Copyright © Physiologia Plantarum 2000PHYSIOLOGIA PLANTARUM 110: 152–157. 2000Printed in Ireland—all rights reser6ed ISSN 0031-9317

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Regulation of flower pigmentation and growth: Multiple signalingpathways control anthocyanin synthesis in expanding petals

David Weiss*

The Kennedy-Leigh Centre for Horticultural Research and The Otto Warburg Center for Biotechnology in Agriculture, Faculty ofAgricultural, Food and En6ironmental Quality Sciences, The Hebrew Uni6ersity of Jerusalem, P.O. Box 12, Reho6ot 76100, Israel*E-mail: weiss@agri.huji.ac.il

Received 31 January 2000; revised 4 May 2000

induction of anthocyanin gene transcription and for accumula-Anthocyanins are the major flower pigments in higher plants.tion of the pigment in the developing corolla. It was alsoIn most cases, anthocyanin accumulation is an integral part of

flower development and the processes of petal pigmentation shown that the effect of these signals is not specific for thepigment’s biosynthesis, they also control petal cell expansionand cell expansion are tightly linked. Activation of the an-and induce the expression of genes from various pathways.thocyanin pathway during petal development requires a com-

plex interaction between environmental and developmental These results support a model in which GAs, light and sugarspromote the activity of master transcription regulators thatsignals. Using Petunia hybrida flowers as a model, some of

these signals were identified and characterized. Gibberellins control various pathways to complete the entire process offlower development.(GAs), sugars and light were shown to be required for the

tion of the anthocyanin pathway in flowers have beenstudied in several model plants including Petunia hybridaand Antirrhinum majus (Martin and Gerats 1993, Mol et al.1998). These plants provide mutants at various steps of thepathway as well as at the regulatory machinery and this hasenabled the pathway’s characterization. Most of the en-zymes involved in anthocyanin biosynthesis have been char-acterized and the corresponding genes cloned (Holton andCornish 1995). In addition, several transcription factorsregulating the anthocyanin pathway in flowers have beenidentified, cloned and characterized (Mol et al. 1998).

Activation of the anthocyanin pathway and accumulationof the pigment during petal development require a complexarray of environmental and developmental signals (Mol etal. 1996). Several reviews have detailed the genetic regula-tion of flower pigmentation and the transcription factorsinvolved (Martin and Gerats 1993, Holton and Cornish1995, Mol et al. 1998). The present review describes the roleof environmental and internal signals in the regulation ofanthocyanin synthesis and petal growth during flowerdevelopment.

Introduction

The ability of plants to prosper throughout millions of yearsof evolution has been strongly dependent on the constantdevelopment of new showy traits. This has led to thecreation of splendid color patterns generated mainly by theflower petals. Two major groups of pigments are producedin the petals: anthocyanins and carotenoids. Anthocyaninsare water-soluble pigments accumulating in the vacuole ofpetal epidermal cells and confer colors ranging from orangeto violet (Martin and Gerats 1993). In most plants, an-thocyanin synthesis in the flowers is under developmentalregulation and its accumulation coincides with petal growth(Mol et al. 1996). In the early stages, petals of most flowersare green due to the presence of chlorophyll in the tissue,and they exhibit a slow growth rate, resulting mainly fromcell division (Weiss and Halevy 1989). Anthocyanin accumu-lation usually occurs at later stages of petal development.These stages are characterized by rapid growth, resultingonly from cell expansion (Martin and Gerats 1993, Ben-Nis-san and Weiss 1996). The anthocyanin biosynthetic pathwayhas been studied intensively in the last 3 decades (Koes et al.1994). The physiological, biochemical and molecular regula-

Abbre6iations – chi, chalcone flavanone-isomerase gene; chs, chalcone synthase gene.

Physiol. Plant. 110, 2000152

The role of anthers and gibberellin

Anthers are required to complete various steps at the laterstages of flower development, including petal growth, anddevelopment of ovaries and styles (Pharis and King 1985).Several studies have indicated that one of the signals in-volved might be gibberellin (GA). Removal of the anthersfrom several flowers prevented those flowers’ developmentand application of GA restored the anthers’ effect (Plack1958, Moham Ram and Rao 1984, Bala et al. 1985, Weissand Halevy 1989). Petal growth in several plant species isassociated with a transitory increase in GA content (Pharisand King 1985). Koning (1985) found a sharp increase inGA levels in Gaillardia petals at the start of the corolla’s fastgrowth stage, followed by a decrease later on. In the Ara-bidopsis GA-deficient mutant, ga1-1, petal growth is ar-rested and the defect can be completely eliminated if GA isapplied to the young flower buds (Goto and Pharis 1999).The tomato GA-deficient mutant, gib-1, initiates flowerbuds, but floral development is not completed unless themutant is treated with GA (Jacobsen and Olszewski 1991).Physiological, biochemical and molecular evidence suggestthat GAs are produced in the developing anthers (Pharisand King 1985, Weiss et al. 1995, Itoh et al. 1999, Rebers etal. 1999). The GA-biosynthetic gene, GA1, that encodesent-kaurene synthase, is primarily expressed in the anthersof Arabidopsis flowers (Silverstone et al. 1997). Promoter-GUS fusion experiments showed that two of the ArabidopsisGA 20-oxidase genes (GA20ox1 and GA20ox2) are ex-pressed in pollen grains (P. Hedden, University of Bristol,personal communication). The tomato GA 20-oxidase gene,is expressed in the tapetum cells (Rebers et al. 1999) and theexpression of the tobacco GA-3b-hydroxylase gene is re-stricted to tapetum cells and pollen grains (Itoh et al. 1999).

Anthocyanin accumulation is an integral part of flowerdevelopment in most plants (Martin and Gerats 1993) andseems to be regulated by the same factors that control petalgrowth. The role of anthers and GA in the regulation ofcorolla growth and anthocyanin synthesis was studied inPetunia hybrida. Removal of anthers from young greenflower buds, inhibited corolla growth and pigmentation(Weiss and Halevy 1989). Application of GA3 replaced theanthers in their effect on both processes. These phenomenawere also found with detached young flower buds grown insucrose solution (Weiss et al. 1995). In detached corollas,isolated at the early-green stage and grown in vitro insucrose medium, GA3 promoted growth and was essentialfor anthocyanin synthesis (Weiss and Halevy 1989). At laterstages, after the transition to the phase of rapid elongation,the corolla is no longer dependent on the anthers or exoge-nous GA for growth and pigmentation. GA may be re-quired only for the initiation of these processes but not fortheir maintenance. Analysis of endogenous GAs in youngpetunia anthers and corollas indicated the presence of thebiologically active GA1 and GA4. Exogenous application ofthese GAs to detached corollas promoted growth and in-duced anthocyanin accumulation (Weiss et al. 1995). It wasconcluded that GAs are produced in the developing anthersand transported to the corollas where they induce growthand pigmentation.

GAs control anthocyanin accumulation in attached anddetached petunia corollas through the induction of an-thocyanin biosynthetic gene expression (Weiss et al. 1992,1995). Analysis of P. hybrida mutants affected in theirability to regulate pigmentation indicated that anthocyaninbiosynthetic genes can be divided into two classes and thateach class is likely to be controlled by different regulators(Mol et al. 1998). The early biosynthetic genes (first class)are chalcone synthase (chs), chalcone flavanone-isomerase(chi ) and flavanone 3-hydroxylase ( f3h), and the latebiosynthetic genes (second class) are dihydroflavonol reduc-tase (dfr), anthocyanidin synthase (as), UDPG flavonoid3-O-glucosyltransferase (uf3gt) and ramnosyl transferase(rt). To coordinately activate all biosynthetic genes duringflower development, the different regulators need to act inconcert, and may be governed by a common signal. GAseems to be a good candidate, since it induces genes fromboth classes (chs, chi, dfr, as and rt) with similar kinetics(Weiss et al. 1995). Run-on transcription assays and analysisof transgenic petunia plants transformed with the GUSreporter gene fused to the chs or chi promoters indicate thatthe hormone induces anthocyanin biosynthetic gene expres-sion by regulating transcription initiation (Weiss et al. 1992).Several observations suggest that GA controls anthocyaningene transcription indirectly: the effect of GA on chs mRNAaccumulation is observed several hours after its applicationand its induction is inhibited by the protein synthesis in-hibitor, cycloheximide (Weiss et al. 1992). Both observationssuggest that GA first induces the synthesis of trans-actingfactors which in turn, activate the entire anthocyanin path-way. It was recently found that the hormone promotes theexpression of two regulators of the anthocyanin pathway inpetunia flowers: a basic helix-loop-helix (bHLH) protein(an1) and a MYB-type protein (an2) (J Kooter, Free Uni-versity of Amsterdam, personal communication) both regu-lating late biosynthetic genes.

The early events in the GA-signal transduction werestudied in petunia corollas using agents that inhibit orpromote specific steps in signal transduction (Leitner-Daganand Weiss 1999). The results of this study suggest thatcalcium from internal, but not external sources is involvedas a second messenger. The effect of calcium seems to bemediated by the calcium-binding protein, calmodulin. It wasalso shown that protein dephosphorylation is required forGA-induced gene expression. However, the sequence ofevents and the precise relationship between these compo-nents are still unknown.

The effect of GA is not specific to the induction of corollapigmentation or to the activation of the anthocyanin biosyn-thetic genes: GA also induces corolla growth (Weiss andHalevy 1989). GA3 has been shown to promote variousprocesses in the developing corolla, including respirationand the expression of genes from primary metabolic path-ways (Ben-Nissan and Weiss 1995). GA3 also induces theexpression of the gibberellin-induced gene1 (gip1). Severalindirect pieces of evidence connect gip1 with the process ofGA-induced corolla cell expansion (Ben-Nissan and Weiss1996). GA may induce an entire developmental program atthe later stages of corolla development, probably via theactivation of master regulatory genes. These factors may

Physiol. Plant. 110, 2000 153

induce genes from various pathways, leading to corollagrowth and pigmentation (Fig. 1). A MYB-type regulatorygene, myb92, was cloned from petunia corolla and its ex-pression was found to be induced by GA in a primaryfashion (rapid induction and no sensitivity to cyclohex-imide). Although myb92 may be a master regulator in theGA-signal cascade, its target DNA sequences are still un-known (L. Mur. 1995. Thesis, Vrije University, Amsterdam,The Netherlands).

The role of GA in petal growth has been demonstrated inmany plants and seems to be a general phenomenon (Pharisand King 1985). However, it is not yet clear how general theeffect of the hormone is on petal pigmentation. GA3 pro-motes anthocyanin accumulation in detached Hyacinthusorientalis (Hosokawa et al. 1996) and ‘Baccara’ rose flowers(Zieslin et al. 1974). The hormone also induces pigmentationin expanding cucumber petals. In this case however, GA3

induces the synthesis of carotenoid pigments and accumula-tion of the chromoplast-specific protein, CHRC (Vainsteinet al. 1994). In Phlox flowers on the other hand, GA inhibitsanthocyanin synthesis in the petals (D. Weiss, unpublisheddata). In this latter plant, anthocyanins accumulate at theearly stages of flower development, before the rapid expan-sion of the petals. It is therefore possible that GA controlspigmentation only in those cases where pigment accumula-tion is directly tied to petal cell expansion.

The role of light

Light regulation of anthocyanin synthesis is often used tostudy photomorphogenesis. Many studies have shown thatlight induces anthocyanin accumulation in green tissues andcultured cells via the activation of anthocyanin biosyntheticgenes (Mol et al. 1996). UV-A and blue light control chsgene expression in Arabidopsis leaves (Christie and Jenkins1996). White and red light induce anthocyanin accumulation

and anthocyanin biosynthetic gene transcription in eggplanthypocotyl (Toguri et al. 1993) and tomato seedlings (Bowleret al. 1994).

Although anthocyanins are the most abundant pigmentsin flowers, very little is known about the role of light incontrolling their synthesis in the perianth. Several studieshave shown that under low light intensity, plants developpale flowers with a low level of anthocyanins (Biran andHalevy 1974, Griesbach 1992). The mechanism by whichlight affects anthocyanin synthesis was studied in petuniacorollas (Weiss and Halevy 1991, Moscovici et al. 1996).When detached petunia flowers were grown in vitro, in thedark, corolla growth, anthocyanin accumulation and chsexpression were strongly inhibited. However, if attachedyoung flowers were covered on illuminated plants, corollasdeveloped normally and the levels of chs transcript andanthocyanin were only slightly reduced. It was concludedthat the green leaves perceive the light and transmit a signalto the corolla (Moscovici et al. 1996). The nature of thetransmitted signal is still unknown but it was shown not tobe GA. While GA is only required at early stages of corollamaturation, light promotes pigmentation and growth ateven later stages of corolla development.

The promotion of petunia corolla pigmentation and chsgene expression is photon-flux dependent. Continuous illu-mination at relatively high-energy fluxes was necessary formaximum levels of gene expression and pigmentation and itwas suggested that high irradiance reactions (HIR) areinvolved (Weiss and Halevy 1991). Through the use ofvarious inhibitors, it was shown that photosynthesis doesnot significantly contribute to the leaf-mediated light re-sponses. Blue light and red light had similar effects ontranscript accumulation, whereas the effect of green lightwas slightly lower (Moscovici et al. 1996). The effect of redlight might be mediated by phytochrome and that of blueand green light by the blue light/UV-A photoreceptor, cryp-tochrome (Lin et al. 1995).

Fig. 1. A model describing the role ofGAs, light and sugar in the regulationof petunia corolla growth andpigmentation. According to the model,these 3 signals interact to inducemaster regulators, controlling genesfrom various pathways leading tocorolla cell expansion andpigmentation. GAs (GA1 and GA4)are produced in the developinganthers and relocated to the corollas.Light is perceived by the green leaves(phytochrome [PHY] andcryptochrome [CRY] mediated highirradiance reactions [HIR]) andinduces the production of anunknown mobile signal that moves tothe corolla to induce pigmentationand growth. Light is also required forphotosynthesis and sugar productionin leaves. Sugars are then transportedto the corollas and act as a signal viathe hexokinase (HXK) pathway toenhance the effect of the GA andlight signals.

Physiol. Plant. 110, 2000154

The role of leaves in the light regulation of petal pig-mentation and growth is probably not unique to petunia.Biran and Halevy (1974) showed that covering rose flow-ers does not inhibit petal growth and pigmentation,whereas covering or removing leaves decreases flower freshweight and anthocyanin content. Direct illumination ofEustoma grandiflorum flowers is not required for chs ex-pression and anthocyanin accumulation. These processesare controlled by illumination of the green leaves (Kawa-bata et al. 1995, Moscovici et al. 1996). Similar resultswere also found in Quisqualis indica (D. Weiss, unpub-lished results).

In green tissues, anthocyanin acts as a protective UV-absorbing pigment (Jenkins et al. 1995). UV-B radiationinduces anthocyanin synthesis in leaves of various plantspecies (Chalker-Scott 1999), including Arabidopsis(Jenkins et al. 1995) and petunia (Koes et al. 1989). Theeffect of UV radiation on petal pigmentation is less clear.UV radiation promotes anthocyanin accumulation in Kan-garoo Paw flowers (Ben-Tal and King 1997) and in apple,UV-B is required for normal flower pigmentation (Donget al. 1998). However, in many other flowers, includingpetunia, Geranium, Phlox, Impatiens, Lobelia (Klein 1990)and Antirrhinum majus (D. Weiss unpublished results), UVradiation has no effect on anthocyanin synthesis.

The role of sugars

Flowers of most plants are heterotrophic and thereforerequire imported carbohydrates for their development. Theflower bud is a major sink for assimilates under favorablegrowth conditions, whereas a shortage of carbohydratesoften leads to the arrest of flower development (Halevy1987). The use of sugars to promote flower developmentand pigmentation is common practice, and most pulsingand holding solutions applied to cut flowers include su-crose (Suc) (Halevy and Mayak 1981). Increased Suc con-centration enhanced petal growth and pigmentation indetached flowers of Eustoma grandiflorum (Kawabata etal. 1995), Hyacinthus orientalis (Hosokawa et al. 1996),Liatris spicata (Han 1992) and rose (Kuiper et al. 1991).Several studies have shown that sugar level increases dur-ing petal development to a maximum at the stage of rapidcell expansion (Tsukaya et al. 1991, Bieleski 1993,Clement et al. 1996). The role of sugars in flower develop-ment may be multifunctional: they can act as an energysource (Moalem-Beno et al. 1997), as osmotic regulators(Ho and Nichols 1977, Bieleski 1993), as precursors formetabolic processes and as a signaling molecule (Neta-Sharir et al. 2000). In the early stages of daylily flowerdevelopment, petals accumulate fructan that later, at thestage of rapid cell expansion, is hydrolyzed to fructose(Fru) (Bieleski 1993). The increased osmoticum in thepetals was suggested to be the driving force for their ex-pansion. Ho and Nichols (1977) suggested that solublesugars in rose flowers are important as osmotically activesubstances in promoting petal growth. The mechanism bywhich sugars regulate petal growth and pigmentation wasstudied in petunia flowers. When detached petunia corol-

las were grown in vitro, they elongated and became pig-mented only in the presence of Suc and GA3 in the light(Weiss and Halevy 1989). Both GA3 and Suc were re-quired for transcription of the chs gene: GA3 was essentialfor its induction and Suc enhanced the effect of the hor-mone. Other metabolic sugars, such as glucose (Glc) andFru had the same effect as Suc (Weiss et al. 1992). Theeffect of sugars on chs expression is not via modificationof the osmotic potential of either the corolla cells or thegrowing solution. The nonmetabolized 3-O-methylglucose,which is taken up by the cells, and mannitol, which isnot, have no effect on corolla growth, anthocyanin syn-thesis or chs expression (Moalem-Beno et al. 1997). It wasalso shown that the effect of sugars in the induction ofgene expression is not dependent on their metabolism inglycolysis or on changes in phosphate level (Neta-Sharir etal. 2000). Many studies have shown that sugars act assignal molecules in higher plants, and in most cases, sugarphosphorylation by hexokinase is required to initiate sig-nal transduction (Jang and Sheen 1997). Several pieces ofevidence support sugars’ signaling role in petunia corollasand the possible involvement of hexokinase as the sugarsensor. Mannose, which is inefficiently metabolized but isphosphorylated by hexokinase at an efficiency similar toGlc, is as effective as Glc in promoting gene expressionand pigmentation. 2-Deoxyglucose, which is a substratefor hexokinase but is not metabolized in glycolysis, alsopromotes gene expression. On the other hand, mannohep-tulose, a competitive inhibitor of hexokinase, completelyabolishes the promotive effect of Glc (Neta-Sharir et al.2000).

The possibility that sugars play a signaling role in theactivation of chs expression is supported by reports show-ing that in Arabidopsis and soybean leaves, sugars regulatechs expression directly (Tsukaya et al. 1991, Sadka et al.1994). Since the increase in chs expression in the develop-ing petunia corolla coincides with an increase in hexoselevels, it was suggested that chs is regulated directly bychanges in intracellular sugar levels (Tsukaya et al. 1991).Other studies, on the other hand (Weiss et al. 1992,Moalem-Beno et al. 1997), have shown that increased in-tracellular sugar levels in petunia corollas are not suffi-cient to induce chs expression, and both GA and sugarsare required for the gene’s induction. The sugar signalseems to interact with the GA signal in petunia flowers,since it specifically promotes the expression of GA-in-duced genes such as chs and gip1 but not that of othergenes (Neta-Sharir et al. 2000). Sugars may promote theactivity of an upstream component in the GA-signal-trans-duction pathway, or induce a specific trans-acting factor,which, in turn, induces various GA-induced genes. Indeed,the chs promoter contains a region that shares homologywith the ‘sugar box’ of the sweet potato sporamin pro-moter (Tsukaya et al. 1991).

The promotive effect of sugars on petal growth andpigmentation seems to be a general phenomenon. How-ever, it is still not clear whether sugars act in all cases asspecific signaling molecules to promote gene expression, orvia another mechanism.

Physiol. Plant. 110, 2000 155

Concluding remarks

Petal development can be divided into two phases: a first,slow-growth phase, resulting mainly from cell division and asecond, rapid-growth phase, resulting only from cell expan-sion (Martin and Gerats 1993). In most plants, petal pig-mentation takes place during the second phase of theirdevelopment, and pigment accumulation is tightly linked tothe process of cell expansion. Various works have shownthat anthocyanin synthesis and petal growth are regulatedby the same environmental and internal signals, but most ofthese studies focused on the horticultural aspects of thisphenomenon. Three signals were identified and character-ized in Petunia flowers: GA, light and sugars. These signalscontrol anthocyanin synthesis, corolla cell expansion andthe expression of genes from various pathways (Weiss andHalevy 1989, Ben-Nissan and Weiss 1995, 1996, Moscoviciet al. 1996, Neta-Sharir et al. 2000). While GA is requiredonly for the initiation of these processes at the early stage offlower development (Weiss and Halevy 1989), sugars andlight are needed to maintain growth and pigmentationthrough the entire process of corolla development(Moscovici et al. 1996). The results of these studies supporta model in which the GA, light and sugar signals interact tocontrol master transcription regulators that act upstream ofthe specific regulators of the anthocyanin pathways and ofother pathways involved in cell expansion (Fig. 1). Theactivation of these pathways during the transition to thesecond phase of development is required to complete theprocess of flower growth and opening.

The Arabidopsis nap gene is one good candidate to play arole in the regulation of the transition to the second phaseof corolla development. The expression pattern of nap andthe phenotypes caused by its misexpression suggest that inpetals it controls the transition from growth by cell divisionto growth by cell expansion. nap expression seems to beregulated by a diffusible signal produced by the stamens(Sablowski and Meyerowitz 1998). Since Arabidopsis petalelongation requires GA (Koornneef and van der Veen 1980)and GAs are produced in the anthers (Silverstone et al.1997), it is possible that nap expression is indeed induced bythe hormone. nap is an immediate target of the floralhomeotic class B proteins, AP3 and PI, but their expressionis not sufficient for its activation: while AP3/PI expressionstarts before petal formation, nap is transiently induced laterduring petal development. It is possible that the nap pro-moter requires a GA-induced trans-acting element for itstemporal regulation in addition to the AP3/PI heterodimerfor its spatial activation.

The petunia MADS box gene, fbp2, regulates floral organidentity at whorls 2, 3 and 4. Inhibition of fbp2 expression intransgenic petunia plants resulted in the development ofhighly aberrant flowers that possess a green corolla ofreduced size (Angenent et al. 1994). Expression of the chsgene in the green corollas is down regulated and could notbe induced by GA3. FBP2 and GA may control chs throughdifferent pathways: FBP2 is required for chs spatial expres-sion while GA is required for its temporal activation.

Thus, it is possible that the activation of master regula-tors, which control the transition of petals to the phase of

cell elongation and pigmentation, requires a floral homeoticprotein (Zachgo et al. 1995) to act in concert with GA, lightand sugar. Together, these factors define the place and timefor the expression of the various downstream genes. Futurework should focus on the identification of such masterregulators and the characterization of their interaction withenvironmental and developmental signals.

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Edited by C. H. Bornman

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