Anthocyanins and betalains: evolution of the mutually exclusive pathways

Anthocyanins and betalains: evolution of the mutually exclusive pathways

plan cience ELSEVIER Plant Science 101 (1994) 91-98 Review article Anthocyanins and betalains: evolution of the mutually exclusive pathways Helen A...

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plan cience ELSEVIER

Plant Science 101 (1994) 91-98

Review article

Anthocyanins and betalains: evolution of the mutually exclusive pathways Helen A. Stafford Department of Biology, Reed College, Portland, OR 97202, USA Received 31 May 1994; revision received 20 June 1994; accepted 21 June 1994

Abstract

The curious fact that the anthocyanidin and the betalain pathways, each leading to both yellow and red pigments, have never been found in the same plant is examined from a genetic and evolutionary point of view. The betalain products, alkaloids called betacyanins and betaxanthins, are found only in one group of Angiosperms, the Caryophyllales, while anthocyanins are widely distributed. However, their distribution within the plant and both their vegetative and reproductive functions are essentially identical. Whereas the functions in pollination and seed dispersal of both pigments are well known, their vegetative functions are ill-defined.- The structural and many of the regulatory genes of the anthocyanin pathway are now well known, but our knowledge of the betalain pathway is severely restricted. Future needs in physiological and molecular genetic studies of the evolutionary mechanism of the mutual exclusion of these two pathways, as well as the vegetative functions of the two groups, are emphasized in this review.

Keywords: Anthocyanin; Betalain; Betacyanin; Betaxanthin; Enzymes; Molecular genetics

1. Introduction Anthocyanidin and betalain derivatives have never been found in the same plant, but their distribution in both vegetative and reproductive tissues is similar. Betalains are found in a small group of Angiosperms closely related to the Ranunculaceae, the Caryophyllales [1], whereas anthocyanidins are widespread in Angiosperms. The similarity in development, tissue distribution and function indicates that the same or comparable regulatory genes are involved, even though the biosynthetic pathways are different. Does this mean that the regulatory genes must be the same

or similar? How did this similarity in regulation and mutual exclusion of these two different biosynthetic pathways evolve?

2. Biosynthetic pathways Betalains are highly colored alkaloids derived from tyrosine via the condensation of betalamic acid with another DOPA derivative such as cyclodopa to produce red colored betalains, or via the condensation of betalamic acid with an amino acid to produce yellow betaxanthins [2,3] (see Fig. 1 for a hypothetical pathway). Except for a tyrosinase activity to produce L-DOPA, the subsequent,

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H.A. Staf/brd/Plant Sci. 101 (1994) 91-98

luteolinidin as shown in Fig. 2, is known up through the level of flavan 3,4-diols or flavan 4ols, plus the final glycosylation and acylation of the aglycone (only the 5,7-hydroxy pathway is

<)OC H tyrosinc 140 L-DOPA N ~ k

phenylalanine

acetate -ooc H

O +

malonyl-CoA

p-coumaroyl -CoA CHS

I c~ 1to OH cyclo-d~pa

J

H betalamicacid

U

I 3'-hydroxylase OH

~/,--proline

cYx~

HO

"N- ~CO0 OH

o flavanone

H

H

betanidin

~

H

indicaxanthin

Fig. 1. Postulated overall biosynthetic pathway to betalains [2,3]. DOPA = 3,4-dihydroxyphenylalanine. Betanidin, a betacyanin, (red, absorption peak at about 535 nm); indicaxanthin, a betaxanthin, (yellow, absorption peak at about 480 nm) as examples of betalains.

postulated biosynthetic steps, possibly as an enzyme complex ('betacyanin or betaxanthin synthase'), have not been demonstrated in cell-free extracts. A mechanism involving, an extradiol cleavage of DOPA to produce betalamic acid is discussed by Piatelli [3]. High tyrosine utilization and enhancement of betacyanin accumulation upon feeding tyrosine indicate a strictly compartmented metabolic pathway. Tyrosine is a better precursor than DOPA [4], an indication of a multienzyme complex. Anthocyanidins, on the other hand, are derived from a condensation of three molecules of malonyi-CoA from the acetate pathway with one of p-coumaroyl-CoA from the phenylpropanoid pathway, in a series of 5-6 steps at the C15 level to produce an aglycone. The cell-free enzymology of this anthocyanidin pathway to cyanidin and

3-OH-flavanone (dihydmflavonol) l -NADPHreductaseflavan-3,4-diol

l flavan-4-ol

I 'AS'

[ 'AS'

anthocyanidin (3-OH)

anthocyanidin (3-deoxy) OH

OH Cyanidin

OH

OH Luteolinidin

Fig. 2. Pathway to anthocyanin aglycones with 3',4'-hydroxy B-rings: cyanidin (red, absorption peak at about 550 nm as an aglycone to 525 nrn as a glycoside); luteolinidin (yellow-orange, absorption peak at about 490 to 485 nm as glycosides) [5]. CHS (cbalcone synthase) plus CHI (chalcone isomerase) produce the basic flavanone molecule. Two hydroxylases, adding hydroxylgroups at the 3'- and 3-positions and an NADPH reductase are subsequently involved. 'AS' represents the postulated enzyme(s) that convert(s) flavanols to anthocyanidins. Subsequent acylation and glycosylation steps are not shown.

H.A. Stafford~Plant Sci. 101 (1994) 91-98

shown) [5]. The steps(s) converting the 3,4-diol or 4-ol to form an anthocyanidin, an 'anthocyanidin synthase' ('AS'), has not yet been demonstrated in cell-free extracts; the 5-deoxy pathway enzymes are poorly known. Since other flavonoids, including proanthocyanidins [6], are found in at least some members of the betalain containing families of the Caryophyllales, this hypothetical 'anthocyanidin synthase' is the only enzyme(s) missing. Both betalain [4] and anthocyanidin pathways [7] involve terminal glycosidation and acylation steps. The absorption spectra of the aglycones are strikingly similar [2], betacyanins being similar to cyanidins and betaxanthins to 3-deoxy anthocyanidins (Figs. 1 and 2). Glycosylation shifts the spectral peaks to shorter wavelengths. The similar

93

spectra permit both types of compounds to function in the same way in pollination and seed (fruit) dispersal. Zwitterionic structures are known in both groups [2,8]. Both pathways require the shikimate pathway to provide an amino acid as a precursor, tyrosine versus phenylalanine. High DOPA contents are found in some culture lines of Chenopodium rubrum that accumulate high concentrations of betacyanins, but DOPA accumulates in some non-betalain species also [4]. 3. Distribution within taxonomic groups of angiosperms Ten of the families in the Caryophyllales (sometimes called the Centrospermae) synthesize

Table 1 Basic flavonoids and betalains in families of the order CaryophyUales [6], listed approximately in the phylogenetic order according to Cronquist [1]

Caryophyllaceae Dianthus caryophyllus Silene dioica Molluginaceae Phytolaccaceae Phytolacca americana Portulacaceae Portulacca grandiflora Baselliaceae Nyctaginaceae

Anthocyanins

PAs*

Flavones

Flavonols

+

+

+

+

+

+

+

Betacyanins

Betaxanthins

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

-t-

Bougainvillea glabra Mirablis jalapa mmaranthaceae

Amaranthus caudatus Amaranthus tricolor Celosia plumosa Gomphrena globosa lrestine herbstii Chenopodiaceae Beta vulgaris

Chenopodiurn rubrum Aizoaceae Glottiphyllum longum Cactaceae Opuntia ficus-indica Opuntia dillenii Phyllocactus hybrida Dideraceae apAs,

proanthocyanidins.

+

+

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H.A. Stq/Jbrd/Plant Sci. 101 (1994) 91-98

only betalains (including Phytolaccaceae and Amaranthaceae), whereas two (Moluginaceae and Caryophyllaceae) contain only anthocyanidin derivatives [1] (Table 1). So far, only betacyanins are found in the Phytolaccaceae, while all other families contain betaxanthins in addition. All members of the Caryophyllales, however, are characterized by unusual sieve-tube plastids and a high ferulic acid content in their cell walls, and many are C4 photosynthetic plants [1,2]. D N A - R N A hybridization studies indicate a close relationship for the betalain families examined; the Caryophyllaceae containing Dianthus and Silene species showed the closest relationship to the betalain families [2]. No other plant group forms betalains, but similar compounds have been identified in some Basidiomycetes [2,3]. Anthocyanidins and their derivatives are found in all Angiosperms. 4. Distribution within plants

Both chemical groups are accumulated in the vacuoles of vegetative and reproductive tissues, and both are found mainly in epidermal and subepidermal tissues [10,11]. Their presence in reproductive tissues such as petals, seeds and fruits is generally 'constitutive' at defined developmental stages, but their presence in vegetative tissues appears to be regulated to a greater extent by environmental conditions. Methods for distinguishing between anthocyanidins and betalains during tissue extractions are discussed by Mabry [2]. 5. Functions in tissues

5.1. Reproductive functions Both these pigments have similar functions in pollination, seed and fruit distribution. Anthocyanin involvement, however, is much better studied than that of betalains. McClure [10] briefly compares their functions. Insects and birds are attracted by differently colored reproductive structures; bees mainly by blues and yellows, humming birds to red [12]. Anthocyanins, 3-deoxy-, as well as 3-hydroxy-, types, span a wider range of colors ranging from yellow, orange, to red and

blue. Except for some members of the Cactaceae, betacyanins lack bluer colors, but betaxanthins provide the yellows and betacyanidins, the reds and purples. Both carotenoids and betaxanthins have been identified in yellow cell cultures of Portulaca grandiflora [13]. Flavonols are also present. The functional relationship of the yellow betalains (as localized UV-honey guides) to carotenoids (as distant attractors) could be the same as postulated for flavonols [12]. So far, no 3deoxyanthocyanins, also yellow to orange pigments, have been identified in the Caryophyllales.

5.2. Vegetative functions Functions in vegetative tissues, on the other hand, are less well defined. Local accumulations in necrotic areas of these secondary compounds in response to specific pathogens and injury in vegetative tissues represent potential defense functions. Recent evidence in sorghum indicates that 3deoxyanthocyanins can function as phytoalexins [14]. No such definitive study is available for betalains, but betalains do form in wounded tissues and have been implicated as a defense mechanism against viral infection (Sosnova, 1970, cited by Piatelli [3]) and fungal induced 'damping offf [2]. Both of these secondary products can inhibit indoleacetic acid (IAA) oxidase as demonstrated in cell-free studies, but any in vivo significance is unknown [3]. Anthocyanin accumulation resulting from cold temperatures, dehydration, sugar accumulation, and deficiency in metals such as phosphate are commonly described as stress markers in vegetative tissues [15]; the specific benefits of these responses, if any, have not been well identified [10]. Does this represent just a breakdown of normal regulation, or can functions be found for this expensive production? Potential roles of both of these secondary products as photoreceptors and as filters need to be examined. Green light effects in biological systems have recently been reviewed by Klein [16]. Green light has been reported to repress plant growth. Both anthocyanins and betacyanins could function as green light photoreceptors attached to proteins. Accumulation of only small amounts would be needed for this purpose. If accumulated in

H.A. Stafford~Plant Sci. 101 (1994) 91-98

larger amounts, they could also function as filters, preventing green light effects. Although anthocyanins are sometimes reported as protective UVfilters, their major absorption peaks are in the 465-560-nm range and would argue against this as a primary function. Both seedling hypocotyls and leaves of some plants in autumn accumulate large quantitites of either of these red pigments in epidermal tissues upon lowered temperature and/or increased sucrose concentrations. Although not effectively proven, the synthesis is probably de novo. No valid physiological significance for these colorations has been advanced, but a filter function altering a green light effect should be considered. For instance, one could speculate that if green light mobilized sugar transport towards apical parts of a plant, blocking of this stimulus might permit increased transport to the roots, a useful response to sudden lowered temperatures for some trees in autumn.

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Phytolacca are the most studied betalain containing plants [11,13,23]. Both pathways are stimulated by 2,4-Dichlorophenoxyacetic acid (2,4-D) (or IAA), cytokinin and light. Gibberellic and abscisic acids both inhibit and stimulate these pathways [24-26]. Inhibition by benzylaminopurine (BAP) in both Phytolacca and Amaranthus cultures was considered to be due to a lessened availability of tyrosine, the biosynthetic precursor of betacyanins [27]. 2,4-D, on the other hand, stimulated betacyanin accumulation in Phytolacca americana, apparently by increasing the conversion of tyrosine to betacyanin [28]. The ratio of 2,4-D to a cytokinin was important in maintaining stable color types in cultures of beets, Beta vulgaris [9]. Stimulation of anthocyanin accumulation in carrot cell suspension cultures by 2,4-D was associated with an increase in phenylalanine ammonia-lyase (PAL) [22]. Competition with protein synthesis for phenylalanine and tyrosine would be expected in both cases.

6. Regulatory controls 6.1. Light control Phytochrome control via gene activation has been demonstrated in both betalain and anthocyanin biosynthesis of seedlings [3,15,17,18]. UV light and red light, via phytochrome, are inducers. Light is an obligatory requirement for betalain biosynthesis in some species, but betalains can accumulate in the dark in other species, and even in different lines of one species [2,4]. Dark culture of cells with anthocyanins, previously induced by light, caused a slow pigment 'degradation', but repigmentation occurred rapidly in l!ght [19]. Although studies have not been as comprehensive as with anthocyanins [18], phytochrome, UVB and blue light all appear to be involved also in the betalain pathway. Interactions with hormonal effects are discussed below. 6.2. Hormonal control Both types of pigments are found in growing as well as stationary phase cell cultures. Both are affected by the addition of hormones involving interactions with light requirements. Recent studies of anthocyanin producing plants include carrot, maize and petunia [20-22]. Amaranthus and

6.3. Structural genes Structural genes for the basic enzymes of the anthocyanin pathway at the Cl5 level (Fig. 2) have been identified mainly in maize, Antirrhinum and petunia. Loci have been defined mutationally and clones isolated by molecular strategies [21,25,29]. This includes a2 of maize and candida of Antirrhinum, genes for 'anthocyanidin synthase'. This as yet undemonstrated enzyme step (or steps) is believed to involve an hydroxylation, and shows over 84% similarity with other flavanone 3-~hydroxylases, 2-oxoglutarate-dependent dioxygenases, from many different sources. Structural genes for the major enzymes at the CI5 level, Y, L A, and R, and a multiple allele of A,a vat" have also been identified in Dianthus caryophyllus, carnation, an anthocyanincontaining member of the Caryophyllales order [30]. A cDNA library from Dianthus has been reported [31]. Genes P, N and M, involved in hydroxylation and glycosidation steps at the C15 level, have been studied in Silene dioica [5]. Information is sparse for the betalain pathways. In Portulaca grandiflora, multiple alleles of gene C have been distinguished that manifest three color shades [3]. Betacyanins appear to be dominant

96

H.A. Staffbrd/Plant Sci. 101 (1994) 91-98

over betaxanthins; white forms were recessive to all colored forms. Segregating genotypes of an R locus involved in betalain coloration in hypocotyls of beet, Beta vulgaris, have been studied [32]; gene R for hypocotyl color was localized on chromosome 11 [3]. No clones have been reported. No structural genes have been reported for the flavone and flavonol pathways known to exist in these non-anthocyanin plants (Table 1). 6.4. Regulatory genes Regulatory genes have been studied mainly within anthocyanin producing species, with transposon tagging as the major technique to isolate the genes. The tissue-specific pathways to 3-hydroxy-anthocyanidins are controlled by genes R/B, C1/PI in maize, by delia, eluta and rosea in Antirrhinum, and by anl,2,4,11 in petunia [29,33,34]. The pathway to 3-deoxy-anthocyanidins, and possibly 3-deoxyproanthocyanidins [5,35], in the pericarp of maize is regulated by the P locus [36]. Delia in Antirrhinum shows extensive homology to the cDNA of R in maize [33]. Conserved domains of transcription factors controlling anthocyanin accumulation (myb and myc type transcription factors) have been identified in maize, petunia and Antirrhinum, an indication that a common gene family regulates pigmentation pattern in diverse plant species [29,33,34]. The evolution and the importance of the variations in the promoter sequences of the different sets of the target, structural genes in the regulation by the above genes are discusssed by Quattrocchio et al. [37] and Koes et al. [38]. No information is available regarding regulatory genes of either the Dianthus or Silene anthocyanin-forming group or for the betalain-containing families of the Caryophyllales. What regulates the regulatory genes remains a mystery.

7. Evolutionary speculations and needed future explorations Speculations about the evolution of betalains and the mutual exclusion of the betalainanthocyanidin pathways can be made [1,39,40]. Did the loss of the 'anthocyanidin synthase' of the

anthocyanin pathway occur first, with a subsequent independent origin of the betalain pathway? Or, did the betalain pathway evolve concurrently, with the ultimate loss of the 'anthocyanidin synthase', now unnecessary since the vegetative and reproductive functions could be adequately performed by the betalain pigments? One possibility is that the 'anthocyanase synthase' step, the step(s) unique to the anthocyanidin pathway, might be inhibited by one of the betalain end-products. No experimental data are available, however, concerning such an inhibition, but betalains have been found to inhibit viral infections and enzymes such as IAA oxidase [3]. Or could the genes have been acquired from the few Basidiomycetes with betalain-type pigments? Neither the enzymology or the regulation of these pigments in fungi are known. In the above models, retention of the entire regulatory system in the Caryophyllales for anthocyanins, or a comparable system, would be required by the betalain-containing groups to explain the similarity of tissue distribution and functions. Would the signal pathways for a common function, such as for defense in vegetative tissue or pollination in flowers, be expected to be conserved during the evolution of the betalain pathway? Can the information about specific promoter sequences that have been identified in enzymes of the anthocyanin pathway to which myc and myb proteins bind as transcription factors, permitting their coordinate activation, be used to explore the regulation of the betalain pathway [25,34-38]? Since only the ~anthocyanidin synthase' is missing, perhaps emphasis should be placed on the regulatory genes such as R, delia and the an series, and the promoter sequences of the structural genes a2, ant17, and candida [29,38]. Unfortunately, neither the critical 'anthocyanidin synthase' nor the 'betalain synthase' have been demonstrated in cell-free systems. Are there related tyrosine derived pathways that might be helpful in understanding the betalain pathway? A better knowledge of the fundamental functions of both these secondary products in vegetative tissues may be central to an understanding of the evolution of these mutually exclusive products. Basic

H.A. Stafford~Plant Sci. 101 (1994) 91-98

research is necessary both in the enzymology and in the molecular genetics of the betalain pathway to explain this curious evolutionary problem. References [1] A. Cronquist, The Evolution and Clasification of Flowering Plants, New York Botanical Gardens, NY, 1988, 555 pp. [2] T.J. Mabry, Betalains, in: E.A. Bell and B.V. Charwood (Eds.), Encyclopedia of Plant Physiology, New Series, Vol. 8, Secondary Plant Products. Springer-Verlag, Berlin, 1980, pp. 513-533. [31 M. Piatelli, The Betalains: structure, biosynthesis, and chemical taxonomy, in: E.E. Conn (Ed.), The Biochemistry of Plants Vol. 7, Academic Press, 1981, pp. 557-575. [4] M. Bokern, V. Wray and D. Strack, Accumulation of phenolic acid conjugates and betacyanins, and changes in the activities of enzymes involved in ferulyglucose metabolism in cell-suspension cultures of Chenopodium rubrum L. Planta, 184 (1991) 261-270. [5] H.A. Stafford, Flavonoid Metabolism. CRC Press, Boca Raton FL, 1990. pp. 298. [6] D.E. Giannasi, Flavonoids and Evolution in the Dicotyledons, in: J.B. Harborne (Ed.) The Flavonoids. Advances in Research since 1980, Chapman & Hall, New York, 1980, pp. 479-504. [7] W. Heller and G. Forkmann, Biosynthesis, in: J.B. Harborne (Ed.) The Flavonoids. Advances in Research Since 1980, Chapman & Hall, New York, 1988, pp. 399-425. [8] J.B. Harborne and J. Grayer, The anthocyanins in: J.B. Harborne (Ed.), The Flavonoids. Advances in Research Since 1980, Chapman & Hall, New York, 1988, pp. 1-20. [9] P.-A., Girod and J-P. Zyrd, Secondary metabolism in cultured red beet (Beta vulgaris L.) cells: differential regulation of betaxanthin and betacyanin biosynthesis. Plant Cell, Tissue, Organ Culture, 25 (1991) 1-12. [10] J.W, McClure, Physiology and functions of flavonoids, in: J.B. Harborne, T.J. Mabry and H. Mabry (Eds.), The Flavonoids, Academic Press, New York, 1975, pp. 970-1055. [11] J. Bianco-Colomas and M. Hugues, Establishment and characterization of a betacyanin producing cell line of Amaranthus tricolor: inductive effects of light and cytokinin. J. Plant Physiol., 136 (1990) 734-739. [12] J.B. Harborne, Introduction to Ecological Biochemistry. Academic Pres, New York, 1982, pp. 1-278. [13] H. B6hm, L. B6hm and E. Rink, Establishment and characterization of a betaxanthin-producing cell culture from Portulaca grandiflora. Plant Cell, 26 (1991) 75-82. [14] B.A. Snyder and R.L. Nicholson, Synthesis ofphytoalexins in sorghum as a site-specific response to fungal ingress. Science, 248 (1990) 1636-1639. [15] G. Hrazdina, Anthocyanins. in: J.B. Harborne, T.J.

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