In vivo grapevine anthocyanin transport involves vesicle-mediated trafficking and the contribution of anthoMATEtransporters and GST

Camila Gomez1, Genevieve Conejero2, Laurent Torregrosa3, Veronique Cheynier1, Nancy Terrier1 and Agnes Ageorges1,*

1INRA, UMR1083 Sciences pour l’œnologie, F-34060 Montpellier, France,2INRA, UMR5004 Biochimie et Physiologie Moleculaire des Plantes, Plateforme Histocytologie et Imagerie

Cellulaire Vegetale, F-34398 Montpellier, France, and3INRA SupAgro, UMR1097 DIA PC, F-34060 Montpellier, France

Received 22 April 2011; accepted 19 May 2011; published online 18 July 2011.*For correspondence (fax +33 (0) 499 612 857; e-mail ageorges@supagro.inra.fr).

SUMMARY

In cells, anthocyanin pigments are synthesized at the cytoplasmic surface of the endoplasmic reticulum, and

are then transported and finally accumulated inside the vacuole. In Vitis vinifera (grapevine), two kinds of

molecular actors are putatively associated with the vacuolar sequestration of anthocyanins: a glutathione-S-

transferase (GST) and two MATE-type transporters, named anthoMATEs. However, the sequence of events by

which anthocyanins are imported into the vacuole remains unclear. We used MYBA1 transformed hairy roots

as a grapevine model tissue producing anthocyanins, and took advantage of the unique autofluorescence of

anthocyanins to study their cellular trafficking. In these tissues, anthocyanins were not only visible in the

largest vacuoles, but were also present at higher concentrations in several vesicles of different sizes. In the cell,

small vesicles actively moved alongside the tonoplast, suggesting a vesicular trafficking to the vacuole.

Subcellular localization assays revealed that anthoMATE transporters were closely related with these small

vesicles, whereas GST was localized in the cytoplasm around the nucleus, suggesting an association with the

endoplasmic reticulum. Furthermore, cells in hairy roots expressing anthoMATE antisense did not display

small vesicles filled with anthocyanins, whereas in hairy roots expressing GST antisense, anthocyanins were

accumulated in vesicles but not in the vacuole. This suggests that in grapevine, anthoMATE transporters and

GST are involved in different anthocyanin transport mechanisms.

Keywords: anthocyanin, compartmentation, grapevine, transport, autofluorescence.

INTRODUCTION

The anthocyanins constitute a major flavonoid group that is

widespread in plants, and in the foods made from them.

They are pigments responsible for the characteristic red,

blue and purple colors of many plant tissues (Harborne

and Williams, 2000), which play a role in plants as visual

attracters for pollinators and seed disseminators, and may

be involved in protection against biotic and non-biotic

stress (Harborne and Williams, 2000; Winkel-Shirley, 2001).

Anthocyanins are synthesized by multi-enzyme complexes

that are localized at the cytoplasmic face of the endoplasmic

reticulum (ER) (Winkel-Shirley, 1999; Winkel, 2004), and then

transported to the large vacuole (Grotewold, 2004).

In some plants, anthocyanins are found in highly pig-

mented bodies located inside the vacuole (Markham et al.,

2000; Lin et al., 2003; Zhang et al., 2006; Poustka et al., 2007).

These anthocyanin aggregates, referred to as anthocyaninc

vacuolar inclusions (AVIs), would allow color intensification

by concentrating anthocyanins above the levels that are

possible in vacuolar solution (Markham et al., 2000). In Vitis

vinifera (grapevine) suspension cultures, AVIs selectively

accumulate acylated anthocyanins (Conn et al., 2003, 2010),

suggesting that AVIs serve a specific role in aggregating or

sequestering anthocyanins with particular modifications.

The mechanism of anthocyanin transport to the vacuole

has long been debated, with numerous models proposed

such as membrane vesicle-mediated or membrane trans-

porter-mediated transport (Kitamura, 2006; Grotewold and

Davies, 2008; Zhao and Dixon, 2009). To date, several

960 ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd

The Plant Journal (2011) 67, 960–970 doi: 10.1111/j.1365-313X.2011.04648.x

molecular players involved in the sequestration of anthocy-

anins have been identified. The importance of glutathione-

S-transferases (GSTs) for anthocyanin transport has been

demonstrated in Zea mays (maize) BZ2 (Marrs et al., 1995),

petunia AN9 (Alfenito et al., 1998) and Arabidopsis TT19

(Kitamura et al., 2004), where mutations in the encoding

genes led to a reduction in anthocyanin accumulation and

a pigment mislocalization. Although a mechanism similar

to detoxification processes was proposed for anthocyanin

(Marrs et al., 1995), no anthocyanin-glutathione conjugate

has been observed in vitro in the presence of GST. This

protein is therefore purported to act as a cytoplasmic

anthocyanin carrier protein in vivo (Mueller et al., 2000;

Mueller and Walbot, 2001). Two major transporter families,

the ABC-C and MATE families would be also involved in

anthocyanin transport in maize (Goodman et al., 2004),

Arabidopsis (Marinova et al., 2007) and grape (Gomez et al.,

2009). However, the involvement of these different proteins

in anthocyanin transport to the vacuole is still unclear.

Evidence has emerged in favor of vesicle-mediated traf-

ficking, where vesicles filled with anthocyanins or other

flavonoids travel from the ER to the tonoplast (Grotewold

and Davies, 2008). In lisianthus and Arabidopsis, anthocya-

nins are localized in vesicles that resemble ER bodies, which

may play a role in transport to the vacuole (Zhang et al.,

2006; Poustka et al., 2007). In Brassica tapetum cells, flavo-

noids have been localized first in the ER network and then

in ER-derived tapetosomes for their delivery to the pollen

surface, suggesting the existence of flavonoid vesicle traf-

ficking (Hsieh and Huang, 2007).

In grapevine, a GST-like gene was found to be upregulat-

ed in red berries (Ageorges et al., 2006). It complemented

the maize mutant BZ2, suggesting its involvement in antho-

cyanin transport (Conn et al., 2008). Recently Gomez et al.

(2009) identified two MATE proteins, anthoMATE1 (AM1)

and anthoMATE3 (AM3), which specifically mediated acyl-

ated anthocyanin transport in vitro. AM1 and AM3 were

localized mainly at the tonoplast and in membrane vesicles

attached to the nucleus (Gomez et al., 2009), suggesting that

they participate in the transport of cytoplasmic-synthesized

anthocyanins across the tonoplast. However, these obser-

vations were performed in grapevine hairy roots not accu-

mulating anthocyanins, and drawing definitive conclusions

requires further investigation in tissues accumulating

anthocyanins. The ectopic expression of anthocyanin tran-

scription factor MYBA1 in grapevine plants and hairy roots

led to the production and vacuolar storage of anthocyanins,

associated with the overexpression of GST and AM3

(Cutanda-Perez et al., 2009). The presence of AVIs was

observed, along with intense coloration and a high accu-

mulation of anthocyanins in the transformed tissues.

Recently, in V. vinifera cell suspension cultures treated with

sucrose and jasmonic acid, white-light microscopy studies

showed that small vesicles (termed AVIs, despite not

definitively being vacuolar vesicles) could proceed from

the cytoplasm across the tonoplast, and were able to fuse

intravacuolarly to give fewer, larger AVIs as the cells mature

(Conn et al., 2010). Overall, the mechanisms by which

anthocyanins and other flavonoids are imported from the

cytoplasmic surface of the ER to the vacuole are not yet

clearly identified (Grotewold, 2004; Zhao and Dixon, 2009).

In this study, we investigated the relationship between the

anthocyanin compartmentation and the subcellular locali-

zation of anthocyanin transporters to gain a further under-

standing of the relationship between transport and the

sequestration of these pigments. We identified vesicle-like

bodies of different sizes filled with anthocyanins. Inside the

cell, the small ones, localized in the cytoplasm, moved

actively alongside the tonoplast, suggesting a vesicular

trafficking. We also investigated the subcellular localization

of GST and anthoMATE transporters (AMs), and the impact

of antisense construction for these two genes in relation to

anthocyanin compartmentation. These observations sug-

gest that in grapevine, anthoMATE transporters and GST are

involved in different anthocyanin transport mechanisms.

RESULTS

Anthocyanins compartmentation in hairy root cells

derived from grapevine tissues producing anthocyanins

In order to characterize the anthocyanin compartmentation,

we generated grapevine tissues producing anthocyanins.

Transformed plantlets regenerated after the expression of

MYBA1 in embryogenic calluses (Cutanda-Perez et al., 2009)

displayed red coloration and accumulated anthocyanins.

From theses plantlets, we generated transgenic hairy roots

(HRs) expressing, under the cauliflower mosaic virus 35S

promoter (p35S), either GFP alone (control HR) or GFP fused

with HDEL (HR-GFPHDEL). HDEL corresponds to an ER

retention signal sequence that can be used as an ER marker

(Haseloff et al., 1997; Batoko et al., 2000). These transgenic

HRs harbored a violet-brown coloration that became

noticeable as soon as the root length reached a few mm.

They presented a thin diameter (1–2 mm) and displayed

reduced growth and secondary branching compared with

HRs generated from wild-type plantlets (Cutanda-Perez

et al., 2009). Transverse sections of these transgenic HRs

revealed that the epidermis and the vascular cylinder were

the most intensely colored tissues, whereas the cortex

parenchyma appeared colorless, with only a few randomly

located red cells (Figure 1a).

Wide-field microscopic observations in the pigmented

epidermal cells showed that the coloration was essentially

the result of an accumulation of anthocyanins in the large

vacuoles, which tended to occupy most of the cellular

volume (Figure 1b,c). In these pigmented cells, we observed

a high density of spherical structures, which appeared much

more intensely colored than the whole vacuole (Figure 1b,c).

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These colored structures look like vesicles and can be

classified into two groups according to their size: group I

(G.I) corresponds to very small structures (0.2–0.5 lm in

diameter), and group II (G.II) corresponds to spherical

structures larger than 1 lm in diameter. Most of the

pigmented cells accumulated anthocyanins simultaneously

in vacuoles, and in G.I and G.II structures (Table 1). One-

third of pigmented cells contained anthocyanins only in

vacuoles and in large G.II structures. By contrast, almost no

cell accumulating anthocyanins only in vesicle-like struc-

tures or exclusively in vacuoles was found (Table 1). Obser-

vations of a pigmented cell in different z-sections confirmed

the presence of several G.I structures that actively moved

inside the cytoplasm (Video Clip S1). The G.II structures,

which are very heterogeneous in size (ranging from 1 to over

15 lm), were motionless, and their localization was less

clear.

In order to determine the subcellular localization of G.I

and G.II anthocyanin-filled structures, these tissues were

examined using confocal microscopy. Anthocyanins exhibit

autofluorescent properties when excited at 543 nm (Poustka

et al., 2007). Using this autofluorescence, we observed a

similar organization to that observed in Figure 1b,c (Fig-

ure 1d). We detected numerous small vesicle-like structures

(G.I) around what we supposed to be the vacuole, and a few

large structures (G.II) localized against a boundary, assumed

to be the tonoplast, and close to G.I (Figure 1d). However, in

our experimental conditions, the subcellular localization of

(a)

(b)

(h)

(c) (d)

(g)(f)(e)

Figure 1. Wide-field and confocal microscopy images of grapevine hairy roots (HRs) producing anthocyanins. Wide-field microscopy images of control HR

transverse section (a) and pigmented epidermal cell (b) showing colored vacuoles and spherical vesicles filled with anthocyanins. Light transmitted images of

control HR longitudinal section (c) and image of vesicles in the same cell (d) showing anthocyanin autofluorescence. Visualization of specific signal of HR-GFPHDEL

in a pigmented cell by confocal microscopy (e, red channel for anthocyanins; f, green channel for GFP; g, overlay of both channels). Visualization of lipids by Nile Red

staining in a pigmented cell with epifluorescence microscope (h). Anthocyanins appear inside vesicles of different size (G.I < 1 lm in diameter; G.II > 1 lm in

diameter). G.I vesicles are visible outside the vacuole delimited by the tonoplast (b, c). In HR-GFPHDEL, anthocyanin autofluorescence appears in G.I vesicles

surrounding the nucleus, and is co-localized with the GFP-HDEL fluorescence (g). DNA is stained in blue with DAPI, red autofluorescence is generated by

anthocyanins and green fluorescence by GFP from the GFP-HDEL fusion protein. Scale bars: 0.5 mm (a); 5 lm (e, f and G); 10 lm (c, d and h). Cp, cytoplasm;

ep, epidermis; ER, endoplasmic reticulum; N, nucleus; V, vacuole; vc, vascular cylinder.

Table 1 Distribution of pigmented cells in control (i.e. MybA1-transformed) hairy roots according to the type of subcellularcompartment in which the anthocyanins were accumulated. Cellswere sorted based on the presence of anthocyanins in vacuoles (CV)and/or small (G.I) and/or large (G.II) vesicles

Compartments

% of cellsaccumulatinganthocyanins

CV + G.I + G.II 68.4 � 10.5a

CV only 1.3 � 1.5b

G.II only 0CV + G.II 30.2 � 7.8a

G.I + G.II 0

Results are expressed as a percentage of the total number of cellsobserved containing anthocyanins. Values with different letters (a, b)indicate significance at P < 0.05. Values are means � SDs from thepercentage of n epidermal cells analysed from the independent hairyroot lines (n1 = 297, n2 = 244, n3 = 237).

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G.II was difficult to establish. Using HR-GFPHDEL, in which

the GFP-HDEL marker is retained in the ER, we confirmed the

cytoplasmic localization of G.I (Figure 1e,f,g). The specific

green fluorescence from the GFP-HDEL marker was detected

in a network surrounding the nucleus, and we also observed

red autofluorescence provided by G.I filled with anthocya-

nins surrounding the nucleus (Figure 1e,f). Observations

under both the red and green channels with confocal

microscopy showed the co-localization of some G.I with

the ER marker, suggesting that G.Is can be considered as

ER-derived bodies (Figure 1g). Subsequently, colored cells

of control HRs were stained by Nile red dye (Greenspan

et al., 1985). We clearly observed a yellow signal associated

with G.I (Figure 1h). By contrast, no signal was detected

around large G.II, suggesting that G.I and G.II do not have

the same lipidic composition.

In order to confirm the motion of G.I and G.II structures,

we used the spinning-disc confocal microscopy system,

which enables confocal observations of very fast processes

in living cells over a long time period (Graf et al., 2005).

Video Clip S2 presents a 4D reconstruction across the cell,

moving along the optical z-axis for 45 min. We observed G.II

structures localized in the middle of the cell that remained

motionless over the 45-min period. During the same period,

small G.Is moved actively and remained localized at the

edge of the cell (Video Clip S2). Crossing the cell, we

observed small vesicle-like structures moving around and

against large G.II structures. Overall, anthocyanins are

compartmentalized in vesicle-like structures of different

sizes: (i) small G.I structures surrounding the vacuole and

exhibiting active movement inside the cytoplasm; and (ii)

large G.II structures (up to 15 lm in diameter) that do not

move, the localization of which is unclear.

Localization of AMs and GST in epidermal cells

from HRs producing anthocyanins

To elucidate the involvement of anthoMATE transporters and

GST in anthocyanin transport, we used MYBA1 transformed

plantlets to generate transgenic HRs expressing under p35S

genes encoding GFP protein fusions of AM1 (HR-AM1GFP),

AM3 (HR-AM3GFP) and GST (HR-GSTGFP). In parallel, the

subcellular localization of these proteins was performed by

immunofluorescent labeling using anti-AM and anti-GST

antibodies in HRs producing anthocyanins (HRMYBA1).

From control sample observations, we confirmed that the

green fluorescence detected in these experiments was highly

specific to the GFP fusion protein or antibodies (Figure S1).

Moreover, immunolabeling with anti-AM and anti-GST anti-

bodies in HRs containing no anthocyanin revealed only a

very weak fluorescent signal (Figure S2). In some cases,

minimal autofluorescence was also seen at the cell wall,

possibly representing other autofluorescent compounds.

Confocal microscopy was first performed in HRMYBA1

after immunofluorescent labeling of AM transporters using

anti-AM antibody (Figure 2a). The anti-AM antibody recog-

nized both AM1 and AM3 proteins (Gomez et al., 2009). The

specific green fluorescent labeling surrounded the vacuole

as green dots, and appeared to be associated with the red

autofluorescence of small G.I structures (Figure 2a). Similar

observations were obtained by confocal microscopy per-

formed in HR-AM3GFP (Figure 2b). The green fluorescent

labeling provided by the AM1GFP fusion protein appeared

discontinuous around the cell wall, and did not clearly reside

on the tonoplast (Video Clip S3 showing a 3D imaging

rotation). Similar results were obtained for both constructs.

These observations suggest that in cells accumulating

anthocyanins, both AMs are present on endomembranes,

probably associated with small G.I vesicle-like structures

close to the tonoplast.

In HRMYBA1, the immunofluorescent labeling of GST

using anti-GST antibody was broadly located in the cyto-

plasm, which occupied a limited volume alongside the cell

wall (Figure 2c). The signal was not tightly linked with the

small G.I, and was more diffuse than those observed with

anti-AM antibody (Video Clips S3 and S4). Confocal micros-

copy analysis of HR-GSTGFP corroborated its cytoplasmic

localization, and a signal was detected around the nucleus,

suggesting a tight association of GST with the ER (Figure 2d;

Video Clip S4).

(a)

(b)

(c)

(d)

Figure 2. anthoMATE transporters (AMs) and glutathione-S-transferase

(GST) subcellular localization in hairy roots producing anthocyanins. Confo-

cal microscopy images after immunodetection assays of AM in colored cells

of HR-MYBA1 (a) and visualization of GFP in HR-AM3GFP cells (b). AMs

surround the vacuole and are associated with G.I vesicles (a, b). Immunode-

tection assays of GST in HR-MYBA1 (c) and visualization of GFP in HR-

GSTGFP cells (d). GST is distributed throughout the cytoplasm and around

the nucleus. Red autofluorescence is generated by anthocyanins, and green

fluorescence is generated either by a secondary anti-rabbit IgG conjugated

with green fluorescent Alexa Fluor 488 (a, c) or by GFP fusions (b, d). DNA is

stained in blue with DAPI. Scale bars: 5 lm. Cp, cytoplasm; N, nucleus.

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Immunodetection of AMs and GST in epicarp cells

of grape berries

To validate the results obtained in transgenic HRs in berries,

we performed immunofluorescent labeling using anti-AM

and anti-GST in fixed sections of Syrah mature grape ber-

ries. Immunofluorescent labeling was never detected when

secondary antibody was used alone (Figure S1). Under

wide-field microscopic observations, the cells of berry skin

displayed numerous colored vesicle-like structures of dif-

ferent sizes (Figure 3a). Several small anthocyanin-filled

structures were observed in the small cytoplasmic space

bordering the large vacuole, suggesting that epicarp cells

have an anthocyanin compartmentation similar to that ob-

served in HRs producing anthocyanins (Figure 3a). Confocal

microscopy of sections of mature red berries revealed that

the AM signal surrounded the vacuole (Figure 3b). More-

over, we detected a high signal associated with small

spherical structures similar to G.I in HRs (Figure 3b). Finally,

immunodetection of GST in the epicarp cells of red berries

showed that this protein was distributed throughout the

cytoplasm and around the nucleus (Figure 3c). Together,

these results obtained in mature berries corroborate the

spatial distribution of AMs and GST in transgenic HRs pro-

ducing anthocyanins.

Subcellular structures of epicarp cells of grape berries

containing anthocyanin

To elucidate the anatomy of these anthocyanin-filled struc-

tures, microscopic observations were completed by trans-

mission electron microscopy (TEM) observations carried out

on transverse microsections of epidermal red cells of ber-

ries. Cells contained one or just a few large vacuolar com-

partments (Figure 3d,h). The cytoplasm of these cells had

large numbers of mitochondria, chloroplasts and vesicles

(a)

(d)

(e) (f)

(g)

(h)

(b) (c)

Figure 3. Immunolocalization of anthoMATE transporters (AMs) and glutathione-S-transferase (GST) in mature red berries, and ultrastructure of epicarp cells.

Wide-field microscopy images of epicarp from mature Syrah berries embedded in paraffin showing various red vesicles G.I and G.II in cells without staining (a).

Confocal microscopy images of epicarp cells after immunodetection assays of AM (b) and GST (c) in red Syrah berry. The green specific signal of AMs are associated

with G.I structures, surrounding the vacuole, and G.II structures (b). The green specific signal of GST is detected throughout the cytoplasm and around the nucleus

(c). DNA is stained in blue with DAPI. Red autofluorescence is generated by anthocyanins, and green fluorescence by a secondary anti-rabbit IgG conjugated with

green fluorescent Alexa Fluor 488. Ultrastructural evaluation by TEM (d–h): general view of a long cell from the epicarp showing various vesicles in the cytoplasm

(d, h); close-up image of a cytoplasmic region closed to the tonoplast showing G.I vesicles (e). Visualization of G.I under high magnification showing a membrane

around these structures (f). Part of an epicarp cell showing G.I structures penetrating the vacuole (g, h). Scale bars: 20 lm (a); 5 lm (b and c); 1 lm (d and h); 0.5 lm

(d insert); 100 nm (e and f); 500 nm (g). Chl, chloroplast; Cy, cytoplasm; CW, cell wall; m, mitochondria; N, nucleus; V, vacuole.

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ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 960–970

alongside the vacuolar membrane (Figure 3d,e). The cyto-

plasmic vesicle size was around 200–300 nm (Figure 3e).

The shape, the size and the location of these cytoplasmic

vesicles were similar to the anthocyanin-filled structures

observed under wide-field microscopy (Figure 3a). These

cytoplasmic vesicles were clearly surrounded by an elec-

tronic-dense membrane (Figure 3e,f). As anthocyanins were

more or less solubilized during the dehydratation step, with

ethanol required for the sample preparation of TEM, the

anthocyanin-filled bodies appeared in some cases to be non-

osmiophilic. At higher magnification, we observed small

cytoplasmic vesicles bridging the tonoplast, penetrating

by endocytosis into the large vacuole (Figure 3g,h). In our

conditions and in the observed cells, no intravacuolar

membranous fragment was observed.

Hairy roots producing anthocyanins and expressing

antisense AMs and GST

Transgenic HRs expressing, under p35S, genes encoding

antisense AM3 (HR-AM3AS) and GST (HR-GSTAS) were

generated from MYBA1-transformed plantlets. The expres-

sion of AM1, AM3 and GST was evaluated to confirm the

repression of these genes in HR-AM3AS and HR-GSTAS

when compared with control HR (Figure 4). HR-AM3AS lines

exhibited 8.3-fold less AM1 expression and 5.6-fold less AM3

expression than control HR lines (Figure 4). HR-GSTAS lines

exhibited four-fold less GST expression than control HR

lines (Figure 4). Wide-field and confocal microscopy obser-

vations performed in HR-AM3AS, HR-GSTAS and control

HR, showed altered anthocyanin compartmentation in

HR-AM3AS and HR-GSTAS compared with control HR

(Figure 5). In cells of control HR, anthocyanins accumulated

in large vacuoles and in small G.I and large G.II structures

(Figures 5 and 6a). In HR-AM3AS, anthocyanins were mainly

accumulated in the large vacuole, and in few G.II structures

(Figures 5 and 6b,c). Moreover, no G.I structures were

observed in HR-AM3AS, suggesting that the G.II structures

are not dependent on the presence of G.I structures. In

contrast, in HR-GSTAS, virtually no colored vacuoles were

detected in epidermal cells, and anthocyanins were mostly

found in G.I and G.II structures (Figures 5 and 6d,e). These

results indicate that AMs and GST are involved in different

anthocyanin transport mechanisms.

DISCUSSION

To elucidate the anthocyanin compartmentation and the

relationship between transport and sequestration of these

Figure 4. Quantitative real-time PCR expression of AM1, AM3 and GST.

Transcript levels of AM1 (a) AM3 (b) in HR-AM3AS and control HR, and GST (c)

in HR-GSTAS and control HR. Gene expression was normalized with the

expression of VvEF1-a. Data are means of results obtained from three

independent transformed lines (two for the control) with error bars indicating

SD.

Figure 5. Effect of antisense expression of AM3 and GST on the compart-

mentation of anthocyanins in epidermal cells. Percentage of cells accumulat-

ing anthocyanins in colored vacuoles (black), G.I vesicles (white) and G.II

vesicles (grey) from control hairy root (HR), HR-AM3AS and HR-GSTAS. Data

are mean values � SDs from the percentage of n epidermal cells analysed

from three independent HR lines (control HR, n1 = 297, n2 = 244, n3 = 237; HR-

AM3AS, n1 = 42, n2 = 51, n3 = 45; HR-GSTAS, n1 = 40, n2 = 36, n3 = 31).

*Significant difference between control and transformed hairy roots

(P < 0.01).

(b)(a)

(c) (e)

(d)

Figure 6. Grapevine hairy roots (HRs) expressing antisense AM3 and GST.

Wide-field microscopy images of control HR (a), HR-AM3AS (b) and HR-

GSTAS (d). Confocal microscopy images of HR-AM3AS (c) and HR-GSTAS (e).

Epidermal cells in control HR exhibited anthocyanin accumulation in colored

vacuoles, in G.I and G.II vesicles (a). Epidermal cells in HR-AM3AS displayed

anthocyanin accumulation in large dark vacuoles; no G.I vesicles were

detected (b and c). Epidermal cells in HR-GSTAS displayed anthocyanins

compartmentalized in G.I and G.II vesicles; no colored vacuole was observed

(d and e). Red autofluorescence is generated by anthocyanins. Scale bar:

5 lm. V, vacuole.

Anthocyanin trafficking in grapevine 965

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 960–970

pigments, we developed a strategy using in vivo imaging

microscopy in grape berries naturally accumulating antho-

cyanins, and in transgenic grapevine HRs producing antho-

cyanins. Both tissues accumulate anthocyanins at high

levels, and the same anthocyanins are present in both tissue

types, although in different proportions (Cutanda-Perez

et al., 2009). Wide-field microscopy and confocal micros-

copy analysis of these tissues showed identical patterns of

anthocyanin compartmentation, indicating that transgenic

HRs accumulating anthocyanins are an appropriate model to

study mechanisms involved in anthocyanin accumulation in

grape berry.

Tissues producing anthocyanins often have unique sub-

cellular structures called ‘anthocyanoplasts’ or AVIs (Pecket

and Small, 1980). In grapevine tissues, anthocyanins are

present in two types of structures, differing mainly by their

diameter and their location inside the cell. This is in accor-

dance with observations of Conn et al. (2010), who observed

small to large red bodies in grape suspension cells. However,

both were called AVIs by the authors, despite the fact that

some of the vesicles were observed outside of the vacuole. In

lisianthus flowers, Zhang et al. (2006) reported anthocyanin

accumulating as vesicle-like bodies in the cytoplasm, which

were considered as pre-vacuolar compartments. In Arabid-

opsis and maize, cytoplasmic vesicles were also identified in

cells accumulating high levels of anthocyanins (Grotewold

et al., 1998; Irani and Grotewold, 2005; Poustka et al., 2007).

In the literature, there are some contradictory microscopic

observations showing the presence (Small and Pecket, 1982;

Jasik and Vancova, 1992; Poustka et al., 2007) or absence

(Nozue et al., 1993; Markham et al., 2000) of membranes

surrounding the anthocyanin-filled vesicles.

In the present study, the color of the cytoplasmic vesicle-

like structures appeared to be red, suggesting that inside the

G.I, anthocyanins were in acidic conditions compared with

cytoplasmic neutral pH (Tanaka et al., 2008). In addition, Nile

red staining indicates the presence of lipids in G.I structures.

All these observations are in favor of the presence of a

membrane surrounding these structures in order to main-

tain the acidic conditions. Moreover, AM proteins, possess-

ing hydrophobic transmembrane domains, were closely

associated with G.I structures, and would suggest that G.Is

are membranous organelles. This corroborates the obser-

vations performed by TEM that in grapevine tissues

producing anthocyanin, we could clearly observe a mem-

brane surrounding the small cytoplasmic structures. These

cytoplasmic G.I structures could correspond to small

vesicles previously described in maize and Arabidopsis

(Grotewold et al., 1998; Irani and Grotewold, 2005; Poustka

et al., 2007).

By contrast with the cytoplasmic vesicles, and within the

set of cells observed, we did not detect any electronic-

dense membranous structures inside the vacuole. A similar

conclusion was made regarding lisianthus AVIs, for which

no membrane encompassing the AVIs inside the vacuole

could be detected, despite the complex forms of AVIs

observed (Zhang et al., 2006). In the literature, the anatom-

ical identity of these vesicles is not firmly established.

According to some authors, the ontogenesis of larger

anthocyanin-filled vesicles from smaller ones is reminiscent

of autophagy, as has already been described in maize (Irani

and Grotewold, 2005). Recently, Pourcel et al. (2010) also

suggested a role for autophagy in the formation of AVIs

in Arabidopsis. The origin of these large vesicles is thus

questionable. In grapevine tissues, the fast movements of

small G.I structures near the large G.IIs observed inside the

cell (Video Clip S2) suggest that small G.Is coalesce into

larger G.II structures, as reported in lisianthus, Arabidopsis

and grapevine suspension cells (Zhang et al., 2006; Conn

et al., 2010). However, the presence of G.IIs and the lack of

cytoplasmic G.I structures in HR-AM3AS suggest that some

G.II structures can be formed independently of the presence

of cytoplasmic G.I. The formation of large anthocyanin-

filled vesicles in grapevine tissues is thus probably the

result of different parallel mechanisms, which co-exist in

the cell.

The presence of numerous small anthocyanin-filled

structures in grapevine tissues producing anthocyanins

could be related to AM transporters. In these tissues, AM

transporters were systematically closely associated with G.I

structures. These cytoplasmic anthocyanin-filled vesicles

would then suggest the occurrence of ER-derived vesicular

transport of anthocyanins (Pecket and Small, 1980; Grote-

wold et al., 1998; Irani and Grotewold, 2005; Zhang et al.,

2006). Anthocyanin vesicular trafficking has been previ-

ously demonstrated in Arabidopsis when it accumulates

large levels of anthocyanins, as well as in lisianthus flower

petals (Zhang et al., 2006; Poustka et al., 2007). These

authors postulated that small vesicles linked to the ER

gradually developed and enlarged in the cytoplasm to

vesicles of various sizes before merging with the central

vacuole to deliver anthocyanins (Zhang et al., 2006; Poustka

et al., 2007; Conn et al., 2010). In grapevine tissues, our

in vivo images of dynamic processes also show that small

vesicles coalesce into larger vesicles. Unfortunately, the

time scale used in the video did not allow the detection of

modifications in the proportion of G.I to G.II structures, or

an increase in the volume of G.II as already observed in

previous studies (Irani and Grotewold, 2005; Conn et al.,

2010). These results raise questions about the origin of

these colored vesicles, and about the way flavonoids are

targeted to them. In vesicle-mediated transport, the traf-

ficking of proteins has been well studied and documented

in plants (Neuhaus and Paris, 2005). In contrast, the

trafficking of phytochemicals is poorly understood (Grote-

wold, 2004). In grapevine, the overexpression of flavonoid

transcription factors MYBA1, MYBPA1 and MYBPA2 in HRs

(Cutanda-Perez et al., 2009; Terrier et al., 2009) induced

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ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 960–970

the expression of a set of genes associated with cellular

trafficking. Further work is clearly needed to elucidate if

anthocyanin vesicular transport takes advantage of protein

secretion machinery or develops alternative secretory

pathways.

According to previous studies (Marrs et al., 1995; Alfenito

et al., 1998; Kitamura et al., 2004), GST is one of the proteins

related to vacuolar transport of flavonoids. In grapevine

tissues accumulating anthocyanins, GST was localized in the

cytoplasm. Moreover, in HR-GSTAS, anthocyanins were

accumulated in G.I and G.II vesicles only, and not in the

vacuoles. This is in accordance with the recent results of

Kitamura et al. (2010), who also reported flavonoid accumu-

lation in small vacuolar-like vesicles in tt19 mutants. These

observations suggest that GST is not involved in the

formation of the flavonoid-filled vesicles, but is required

for the accumulation of anthocyanins into the vacuole. The

modified cellular anthocyanin compartmentation in HR-

GSTAS demonstrated the existence of anthocyanin trans-

port from the ER to the tonoplast, involving GST, in

grapevine, as had already been hypothesized in some other

plants. Nevertheless, to date the functional role of GST in the

vacuolar anthocyanin sequestration has not been clearly

described.

Two models have been classically proposed to explain

how flavonoids traffic from the surface of the ER to the

tonoplast: ligandin transport (LT) and vesicular transport

(VT) (Grotewold and Davies, 2008). According to these

models, GST would be associated with the LT mechanism,

as already described in some plants, and AMs would be

more closely related to the VT mechanism. Nevertheless,

a role of AMs in the LT mechanism cannot be excluded

because AMs were also localized to the tonoplast in cells

that do not accumulate flavonoids (Gomez et al., 2009). In

addition, HR-AM3AS still accumulate anthocyanins,

suggesting that other tonoplast transporters besides AMs

are involved, as already suggested by the specificity of AM

transporters towards acylated anthocyanins (Gomez et al.,

2009). Furthermore, the subcellular localization of GST in

the cytoplasm and around the nucleus where biosynthesis

takes place suggests that it may be involved in the escort

of anthocyanins to the tonoplast. Tissues expressing

antisense GST or AM genes still accumulate anthocyanins

not only in the cytoplasm in these cases, suggesting that

both LT and VT would coexist in HR cells. To date, GST and

transporters have been identified in the maize anthocyanin

pathway (BZ2 and MRP3) and in the Arabidopsis flavonoid

pathway (TT19 and TT12). In maize, the authors observed a

similar disruption in anthocyanin vacuolar accumulation

in both the bz2 mutant (Marrs et al., 1995) and in the

ZmMRP3 mutant (Goodman et al., 2004), suggesting that a

VT mechanism may not exist in maize, and that both GST

and MRP3 are involved in an LT mechanism. On the other

hand, in Arabidopsis seed coat cells, Kitamura et al. (2010)

observed an accumulation of PA derivatives in small

vesicles in tt19 mutants and at the cytoplasmic face of

the tonoplast in tt12 mutants, whereas flavonols were

located in the cytosol instead of inside tapetosomes in

pollen cells in both mutants (Hsieh and Huang, 2007).

Therefore, it seems that the coexistence of VT and LT

mechanisms, and the involvement of GST and transporters

in each type of mechanism, would be cell-type and/or

flavonoid-class dependant, and cannot be generalized in

the plant kingdom.

In grapevine, HRs expressing MYBA1 produce mainly

acylated anthocyanins, which are not the major form of

anthocyanins found in grape berries (Cutanda-Perez et al.,

2009). In lisianthus petals, AVIs extracted from vacuoles

selectively bind acylated diglycosides, which are the minor

form of anthocyanins in flowers (Markham et al., 2000). In

Arabidopsis, the over-accumulation of anthocyanins after

PAP1 overexpression or light stress leads to the pre-dom-

inant accumulation of acylated anthocyanins (Tohge et al.,

2005; Luo et al., 2007). In Solanum lycopersicum (tomato),

the overexpression of anthocyanin transcription factors

ANT1 and Rosea1 leads to the accumulation of acylated

anthocyanins (Mathews et al., 2003; Butelli et al., 2008).

Therefore, we cannot exclude that in our study, the over-

accumulation of anthocyanins led to a modification of a part

of the pool of anthocyanins followed by a vesicular traffick-

ing to the vacuole. This mechanism may be closely related to

the rapid removal of potentially toxic material from the

cytoplasm, and may overlap with other trafficking processes

(Snyder and Nicholson, 1990; McLusky et al., 1999; Nielsen

et al., 2004; Field et al., 2006). Therefore, the accumulation of

anthocyanins in plant tissues may be a hallmark of plant

stress, even if the role of flavonoids in the stress response is

still poorly understood (Winkel-Shirley, 2002).

Using non-destructive techniques, we were able to exam-

ine living cells containing anthocyanins, and obtained

in vivo images of dynamic processes. Convincing evidence

exists for at least GST-mediated transport, vesicle trafficking

and anthoMATE transporter mechanisms for the final vac-

uolar accumulation of anthocyanins in grapevine. The

existence of other putative transporters and the interaction

between GST with its ligand and/or tonoplastic transporters

remain to be elucidated in order to precisely characterize

these two mechanisms. Several questions concerning the

origin, structure, composition and behavior of anthocyanin-

containing vesicles remain. The characterization of these

vesicles and of their cargo should help, and may provide

additional insights into engineering this metabolic pathway.

EXPERIMENTAL PROCEDURES

Plasmid construction

The AM1-GFP, AM3-GFP and GST-GFP fusions were obtained byGateway cloning (Invitrogen, http://www.invitrogen.com). The stop

Anthocyanin trafficking in grapevine 967

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 960–970

codon-less AM1, AM3 and GST cDNAs were amplified using high-fidelity Taq Polymerase (Advantage-HF2 PCR kit; Clontech, http://www.clontech.com), according to the manufacturer’s instructions.The primers used for AM1 (accession no. GB FJ264202) and AM3(accession no. GB FJ264203) amplification are described in Gomezet al. (2009), and the following primers were designed for GST(accession no. GB AY971515): forward 5¢-CACCATGGTGATGAAGGTGTATGGCCCA-3¢ and R 5¢-AGCAGCGAGCTCCATGACTTTT-3¢.The resulting PCR products were transferred into pENTRTM/D-TOPO� vector (Invitrogen) and subsequently into pH7FWG2(Karimi et al., 2002) by TOPO and LR clonase reactions, according tothe manufacturer’s instructions.

The AM3 and GST cDNAs were amplified using high-fidelity TaqPolymerase (Advantage-HF2 PCR kit; Clontech), according to themanufacturer’s instructions. The following primers were used:AM3, forward 5¢-CACCATGGAGACACCGCTGCTCAAGAGC-3¢ andreverse 5¢-TCATACATCATCGGCTTCAATCC-3¢; GST forward 5¢-CAC-CATGGTGATGAAGGTGTATGGCCCA-3¢ and reverse 5¢-TCAAG-CAGCGAGCTCCATGACTTTT-3¢. The resulting PCR products weretransferred into pENTRTM/D-TOPO� vector (Invitrogen), and subse-quently into pH2WG7 (Karimi et al., 2002) by TOPO and LR clonasereactions, according to the manufacturer’s instructions.

Plant transformation

All transgenic HRs have been generated from MYBA1-transformedgrapevine plantlets (described in Cutanda-Perez et al., 2009). Theinduction and culture of transgenic HRs in grapevine were per-formed as described by Torregrosa and Bouquet (1997), withmodifications reported in Cutanda-Perez et al. (2009). For eachtransformation, four independent HR clones arising from separateinduction experiments were selected. Microscopy observationswere performed after two rounds of subculture.

DNA was extracted from 100 mg of fresh tissue using the DNAPlant Mini kit (Qiagen, http://www.qiagen.com). Transformantswere checked by PCR using the hygromycin primers, forward5¢-GCCTGAACTCACCGCGACGTC-3¢ and reverse 5¢-CAGTTTGCCAGTGATACAC-3¢.

Gene expression analysis

RNA was extracted from 150 mg of tissue using RNeasy Plant MiniKit (Qiagen), following the manufacturer’s protocol. QuantitativePCR analysis was performed as described in Cutanda-Perez et al.(2009). Gene transcripts were quantified upon normalization toVvEF1-a as the internal standard. Results are reported as 2)DCt,where DCt is the cycle threshold required to the log phase ofamplification for the experimental gene minus the same measurefor VvEF-a (Livak and Schmittgen, 2001). Primers used for amplifi-cation of GST and AM3 are described in Cutanda-Perez et al. (2009)and Gomez et al. (2009), respectively. All primer pairs were deter-mined to have equal amplification efficiency, and the PCR productswere checked by electrophoresis.

Subcellular localization

The anti-AM and anti-GST antibodies were obtained from rabbitsera following immunization with the peptides AALSIRVSNEL-GYGHPRAAK and KLEKVFDVYEQRLSKSR, respectively, preparedby Proteogenix (http://www.proteogenix.fr).

Immunocytolocalization of AM and GST in sections of grape

berries. Grape berry tissue preparation was performed asdescribed in Paciorek et al. (2006). Berries (cv. Syrah) wereharvested at 10 weeks post-flowering to obtain a ripe stage. The

procedure involved a fixation step with paraformaldehyde 4% in10 mM phosphate-buffered saline, pH 7.2 (PBS; 7 mM Na2HPO4,3 mM NaH2PO4, 120 mM NaCl, 2.7 mM KCl), dehydration steps withethanol at increasing concentrations and a 100% butanol bath(5 days). Tissue embedding was performed with successive bathsof increasing concentrations of Safesolv (Labonord, http://www.labonord.com) in butanol, then with Safesolv/Paraplast pure wax(paraffin X-TRA; McCormick Scientific, http://www.mccormicksci-entific.com) baths at increasing pure wax concentrations. Paraplastblocks were cut with a razor blade at 8-lm thickness (Leica micro-tome RM 2265; Leica, http://www.leica.com). Strips were placed ona warm water surface on a heating plate and straightened. The crosssections were transferred on silanized slides and dried completely.Samples were then dewaxed and rehydrated following the conversesteps. After a blocking incubation with BSA (2% in PBS) to reducenon-specific binding, the primary antibody solution (a rabbit anti-AM polyclonal antibody 1:200, or a rabbit anti-GST antibody 1:500)was gently poured on the microscope slides. After an overnightincubation at 4�C, the sections were washed with PBS (three timesfor 10 min each) and incubated in the secondary antibody [anti-rabbit IgG F(ab¢)2 fragment conjugated to the Alexa Fluor 488fluorochrome (Invitrogen)] for 1 h 30 min at room temperature(25�C) in the darkness. The secondary antibody was then removedand after washing in PBS, a 2-lM 4¢,6-diamidino-2-phenylindole(DAPI) solution (Sigma-Aldrich, http://www.sigmaaldrich.com) wasused to specifically stain the nuclei. The sections were mounted inMowiol anti-fading medium.

Immunocytolocalization of AM and GST in HRs. Cross sec-tions (50 lm) were obtained from fresh HRs using a vibratome(MICROM HM650V; Thermo Scientific, http://www.thermo.com),and lightly fixed for 1 h in paraformaldehyde 4% in 10 mM PBS insmall glass dishes. After washing with PBS, the sections were pre-treated with a BSA solution (3%) for h to reduce non-specific bind-ing. The sections were incubated in the polyclonal antibodiessolutions (a rabbit anti-AM polyclonal antibody 1:200 or a rabbitanti-GST antibody 1:500) overnight at 4�C. After washing with PBS,the secondary antibody Alexa488-conjugate was applied for 1 h(1:400) in the darkness. Then, after washing with PBS, sections weremounted in Mowiol antifading medium on fluoro-slides with twocircles (ESCO, http://www.escocorp.com). Controls were included inorder to show the specificity of the signal, some sections weretreated with neither anti-AM (or anti-GST) nor secondary antibody(autofluorescence), and others were treated with the secondaryantibody only.

Light microscopy and laser scanning confocal microscopy

Cross sections (50 lm) were obtained from fresh HRs using avibratome (MICROM HM650V; Thermo Scientific), dipped in PBSand directly observed on a Leica DM6000 wide-field microscopewith a PL APO objective 40 · (NA: 1.25). The pictures and videos(10 frames s)1; Video Clips S1–S4) were obtained with VOLOCITY-

ACQUISITION 5.1.0 (Perkin Elmer, http://las.perkinelmer.com).The sections were dipped in Nile red solution (2 lg ml)1) for

10 min in the dark, and were then observed after washing with LeicaDM 6000 (transmitted light and epifluorescence; filter I3 and TX,excitation 450–490 and 540–580 nm, emission 515–800 and 608–682 nm, respectively) to visualize the neutral or membrane lipids inyellow or red, respectively.

The confocal microscope imaging was performed on the Mont-pellier RIO Imaging Platform (http://www.mri.cnrs.fr), with a confo-cal laser scanning Axiovert 200M 510 META microscope (Zeiss,http://www.zeiss.com). Excitation was provided by a 405-nm laser

968 Camila Gomez et al.

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 960–970

for the DAPI stain (bandpass 422–476 nm), a helium-neon 543-nmlaser for the autofluorescence of anthocyanins (long pass 585 nm)and a 488-nm Argon laser for the Alexa 488 and GFP (bandpass 505–530 nm), using multitracking mode (fast line-wise switchingbetween excitation laser lines) to avoid the simultaneous excitationof, and hence emission from, the fluorophores (crosstalk).

The sections were observed with Plan Neofluar 25 · /0.8 or C-Apochromat 40 · /1.2 Zeiss objectives. Pictures were processedusing the Zeiss LSM IMAGE BROWSER software and PHOTOSHOP CS3(Adobe, http://www.adobe.com). Three-dimensional pictures wereobtained with VOLOCITY VISUALIZATION 5.0.2 (Improvision; a PerkinElmer Company). The 3D video was created from Z-stack sectionsfrom the confocal microscope with the VOLOCITY VISUALIZATION usingvarious rotations of 3D rendering. The video 4D (time lapse) wasobtained with a Cell Observer SD Zeiss (multidimensional fastconfocal image acquisition or spinning disk) with a 532-nm laser.

Transmission electron microscopy (TEM)

Mature grape berry samples (epicarp) were immersed in 2.5% glu-taraldehyde in Sorensen’s buffer (0.1 M, pH 7.4) overnight at 4�C,and then rinsed in Sorensen’s buffer and post-fixed in 0.5% osmicacid for 2 h in the dark and at room temperature. After two bufferrinses, tissues were dehydrated in a graded ethanol series (30–100%). Tissues were then embedded in EmBed 812 resin using anAutomated Microwave Tissue Processor for Electronic Microscopy(Leica EM AMW). Ultra-thin sections (80 nm; Leica-Reichert Ultra-cut E) were collected at different levels of each block, and counter-stained with uranyl acetate. Observation and photography wereperformed using a Hitachi 7100 transmission electron microscope(http://www.hitachi.com) in the Centre de Ressources en ImagerieCellulaire de Montpellier, France.

ACKNOWLEDGEMENTS

The authors especially thank M. Lartaud and E. Jublanc, PHIV,Montpellier Rio Imaging, IFR 127 DAPHNE and E. Jullien from Zeissfor their helpful assistance with confocal and spinning-discmicroscopy. The authors are also grateful to C. Cazevieille andC. Sanchez for their technical assistance and interpreting dataconcerning ultrastructural evaluation. The authors thank N. Paris forproviding GFP-HDEL and C. Tesniere for providing the berries to beused in the immunocytolocalization assays, and I. Gil and G. Lopezfor their assistance in the handling of the in vitro plant material. Thiswork was supported by the European Community (STREP projectFLAVO–FOOD–CT–2004–513960) and by INRA.

SUPPORTING INFORMATION

Additional supporting information may be found in the online ver-sion of this article:Figure S1. Immunofluorescence and GFP controls by confocalmicroscopy.Figure S2. anthoMATE transporters (AMs) and glutathione-S-trans-ferase (GST) subcellular localization in hairy roots not expressingMybA1.Video Clip S1. Bright-field microscopy observation of a pigmentedcell from control hairy roots, showing small vesicles movingactively within the cytoplasm, and large vesicles located withinthe vacuole but remaining motionless. During acquisition, the focuswas successively adjusted to the cell wall, the cytoplasm and themiddle of the vacuole.Video Clip S2. Four-dimensional acquisition using spinning-discconfocal microscopy (Cell Observer SD Zeiss) of an epidermal cell ofcontrol hairy roots from Z-stack optical sections over a period of

45 min. The red autofluorescence of anthocyanins is obtained witha 532-nm laser.Video Clip S3. Three-dimensional rendering of an epidermal cell inHR-AM1GFP from Z-stack optical sections, made with VOLOCITY

VISUALIZATION software. The video shows a rotation of this 3Dreconstruction for a better visualization of the specific green signalof GFP in the cellular volume. The red autofluorescence of antho-cyanins and the green fluorescence of the AM1-GFP fusion are seenusing laser multitracking at 543 and 488 nm, respectively, on theconfocal microscope.Video Clip S4. Three-dimensional rendering of an epidermal cell inHR-GSTGFP from Z-stack optical sections, made with VOLOCITY

VISUALIZATION software. The video shows a rotation of this 3Dreconstruction for a better visualization of the specific green signalof GFP in the cellular volume. The red autofluorescence of antho-cyanins and the green fluorescence of the GST-GFP fusion are seentogether with the DAPI-stained nucleus using laser multitracking at405, 543 and 488 nm, on the confocal microscope.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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