Chapter 1 Flavonoids: a colorful model for the regulation and … · pigmentation gene PhAN2 in petunia species with colored and white flowers, showed that the color change by the

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Vacuolar acidification: mechanism, regulation and function in petunia flowers

Verweij, C.W.

2007

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7

Chapter 1

Flavonoids: a colorful model for the regulation and evolution of

biochemical pathways

Ronald Koes, Walter Verweij and Francesca Quattrocchio

This article has been published in modified form in Trends in Plant Sciences. May

2005;10 (5): 236-42

Abstract

For more than a century, the biosynthesis of flavonoid pigments has been a favorite of

scientists to study a wide variety of biological processes like inheritance and transposition,

and became one of the best-studied pathways in nature. The analysis of pigmentation

continues to provide insights into new areas, such as the channeling and intracellular

transport of metabolites, regulation of gene expression and RNA interference. Moreover,

because pigmentation is studied in many species, it provides unique molecular insights into

the evolution of biochemical pathways and regulatory networks.

Chapter 1

8

Synthesis and function of flavonoid pigments

The pigments that color most flowers, fruits and seeds are flavonoid secondary metabolites.

Flavonoids are synthesized by a branched pathway that yields both colorless compounds

(e.g. flavonols) and colored pigments such as anthocyanins, polymeric phlobaphenes and

proanthocyanidins (PAs) (Figure 1).

The different flavonoids have several biological functions, including protection against UV-

light and phytopathogens (phytoalexins in legumes), signaling during nodulation, male

fertility, and auxin transport (Mol et al., 1998; Winkel-Shirley et al., 2002). It was long

suspected that the coloration of flowers is a visual signal to attract visits of pollinating

animals. Recent field studies with nearly isogenic Mimulus lines showed that a (carotenoid-

based) color change can indeed lead to a switch from bumblebee to hummingbird-

pollination, which may result in genetic isolation and ultimately speciation (Bradshaw et

al., 2003). Whether pigmentation (or other floral traits) plays an equally important role in

other species is, however, under debate (Brown et al., 2002). Analysis of the flower

pigmentation gene PhAN2 in petunia species with colored and white flowers, showed that

the color change by the an2 mutation happened after their genetic isolation and thus was, at

least in this case, not the prime cause of speciation (Quattrocchio et al., 1999).

The pigmentation pathways provide a natural reporter gene system that has been used to

study a wide variety of processes since the works of Gregor Mendel. This chapter describes

the molecular analysis of genes involved in pigmentation and genes involved in vacuolar

pH, providing new insights into the intracellular transport of metabolites and the regulation

of biochemical pathways. Curiously, pigmentation and pH regulation are found to be

intimately linked with the control of several other processes, including cell morphogenesis

(hair formation) and the biogenesis and physiology of vacuoles.

Biosynthesis and transport

Most of the flavonoid enzymes are recovered in the “soluble” cell fractions, and immuno-

localization experiments suggest that they may be loosely bound to the endoplasmatic

reticulum, possibly in a multi-enzyme complex (Saslowsky et al., 2001; Winkel-Shirley et

al., 2001) whereas the pigments themselves accumulate in the vacuole (anthocyanins and

PAs) or the cell wall (phlobaphenes) (Grotewold et al., 2004). In maize, the vacuolar

sequestration of anthocyanins requires an MRP-type (multidrug resistance associated

Introduction

9

protein) of transporter on the tonoplast membrane, which expression is co-regulated with

the structural anthocyanin genes (Goodman et al., 2004). MRPs are often referred to as

glutathione S-conjugate (GS-X) pumps, because they transport a variety of glutathione

conjugates. Indeed, Glutathione S-Transferase-like (GST) proteins have been shown to be

required for vacuolar sequestration of pigments in maize Bronz2 (ZmBz2), petunia

anthocyanin 9 (PhAN9) and Arabidopsis Transparent Testa 19 (AtTT19) (Marrs et al.,

1995; Alfenito et al., 1998; Kitamura et al., 2004). Because anthocyanin-glutathione

conjugate(s) could never be found, it was proposed that these GSTs might deliver their

flavonoid substrate directly, without glutathionation, to the transporter (Mueller et al.,

2000). AtTT12 of Arabidopsis encodes a protein of the MATE (multidrug and toxic

compound extrusion) group of transporters that is required for vacuolar localization of PAs

in the testa of the seed (Debeaujon et al., 2001). Although MATE transporters are not

known to require glutathionated substrates, the vacuolar localization of PAs does require

the GST-like protein AtTT19 (Kitamura et al., 2004). PA precursors in tt19 mutants appear

to accumulate in vesicle-like structures, rather than freely in the cytoplasm, suggesting that

AtTT19 does not act in the transmembrane transport as such, but in subsequent transport of

the vesicles to the large vacuole (Kitamura et al., 2004). In tomato, an AtTT12-like

membrane transporter and a PhAN9-AtTT19-like GST were identified that are co-regulated

with other structural anthocyanin genes and probably involved in vacuolar sequestration of

anthocyanins (Mathews et al., 2003).

Interestingly, PhAN9 and AtTT19 belong to a different GST class than ZmBZ2, suggesting

that the GSTs were, like the transporters (MATE of MRP-type), recruited two times

independently during evolution. Because GST mutants in maize (bz2), petunia (an9) and

carnation (fl3) can be complemented by both PhAN9 and ZmBZ2, it appears that these

distinct GSTs fulfill similar roles (Alfenito et al., 1998; Larsen et al., 2003). Whether the

MATE and MRP transporters are also functionally exchangeable has not been tested, and

thus it cannot be excluded that both transporters are required together.

Chapter 1

10

phenylalanine 4-coumaroyl-CoA

CHS

CHI

F3H

chalcone

naringenin

dihydrokaempferol

dihydromyricetin

delphinidinpelargonidin

dihydroquercitin

cyanidin

vacuolar anthocyanin

leucocyanidin leucodelphinidinleucopelargonidin

flavan 4-ol

ph

lob

ap

hen

es in c

ell

wall

LARcatechin

polymerization & transport

5GTs, RTs, ATs, MTs etc.

decorated anthocyanin

DFRDFR

DFR

DFR

LDOX LDOXLDOX

GST

flavonols

TRANSPORTER

epicatechinANR

GST

colorless

proanthocyanidin

CE

brown proanthocyanidin

derivative

F3’H F3’5’H

TRANSPORTER

vacuole

vacu

ole

3GT 3GT3GT

anthocyanin 3-glycoside

Figure 1. Biosynthesis of flavonoid pigments. The basic skeleton of all flavonoids consists of three

aromatic rings and is generated by the enzymes chalcone synthase (CHS) and chalcone isomerase

(CHI). Oxidation of the central ring by flavonoid 3-hydroxylase (F3H) yields a dihydroflavonol

(dihydrokaempferol), which can be hydroxylated on the 3’ or 5’ position of the B-ring by flavonoid

3’-hydroxylase (F3’H) and/or flavonoid 3’5’hydroxylase (F3’5’H) yielding precursors of orange

(pelargonidin), red-magenta (cyanidin) and purple-mauve (delphinidin) anthocyanin pigments. Some

plant species (e.g. rose, carnation) cannot make purple colors because they lack F3’5’H and/or F3’H.

Dihydroflavonols are converted by dihydroflavonol reductase (DFR), leucoanthocyanidin oxidase

(LDOX) and a 3-glucosyl transferase (3GT) to yield an anthocyanin 3-glucoside that can be further

substituted by glucosyl-, rhamnosyl-, acyl- and/or methyltransferases, resulting in “decorated”

anthocyanins with different colors. Transport of the end product to the vacuole requires a gluthathion

transferase (GST) and a specific transporter localized in the vacuolar membrane.

Proanthocyanidin (PA) synthesis starts in some species with the reduction of leucoanthocyanidin by a

leucoanthocyanidin reductase (LAR) (Tanner et al., 2003). Arabidopsis, however, seems to lack a

LAR gene and PA synthesis branches of from the anthocyanin pathway after the LDOX step via an

anthocyanidin reductase encoded by AtBANYULS (AtBAN) (Abrahams et al., 2003; Xie et al.,

2003). The resulting flavan 3-ols, catechin or epicatechin, are transported into the vacuole via a GST

and a transporter. Polymerization is thought to take place inside the vacuole via a condensing enzyme

(CE). The following steps that result in a brown PA derivative are not well understood and may rely

in part on products of the TANNIN-DEFICIENT SEED (TSD) loci from Arabidopsis (Abrahams et al.,

2002). The inset photographs show some examples of colored tissues of a wild type (on the right) and

mutant tissue lacking pigments (on the left) Bottom right: anthocyanins accumulating in the aleurone

of maize kernels and petunia flowers. Bottom left: Proanthocyanidins pigments in wild type and ttg2

mutant Arabidopsis seeds. Top left: Brown phlobaphene pigments in the pericarp from maize (Note

the variegation in the right kernel, which is due to reversions of an unstable p allele).

Introduction

11

Regulation of pigment synthesis

In most plants, pigmentation is limited to some specific tissues and is regulated by external

and internal factors, such as light (see e.g. Procissi et al., 1997; Piazza et al., 2002) and the

clock (Harmer et al., 2000). Most of the regulation of pigment synthesis occurs by a

coordinated transcriptional control of the structural genes (Mol et al., 1998). Some genes

are in addition subject to post-transcriptional control (Pairoba et al., 2003), but for most

genes this has not been properly investigated. Via mutation analysis numerous regulators

have been identified that control transcription of the structural genes in Arabidopsis, maize

and petunia (Figure 2).

Figure 2. Regulatory genes involved in pigment synthesis in Arabidopsis, petunia and maize. The

diagram shows the involvement of various types of transcription regulator in pigment (anthocyanin,

PA and phobaphene) synthesis, hair formation, mucilage production and pH regulation. Most proteins

function as positive regulators (+) while others are inhibitors (-). Abbreviations of gene names:

Arabidopsis thaliana (At): TRANSPARENT TESTA GLABRA1 (AtTTG1) (Walker et al., 1999),

TRANSPARENT TESTA2 (AtTT2), AtTTG2 (Johnson et al., 2002), PRODUCTION ANTHCYANIN

PIGMENT1 (AtPAP1) and AtPAP2 (Borevitz et al., 2000), GLABROUS2 (AtGL2) and, WEREWOLF

(AtWER), AtTT8 (Nesi et al., 2000), GL3 and ENHANCER OF GLABROUS3 (EGL3) (Zhang et al.,

2003), AtTT16 (Nesi et al., 2002), AtTTG2 (Johnson et al., 2002), TT1 (Sagasser et al., 2002). Petunia

(Ph): ANTHOCYANIN11 (PhAN11) (de Vetten et al., 1997), ANTHOCYANIN2 (PhAN2)

(Quattrocchio et al., 1999), PhAN1 (Spelt et al., 2000), PhJAF13 (Quattrocchio et al., 1998), PhPH3

(Chapter 3), PhPH4 (Quattrocchio et al., 2006, Chapter 2) and PhMYBx (van Houwelingen et al.,

1998) (A. Kroon PhD thesis, Vrije Universiteit, Amsterdam). Maize (Zm): PALE ALEURONE

COLOR (ZmPAC) (Carey et al., 2004), COLOURLESS1 (C1), PURPLE LEAF (ZmPL), RED (ZmR),

BOOSTER (ZmB), PERICARP COLOR (P) (Mol et al., 1998).

Role of MYB, HLH and WD40 proteins in transcription regulation

In all analyzed species, the common denominators in the regulation of structural

anthocyanin and PA genes are transcription factors with MYB or helix-loop-helix (HLH)

domains and a WD40 protein (Figure 2). Yeast two-hybrid (Y2H) assays indicate that these

proteins can mutually interact, indicating that they are part of one transcription activation

+ or - regulator Arabidopsis petunia maize

anthocyanin PA hairs mucilage anthocyanin PA pH seedcoat anthocyanin phlobaphene

+ WD40 TTG1 TTG1 TTG1 TTG1 AN11 AN11 AN11 AN11 PAC PAC

+ HLH-1 GL3/EGL3 GL3/EGL3 GL3/EGL3 JAF13 JAF13 JAF13 B/R

+ HLH-2 TT8 AN1 AN1 AN1 AN1

+ MYB-R2R3 PAP1 or 2 TT2 GL1/WER MYB61 AN2/AN4 PH4 PH4 C1/PL P

+ WRKY TTG2 TTG2 TTG2 PH3

+ Zn-finger TT1

+ MADS TT16

- MYB-R3 CPC/TRY MYBX MYBX

- HLH2 IN1

target genes CHS, DFR, TT12 (MATE), ? MUM4 CHS, DFR, AN9 (GST) Differentials ? CHS, A1, Bz2 (GST) ect.

TT19 , BAN ect. Chapter 4

Chapter 1

12

pathway (Figure 3). Experiments in which the activity of these regulators could be

controlled post-translationally -by fusing them to the ligand-binding domain of the

glucocorticoid receptor- indicated that they activate transcription of structural genes

directly, without synthesis of “intermediate” regulators (Spelt et al., 2000; Baudry et al.,

2004). Although the MYB ZmC1 can bind to regulatory elements in the promoters of

structural anthocyanin genes, it is insufficient for transcription activation, and even when

the DNA-binding activity of ZmCI is enhanced, its HLH partner (ZmR or ZmB) remains

essential (Sainz et al., 1997; Lesnick et al., 1998; Grotewold et al., 2000; Hernandez et al.,

2004). The HLH regulators could not be shown to bind DNA, but might activate

transcription by (i) recruiting an unknown factor that binds to a second cis-element

promoter of the anthocyanin gene (Figure 3) and (ii) by freeing ZmC1 from an inhibitory

protein (Hernandez et al., 2004).

WD40 proteins

The WD40 regulators are highly conserved, even in organisms that cannot synthesize

anthocyanins (algae, fungi, animals) and the human homolog, HsAN11, can at least in part

replace PhAN11 in functional assays (de Vetten et al., 1997). The precise molecular

function of these WD40s is, however, still unclear. Arabidopsis ttg1 and petunia an11

mutants can be (partially) rescued by high expression of their HLH or MYB partners,

suggesting that the WD40 somehow activates the downstream MYB and HLH protein

complex post-translationally (de Vetten et al., 1997; Lloyd et al., 1992; Zhang et al., 2003).

In petunia petals most of the PhAN11 protein is in the cytosol (de Vetten et al., 1997) and

the Perilla homolog PfWD also localized to the cytosol when expressed in onion cells.

However, when co-expressed with its HLH partner (PfMYC), some PfWD protein enters

the nucleus, suggesting that it could be directly involved in transcription activation

(Sompornpailin et al., 2002). When expressed in yeast, AtTTG1 boosts transcription driven

by a MYB (AtTT2) and HLH protein (AtTT8). Interestingly, addition of a transcription

activation domain to AtTTG1 enhanced its effect, suggesting that AtTTG1 may be present

in the transcription complex (Baudry et al., 2004). However, because the effect on protein

stability, localization or folding was not analyzed, other explanations cannot be ruled out

completely. By contrast, human HsAN11 was found in a protein kinase complex that

Introduction

13

controls the activity of glycogen synthase by phosphorylation, suggesting a role in signal

transduction (Skurat et al., 2004).

Based on the observations summarized above, simple models have been proposed for the

activation of structural pigmentation genes (Figure 3), and similar models have been put

forward for MYB, HLH and WD40 proteins controlling hair formation in Arabidopsis

(Szymanski et al., 2000; Pesch et al., 2004; Figure 2). These models are perhaps better seen

as working hypotheses and are likely to undergo many modifications in the near future. One

potential problem is that Figure 3 assumes a 1:1:1 stoichiometry of MYB, HLH and WD40

proteins. In yeast, HLH proteins can form dimers, not through the HLH domain as one

might expect but via a region just downstream (Zhang et al., 2003; A. Kroon (2004), PhD

thesis Vrije Universiteit Amsterdam), and some MYBs (AtTT2) can form homodimers

(Baudry et al., 2004). Thus, in vivo the transcription complex may include multiple HLH,

and MYB proteins.

Figure 3. Model depicting the role of MYB, HLH and WD40 regulators (in petunia AN2, AN1 and

AN11, respectively) in transcription activation of a structural pigmentation gene DFR. (Some) of the

MYB proteins control transcription of (some) of the HLH factors, and subsequently form a complex

that also involve the WD40 protein. Whether the WD40 protein is also present in the transcription

complex on the promoter of the structural gene (DFR) is unclear (as indicated by the question mark).

The small R3-MYB (dotted circle) acts as an inhibitor, most likely by sequestration of the HLH

protein into an inactive complex.

Based on their sequences, the HLH type regulators group into multiple phylogenetic clades;

one includes PhJAF13, AmDELILA, ZmLC and ZmR, whereas PhAN1 and AtTT8 are in a

distinct clade (Spelt et al., 2000; Nesi et al., 2000). The pigmentation MYBs may also

constitute two distinct clades, represented by the pairs AtPAP1-PhAN2 and AtTT2-ZmC1,

respectively (Nesi et al., 2001). In functional assays MYBs and HLHs of distinct clades are

often exchangeable (Spelt et al., 2000; Quattrocchio et al., 1998; Zimmermann et al., 2004),

but it should be noted that the high expression levels that are generally used can hide

R2R3-MYB

HLH

HLH

R2R3-MYB WD40 ?

DFR ?

R3-MYB

Chapter 1

14

substantial differences (see e.g. Spelt et al., 2002, Liu et al., 1998). Nevertheless, some

MYB and HLHs display clear functional differences, even when overexpressed. For

example, (i) Arabidopsis ttg1 mutants can be complemented by (over)-expression of the

HLH factors ZmR (Lloyd et al., 1992), AtGL3 and AtEGL3 (Zhang et al., 2003), but not

AtTT8 (Nesi et al., 2001), (ii) the HLH PhAN1 can boost the activity of ZmP, a MYB

regulator of phlobaphene synthesis, in petunia cells, but ZmR and PhJAF13 cannot (Spelt et

al., 2000). Furthermore, the phenotype of an1 mutants shows that PhJAF13 cannot replace

PhAN1 in vivo, even though both are co-expressed (Spelt et al., 2000; Quattrocchio et al.,

1998).

These findings may be explained in two ways that are not mutually exclusive. One

possibility is that the in vivo transcription complex indeed includes multiple MYB and HLH

proteins that differ in function and evolutionary origin. The other possibility is that the

transcription complex contains one member of each class, and that during evolution

multiple MYBs and HLHs were recruited independently to fulfill the same function. It is

worth mentioning that a single amino acid change can turn the HLH factor AtATR2, a

regulator of tryptophan synthesis, into an inducer of pigmentation (Smolen et al., 2002).

Specificity of MYB/HLH complexes

Yeast two-hybrid screens showed that the conserved N-terminal domain of PhAN1 could

interact with 4 distinct MYB proteins expressed from petunia petal cells. This includes

PhAN2, PhMYBx and two new MYB proteins named PhMYBa and PhMYBb. PhMYBa is

identical to PhPH4, which is isolated by transposon display simultaneously. Mutations in

PhMYBa/PH4 do not affect expression of genes in the anthocyanin biosynthesis pathway

(Chapter 2 of this thesis) but shows a mild seed coat phenotype and a shift in pH value of

the crude flower extract, indicating that this MYB is involved in different phenomena than

pigmentation.

The PhMYBb amino acid sequence shows high similarity with PhAN2 and its mRNA is

found in petals and tubes. Expression analysis in petal tissue showed that PhMYBb

expression is independent from PhAN1, PhAN2 or PhPH4 and is the best candidate for

PhAN2-like activity in tubes and anthers. an2 mutant petals are not completely depleted of

anthocyanin production what can be assigned to the residual AN2 like activity of PhMYBb.

Introduction

15

Plants in which PhMYBb is down-regulated do not show a clear phenotype and therefore

we only have indirect evidences of its function in the establishment of pigmentation pattern

Another MYB, cloned by DNA homology with PhMYBb, is PhMYBb2 and shows

expression in flower tubes (stages 2-5) and in anthers during developmental stages 1-2 (A.

Kroon (2004), PhD thesis Vrije Universiteit Amsterdam). MYBb2 is encoded by the

regulatory locus PhAN4, a regulator of anthocyanin biosynthesis in anthers and was also

shown to be capable of interact PhAN1 and PhJAF13 in an Y2H assay (unpublished data).

The expression pattern of PhMYBb and PhMYBb2/AN4 in different pigmented tissues

overlaps but is not identical. PhMYBb2/AN4 knockdown plants have white anthers showing

that PhMYBb2/AN4 has a function in activation of structural genes of the anthocyanin

biosynthesis pathway in anthers.

Thus, PhAN1 mutually interacts with multiple MYB proteins, which are involved in

different biological processes; PhAN2 and PhAN4 are involved in anthocyanin biosynthesis

and PhPH4 in vacuolar acidification. This made us conclude that AN1 regulates these

specific processes (in the same epidermal cell) by recruiting a different MYB protein in the

WD40/HLH/MYB protein complex. Indeed, it has been shown in leaf co-bombardment

experiments that PhAN1/PhAN2/PhJAF13 together induce the DFR promoter. However,

when PhPH4 instead of PhAN2 is co-expressed, no activation of the DFR promoter has

been measured indicating that the MYB factor determines the specificity of this complex

(Quattrocchio et al., 2006; chapter 2; Spelt et al., 2002)

Other activators

The synthesis of PAs in Arabidopsis seeds requires the induction of DFR, ANR (encoded by

AtBAN) and the transporter AtTT12 by the MYB AtTT2 and the HLH AtTT8 (Nesi et al.,

2000 and 2001). Through the analysis of Arabidopsis mutants, several more regulatory

proteins have been identified that control PA synthesis. This includes transcription factors

with a MADS box (AtTT16), a Zn-finger (AtTT1), or a WRKY domain (AtTTG2; Johnson

et al., 2002; Nesi et al., 2002; Sagasser et al., 2002; Figure 2). Currently it is unclear, if and

how these factors co-operate with the MYB, HLH and WD proteins. AtTT16 and AtTT1

are required for PA synthesis in a particular region of the testa only, but do not control

pigmentation in other tissues (Nesi et al., 2002, Debeaujon et al., 2003; Sagasser et al.,

2002). Moreover, in tt1 mutants AtBAN expression is downregulated, but AtDFR and the

Chapter 1

16

earlier structural genes remain normally expressed (Sagasser et al., 2002). Thus, if TT1

directly co-operates with MYB, HLH and WD40 proteins, it does so only for activation of

AtBAN, not AtDFR. The action of AtTT16 and AtTTG2 also seems confined to genes that

act late in PA synthesis, after the split from the anthocyanin pathway, but detailed

molecular data are currently lacking (Nesi et al., 2002; Debeaujon et al., 2003).

Differential activation of distinct pigmentation pathways

Despite the plethora of regulatory genes and mutants identified, it is still largely unclear

how a cell determines which class of pigment it synthesizes. In part, this happens by

competition of distinct pathways for a common substrate. For example, loss of ANR

activity and PA synthesis enhances anthocyanin accumulation in Arabidopsis seeds (Albert

et al., 1997; Xie et al., 2003). Differential transcription of structural genes in distinct

branches of the pathway is likely to play a major role, but this has only been studied in

maize, not in petunia or Arabidopsis.

In the aleurone of maize seeds the structural anthocyanin genes are activated by a MYB

(ZmC1), WD40 (ZmPAC) and HLH (ZmR or ZmB) triad, whereas phlobaphenes are

synthesized in the pericarp and the chaff of the maize cob under the control of the MYB

ZmP (Figure 2). Probably, ZmP cooperates with a WD40 partner, encoded by ZmPAC1

and/or a paralogous gene (Selinger et al., 1999), but no HLH partner is known. ZmP and

ZmC1 bind to the same sites in the DFR (ZmA1) promoter, but ZmC1 needs an HLH

partner, ZmR or ZmB, for transcription activation, whereas ZmP does not (Sainz et al.,

1997; Grotewold et al., 2000; Pooma et al., 2002; Hernandez et al., 2004). However, the

3GT gene (ZmBZ1), which is required for anthocyanin synthesis only, cannot be activated

by ZmP because it lacks ZmP binding sites, but still responds to the combination of ZmC1

and ZmR or ZmB (Hernandez et al., 2004). Thus, the specificity of ZmC1 and ZmP is

determined by their capability to interact with HLH partners in combination with cis-

elements in specific target promoters (Hernandez et al., 2004).

Regulation of the regulators

In general, the WD40 regulators are expressed more or less ubiquitously (de Vetten et al.,

1997; Walker et al., 1999; Carey et al., 2004), whereas the expression of the MYB and

HLH factors and, consequently, the structural genes is limited to pigmented tissues (Mol et

Introduction

17

al., 1998). So far, only few of the factors that control the expression of the regulatory genes

have been identified and a comprehensive view is still lacking. The transcription factor

AtFUSCA3 (AtFUS3) regulates a variety of processes during Arabidopsis embryogenesis,

in part by limiting the expression domain of AtTTG1 (Tsuchiya et al., 2004), but it is

unclear how (in)direct this regulation is.

Several observations indicate that (some of) the identified MYBs may have a dual function

as (i) a direct activator of structural genes, together with a HLH partner, and (ii) as an

activator of the gene(s) encoding the HLH regulator (Figure 1). This explains why PhAN2,

AtTT2, AtPAP1 or AtPAP2 alone are sufficient for ectopic activation of structural genes in

transgenic plants (Quattrocchio et al., 1998; Borevitz et al., 2000; Nesi et al., 2001),

whereas in transient assays –which measure in the short time span only relatively direct

interactions- an HLH factor must be co-expressed from a separate gene construct

(Quattrocchio et al., 1998; Zimmermann et al., 2004). Constitutive expression of PhAN2

and AtTT2 in transgenic plants indeed activates mRNA expression of their HLH partners

(PhAN1 and AtTT8), but because an2 and tt2 loss of function mutations do not alter HLH

gene expression, the biological relevance of this finding remained unclear (Spelt et al.,

2000; Nesi et al., 2001).

In petunia, PhAN4 is required for expression of PhAN1, but not PhJAF13, in anthers (Spelt

et al., 2000). Because an4 mutants can be fully complemented by ectopic expression of

PhAN2, PhAN4 was thought to encode a similar MYB protein (Quattrocchio et al., 1998;

Spelt et al., 2000). Recent experiments show that PhAN4 indeed encodes a MYB protein

with high similarity to PhAN2, providing conclusive evidence for a role of (some) of the

MYBs as regulators of the HLH genes (A. Kroon (2004), PhD thesis Vrije Universiteit,

Amsterdam, see http:// www.bio.vu.nl/vakgroepen/genetica).

The TT8 promoter activity is negatively affected in gl3/egl3 mutants and thus, these

proteins control the activity of the TT8 promoter. The TT8 promoter activity was also

greatly reduced in a tt8 mutant background, indicating that TT8 regulates its own

expression in a positive feedback mechanism (Baudry et al., 2006).

PhMYBx of petunia encodes a single repeat MYB protein that can bind the HLHs PhAN1

and PhJAF13 in yeast cells. It is structurally similar to AtCPC and AtTRY and acts as an

inhibitor as deducted from experiments in which constitutive expression of PhMYBx down-

regulated anthocyanin synthesis (A. Kroon (2004), PhD thesis Vrije Universiteit,

Chapter 1

18

Amsterdam), similar to the action of some dominant negative mutant alleles of ZmC1

(Chen et al., 2004). Because PhMYBx mRNA expression is partially down regulated in an1

and an11 petals, it might be part of an auto-regulatory loop that modulates PhAN1 activity.

However, why PhAN1 activity needs to be modulated is unclear, because over expression

of PhAN1 does not result in any obvious alterations (Spelt et al., 2000).

In maize, distinct alleles of ZmP are found that condition pigmentation of either the

pericarp (P-rw), the chaff of the cob (P-wr) or both (P-rr). Interestingly, introduction in

maize plants of P-rr and P-wr (hybrid) promoter-cDNA constructs resulted in all cases in a

P-rr phenotype (red pericarp and red cob). In subsequent generations, these transgenics

switched at a low frequency from P-rr to a P-wr phenotype, which correlated with

increased methylation of the transgene (Cocciolone et al., 2001). Thus, P-wr is controlled at

least in part by an epigenetic mechanism, which may be similar to that inactivating ZmPL

in some (epi)alleles (Hoekenga et al., 2000). Whereas P-rr contains a single MYB gene, the

P-wr allele contains six tandemly repeat gene copies, suggesting that this complex structure

is required for epigenetic silencing in the pericarp (Chopra et al., 1998; Chopra et al.,

2003).

Integration into the regulatory web

We previously proposed that the anthocyanin-specific branch of flavonoid metabolism

originates from the birth of new structural genes that were placed under the control of pre-

existing regulators (Koes et al., 1994; Quattrocchio et al., 1998). The strong conservation of

the WD40 regulator even in mammals showed that (some of) the regulators are much older

than the pathway itself, and therefore may co-regulate other (older) processes. It is now

well established that the WD40 and HLH factors regulate besides pigmentation several

other seemingly unrelated processes, by associating with MYB proteins with a more

specific function.

In Arabidopsis, TTG1, TTG2, GL3 and EGL3 control besides pigment synthesis also the

formation of mucilage in the seed and the development of hairs on the aerial plant body. In

addition, ttg1, ttg2 and gl3 egl3 mutants form ectopic root hairs, indicating that in roots the

wild type genes promote a non-hair (atrichoblast) cell fate (Pesch et al., 2004). For the

specification of (non)-hair cell fate TTG1, GL3 and EGL3 interact with specific MYB

transcription activators, WEREWOLF (AtWER) and AtGL1, in a manner that closely

Introduction

19

resembles the activation of pigmentation genes (Payne et al., 2000; Bernhardt et al., 2003;

Zhang et al., 2003; Pesch et al., 2004). The formation of seed mucilage requires yet another

MYB protein (AtMYB61), but it is not fully (dis) proven that it functions in the AtTTG1-

AtGL3-AtEGL3 pathway (Penfield et al., 2001). Strikingly, in petunia or maize neither KO

mutations nor ectopic expression of any of pigmentation regulators affects trichome

formation in their host (de Vetten et al., 1997; Carey et al., 2004), even though expression

of ZmPAC or ZmR rescues the hair defects in Arabidopsis ttg1 mutants (Carey et al., 2004;

Lloyd et al., 1992).

In petunia, PhAN1 and PhAN11 affect multiple processes in the seed coat epidermis (Spelt

et al., 2002). After fertilization, ovules develop into seeds and increase several-fold in size.

During expansion of the seed coat, the epidermal cells do not divide, but increase several-

fold in size, and acquire a brown color presumably due to PA accumulation.. In an1 and

an11 seeds, the epidermal cells are yellow (blocked PA synthesis) and instead of growing

as large as wild type, they divide 1 or 2 times. Whether the primary effect of PhAN1 and

PhAN11 here is to suppress cell division, to stimulate cell growth, or both is unclear.

However, the process of cell size seems less conserved than PA accumulation in the seed

coat, since this process is observed in many species.

Curiously, the additional processes that have been identified so far seem limited to small

sets of species, and therefore seem to represent relatively new “acquisitions” during

evolution; in petunia, AN1, AN11 regulate another process, which is the acidification of

vacuoles. This pathway was initially seen in petunia petal cells but now there are

indications that this pathway is conserved among other plant species. Vacuolar acidification

is required for PA pigmentation in seed coat cells as deducted from Arabidopsis aha10 and

petunia ph5 mutant phenotype (yellow seeds; Chapter 5). Since PA synthesis is an older

process than anthocyanin biosynthesis we speculate that the control of vacuolar pH evolved

simultaneously with PA synthesis. A process, which is closely related to vacuolar pH

regulation, is “fading”. Flowers of mutants, which fail to acidify vacuoles in petal cells

initially color red but during aging, the flowers loose the pigment molecules and become

white. Little is known about this phenomenon but it requires high vacuolar pH, specific

anthocyanin composition and the “Fading” locus. In Brunfelsia latifolia (yesterday-today-

tomorrow) fading seems to be an active process since treatment with cyclohexamide, a

translation inhibitor, prevents degradation of anthocyanin molecules (Vaknin et al., 2005).

Chapter 1

20

The vacuolar pH affects flower color

The absorption spectrum of soluble anthocyanin molecules depends on the pH; in acidic

conditions anthocyanins have a reddish color and in alkaline conditions they appear more

bluish. Because anthocyanins are sequestrated into the vacuole, the vacuolar pH is an

important factor for the final flower color. Direct measurements (by using microelectrodes)

of the vacuolar lumen of Morning glory cv petals showed indeed the correlation between

the coloration of the flowers and the vacuolar pH (Yoshida et al., 2003). In Morning glory,

the Na+/H

+ pump PURPLE sequestrates sodium in the vacuole, in exchange for protons and

thereby reduces the pH gradient across the tonoplast. Mutations affecting the function of

this protein result in red flowers with acidic vacuoles (Fukada-Tanaka et al., 2000).

Young wild type petunia buds have a “basic” (pH ~6.3) vacuolar lumen. At the time that

the flower bud will open, vacuoles of petal epidermal cells undergo an acidification step

resulting in a low vacuolar pH (pH ~5.3) and a reddish color. We identified 7 loci (PH1-

PH7), which are involved in this acidification process since mutants ph1-ph7 fail to

undergo such an acidification process and the flowers have a higher vacuolar pH and

appear bluish. The anthocyanin regulatory factors AN1, AN2 and AN11 were found to

regulate besides anthocyanin biosynthesis, also this vacuolar acidification pathway (Spelt et

al., 2000 and 2002). In first instance this was overseen since an1, an2 and an11 mutants

lack anthocyanin production and thus the pH shift was not displayed (Figure 4). The direct

measure of the crude petal extract showed a pH increase of ~ 1 pH unit in comparison to

wild type (pH ~5.3 for wild type and pH ~6.3 for an mutants). Because mutations in

structural genes of the anthocyanin pathway (e.g. AN3, encoding F3’H) do not exhibit such

a pH shift, the absence of pigment molecules cannot account for the pH difference.

PH wild type ph mutant an mutant

pH

~6

.3

pH

~6

.3

pH

~5

.3

Figure 4. Flower phenotypes of PH wild type, ph mutant and an mutant plants. The crude petal

homogenate shows an increased pH in ph and an mutants when compared to wild type (pH ~6.3 for

ph and an mutants vs pH 5.3 for wild type). Phenotypes of wild type and ph mutant are in a

background that synthesizes 3RGac5G-sunstituted anthocyanins and results in a clear red-to-blue-

shift. an mutants are in background that synthesize cyanidin-type anthocyanins because of mutations

in RT (Rhamnosil Transferase) and Hf1 (F3’5’H).

Introduction

21

The cytoplasmatic pH is thought to be fairly constant since many enzymatic reactions

would be affected by large variations. The pH in the cytoplasm is nearly neutral (pH 7.0-

7.5) whereas the pH of the vacuolar lumen is more variable, depending on the function of

the specific vacuole. The construction of the pH gradient across the vacuolar membrane is

an active process, achieved and maintained by proton pumps; the vacuolar (H+)-ATPases

(v-ATPases), which uses ATP as energy source and the vacuolar H+-pyrophosphatase

(H+PPase), which is pyrophosphates powered (Maeshima et al., 2001).

Double mutants of anthocyanin biosynthetic regulatory genes (AN1 and AN11) and PH

genes (PH3 and PH4) do not show an additional pH shift of the crude petal extract,

suggesting that the mutations affect a single pathway. Molecular analysis showed that PH4

and PH3 encode transcription factors (Chapter 2 and 3, respectively), that act together with

AN1 and AN11 to activate expression of target genes involved in the vacuolar acidification

pathway. One of these target genes is PH5, which encodes a proton pumping P-ATPase.

Since PH5 is localized on the vacuolar membrane we presume that PH5 functions as proton

pump that across the vacuolar membrane and rather than the plasma membrane as reported

for all other members of this family of ATPases (a detailed characterization of PH5 in

Chapter 5).

PH6 is a special allele of AN1 (an1G621

), which over-expresses (about 25-fold compared to

wild type level) a truncated AN1 protein, lacking the bHLH domain. an1G621

is still able to

induce genes of the anthocyanin biosynthesis pathway but is unable to induce structural

genes of the vacuolar acidification pathway, resulting in a ph phenotype similar to that of

ph1-ph5 (Spelt et al., 2002). Cloning and characterization of PH2 revealed that it encodes a

protein kinase with similarity to STE20 from yeast. STE20 is a serine/threonine kinase,

which acts in the early steps of the MAP kinase signal transduction pathway (Dan et al.,

2001). Since all identified PH genes and a set of newly identified transcripts (see Chapter

4), regulated by AN1, PH3 and PH4, function independently from PH2, PH2 could operate

in a separate acidification pathway or in the modification of proteins in this same pathway.

Outline of this thesis

The goal of this PhD research project was to understand how vacuolar pH is regulated in

petunia petal cells. We therefore set out to isolate PH3, PH4 and PH5 by transposon

Chapter 1

22

tagging and we also performed mRNA profiling experiments in mutant petals in order to

identify down stream genes in this, yet unexplored, vacuolar acidification pathway.

The characterization of the MYB transcription factor PH4 is described in Chapter 2 in

which we show that AN1, together with PH4, is able to induce expression of a set of genes

in the pH regulating pathway.

Chapter 3 describes the WRKY transcription factor PH3, which is genetically controlled

by AN1 and PH4. Mutations in PH3 result in bluish petal caused by an increased vacuolar

pH. PH3 shows high similarity to AtTTG2, which is involved in leaf trichome initiation and

PA accumulation in seeds. The result of gene swapping experiments indicate that TTG2 can

induce the vacuolar pH pathway in petunia but whether PH3 is able to complement ttg2

mutations in Arabidopsis remains to be tested.

Chapter 4 describes how we identified, by mRNA profiling approach (micro-array and

cDNA-AFLP), a set of genes regulated by AN1, PH4 and PH3. This yielded some 12

transcripts with a possible function in the vacuolar acidification pathway. For two of those

transcripts, gene specific silencing proved the involvement in pH regulation. The full

analysis of one of the target genes, MACF55 (described in Chapter 5) showed that it

encodes a P3A-type ATPase and that it is identical to PH5. MACF55/PH5-GFP is localized

on the tonoplast in cowpea protoplasts suggesting that this protein is responsible for the

acidification of the vacuole by pumping protons from the cytosol into the vacuolar lumen.

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