Developmental Cell Supplemental Information Iron-Dependent ...

Developmental Cell

Supplemental Information

Iron-Dependent Callose Deposition

Adjusts Root Meristem Maintenance

to Phosphate Availability

Jens Müller, Theresa Toev, Marcus Heisters, Janine Teller, Katie L. Moore, Gerd Hause,

Dhurvas Chandrasekaran Dinesh, Katharina Bürstenbinder, and Steffen Abel

Figure S1, related to Figure 1

−Pi−Fe

−Pi+Fe Col-0 ShaBaypdr2

lpr1

lpr2

pdr2

lpr1

lpr2

A

50 h

0 h

50 h

50 h

Col-0

+Pi+Fe −Pi+Fe

pdr2

−Pi+Fe+Pi+Fe

lpr1lpr2

−Pi+Fe+Pi+Fe

Perls/DAB

(Fe3+)

Turnbull/DAB

(Fe2+)

Fe (

ng/m

g F

W)

Col-0 pdr2 lpr1lpr2

a a

b

cb

d

e

d

f fg

h hh

B

a

Col-0 pdr2 lpr1lpr2

12 h

17 h

20 h

27 h

50 h

100 h

0 h

−Pi+Fe

+Pi+Fe

−Pi−Fe

+Pi−Fe

Col-0

0 h 50 h

−Pi+Fe

−Pi+Fe

C

D

Col-0 pdr2 lpr1lpr2

SUPPLEMENTAL INFORMATION


lpr1lpr2 lpr1lpr2pdr2

SCN TAC

Col-0

SCN TAC

+Pi+Fe

−Pi+Fe

SCNRAM RAM


Col-0 ShaBaypdr2

lpr1

lpr2

pdr2

lpr1

lpr2

0 h

20 h

40 h

−Pi+Fe

A

Root C

ap

Epid

erm

is

Cort

ex

Endoderm

is

Root C

ap

Epid

erm

is

Cort

ex

Endoderm

is

QC

E

C

QC

C

E

Col-0 pdr2

C

−Pi−Fe

Col-0 pdr2

−Pi−Fe+Pi+Fe +Pi+Fe

B


p35S::LPR1

Col-0 pdr2 lpr1 #26 #39 #41

+Pi

−Pi

A E

+Pi

−Pi

B

+Pi

−Pi

D

Aniline Blue Staining

+Pi

−Pi

C

Perls Staining

10

30

20

Col-0 pdr2 lpr1 #26 #39 #41

1o

Root Length

(m

m)

p35S::LPR1

103

102

10

1

0.1

0.01

F

Rel. E

xpre

ssio

n

H

(nkata

l m

g-1

)

G

(kD

a)

120

85

0.1

0.5

0.4

0.3

0.2

0

Actin


A

B

Fet3p LPR2 LPR2/Fet3p (T1 Cu site)

T1 T2/T3

C Col-0 pUBQ10::SPLPR1~GFP~LPR1 #2GFP

PI

Merge

GFP

PI

Merge

pUBQ10::SPLPR1~GFP~LPR1 #6 pUBQ10::SPLPR1~GFP~LPR1 #8

D283

D409

H413

E185

H489

C484

D372

D464

E271

H568 H466

C563

LPR2

Fet3p

T1

Fe(II)


Col-0 irt1-1

+Pi+Fe

−Pi+Fe

SUPPLEMENTARY FIGURE LEGENDS

Figure S1, related to Figure 1.

Pi-dependent and genotype-dependent dynamics of Fe accumulation and distribution in

primary root meristems.

(A) Fe accumulation and distribution in primary roots. Seeds were germinated on +Pi+Fe

medium (4 days) and transferred to –Pi+Fe agar prior to Perls/DAB staining. The upper and

lower panels of both time points (0 hr and 50 hr) show the region near the hypocotyl junction and

the root apical region, respectively. Scale bar, 0.5 mm. The higher magnification panels in the

lower two rows show Pi-dependent dynamics of Fe accumulation and distribution in primary

root tips. Prior to Fe staining (Perls/DAB staining) of root tips, seeds were grown as above and

transferred to −Pi+Fe or −Pi−Fe medium (50 hr). Scale bar, 100 µm.

(B) Fe accumulation in whole roots. Seedlings were germinated for 4 days on high Pi (+Pi+Fe)

agar and subsequently transferred to low Pi (−Pi+Fe) medium for up to 3 days prior to Fe

quantification by the spectrophotometric BPS method. In a second experiment, seedlings were

transferred from high Pi control medium (+Pi+Fe; 2.5 mM Pi, 50 µM Fe) to high Pi medium

containing 500 µM Fe. Mean root Fe concentrations (ng Fe/mg root fresh weight) are given

(±SE, n=7-8, p<0.05, Student’s t test).

(C) Comparison of Perls/DAB staining, which detects Fe3+ and some Fe2+, and Turnbull/DAB

staining, which is specific for Fe2+, in Col-0, pdr2 and lpr1lpr2 root tips. Seedlings were

germinated for 4 days on +Pi+Fe agar and subsequently transferred to +Pi+Fe or −Pi+Fe

condition for additional 3 days. Scale bar, 100 µm.

(D) Perls staining (no DAB intensification) of Fe in root tips upon transfer of 4-days-old

seedlings from control agar to the indicated media. Upper panels: Fe detection in the root SCN

after transfer from +Pi+Fe to −Pi+Fe medium for up to 100 hr. The extended time series

contains some data presented in Figure 1D. Scale bar, 25 µm. Center panels: Fe detection in the

root SCN after transfer from +Pi+Fe medium to the indicated media without Fe to demonstrate

specificity of the Perls method. Scale bar, 25 µm. Lower panels: Fe detection in primary root

tips (low magnification; scale bar, 100 µm) and the SCN (high magnification; scale bar, 25 µm)

of Col-0 roots after transfer from +Pi+Fe to −Pi+Fe medium (0 hr and 50 hr). Roots were

cleared using chloral hydrate solution.


Fe deposition in root meristems of lpr1lpr2 and lpr1lpr2pdr2 seedlings.

Semi-thin (1 µm) longitudinal sections of Perls/DAB-stained root tips of lpr1lpr2, lpr1lpr2pdr2,

and Col-0 seedlings after transfer from +Pi+Fe to –Pi+Fe medium (20 hr). RAM: overview

images of the RAM. SCN: details of the SCN. TAC: Details of transit amplifying (TA) cells.

Scale bars, 25 µm. The image of the Col SCN (–Pi+Fe) is also shown in Figure 3D.


Consequences of Pi deprivation on root meristem organization.

(A) Electron micrographs of ultra-thin (90 nm) longitudinal sections of the SCN (lower panels)

and proximal meristem of root tips (upper panels) derived from Col-0 and pdr2 seedlings

experiencing Pi deprivation. Radial root cell layers are indicated (upper panels); and cell types

of the SCN, quiescent center (QC), endodermal cell (E), cortical cell (C), are labeled in yellow

(lower panels). Scale bar, 5 µm.

(B) Propidium iodide (PI) staining of root tips upon transfer of 4-days-old seedlings from +Pi+Fe

to −Pi+Fe medium for up to 40 hr. Red arrows point to dead QC cells in pdr2 roots and to

irregularly shaped stem cells in Col-0 and Sha root meristems. Yellow arrowheads point to

prematurely formed middle cortex cells in Col-0 and Sha root tips. Scale bar, 50 µm. Pie charts:

Statistical analysis of cell death events upon transfer (3 days) from +Pi+Fe to −Pi+Fe condition

in Col-0, pdr2 and lpr1lpr2. The graphs show the percentage of primary roots revealing cell

death within the QC, compared to the occurrence of random cell death events in the surrounding

SCN (no cell death in the QC) and to the complete absence of cell death events in the meristem

(n=71 for Col-0, n=56 for pdr2, n=22 for lpr1lpr2).

(C) Aniline blue staining of callose in Col-0 and pdr2 primary root tips. Seedlings were

germinated for 4 days on +Pi+Fe agar and subsequently transferred for 2 days to +Pi+Fe or

−Pi−Fe medium. Scale bar, 100 µm.


Phenotypes and molecular analysis of CaMV p35S::LPR1 overexpression lines in

comparison with wild-type, pdr2 and lpr1 plants.

(A, E) Seedlings of the indicated genotypes (A) and primary root length (E) after germination

and growth on +Pi+Fe (+Pi) or −Pi+Fe (−Pi) medium for 5 days. Overexpression of LPR1

(p35S::LPR1) caused partial gene silencing and produced progeny of variable primary root

length on –Pi medium. p35S::LPR1 seedlings with a short primary root were selected for the

analyses. Shown are the means of 3 independent experiments (±SD, n=30-40).

(B-D) Root tip morphology (B), Fe detection (C), and callose deposition (D) in primary roots of

the indicated genotypes after germination and growth on +Pi or −Pi medium for 5 days. Scale

bars, 500 m (B) and 100 m (C, D). Data for line #41 are also shown in Figure 5B.

(F-H) Relative LPR1 mRNA expression levels as determined by quantitative RT-PCR (F),

immunoblot analysis of LPR1 protein expression using a peptide-specific anti-LPR1 antibody

(triangle), the upper non-specific band indicates equal loading (G), and ferroxidase activity in

root extracts (H) of the indicated genotypes on +Pi medium. Shown are the means of 3

independent experiments (±SE).


Structural modeling of LPR proteins, prediction of putative Fe2+ binding sites, and cell wall

targeting of GFP~LPR1.

(A) Multiple sequence alignment of Fet3p, Fet5p, LPR1 and LPR2 proteins using the MAFFT

algorithm (Katoh et al., 2002). Highlighted are identical and conserved amino acid residues

(black and grey), conserved His and Cys residues that form the trinuclear (T2/T3) Cu cluster

(blue) and mononuclear (T1) Cu site (olive), and the predicted acidic amino acid residues

binding the Fe2+ substrate (red).

(B) 3D structure of Fet3p (left) and homology model of LPR2 (center). Ribbon representation of

Phyre2 (Kelley and Sternberg, 2009) modeled LPR2 is shown, which adopts the tripartite

cupredoxin fold, containing three Greek key -barrel cupredoxin domains similar to other

multicopper oxidases (MCOs). The trinuclear (T2/T3) Cu cluster of the LPR structures (see

Figure 5E for LPR1) were predicted by 3DLigandSite (Wass et al., 2010) and shown as colored

spheres. The predicted T1 Cu and Fe2+ binding sites of LPR2 (right). The predicted amino acid

residues (sticks) of LPR2 (orange) and Fet3p (green) are presented together with the metal ions

(spheres), T1 Cu (purple) and Fe2+ (red), in structural superimpositions of LPR2 and Fet3p.

(C) Cell wall localization of GFP~LPR1 in transgenic pUBQ10::SPLPR1~GFP~LPR1 lines.

Because all lines showed only low GFP fluorescence, laser intensity was set to a level inducing

autofluorescence in untransformed wild-type controls (Col-0). Under these conditions, all

pUBQ10::SPLPR1~GFP~LPR1 lines tested, but not the control seedlings, showed a distinct GFP

signal in cell walls (red arrows). Cell wall localization was confirmed by co-staining with

propidium iodide (PI) (right panels). This panel contains some data presented in Figure 5I (line

#2). Scale bar 20 µm.


Fe distribution in root meristems of wild-type and irt1 seedlings.

Fe staining (Perls/DAB) of Col-0 and irt1-1 root tips (overview and detail) after seedling (4-

days-old) transfer from +Pi+Fe to +Pi+Fe (+Pi) or −Pi+Fe (−Pi) medium (2 days). Staining time

was decreased compared to images in Figure 1 and Figure S1 to avoid oversaturation. Contrast

and brightness in higher magnification images were increased for better visualization of Fe

staining in the SCN. Arrows point to the QC. Scale bar 100 µm.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Generation of Transgenic Arabidopsis Lines. For CaMV p35S::LPR1 transgenic Arabidopsis

thaliana (Col-0) plants, the LPR1 coding region was amplified from Arabidopsis cDNA (Col-0)

using the primer pair:

5’- CACCATGGAATCTCTGTTGTGTCGG-3’ /

5’- TCAAATGATGACCTTAAGCGG-3’.

The amplified LPR1 cDNA fragment (1,746 bp) was cloned into the pENTR/D-TOPO vector

(Invitrogen). After sequence verification, the LPR1 cDNA was mobilized into the pB7WG2

binary vector (Karimi et al., 2002), which contains the CaMV 35S promoter-driven expression

cassette, using Gateway LR clonase II (Invitrogen). To generate CaMV

p35S::SPLPR1~eGFP~LPR1 and pUBQ10::SPLPR1~eGFP~LPR1 overexpression lines, the

predicted 84 bp sequence coding for the LPR1 signal peptide (SPLPR1) was amplified using the

primer pair:

5’- ATGGAATCTCTGTTGTGTCGG-3’ /

5’-CTCCTCGCCCTTGCTCACTCCGCACGTGCT-3’.

cDNA fragments of eGFP and LPR1 (without signal peptide) were generated with primer pairs:

5’-AGCACGTGCGGAGTGAGCAAGGGCGAGGAG-3’ /

5’-GTTGGTCCTCGAGCTCCTTGTACAGCTCGTCC-3’ and

5’- GGACGAGCTGTACAAGGAGCTCGAGGACCAAC-3’ /

5’-TCAAATGATGACCTTAAGCGG-3’.

The SPLPR1~eGFP~LPR1 cDNA fragment was generated, mobilized into the pENTR/D-TOPO

vector, and transferred into the pB7WG2 binary vector (Karimi et al., 2002) or the pUBQ10

destination vector (Grefen et al., 2010). To generate transgenic lines expressing the GUS

reporter under control of the LPR1 promoter (pLPR1Col::GFP~GUS), a 2.3 kb fragment of the

LPR1 promoter was amplified from genomic DNA using the primer pair:

5’- CCTAGAATGTTATTGATGTTTCTT-3’ /

5’-TTCTGACAAGTCAATTCAGTTTTGA-3’.

Using GATEWAYTM technology, the LPR1 promoter fragment was transferred into the

pBGWFS7 binary vector, which provides the coding sequence of -glucuronidase (GUS). All

constructs were transformed into wild-type plants (Col-0) by conventional floral-dip infiltration

methods using Agrobacterium tumefaciens strain GV3101(Clough and Bent, 1998).

Transformants were selected on culture media containing Basta® (Sigma-Aldrich), and all

experiments were carried out using T3 or T4 plants harboring a single T-DNA insert.

Transient Expression in Tobacco. Agrobacterium-mediated transient transformation of tobacco

leaves (Nicotiana benthamiana) was performed as described (Burstenbinder et al., 2013).

Determination of Root Fe Content. Fe content of whole roots was determined by a

spectrophotometric assay (Tamarit et al., 2006). Entire roots were removed, frozen in liquid

nitrogen (approx. 50 mg FW), ground to a fine powder, and extracted overnight in 350 l 3%

(v/v) nitric acid at 95 oC. After treatment of the cleared lysate with Na-ascorbate to reduce iron,

the Fe2+ concentration was determined after complex formation with bathophenanthroline

disulfonic acid (BPS) (Sigma-Aldrich) at 535 nm.

Electron Microscopy. Seedlings were fixed (4 hr) in 100 mM Na-cacodylate buffer (pH 7.2),

4% (v/v) glutaraldehyde. After postfixation (30 min) in the same buffer and 1% (w/v) OsO4,

plants were dehydrated in a graded ethanol series (10-30-50-70-90-100%), which included a 1%

(w/v) uranyl acetate staining step at 70% ethanol (1 hr). Roots were infiltrated with Spurr’s

epoxy resin (EM0300, Sigma-Aldrich) by incubation in a series of EtOH dilutions according to

the manufacturer’s protocol. After polymerization at 70 oC (12 hr), ultra-thin sections (90 nm)

were prepared with an Ultramicrotome S (Leica), stained with uranyl acetate and lead citrate

using an EM Stain (Leica) and imaged with a Zeiss Libra 120 TEM operating at 120 kV. Images

were taken by a Dual-Speed on axis SSCCD camera BM-2k-120 (Moorenweis). Immunogold

labeling of callose was performed using a monoclonal -1,3-glucan antibody (#400-2,

Biosupplies) in combination with a goat anti-mouse 10 nm gold conjugated secondary antibody

(#G7777, Sigma-Aldrich). Sections were poststained with uranyl acetate and lead citrate and

imaged as above.

NanoSIMS Analysis. Roots were fixed as described for electron microscopy albeit the uranyl-

acetate staining step was omitted. Root sections (1 µm) were deposited onto a droplet of water on

Thermanox coverslips coated with 20 nm of platinum and stretched flat on a hotplate.

Subsequently, the samples and substrates were coated with 10 nm platinum to ensure

conductance. High resolution secondary ion mass spectrometry analysis was done using a

CAMECA NanoSIMS 50 (CAMECA, France). A 16 keV Cs+ ion beam focused to

approximately 100 nm with a beam current of 1.4-1.7 pA was scanned over the section surface.

The resulting negative secondary ions generated during this process were analyzed according to

their mass using a double focusing mass spectrometer. The five detectors were carefully tuned

using standards of GaP and steel to detect 16O-, 12C14N-, 31P-, 31P16O- and 56Fe16O- taking care to

avoid mass interferences (Moore et al., 2012). Iron was detected as 56Fe16O- as described

previously (Moore et al., 2012). For each area a dose of 1 x 1017 Cs+ ions cm-2 was implanted by

continuously scanning a large defocused beam to remove the platinum coating and ensure that

the sample was at steady state. Images (50 x 50 µm) were acquired at a resolution of 256 x 256

pixels. Dwell times were 60 ms per pixel and for each region of interest, several sequential

images were taken to improve the statistics as the 56Fe16O- signal was quite low. The stack of

images was then summed together using ImageJ and the OpenMIMS plug-in (Harvard) and are

presented as a single image and as a red green color merge.

GUS Staining. Four independent transgenic pLPR1Col::GUS lines (single insert) were identified

and grown for 4 days on +Pi+Fe medium before transfer to the indicated media. GUS staining

was performed by incubation of seedlings in GUS-staining solution (50 mM Na-phosphate,

pH7.2; 0.5 mM K3Fe(CN)6; 0.5 mM K4Fe(CN)6; 2mM X-Gluc; 10mM EDTA; 0,1% TritonX) at

37°C (Wong et al., 1996).

ROS Imaging. The presence of ROS was determined in root tips as described (Freeman et al.,

2004). Seedlings were stained with 10 µM Carboxy-H2DCFDA (Invitrogen) for 10 min and

subsequently imaged without removal of the dye to allow ROS detection at cell walls.

For controls, roots were briefly rinsed to remove extracellular dye. The ability of the dye to

report apoplastic ROS formation was confirmed by application of 10 mM H2O2, which

significantly increased intracellular and cell wall localized fluorescence. For superoxide staining,

seedlings were incubated for 45 min in a 0.5 mg/ml Nitro Blue Tetrazolium (NBT) (AppliChem)

solution in 100 mM Na-phosphate buffer, pH 7.2 (Tyburski et al., 2009).

LPR1 mRNA Expression Analysis. Total RNA was prepared from about 100 mg seedlings by

using the RNeasy Plant Mini Kit (Qiagen). RNA samples were DNase treated (DNase set;

Qiagen) and quantified. cDNA was prepared using 4-5 g total RNA, which was reverse

transcribed by using oligo(dT) with a First Strand cDNA Synthesis Kit (Fermentas) according to

the manufacturer’s protocol. Quantitative real-time PCR was performed with diluted (1:10) first

strand cDNA template on a 7500 Fast Real-Time PCR System with Fast SYBR Green Mix

(Applied Biosystems). The following LPR1 specific primers were used:

5’-GCCGACGCGTGGTTCACTGCC-3’ and 5’-AGGCGCCTACCAAACCGGCAAGG-3’.

The reported fold induction was analyzed by the ∆Ct method and normalized to the endogenous

PP2A (protein phosphatase 2A) control, using the PP2A primers 5’-

CCTGCGGTAATAACTGCATC-3’ and 5’-TGGTCGACTATCGGAATGAG-3’.

LPR1 Immunoblot Analysis. Polyclonal LPR1 epitope-specific antibodies were raised in

rabbits against a synthetic peptide (LPR1: 402-YPNADVSNA-410) and affinity-purified

(immunoGlobe, Himmelstadt, Germany). Total proteins were extracted from frozen-powdered

roots of 6-days-old seedlings in the extraction buffer [50 mM Tris-HCl, pH 8.0, 100 mM NaCl,

0.5 mM EDTA, 10% (v/v) glycerol, containing complete protease inhibitor cocktail (Sigma-

Aldrich)], using a ratio of 150 l extraction buffer per 100 mg root material. The supernatant was

collected after centrifugation at 20,000 x g for 10 min (4 oC). After determination of the protein

concentration (2D-Quant, GE Healthcare), protein samples were separated in 10% SDS/PAGE

gels and transferred to Protran nitrocellulose membranes (Semi-Dry-Blot, GE Healthcare). After

transfer, membranes were exposed to blocking buffer [1xPBS, 0.05% Tween, 2.5% milk

powder] at room temperature for 2 hr. To detect LPR1, membranes were incubated overnight in

blocking buffer containing 0.1 g/ml affinity-purified, peptide-specific anti-LPR1 antibody at 4

oC. Horseradish-peroxidase-conjugated goat anti-rabbit IgG (BioRad) was chosen as a secondary

antibody, and the Super Signal West Pico Chemiluminescent kit (Thermo Fisher Scientific) was

used to detect signals derived from the antibodies (Alpha Innotech Multiimager III). GFP~LPR1

and GFP were detected using rabbit polyclonal anti-GFP (Sigma-Aldrich). Mouse monoclonal

anti-actin (plant) (Santa Cruz) was used for loading controls. Alternatively, membranes were

stained with Ponceau S to visualize total proteins as loading reference.

Cell Wall Protein Extraction. Cell wall proteins were sequentially extracted form suspension-

cultured Arabidopsis thaliana (Col-0) cells as described (Robertson et al., 1997). Cells were

harvested 7-8 days after subculture by filtration through 3 layers of miracloth and washed three

times with ddH2O (3 ml g-1 fresh weight). The subsequent steps were sequentially performed

with the same cells at 4°C. Cells were stirred for 30 min in 3 volumes of 0.2 M CaCl2, collected

by filtration (miracloth) and washed as above. This step was followed by three more extractions

with 2 mM DTT, 1 M NaCl, and 0.2 M borate, pH 7.5. The borate extraction was carried out at

room temperature. Between extractions cells were washed with ddH2O on the filter as described

above. Aliquots of extracts (15 ml) were dialyzed twice for 1 hr at 4 °C against 400 ml of ddH2O

with a subsequent dialysis overnight. Dialyzed extracts were lyophilized for 2 days. Lyophilized

samples were dissolved in 150-300 µl of protein buffer (20 mM Tris-HCl pH 7,5; 150 mM NaCl;

10% Glycerol; 0,1 mM PMSF; plant protease inhibitor cocktail [Sigma 1:100]). 4 x SDS PAGE

sample buffer was added and 35 µl were loaded on a 10 % SDS Gel.

SUPPLEMENTAL REFERENCES

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B.J., Dinesh, D.C., and Abel, S. (2013). Arabidopsis calmodulin-binding protein IQ67-domain 1

localizes to microtubules and interacts with kinesin light chain-related protein-1. J Biol Chem

288, 1871-1882.

Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated

transformation of Arabidopsis thaliana. Plant J 16, 735-743.

Freeman, J.L., Persans, M.W., Nieman, K., Albrecht, C., Peer, W., Pickering, I.J., and Salt, D.E.

(2004). Increased glutathione biosynthesis plays a role in nickel tolerance in thlaspi nickel

hyperaccumulators. Plant Cell 16, 2176-2191.

Grefen, C., Donald, N., Hashimoto, K., Kudla, J., Schumacher, K., and Blatt, M.R. (2010). A

ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal

stability and native protein distribution in transient and stable expression studies. Plant J 64, 355-

365.

Karimi, M., Inze, D., and Depicker, A. (2002). GATEWAY vectors for Agrobacterium-mediated

plant transformation. Trends Plant Sci 7, 193-195.

Katoh, K., Misawa, K., Kuma, K., and Miyata, T. (2002). MAFFT: a novel method for rapid

multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30, 3059-3066.

Kelley, L.A., and Sternberg, M.J. (2009). Protein structure prediction on the Web: a case study

using the Phyre server. Nat Protoc 4, 363-371.

Moore, K.L., Zhao, F.-J., Gritsch, C.S., Tosi, P., Hawkesford, M.J., McGrath, S.P., Shewry, P.R.,

and Grovenor, C.R.M. (2012). Localisation of iron in wheat grain using high resolution

secondary ion mass spectrometry. J Cereal Sci 55, 183-187.

Robertson, D., Mitchell, G.P., Gilroy, J.S., Gerrish, C., Bolwell, G.P., and Slabas, A.R. (1997).

Differential extraction and protein sequencing reveals major differences in patterns of primary

cell wall proteins from plants. J Biol Chem 272, 15841-15848.

Tamarit, J., Irazusta, V., Moreno-Cermeno, A., and Ros, J. (2006). Colorimetric assay for the

quantitation of iron in yeast. Anal Biochem 351, 149-151.

Tyburski, J., Dunajska, K., and Tretyn, A. (2009). Reactive oxygen species localization in roots

of Arabidopsis thaliana seedlings grown under phoshate deficiency. Plant Growth Regul 59, 27-

36.

Wass, M.N., Kelley, L.A., and Sternberg, M.J. (2010). 3DLigandSite: predicting ligand-binding

sites using similar structures. Nucleic Acids Res 38, W469-473.

Wong, L.M., Abel, S., Shen, N., de la Foata, M., Mall, Y., and Theologis, A. (1996). Differential

activation of the primary auxin response genes, PS-IAA4/5 and PS-IAA6, during early plant

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