beet

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genotypes, various red, yellow, and sugar beet cultivars were analyzed at 4 and 12 weeks after

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germination. Consistent with the developmental data described above (Figure 2), red beets

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accumulated more betalains than yellow beets at both 4 and 12 weeks (Figure 3A and B,

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respectively). Again, the levels of tyrosine were opposite to those of betalains and generally

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higher in yellow than red beets (Figure 3A and B). Badger Torch accumulated the highest

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betaxanthin levels among yellow beets, without detectable accumulation of betacyanins

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(Supporting Information Figure S3), and had relatively low tyrosine at 12 weeks (Figure 3B).

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The sugar beet cultivar, which accumulated very little betalains, accumulated tyrosine at the

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levels equivalent to red beets (Figure 3).

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When the levels of betalains were converted to nmol/gFW (using estimated average

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molecular weights of various betaxanthins and betacyanins as 308 and 550, respectively)58 and

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compared to those of tyrosine for individual lines, betalain/tyrosine molar ratios were between 43

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and 104 (average 68.5 ± 6.8) for red beet genotypes and 0.1 and 8.2 (1.9 ± 0.8) for yellow

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genotypes. When betalain levels were plotted against those of tyrosine for red and yellow

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genotypes, respectively, red beets varieties showed positive correlations between tyrosine and

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betalain contents especially at 12 weeks (R2 = 0.80, Figure 3B). This means that as tyrosine

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concentration increases, more betacyanins are produced in red beets. In yellow genotypes,

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betalain and tyrosine levels positively correlated to some extent at 4 weeks (R2 = 0.41) when

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tyrosine concentration is still low (Figure 3A); however, no correlation was observed at 12

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weeks and higher tyrosine concentrations did not lead to higher betalain accumulation (Figure

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3B).

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To test if a similar negative relationship between betalain and tyrosine contents also

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exists in Swiss chard, betalain and tyrosine contents of white, pink, red, and yellow genotypes

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were analyzed at 12 weeks from hypocotyls. As shown in Figure 4, yellow chard accumulated

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the highest level of tyrosine among the four different chard genotypes tested.

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DISCUSSION

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Previous breeding efforts to increase betalain pigments in table beet have focused primarily on

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half-sib family recurrent selection, which was initiated in the 1970s.10,35,44 Steady gains in

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betacyanin pigment concentration have been realized in the red populations subjected to

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recurrent selection, and these populations have been used commercially for the production of Wang%et%al.%


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betacyanins for the food industry.35 A similar approach was taken with yellow beet, beginning

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about 20 years later. Gains in betaxanthin concentration were realized but at a much lower rate

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and overall more modest than those for betacyanins. Even though it is well understood that the

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typical red beet accumulates both betacyanins and betaxanthins, and that yellow beet

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accumulates betaxanthins, we lacked an explanation for why increasing betaxanthin pigment

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concentration in yellow or red beets was much less successful compared to that for betacyanins.

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This study is the first to suggest an explanation for the relative inefficiency of betaxanthin

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pigment biosynthesis in yellow beet.

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The final concentration of betalains determines the quality of table beets and thus was

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measured during prior breeding programs. This study, however, analyzed developmental changes

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in the levels of betalains and tyrosine in order to understand the relationships between the

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products and its precursor, respectively. While betalain concentration was relatively constant

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throughout plant development, tyrosine concentration increased substantially after 4 weeks

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(Figure 2). Root/hypocotyl tissues of beets initially undergo active cell division followed by

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tissue swelling through continuous cell division and cell expansion from a series of peripheral

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secondary meristems laid down during the early stages of development.62,63 Thus, low overall

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tyrosine levels at 4 week-old beet root/hypocotyl tissues could be due to active utilization of the

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amino acid for protein synthesis during cell division. After 4 weeks, decreased demand of

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tyrosine for protein synthesis during tissue swelling might have led to the elevated accumulation

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of tyrosine (e.g. 12 weeks, Figures 2B and 3).

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Comparison of red and yellow genotypes during beet development (Figure 2) and among

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various beet genotypes (Figure 3) revealed an interesting negative relationship between betalain

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and tyrosine contents: yellow beets, which accumulate less betalain pigment, had much more

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tyrosine than red beets (Figures 2 and 3). Similar results were also observed in yellow vs. red

307%

Swiss chard genotypes (Figure 4). The average betalain/tyrosine molar ratios in 12-weeks-old

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root/hypocotyl were 36 times higher in red than yellow beets (68.5 vs. 1.9). Given that most

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betacyanins are derived from two tyrosine molecules while betaxanthins needs only one (Figure

310%

1), these results strongly support that red beets are much more efficient in converting tyrosine

311%

into betalains than yellow beets. In 12-weeks-old root/hypocotyl tissues, the betalain levels of

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red genotypes are highly positively correlated with tyrosine concentrations (Figure 3B),

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suggesting that tyrosine availability is a key factor for betalain production in red beets. In

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contrast, yellow beets showed essentially no positive correlation between tyrosine and betalain

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contents at 12 weeks (Figure 3B), suggesting that yellow genotypes are limited in converting the

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tyrosine precursor into yellow betaxanthins.

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The limited utilization of tyrosine in yellow beets could be explained by recent molecular

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findings concerning the betalain biosynthetic enzymes.37,38,41,64 Unlike red beets that have full

319%

complements of betalain biosynthetic genes (Figure 5A), yellow beets are homozygous recessive

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at the R locus encoding CYP76AD1, which is responsible for the L-DOPA oxidation to cyclo-

321%

DOPA and hence deficient in red betacyanins (Figure 5B). CYP76AD1 was recently found to be

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a bifunctional enzyme and also to play the major role in the upstream reaction, tyrosine

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hydroxylation to L-DOPA.37,38,64 Although two other homologous enzymes, CYP76AD5 and

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CYP76AD6, act redundantly and partially contribute to the tyrosine hydroxylation reaction37,38,64,

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suppression of CYP76AD5 or CYP76AD6 did not reduce betalain pigmentation in beets, unlike

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that of CYP76AD1.37 Evolutionary analysis also demonstrated that CYP76AD1 and DODA

327%

(referred to as CYP76AD1-α and DODA-α in the paper) together emerged during the evolution

328%

of betalain pigmentation in the core Caryophyllales.65 Thus, yellow beets not only are defective

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Page 17 of 34


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in the L-DOPA oxidation, but also have substantially reduced tyrosine oxidation to L-DOPA,

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which is the committed step of betalain biosynthesis and hence determines overall levels of

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betalain pigments. This genetic constraint, due to the bifunctionality of the R locus, could explain

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the long-observed difficulty in enhancing the yellow betaxanthin pigments to the levels similar to

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the red betacyanins through recurrent selections. Upon L-DOPA feeding, leaves and roots of

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yellow beets accumulated more betaxanthins as compared to water feeding controls (Supporting

335%

Information Figure S4). These data together suggest that the lack of the major tyrosine

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hydroxylation activity derived from R locus is likely the bottleneck of betaxanthin production in

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yellow beet.

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Unlike yellow beets, white-rooted genotypes such as sugar beet, which accumulate very

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little to no betalain pigments (Figure 3), did not exhibit elevated accumulation of tyrosine, and

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this was true for both sugar beet and white Swiss chard (Figures 3 and 4). These results suggest

341%

that production of tyrosine is also down-regulated in white genotypes and that a simple up-

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regulation of the structural genes of the betalain pathway (e.g. CYP76AD1 and DODA) may not

343%

be sufficient to achieve high betalain production in white genotypes. Interestingly,

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overexpression B. vulgaris MYB (Y locus) in the hairy roots of the white Albina Vereduna beet

345%

resulted in red betacyanin accumulation.43 Although the actual betalain contents were not

346%

quantified,43 this result raises a possibility that besides betalain pathway genes (e.g. CYP76AD1,

347%

DODA), BvMYB transcription factor may also be able to induce genes encoding upstream

348%

pathway enzymes and hence elevate the supply of tyrosine.

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Based on our data, different strategies are needed to further improve the levels of

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betalains in various beet genotypes. Given that red beets appear to be efficiently converting

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tyrosine into betalains (betacyanins), further increase in tyrosine production may lead to elevated

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Page 18 of 34

352%

accumulation of betalains in red cultivars (Figure 5A). Plants and microbes generally synthesize

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tyrosine via arogenate or prephenate dehydrogenases (ADH or PDH), respectively, which are

354%

both feedback inhibited by tyrosine.66,67 Our recent study identified tyrosine-insensitive PDH

355%

enzymes from Glycine max (soybean) and Medicago truncatula,15,16 which could be introduced in

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red beet to introduce an alternative tyrosine biosynthetic pathway that avoids feedback

357%

inhibition.

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Yellow beet, on the other hand, first needs the improvement in tyrosine utilization via the

359%

initial tyrosine hydroxylation reaction (Figure 5B). Biosensor-based mutagenesis screening of

360%

CYP76AD1 from Beta vulgaris identified two point mutations (W13L, tryptophan to leucine;

361%

F309L, phenylalanine to leucine) that specifically reduce the L-DOPA to cyclo-DOPA oxidation

362%

activity without affecting the tyrosine to L-DOPA conversion.64 Thus, the introduction of the

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mutated CYP76AD1 enzyme into yellow beet backgrounds through genetic engineering, or by

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targeted mutagenesis of the specific residues in red beet genotypes, will likely boost the

365%

production of betaxanthins. Badger Torch, which has a quite different breeding history than the

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other yellow genotypes of the recurrent selection (see method), had the highest betaxanthin

367%

levels (though still much lower than betacyanin levels in red genotypes, Figure 3), suggesting

368%

that these genetic engineering efforts can be combined with further breeding efforts utilizing

369%

different genetic materials.

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Natural colorants are a multimillion dollar world market, and an increasing number of

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food manufacturers are seeking natural alternatives to synthetic dyes mainly due to some

372%

implicated negative impacts of synthetic dyes on our health68,69 and health benefit of many

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natural colorants.70,71 Currently, natural sources of red colorant for food products include table

374%

beet and paprika, while the natural source of yellow is primarily turmeric. In the U.S., colorants

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Page 19 of 34


375%

fall into two major categories: exempt and certified, with the former being the category for

376%

natural colorants and the latter for synthetic colorants. Synthetic colorants are manufactured by

377%

chemical synthesis, while natural colorants are made by fruits, vegetables, animal, or mineral

378%

sources. Both categories of colorants are covered by federal regulations.10 Betalains remain of

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great interest for natural coloring applications for food as they can be easily produced by fruits

380%

and vegetables and extracted using relatively simple technologies.36 Though they lack the heat

381%

and light stability of synthetic colorants, they can be used in food products where such stabilities

382%

are not required, such as powdered drink and soup mixes, ice cream, yogurt, candy, and many

383%

other products. The primary limitation to the use of betalains has been the economics of their

384%

concentration in plant biomass. As levels of betalain pigments are increased in selected beet

385%

populations by breeding, the cost competitiveness to synthetic dyes also increases, though natural

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colorants are still far more expensive than their synthetic counterparts.10 Thus, innovations that

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would allow for more efficient betalain production, such as by enhancing carbon flow from

388%

tyrosine to betaxanthins through modulating CYP76AD1, would contribute significantly to the

389%

market for natural colorants.

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Page 20 of 34

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SUPPORTING INFORMATION

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Figure S1: Beet tissues harvested from roots/hypocotyls and leaves for metabolite analysis.

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Figure S2: Identification of betacyanin and betaxanthin peaks in the root/hypocotyl extract of

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12-week-old red beets.

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Figure S3: Identification of betaxanthin peaks in the root/hypocotyl extract of 12-week-old

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Badger Torch yellow beets.

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Figure S4: Betaxanthin contents of leaf and root/hypocotyl tissues upon water and L-DOPA

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feeding.

398%

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AUTHOR INFORMATION

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Corresponding Author: *(H.A.M.) E-mail: [email protected]

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ORCID: Hiroshi Maeda: orcid.org/0000-0003-0246-694X

402% 403%

Funding: This project was supported by the Agriculture and Food Research Initiative competitive

404%

grant 2015-67013-22955 of the USDA National Institute of Food and Agriculture.

405% 406%

Disclosures: Author I.G. receives royalties for the use of high pigment table beet germplasm

407%

developed at the University of Wisconsin through the University and the entity PhytoColorants.

408%

409%

ACKNOWLEDGEMENTS

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We thank Matthew Mirkes and Samantha Milota for their help on growing beets and chards,%and

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Sarah Friedrich from the UW-Botany Studio for the help in taking some beet pictures. Wang%et%al.%


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Page 21 of 34


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Plant Mol. Biol. 2002, 48, 361–368. (68)

McCann, D.; Barrett, A.; Cooper, A.; Crumpler, D.; Dalen, L.; Grimshaw, K.; Kitchin, E.;

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Lok, K.; Porteous, L.; Prince, E.; et al. Food additives and hyperactive behaviour in 3-

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year-old and 8/9-year-old children in the community: a randomised, double-blinded,

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placebo-controlled trial. Lancet 2007, 370, 1560–1567.

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Bateman, B.; Warner, J. O.; Hutchinson, E.; Dean, T.; Rowlandson, P.; Gant, C.; Grundy,

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J.; Fitzgerald, C.; Stevenson, J.; Warner, J. The effects of a double blind, placebo

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controlled, artificial food colourings and benzoate preservative challenge on hyperactivity

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in a general population sample of preschool children. Arch Dis Child 2004, 89 (40), 506–

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Kim, J. S.; Ahn, J.; Lee, S. J.; Moon, B. K.; Ha, T. Y.; Kim, S. Phytochemicals and

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antioxidant activity of fruits and leaves of Paprika (Capsicum Annuum L., var. Special)

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cultivated in Korea. J. Food Sci. 2011, 76 (2), 193–198.

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FIGURES

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Figure 1. Biosynthetic pathways of tyrosine and betalains in Beta vulgaris. Betalain pigments, red betacyanins and yellow betaxanthins, are produced from L-tyrosine derived from the shikimate pathway. Tyrosine is first oxidized to L-dihydroxylphenylalanine (LDOPA) by three redundant cytochrome P450 enzymes, CYP76AD1, AD5, and AD6. L-DOPA is further converted by L-DOPA dioxygenase (DODA) and CYP76AD1 to betalamic acid and cyclo-DOPA, respectively, both of which are required for red betacyanin production, while only betalamic acid, together with some amines, is required for yellow betaxanthin production. Yellow beets lack R locus encoding CYP76AD1, while white beets have low activity of MYB1 transcription factor that activates betalain pathway gene expression.

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Figure 2. Developmental changes in beet betalain and tyrosine contents. (A) Betalain contents of root/hypocotyl (left) and leaf (right) tissues harvested at the indicated weeks after germination. Data are means ± s.e.m. (n = 3 independent biological samples). Red genotypes, HiRed and W357B, are indicated by dark and light pink bars, respectively, whereas yellow genotypes, HiYellow1 and HiYellow2, are in dark and light orange bars. (B) Tyrosine contents of root/hypocotyl (left) and leaf (right) tissues harvested at the indicated weeks after germination. Data are means ± s.e.m. (n = 5 independent biological samples). The same colors were used to indicate respective cultivars as in (A)

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Figure 3. Betalain and tyrosine contents in different beet varieties. R87 to 08 (red bars) and Y01 to 11 (yellow bars) are red and yellow beet germplasm, respectively, from recurrent selection. Sugar beet is indicated by white bars, while yellow Badger Torch beet cultivar is in orange diagonal stripe. As in Figure 2, red genotypes, HiRed and W357B, are indicated by dark and light pink bars, respectively, whereas yellow genotypes, HiYellow1 and HiYellow2, are in dark and light orange bars. The bottom panels plot betalain contents (converted into nmol/gFW using estimated average molecular weights of various betaxanthins and betacyanins as 308 and 550, respectively58, y-axis) against tyrosine contents (xaxis) for red and yellow genotypes. (A) Betalain and tyrosine contents of the root/hypocotyl tissues from different beet varieties harvested at 4 weeks after germination. Data are means ± s.e.m. (n = 3 independent biological samples). Red germplasm from years 1987 and 1993 (R87 and R93) had poor germination and could not be included in 4 week-old analysis. (B) Betalain and tyrosine contents of the root/hypocotyl tissues from different beet varieties harvested at 12 weeks after germination. Data are means ± s.e.m. (n = 5 independent biological samples).

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Figure 4. Betalain and tyrosine contents in 12 week-old chard varieties. (A) A representative chard (pink) plant at 12 week-old. The hypocotyl tissue harvested for metabolite analyses is indicated by star. N.D., Not detectable (below detection limit). (B) Betalain contents of hypocotyl tissues from white, pink, red, and yellow chard varieties. Data are means ± s.e.m. (n = 4 independent biological samples). (C) Tyrosine contents of hypocotyl tissues from different chard varieties Data are means ± s.e.m. (n = 4 independent biological samples).

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Limited Tyrosine Utilization Explains Lower Betalain Contents in

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