www.sciencemag.org/cgi/content/full/310/5745/121/DC1

Supporting Online Material for

Arabidopsis H+-PPase AVP1 Regulates Auxin-Mediated

Organ Development

Jisheng Li, Haibing Yang, Wendy Ann Peer, Gregory Richter, Joshua Blakeslee, Anindita Bandyopadhyay, Boosaree Titapiwantakun, Soledad Undurraga,

Mariya Khodakovskaya, Elizabeth L. Richards, Beth Krizek, Angus S. Murphy, Simon Gilroy, Roberto Gaxiola*

*To whom correspondence should be addressed. E-mail: Roberto.Gaxiola@uconn.edu

Published 7 October 2005, Science 310, 121 (2005)

DOI: 10.1126/science.1115711

This PDF file includes:

Materials and Methods Figs. S1 to S13 Tables S1 to S3 References

Arabidopsis H+-PPase AVP1 Regulates Auxin Mediated Organ Development

Jisheng Li1, Haibing Yang1, Wendy Ann Peer 3, Gregory Richter2, Joshua Blakeslee 3,

Anindita Bandyopadhyay 3, Boosaree Titapiwantakun 3, Soledad Undurraga1, Mariya

Khodakovskaya,1 Elizabeth L. Richards3, Beth Krizek4, Angus S. Murphy 3, Simon

Gilroy,2 Roberto Gaxiola1*

Supporting Online Material

Materials and Methods

Plant Materials and Growth Conditions

Control, gain-of-function (AVP1OX) and loss-of-function lines (avp1-1 and

AVP1(RNAi)) used in the study were all in the Arabidopsis thaliana ecotype Columbia-0

background. The AVO1OX lines, AVP1-1 and AVP1-2, were described previously (1).

The loss-of-function mutant, avp1-1, was obtained from GABI-Kat, Koln, Germany

(original assigned number 005D04.) Direct sequencing showed that the T-DNA insertion

of this line localizes to the predicted fifth exon of the AVP1 ORF. The T-DNA insertion

contains the SULr open reading frame for resistance against the herbicide sulfadiazine

(sul; 4-amino-N-2-pyrimidinylbenzene sulfonamide, Sigma Aldrich) (2). For the genetic

analysis of the T-DNA insertion line, T2 seeds were selected in half strength MS

(Murashige and Skoog salt mixture) media supplemented with 11.5mg/L sulfadiazine.

The ratio of resistant to sensitive seedlings was approximately 3/1 (438/145) consistent

with a single integration locus. About one-third (129 from 438) of the herbicide resistant

seedlings displayed a dramatic impairment in their root and shoot development even after

transfer to herbicide free media. PCR analysis using the following primer pairs

demonstrated that these abnormal seedlings were homozygous for the T-DNA insertion

in the fifth exon of the AVP1 ORF. AVP1-specific primer AVP1244 (5’-

CCAATGATAACTTTAGGGGTCAAA-3’) was paired with a T-DNA-specific primer

TDNA245 (5’- CCCATTTGGACGTGAATGTAGACAC-3’) yielding a 740bp fragment,

or with another AVP1-specific primer AVP118 (5’-

GTCGGCGCTGACCTTGTCGGTAAA- 3’), yielding 8228 bp product. Homozygous

avp1-1 plants are not fertile; therefore, the avp1-1 allele was propagated as a

heterozygotes.

Plants were grown in soil or under hydroponic conditions (3) in growth chambers with a

16h light / 8h dark cycle at 21˚C. When aseptic growth was required, plants were grown

in medium containing half strength MS salts, 1% (w/v) sucrose, 0.7% (w/v) agar, at 25˚C

under a 16h light / 8h dark cycle. In the case of the experiments with ethanol inducible

AVP1 (RNAi) lines (see Constructs), 0.25% (v/v) ethanol was added to the growth

medium. The same media was used when either exogenous 5µM IAA (indole acetic

acid), or 5µM NAA (Naphthalene acetic acid), or 25 µM 1-naphthylphthalamic acid

(NPA) were added.

Leaf Size Determination

Rosette leaves were carefully excised with a scalpel blade and the leaf areas were

measured with a Li-Cor 4100 area meter.

Cell Size Determination

In order to determine the number of mesophyll cells per unit area from control and avp1-

1 plants, fully expanded leaves from 35 day old plants grown on agar plates were cleared

with chloral hydrate. Mesophyll cells were then photographed with an Olympus IX70

microscope (Olympus, Melville, NY) equipped with a (IX-SPT) CCD camera and the

area of individual cells measured with Quantity One quantifying software (Bio-Rad,

Hercules, CA). For the evaluation of epidermal cell numbers, fully expanded rosette

leaves from soil grown plants were collected. Chlorophyll was removed by boiling the

leaves in 95% ethanol for 5 min. Leaves were further cleared by boiling them in a

solution containing equal amounts of lactic acid, phenol, glycerol and water. The cleared

leaf blades were mounted in 25% glycerol on microscope slides and photographed with

an Olympus IX70 microscope equipped with a (IX-SPT) CCD camera. Adaxial

epidermis cell numbers in a fixed area (2.5X105 µm2) was scored at different locations in

the leaf blade – the tip, the middle and the base.

GUS Staining

Transgenic plants carrying the CycB1::CDBGUS construct were grown on soil. Leaves

number 5, 6 and 7 were carefully excised and stained with X-Gluc (5-bromo-4-chloro-3-

indolyl-beta-D-glucuronic acid) as described elsewhere (4). Briefly, leaf tissue was first

placed in 90% acetone on ice for 15 min and then in a X-Gluc staining solution (750

mg/ml X-Gluc, 100 mM NaPO4 (pH 7.0), 3 mM K3Fe(CN)6, 10 mM EDTA, 0.1%

Nonidet-P40) under a vacuum for 16 h at room temperature. In the case roots with the

DR5::GUS reporter this process was carried only for 30 mins. The staining solution was

removed and the tissues were placed in 70% ethanol to clear chlorophyll. Samples were

analyzed under an Olympus IX70 microscope and photographed with a (IX-SPT) CCD

camera.

Western Blot Analysis

Microsomal fractions were isolated from 19-d old Col-0 and avp1-1 plants grown in half

strength MS agar plates as described elsewhere (5). The protein concentration was

determined with the BCA protein assay reagent (Pierce, Rockford, IL). 15 µg per sample

were electrophoretically resolved in 12% Tris-HCl SDS gel (Bio-Rad) and transferred to

the Immobilon-P transfer membrane (Millipore, Bedford, MA). The membranes were

incubated for 1.5 h with an antiserum raised against a synthetic peptide corresponding to

the putative hydrophilic loop IV of the AVP1 protein (6) or the 2E7 monoclonal antibody

against V-ATPase (7) or with a polyclonal antiserum raised against Arabidopsis P-

ATPase (8). After 1.5h of incubation with a secondary antibody conjugated with alkaline

phosphatase, the membranes were treated with a NBT/BCIP substrate solution (Roche,

Indianapolis, IN) for staining. Of note, when the NBT/BCIP treatment was extended for

one hour a fine band of about 84 kDa was noticeable in avp1-1 samples. It is likely that

this band could represent the product of the AVP2/AVP1/L1 gene (9).

Membrane Isolation via Discontinuous Sucrose Gradients

Membranes were isolated from root systems of Col-0 and AVP1OX plants as previously

describe (10). Microsomes (1ml) were layered onto a discontinue sucrose (Suc) gradient

(top layer = 4ml of 22% w/v Suc, mid layer = 4ml of 32% w/v Suc, and cushion layer =

4ml of 38% w/v). Gradients were centrifuged at 100,000g (3h at 4°C) in a Beckman SW

28 swinging rotor with a Beckman Coulter Optima L-90K Ultracentrifuge, Fullerton, CA.

P-ATPase Activity

Microsomes were isolated from Col-0, avp1-1, and AVP1-2 seedlings grown in ½

strength MS medium for 16 days as described above. Protein concentration was

determined with the aid of the BCA protein assay kit from Pierce (Rockford, IL) after

TCA precipitation. 50 µl of a microsome suspension with a protein concentration of 0.5

mg/ml wereadded to 450 µl ATPase assay mix (25 mM BTP-MES pH7, 100mM KCl, 4.5

mM MgSO4, 2.5 mM Na2·ATP, 1 mM NaN3, 1 mM NaMoO4, 2 µM gramicidin) with or

without P-ATPase specific inhibitor Na3VO4 400 µM and incubated in a 5 ml glass tube.

After 30 min incubation at 37 ºC, 1.25 ml of Fiske & Subbarow mix (11) was added to

stop the reaction and develop color reaction. A660 was measure with UV160 SHIMADZU

(Japan) spectrophotometer. The net P-ATPase A660 was calculated by deducting the A660

with Na3VO3 from A660 without Na3VO3. P-ATPase activity was reported as µmol of

released Pi per mg microsomal protein per minute.

Constructs

dsRNA Construct. Sense and antisense fragments corresponding to the exon1 of the AVP1

ORF were ligated to the intron1 of the AVP1 genomic sequence in order to generate the

AVP1 (RNAi) cassette. The AVP1 (RNAi) cassette was ligated downstream to the ethanol

inducible promoter pAlcA (12). The pAlcA:AVP1(RNAi):tnos cassette was then cloned

into the Hind III site of the plant transformation vector pBart_AlcR, that contains the

alcR regulator required to activate pAlcA promoter in the presence of ethanol (12). The

pBart_AlcR vector also contains the bar gene (phosphinothricin acetyltransferase) for

selection with herbicide phosphinothricin (Basta). A diagram of the construct is shown

below.

CycB1::CDBGUS construct. The CycB1::CDBGUS reporter construct, that has been

described elsewhere (4), was provided by J. Celenza. However, to allow selection of

transformed plants with gentamycin, the CycB1::CDBGUS cassette was subcloned into

the pPZP222 vector (13). pINDEX3-AVP1 construct. The open reading frame of the

AVP1 gene (14) was cloned into the XhoI site of the pINDEX3 (AF294982) vector (15).

In this vector, the expression of the AVP1 open reading frame is regulated by the addition

of dexamethansone (10 µM) into the medium.

Plant Transformation and Selection

The constructs were introduced into Arabidopsis thaliana Colombia ecotype via the floral

dip method with the Agrobacterium tumefaciens strain GV3101 (16). Plants transformed

with the dsRNA construct were selected on soil after spraying with BASTA (T1). Seeds

resulting from self-pollinated transformants (T2) were scored again for herbicide

resistance on soil. Complete BASTA resistance identified homozygous AVP1 (RNAi)

plants of the T2 progeny. Six independent homozygous lines were obtained. Col-0

plants transformed with the CycB1::CDBGUS were selected in half strength MS agar

medium added with 1% (w/v) sucrose and 80 mg/L gentamycin (T1). Antibiotic resistant

plants were transferred to soil and their seeds (T2) were re-tested in gentamycin

containing medium. From a total of six independent CycB1::CDBGUS homozygous

lines, one was used to cross with the Col-0 and AVP1-1 and AVP1-2 transgenic plants.

The F1 progeny that resulted from these crosses was used to score cell proliferation

activity. Complementation of avp1-1 mutant was carried out by transformation of

heterozygous AVP1/avp1-1 plants with the inducible pINDEX-AVP1 construct via

Agrobacterium-mediated transformation by floral dip method (16). The resultant T1

plants were selected in half strength MS medium supplemented with 50 mg/L

hygromycin, 12.5 µg/L sulfadiazine and 10 µM Dexamethansone. The resistant plants

were transferred and grown in soil. The homozygous identity of T-DNA insertion of the

rescued plants was confirmed by PCR assay.

RT-PCR

RNA from 20-day old seedlings of Col-0 and AVP1 (RNAi) lines (n = 35 per line) grown

in half-strength MS medium, 1% (w/v) sucrose and 0.25 % (v/v) ethanol was extracted

with TRI Reagent (Molecular Research Center Inc, Cincinnati, OH). 10 µg RNA was

incubated with 2 U DNase from a DNA-free kit (Ambion, Austin, TX) at 37°C for

30min. One µg of each RNA sample was used to synthesize cDNA with the

RETROscript kit (Ambion). For each of the PCR reactions, 0.5 µl of the synthesized

cDNA was used as template. Semiquantitative PCR was started at 95 °C for 3 min

followed by 25 cycles of 94 °C (30s), 60 °C (30s). For an internal control, we used 18S

rRNA primers and competimers from a QuantumRNA Universal 18S rRNA kit (Ambion)

at a 1:9 ratio. The following primers were used for the PCR reactions: AVP158 5’-

CCGGATCCATGGTGGCGCCTGCTTT-3’ and AVP194 5’-

GACAAGGTCAGCGCCGACAT-3’.

Immunolocalization

Arabidopsis seedlings (Col0, avp1-1 and AVP1-2) were grown on 1% phytagar plates,

1/2 Murashige and Skoog basal salts, 1% sucrose, pH 4.85, 22°C and 14 h, 100 µmol m-2

s-1. 5 days old seedlings were prepared for immunolocalization following the protocol in

Peer et al., 2004 (17). Anti- AVP1, PIN1, and PIN2 antisera were utilized at dilutions that

produced no signal in the respective knockout mutants. Anti-AVP1, PIN1, PIN2 and P-

ATPase antibodies were used in 1:200, 1:400, 1:250 and 1:300 dilutions, respectively.

Immunofluorescence analysis was done using a confocal laser scanning microscope

(Nikon, Eclipse 800) equipped with an argon laser (488nm) (Bio-Rad). Images were

captured with a SPOT camera and processed using Adobe Photoshop 7.0.

Confocal Imaging of Cell Organization

Cell positions were visualized by confocal imaging of plants stained with 25 µM of the

fluorescent dye FM 4-64 (Molecular Probes, Eugene, OR) diluted in water from a 1 mM

stock in DMSO. For root imaging, seedlings were grown according to Wymer, et al. (18),

stained for 5 mins with dye and visualized using a LSM 510 confocal microscope (Zeiss,

Thornwood, NY) with a 20x, 0.75 numerical aperture, dry objective, or 40 x 1.2

numerical aperture water immersion objective, 543 nm excitation, 543 nm primary

dichroic mirror and >600 nm emission. For shoot and floral meristem imaging of

mutants, the plants were incubated in dye for 15 mins and then imaged as described

above. For wild type plants, the leaves surrounding the meristem were removed using a

dissecting microscope and mounted needles to reveal the meristem. Specimens were then

stained and imaged as above.

Cell Wall, Cytosolic and Vacuolar pH Measurement

Cytosolic, cell wall and vacuolar pH was monitored as described previously (19).

Briefly, for cytosolic measurements, cells were microinjected with the fluorescent pH

sensor 7-bis-(2-carboxyethyl)-5-(and 6) carboxyfluorescein (BCECF) conjugated to a 10

kDa dextran (Molecular Probes, Eugene, OR) and pH monitored by ratio imaging as

detailed in Bibikova et al. (20). Apoplastic pH was monitored by ratio imaging of cells

where a cellulose binding domain peptide-oregon green conjugate was microinjected onto

the wall. Wall pH was then quantified by ratio analysis of the pH-dependent (480 nm)

and pH-independent (440 nm) excitation wavelengths of Oregon Green (Emission 530

nm ±20 nm) according to Fasano et al., 2001. Many unconjugated fluorescent dyes are

accumulated in the plant vacuole (21, 22). Therefore we measured vacuolar pH by

incubating roots for 30 min in the vacuole accumulated fluorescent reporter Orgeon

Green-acetoxy methyl ester. The pH-dependent (480 nm) and pH-independent (440 nm)

excitation wavelengths were selected using interference filters (±20 nm), and emission

was monitored at 520 nm (long pass) using a 510-nm dichroic mirror. Autofluorescence

represented <5% of the 440- or 480-nm excitation signals. Measurements were taken

using a Diaphot 300 epifluorescence microscope (Nikon, Melville, NY) with a 40x dry

0.7 numerical aperture objective and a Sensys cooled CCD camera (Photometrics, Austin,

TX) analyzed using IPLabs spectrum image analysis software (Signal Analytics, Vienne,

VA).

In situ hybridization

Inflorescences, seedlings, and roots were fixed, embedded, sectioned, hybridized, and

washed as described previously (23). Digoxigenin-labeled RNA probes were synthesized

by in vitro transcription. The AVP1 antisense probe was made by linearization of

pRG207 with Asp718 and in vitro transcription with T3 RNA polymerase. The AVP1

sense probe was made by linearization of pRG207 with XbaI and in vitro transcription

with T7 RNA polymerase. pRG207 contains the full-length AVP1 cDNA. This probe

recognized a single band on a DNA gel blot.

Flavonoid localization and identification

Seedlings were grown on 0.25x MS, 1 % phytagar plates and VMT (24). 4 day seedlings

were stained with diphenyl boric acid 2-aminoethyl ether (DPBA) (Sigma) and imaged

using an epifluorescence microscope as previously described (25, 26).

Tables

Table S1. Rosette leaf number. Control and AVP1OX plants (n = 14/line) were grown in soil for 42 days as described in Materials and Methods. Rosette leaves were carefully excised and counted.

Rosette leaf number (Mean ± SD)

Col-0 11.8 ± 1.6 AVP1-1 15.4 ± 2.3 AVP1-2 28.6 ± 6.2

Table S2. Root dry weight. Control and AVP1OX lines were grown hydroponically for

45 days. The roots were dissected and their dry weights were determined after 48 h of

incubation in an oven at 70 °C (n = 8/line).

Root dry weight (Mean ± SD)

Col-0 17.7 ± 6.9 AVP1-1 46.0 ± 14.0 AVP1-2 166.4 ± 14.5

Table S3. AVP1 gene expression level in Col-0 and three independent AVP1 (RNAi)

lines. RNA was extracted from 20-day old seedlings (n = 35 per line) grown in half-

strength MS medium, 1% (w/v) sucrose and 0.25 % (v/v) ethanol. Complementary DNA

was synthesized from 1g of each RNA sample. PCR reaction was carried out for 25

cycles with the primer pairs AVP158 5’-CCGGATCCATGGTGGCGCCTGCTTT-3’ and

AVP194 5’-GACAAGGTCAGCGCCGACAT-3’. Bands were electrophoretically

resolved on 1% agarose gels and visualized with ethidium bromide. The relative

intensity of the bands was determined with the BioRad Quantity One software.

% of Control ± SD

Col-0 100 ± 0.0 AVP1 (RNAi)1 39.6 ± 6.1 AVP1 (RNAi)2 49.8 ± 13.7 AVP1 (RNAi)3 30.7 ± 5.3

Figures

Figure S1. Rosette leaf area of Col-0 and AVP1OX lines

Seven plants from each line were grown in soil as described in Materials and Methods for

45 days. Fully developed leaves (leaf 5 to leaf 10, as indicated) from wild type (white

bars), AVP1-1 (black bars), and AVP1-2 (gray bars) were carefully excised and their areas

determined with the Li-Cor area meter. The areas of AVP1OX rosette leaves were

significantly larger than in wild type (P<0.01) with the exception of leaf number 10 in

AVP1-2 plants (P<0.05).

0

5

10

5 6 7 8 9

10

Are

a (

cm

2)

0

20

40

60

80

100

Tip Middle BaseCell

s p

er

Un

it A

rea

Figure S2. Number of epidermal cells in leaves of control and AVP1OX plants

The number of epidermal cells contained in fixed areas at the tip, middle and base of the

rosette leaves from Col-0 (white bars), AVP1-1 (black bars), and AVP1-2 (gray bars)

plants was determined. Fully expanded rosette leaves were cleared and adaxial epidermis

cells were photographed as described in Materials and Methods. At least 12 areas per

leaf were counted for each line (n = 5).

Note: To determine if leaf size difference was the result of an increase in cell number or

cell size, we scored the number of epidermal cells per fixed area (2.5X105 �m2) of fully

developed rosette leaves (Fig. S2). There was no significant difference in the cell

densities scored for wild type and AVP1OX leaves at the tip, middle and base portions,

indicating that cell size was unaltered in the mutants and suggesting that the larger organs

reflected increased cell number.

Figure S3. GUS activity in developing rosette leaves from Col-0 and AVP1OX lines

The frequency of cells displaying GUS activity was assessed in developing leaves from

F1 GUS expressing offspring of Col-0 (white bars), AVP1-1 (black bars), and AVP1-2

(gray bars) lines (see Material & Methods). Individual leaves (as indicated) were

visualized with the aid of an Olympus IX70 microscope and photographed (n = 6 per

line). The photograph corresponds to light micrographs of GUS-stained 22-day old

rosette leaf No. 8 of Col-0 and AVP1OX plants as indicated. Plants were grown in soil

under 16h light/ 8h dark. Bar, 1 mm.

0

400

800

1200

1600

Leaf 6 Leaf 7 Leaf 8

GU

S S

po

ts/L

eaf

Note: To visualize cell proliferation in wild type and AVP1OX plants, we used a

CycB1::CDBGUS reporter gene. CycB1 is expressed before and during mitosis, making

it an ideal marker for cell proliferation (4, 27). Homozygous wild-type CycB1::CDBGUS

plants were crossed to wild type and AVP1OX plants. GUS staining was monitored at

22-day post germination in expanding leaves of the resulting F1 offspring (Fig.S3). As

reported previously (4), GUS staining was mainly restricted to the leaf base in all of the

plants (Fig. S3). However, there was a larger population of proliferating cells at the base

in the leaves of AVP1-1 and AVP1-2 transgenic plants (Fig. S3), supporting a role for

AVP1 in cell division mechanisms associated with the early stages of organ formation.

Figure S4. T-DNA insertion site in AVP1 and genotypic characterization of T2 seeds

carrying an AVP1 T-DNA insertion. (A) T-DNA insertion site in AVP1 in GABI-Kat

line 005D04 line. LB, T-DNA left border; RB, T-DNA right border. White boxes

represent exons. The T-DNA is not drawn to scale. (1), (2) and (3) indicate the relative

position and direction of primers used to detect genotypes. Bar, 400 bp. (B) Genotypic

characterization of T2 seeds carrying an AVP1 T-DNA insertion. DNA was extracted

from leaf tissue and subjected to two sets of PCR reactions with different sets of primer

pairs. AVP1-specific primer AVP1-244 (3) was paired with a T-DNA-specific primer

TDNA-245 (2), yielding a 740bp fragment (T-DNA-AVP1), or with another AVP1-

specific primer AVP1-118 (1), yielding either an 1379 bp fragment (AVP1) when the T-

DNA was absent, or an 8228 bp product, which was not detected under normal PCR

conditions, when the T-DNA was present. In lanes 1 to 3, the template DNA comes from

three independent abnormal-looking seedlings (shown in the insert). Their genotype was

avp1/avp1. In lanes 4 to 8, the template DNA was from normal looking seedlings (shown

in the insert). The genotypes were deduced as either avp1/+ (Lane 5, 7, 8) or +/+ (Lane 4,

6). Lane 9, DNA from Col-0 plant, with a wild-type +/+ genotype.

Figure S5. Vascular patterning and cell organization were altered in avp1-1 rosette

leaves. Rosette leaves of wild type and avp1-1 plants grown in ½ MS medium were

allowed to reach their full size and cleared with chloral hydrate. Photographs (A) and (B)

were taken under an Olympus SZ-PT stereomicroscope. Photographs (C) and (D) were

taken under an Olympus XI-70 microscope. (A). Rosette leaf of Col-0. Scale bar

represents 1 mm. (B). Rosette leaf of avp1-1. Scale bar represents 1 mm. (C). Mesophyll

cells of Col-0 rosette leaf. Scale bar represents 10 µm. (D). Mesophyll cells of avp1-1

rosette leaf. Scale bar represents 10 µm.

Figure S6. Effects of avp1-1 on meristem structure. Representative images of Wild-

type (A) and avp1-1 (B) shoot apical meristems. (C) floral mersitem of avp1-1 and (D)

wild-type plants. All images were taken using confocal microscopy of tissues stained

with FM 4-64. Scale bars represent 20µm.

Note: Thus, the L1, L2 and L3 layers characteristic of the shoot meristem were present

and of comparable order in both knockout and wild type plants stained with FM4-64 (28)

(Fig. S6A and B). Likewise, at the earliest stages of floral development the organization

of the floral meristem in the knockout (S6C) and wild type (S6D) plants was very similar.

Thus, floral primordia were present in both and appeared alike in structure (compare S6C

and 6D).

Figure S7. Induced phenotypes of AVP1(RNAi) plants resemble avp1-1 mutants.

EtOH-dependent phenotypes in AVP1(RNAi) seedlings (see Material and Methods). (A)

Shoot and root development of Col-0 and AVP1(RNAi) seedlings with extreme

phenotypes of three independent lines (AVP1 (RNAi)-1,-2,-3) grown under a 16-h light

regimen for 7 days on MS salts, 0.7% agar, 1% sucrose plates added with 0.25% EtOH.

Bar, 1 cm. (B) Representative agarose gel showing AVP1 gene expression levels

monitored via RT-PCR (see Material and Methods) in Col-0 and three independent AVP1

(RNAi) lines as indicated. Bands were electrophoretically resolved on 1% (w/v) agarose

gels and visualized with ethidium bromide (see table S3 for quantification). (C-E) Detail

of AVP1(RNAi) seedlings from (A). Bar,1 mm. (F-H) Development of adventitious roots

in AVP1(RNAi) seedlings after transfer to an ethanol-free medium. (F) day one, (G) day

two and (H) day seven. Of note, the ethanol-induced arrest of the primary root

development (white arrow) was irreversible. Bar, 1 mm.

Figure S8. Development of homozygous avp1-1 mutant complemented with

pINDEX3-AVP1 construct. Six day old pINDEX3-AVP1 /avp1-1 seedlings were

transferred to MS madia with 10 µM Dexamethasone. Photograph shows the same plant

at day 1 (inset), day 12 (A) and day 40 (B) after induction. (C) Detail of a flower from

(B).

Note: As would be expected, shoot developmental abnormalities originating at

embryogenesis were still noticeable after DEX treatment. Of note, longer incubations

with periodic additions of DEX resulted in the development of normal plants with fertile

flowers (Fig. S8).

Figure S9. Effects of alterations in AVP1 expression on pH homeostasis.

Cytoplasmic (A) and vacuolar (B) pH was determined at the elongation zone of roots

from Col-0, avp1-1, and AVP1-2 6 day old seedlings using the fluorescent indicator

Oregon Green-Cellulose binding domain conjugates described by Fasano and

collaborators (29). Values represent means ± SE of 5 measurements from ≥10 individual

plants.

Figure S10. AVP1OX seedlings exhibit higher rates of gravitropic bending. Wild type

and AVP1-1 seedlings were grown vertically for 4d on agar containing Murashige Skoog

basal salts, then rotated 90º. Bending root growth was measured at 2 h intervals. Results

are from 3 sets of 20 seedlings each. Error bars represent standard deviations.

Figure S11. Reduced sensitivity to exogenous naphthalene acetic acid (NAA) in

avp1-1 seedlings. Shoot and root development of Col-0 and avp1-1 plants as indicated

grown for 53 days on MS salts, 0.7% agar, 1% sucrose plates added with 5µM

naphthalene acetic acid (NAA) under a 16-h light regimen. Bar, 1 cm.

Figure S12. Altered flavonoid patters in AVP1 loss-of-function mutant seedlings.

(A) Cotyledonary node of Col-0 seedling accumulates quercetin (orange fluorescence).

(Quercetin accumulation is also observed in the stomata in the cotyledons.) (B)

Cotyledonary node of avp1-1 seedling only has red autofluorescence form chlorophyll.

(C) Root-shoot junction (RSJ) and upper root of wild type seedlings accumulate

quercetin. (D) RSJ and upper root of avp1-1 seedlings accumulate about 25% more

quercetin than Col-0 control. (E) The root tip of Col-0 seedlings accumulates kaempferol

(green fluorescence). (F) The root tip of avp1-1 seedlings does not accumulate

flavonoids. Naringenin chlacone appears to have an expanded distribution, but this

reflects the small cell sizes in avp1-1 root tips. A-F: Bar, 100 µm.

Note: In addition, flavonol accumulation observed in wild type shoots was absent in

avp1-1 (Fig S12 A and B). Loss of flavonol accumulation was not reported when auxin

flux was decreased with auxin transport inhibitors (17) suggesting that the reduction in

auxin transport seen in avp1-1 seedlings was unlikely to explain this alteration in flavanol

distribution. Further, there is an increase in the quercetin signal observed in avp1-1 root-

shoot transition zones (Fig. 4 C and D), where flavonols were previously shown not to be

involved in auxin transport to the root apex.

Figure S13. PIN2 immunofluorescence analysis in roots of wild type, AVP1 gain-,

and loss-of-function mutant seedlings. PIN2 localization in root tips of 5 day old Col-0,

avp1-1 and AVP1-1 as indicated. Bar, 50 µm.

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CDB-GUS construct, G.Hagen for the DR5::GUS Arabidopsis line, K.Noonan for cartoon, H. Sze and R.Serrano for the V-ATPase and P-ATPase sera, respectively. This work was supported by grants from NRI USDA CSREES no. 2001-35100-10772 , UCONN Research Foundation, Storrs Agricultural Experimental Station HATCH to RAG; NSF 0132803 and USDA 01-35304-12290 to ASM; DOE grant 98ER20312 to BK; NSF (MCB 02-12099, DBI03-01460) and NASA (NAG2-1549) to SG and a NASA Graduate student fellowship to GLR.