supporting online material forarabidopsis h +-ppase avp1 regulates auxin mediated organ development...
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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: [email protected]
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.