Flavonoids as developmental regulators

Loverine P Taylor1and Erich Grotewold2

Flavonoids,usually regarded as dispensable phytochemicals

derived from plant secondary metabolism,play important roles

in the biology of plants by affecting several developmental

processes.Bioactive?avonoids also signal to microbes,serve

as allelochemicals and are important nutraceuticals in the

animal diet.Despite the signi?cant progress made in identifying

?avonoid pathway genes and regulators,little is currently

known about the protein targets of?avonoids in plant or animal

cells.Recently,there have been advances in our understanding

of the roles that?avonoids play in developmental processes of

plants.The multiple cellular roles of?avonoids can re?ect their

chemical diversity,or might suggest the existence of cellular

targets shared between many of these seemingly disparate

processes.

Addresses

1511Abelson Hall,School of Molecular Biosciences,Washington

State University,Pullman,Washington99164-4234,USA

2206Rightmire Hall,1060Carmack Road,Department of Plant

Cellular and Molecular Biology and Plant Biotechnology Center,

The Ohio State University,Columbus,Ohio43210,USA

Corresponding authors:Taylor,Loverine P(ltaylor@https://www.360docs.net/doc/5f13054314.html,);

Grotewold,Erich(grotewold.1@https://www.360docs.net/doc/5f13054314.html,)

Current Opinion in Plant Biology2005,8:317–323

This review comes from a themed issue on

Physiology and metabolism

Edited by Toni Kutchan and Richard Dixon

Available online1st April2005

1369-5266/$–see front matter

DOI10.1016/j.pbi.2005.03.005

Introduction

Flavonoids are widely distributed throughout the plant

kingdom and are abundant in many?owers,fruits and

leaves.They are characterized by the presence of two

benzene rings(rings A and B,Figure1)that are linked by

a3-carbon bridge(to form chalcones)or by a pyrane or

pyrone ring(ring C,Figure1).On the basis of the position

of and the modi?cations to the A,B and C rings,the

>4000?avonoids discovered to date can be classi?ed into

several classes,including the?avonols,the?avones,the

iso?avones and the anthocyanin pigments.Although the

most visible function of the?avonoids is the formation

of the red and purple anthocyanin pigments,non-

pigmented?avonoid compounds also play central roles

in the biology of plants,serving as signals for pollinators

and for other bene?cial organisms,participating in plant

hormone signaling,facilitating pollen-tube germination,

protecting plants from UV-B,and functioning as phytoa-

lexins and allelopathic compounds[1].Plant species have

exploited the?avonoid chemical diversity in unique

ways,and?avonoids that have important functions in

one plant(e.g.serving as phytoalexins or facilitating

pollen-tube germination)might not serve a similar func-

tion in another.Although powerful genetic tools aided by

the use of in vitro bioassays have identi?ed the bioactive

?avonoids responsible for several phenomena,the in vivo

protein cellular targets of?avonoids remain largely

unknown.This is a serious shortcoming that requires a

creative integration of chemistry and biology to accelerate

future advances in this?eld.

In this review we examine recent advances in the under-

standing of the roles that?avonoids play in developmen-

tal processes of plants,such as auxin transport,pollen

germination and signalling to microorganisms,and dis-

cuss their allelopathic and anti-tumor activities.We also

discuss the intriguing possibility that the apparently

distinct activities of?avonoids in plant and animal cells

could be a consequence of these phytochemicals mod-

ulating the function of similar proteins in both kingdoms

(Figure2).

Flavonoid effects on auxin transport

Since the early experiments of Jacobs and Rubery[2],

which showed that?avonoids competed with the auxin

ef?ux inhibitor1-naphthylphthalmic acid(NPA)for

transporters in vitro,several studies have shown that

various?avonoids can negatively regulate polar auxin

transport in vivo[3 ,4,5].The search for NPA-binding

factors has resulted in the identi?cation of two protein

complexes,one with high-af?nity and the other with low-

af?nity NPA binding sites.The low-af?nity complex

contains a?avonol-sensitive aminopeptidase AtAPM1

that is localized to the plasma membrane[6],whereas

the high-af?nity complex includes various proteins with

homology to human multidrug resistance(MDR)ABC

(ATP-binding cassette)transporters[6,7].In addition to

these transporters,the asymmetric distribution of the

PIN-FORMED1–4(PIN1–4)auxin ef?ux proteins[8–

11]within localized membrane regions is likely to con-

tribute to auxin polar transport.Consistent with a role of

?avonoids in modulating polar auxin transport(Figure2),

Arabidopsis with mutations in the transparent testa4(tt4)

locus that encodes chalcone synthase(CHS),the?rst

committed step to the?avonoid biosynthetic pathway,

display developmental abnormalities that include an

increased number of in?orescences,reduced plant height,

increased secondary root development[4]and delayed

gravitropism[3 ].These phenotypes can be largely complemented by naringenin,an intermediate in the pathway.The observed delay in the gravitropic response is more consistent with reduced basipetal(downward) auxin transport,rather than with the increased auxin transport that is characteristic of tt4roots[5].These results suggest that elevated basipetal auxin transport interferes with the formation of the auxin gradient that is required for the timely gravitropic response[3 ].The mechanisms by which?avonoids interfere with auxin ef?ux,and hence with the establishment of the auxin polar gradient,are not yet clear.Recent?ndings suggest that,in Arabidopsis?avonoid mutants,the localization of some of the PIN proteins is altered[12 ].It remains to be established whether PIN protein localization is directly altered by?avonoids,perhaps by acting on vesicular traf?cking proteins[13],or whether the altered PIN protein movement is a consequence of?avonoid-mediated alterations in auxin transport[12 ].The iden-ti?cation of PINOID(PID)as a serine or threonine (serine/threonine)kinase that controls the polar localiza-tion of PIN1[14 ]suggests that PID could be one possible target for?avonoid action.Flavonoids and other phenylpropanoids are induced in Arabidopsis roots by light[15].Because the production of auxins in the shoot is also light-induced[16],it is possible that the increased accumulation of?avonoids in the roots helps to balance the level of auxin transport.In addition to the participa-tion of?avonoids in the polar transport of auxins in Arabidopsis,?avonoids are involved in the inhibition of auxin breakdown by peroxidases in white clover[17].It is evident from these and several other studies that ?avonoids are nonessential regulators that are responsible for the establishment of auxin polar gradients that in?u-ence multiple developmental programs.However,funda-mental questions remain.Flavonoids are likely to be synthesized by a multi-enzyme complex located on the cytoplasmic surface of the endoplasmic reticulum[18], and are transported to the vacuolar compartment by a combination of transporters[19]and vesicles[20].But, how much free?avonoid remains in the cytoplasm to modulate the traf?cking or the activity of auxin transpor-ters?How widely spread across the plant kingdom is the participation of?avonoids in the establishment of a polar auxin gradient?If the modulation of polar auxin gradients is a general role of?avonoids,do?avonoids function in all plants in a similar fashion or do they target distinct components of the auxin signaling machinery?All these are questions that still need to be investigated. Flavonoids and pollen germination

Reports of‘white pollen’have been noted in species as diverse as bristle cone pine and morning glory,but it was in maize,with its numerous and well-characterized antho-cyanin mutants,that the correlation between pollen ferti-lity and?avonoids was?rst established[21].The subsequent analysis of CHS mutants in maize and petunia revealed that,although viable,?avonoid-de?cient pollen failed to produce a functional pollen tube.The defect was biochemically complemented and fertility was restored by the application of speci?c?avonols to an in vitro suspen-sion of the pollen or to the stigma at pollination[22].The

318Physiology and metabolism

Figure1

Structure of the main classes of flavonoids,in which R1and R2indicate the sites of possible substitutions.OGly indicates a glycosidic linkage. The numbering system of the flavonoid skeleton is indicated on the flavanone structure.For example if R1is hydrogen(H)then the depicted flavonol structure is3,40,5,7,tetrahydroxy flavone,which is commonly known as kaempferol.

?avonol-requirement for functional pollen occurs in mono-cots and dicots as well as in angiosperms and gymnos-perms,which suggests that it might have arisen in an early ancestor of land plants[23].Signi?cantly,Arabidopsis CHS mutants(tt4)are fertile[24],yet display reduced seed set and reduced in vitro pollen germination[25,26].This suggests that tube growth in Arabidopsis pollen can be enhanced by?avonoids,but that other compound(s) might,in part,compensate for the role of?avonoids. The biosynthesis and metabolism of?avonols in anthers and pollen has been well characterized in maize and petunia.Flavonols are synthesized in the tapetal cells

of the anther and are taken up and glycosylated at the 3OH position(Figure1)in the developing pollen grain

[27].The rapid germination response to nanomolar con-

centrations of speci?c?avonols suggests that they have a signaling function,but the molecular partners of pollen ?avonols are unknown.In an effort to identify down-stream targets of?avonol action,Guyon et al.[28] exploited the germination requirement to characterize transcripts that are up-regulated by the?avonol kaemp-ferol(Figure1)during early pollen-tube growth.This approach identi?ed many low-abundance transcripts that encode regulatory motifs such as SHY,an extracellular leucine-rich repeat protein that is required for pollen-Flavonoids as developmental regulators Taylor and Grotewold319

Figure2

Cellular targets of flavonoids in plant and animal cells.1.Flavonoids inhibit auxin(IAA)efflux.2.Flavonols secreted by the tapetum participate in pollen-tube germination and male fertility.3.Flavonoids secreted by plant roots provide signals for symbionts and serve as allelochemicals.

4.Allelochemicals are either taken up or recognized by yet to be identified membrane receptors,which results in the induction of cell death by activating reactive oxygen species.

5.Interactions between flavonoids and the MDR proteins lead to inhibition of drug efflux activity in animals.

tube penetration into the ovules [29 ].Signi ?cantly,the tomato ortholog of SHY (LeSHY)interacts with a pollen receptor kinase (LePRK2)in yeast two-hybrid and pull-down assays [29 ].This suggests that SHY is involved in a signaling pathway that is mediated by ?avonols.Root hairs are tip-growing cells,like pollen tubes,and mutations have been described that affect polar growth in both tissues [30,31].The ?nding that the ?avonoid-de ?cient petunia described above produces signi ?cantly fewer and shorter root hairs than the wild-type plant,in addition to having impaired pollen-tube growth,suggests that ?avonoids play a role in polar growth (Figure 3).The severity of the mutant phenotype fades with time;after the plant is transferred to soil,the number and length of root hairs of the ?avonoid-de ?cient plants approach that of the wild-type (A Bartley and LP Taylor,unpublished).

Although the abundance of most transcripts in ?avonoid-de ?cient pollen peaks 0.5–2hours after ?avonol applica-tion [28],the response is not rapid enough to trigger pollen germination.The ?rst detected response in ger-minating petunia pollen occurs within one minute —this is the conversion of the ‘rescuing ’?avonol aglycone to a

water-soluble galactoside,which is mediated by a pollen-speci ?c UDP-galactosyltransferase [32].Thus,a chemical biology approach is currently being developed that uses photoaf ?nity-tagged ?avonol analogs to identify the pri-mary targets of ?avonol activity in pollen [33].

Flavonoids as signals to microorganisms

The prerequisite to the formation of the nitrogen-?xing nodule,which can be considered to be a plant organ,is the generation of ?avonoid signal(s)that are secreted from the root exudates of the leguminous host.In lieu of any iden-ti ?ed active uptake mechanism,the ?avonoid aglycone is presumed to diffuse into the rhizobial bacteria [34 ],perhaps through porins.Several years ago,genetic analyses identi ?ed the bacterial nodulating D (NodD)protein to be the target of ?avonoid action [35],but almost 20years on physical con ?rmation of this binding has yet to be demon-strated.Nonetheless,the inferred molecular interaction of ?avonoids with NodD results in the rapid transcriptional activation of the bacterial Nod genes [34 ,36]that encode the so-called Nod factors.Nod factors (bacterial lipo-chito-oligosaccharides)initiate root-hair curling and are involved in the subsequent steps of nodule formation.

After initiating the symbiotic dialog,?avonoids might function as positional signals for cell division and/or growth in nodulating white clover roots.This function was inferred because both the induction of a CHS –Gus A fusion and the accumulation of ?avonoids occurred at the site where either puri ?ed Nod factor or nodulating rhi-zobia strains (but not of non-nodulating strains)were applied [37].In situ ?uorescence emission spectra obtained from individual cells identi ?ed 7,40-dihydroxy-?avone to be the ?uorescing compound that marked the site of nodule formation.Unfortunately,this compound was not measured in an earlier study [38],which showed that Nod factors,NPA and speci ?c ?avonoid aglycones all cause a transient perturbation in polar auxin transport at the application site,as measured by the accumulation of an auxin-responsive fusion marker (GH3:GUS).Thus,the molecular interplay of ?avonoids and Nod factors is likely to occur at several stages during nodule ontogeny.An attempt to establish a correlation between the Nod -gene-inducing activity of root exudates and the number of nodules formed,purported to show that the nodulation response could be dissociated from the ?avonoid burst in roots after bacterial inoculation [39].This type of approach raises issues that can only be resolved by the analysis of a true ?avonoid-null mutant of a leguminous species,which unfortunately has not yet been identi ?ed.

Allelopathic and anti-tumor activities of ?avonoids:common targets in different kingdoms?

Flavonoids have long been known to be important nutra-ceutical components of our diet.In part,this is a con-320Physiology and metabolism

Figure

Flavonoid-deficient

Wild-type

Current Opinion in Plant Biology

Root-hair formation and growth are severely retarded in flavonoid-deficient petunia plants.(a)A flavonoid-deficient,male sterile Petunia hybrida mutant [22]produced fewer and shorter root hairs compared with (b)a V26wild-type line after five days of growth.After two weeks of growth,the mutant and wild-type root-hair pattern was similar.The magnified images in the right panels show that the

flavonoid-deficient plants do produce root hairs but that the density and length are highly reduced compared with wild-type.Surface sterilized seeds were plated on MS media solidified with agarose and the plates were placed in the vertical position.Digital images of root growth at five days were obtained using an Olympus CK2inverted microscope.Scale bars represent 500m m.

sequence of the potent anti-oxidant properties displayed by many?avonoids and phenolic compounds[40]. Whether?avonoids have a similar anti-oxidant function in plants is not known,but the recent?nding that the R2R3MYB regulators,which regulate the?avonoid path-way,are themselves reduction–oxidation(REDOX)-con-trolled[41 ]is intriguing and might suggest a link between the REDOX potential of the cell and the control of accumulation of?avonoid compounds.The?avonoid (à)-catechin is a potent phytotoxin that is secreted by the roots of the invasive plant Centaurea maculosa[42 ].In susceptible plants,this allelochemical triggers a wave of reactive oxygen species in the roots that ultimately results in cell death.Resistance to allelochemicals is largely accomplished through detoxi?cation pathways that involve the modi?cation,followed by the secretion or the vacuolar sequestration,of xenobiotics[43]in mechan-isms similar to those used in the normal traf?cking of phytochemicals[44].The direct cellular targets of(à)-catechin and of other allelochemicals remain to be iden-ti?ed.Flavonoids also induce programmed cell death (apoptosis)in several animal model systems[45].Given the central role that apoptosis plays in the development of metazoans[46],the question remains as to whether ?avonoids have the potential to target similar enzymes in plants as they do in animals,with the consequent effect on plant development.

In addition to their anti-oxidant properties,?avonoids have anti-proliferative,anti-tumor and pro-apoptotic activities.This is likely to be a consequence of their effect on several mammalian enzymes[47].For exam-ple,?avonoids have estrogenic activity[48],inhibit protein-tyrosine kinase activity[49,50],and function as allosteric activators of the sirtuin deacetylases (NAD+-dependent deacetylases)that regulate caloric-restricted longevity in yeast,worms,?ies and in human cells[51 ,52,53].A screen for small molecule inhibitors of animal cell sirtuin deacetylases identi?ed sirtinol,a cell-permeable non-natural product[54].Subsequently, the isolation and characterization of a sirtinol-resistant1 (SIR1)mutant in Arabidopsis provided evidence that SIR1functions as a negative regulator of auxin signaling [55].Moreover,sirtinol is proposed to bind to a putative ATP binding site in the SIR1protein.Thus,a potential link is made between auxin signaling,sirtuin-like dea-cetylases and?avonoids.An additional important phar-macological application of?avonoids is their ability to inhibit the P-glycoprotein MDR1,a key component of the MDR response of animal cells to multiple anti-tumor drugs[56].This inhibition of MDR1bears resemblance to the effect that?avonoids have on components of the auxin ef?ux machinery(Figure2).Flavonoids are known to mimic ATP as they bind to a number of nucleotide-binding proteins,and nuclear magnetic resonance ana-lysis has identi?ed the polar groups at3,40and50to be the sites of interaction with the nucleotide-binding

domain(NBD)of a mouse MDR transporter[57 ].Both the?avone aglycone luteolin,and its7-O-glycoside,bind in the same pocket within the mouse MDR protein.This feat can be accomplished because substitutions at the7 position in ring A are1808opposite the binding contacts at3,40and50described above.It is questionable whether anthocyanins(anthocyanidin3-O-glycopyranosides) could be accommodated in this orientation because the 3OH group,the conjugation site for the mono-,di-and tri-glycosides,is directly in the middle of the binding pocket.MDR proteins are conserved across kingdoms,a property that was exploited to isolate a maize MDR gene (ZmMrp3)that is speci?c for anthocyanin transport into the vacuole[19].The results of binding studies with the maize MDR protein and various chemical species of ?avonoids should provide information on the important contact sites.

Conclusions

It is evident that the role of?avonoids in the fundamental aspects of the plant biology goes beyond the decorative or accessory functions associated with their historical classi?cation as‘secondary metabolites’.Genetic ap-proaches,combined with the genomic resources in Arabidopsis,have de?ned numerous?avonoid functions.

They have also ordered genes in the biosynthetic path-way and have facilitated the identi?cation of regulators.

However,these approaches have not yet provided signi?cant insights into how?avonoids function mechan-istically and have not identi?ed the protein targets of ?avonoids inside the cell.

This review has touched on a few new developments in the detection of?avonoid–macromolecular interactions and hopefully will spur more development in this area.

For example,mass spectrometry and high pressure liquid chromatography analysis have made quantitation and structural identi?cation of?avonoids routine;how-ever,a major limitation toward the assignment of a functional role for?avonoids in general,or to a speci?c molecular species,is the inability to detect or to identify ?avonoids in the tissues or the individual cells that express a response.The combination of?uorescent microscopy with sensitive and speci?c detection probes and/or techniques at the cellular and the sub-cellular level is required.Moreover,the time is ripe for the development of creative approaches that use chemical biology to discover the cellular targets and mechanistic action of?avonoids.It is very likely that these studies will reveal that the cellular proteins recognized by?a-vonoids in plants are very similar to those that?avonoids target in animals.

Acknowledgements

This work was supported by grants from the National Science

Foundation(MCB-0130062and MCB0437318)and the US

Department of Agriculture(NRICGP2003-02158)to EG.

Flavonoids as developmental regulators Taylor and Grotewold321

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Flavonoids as developmental regulators Taylor and Grotewold323