Supporting Information: Figs S1-S8, Tables S1–S5, Methods S1
Methods S1
SiCSA RNA isolation and amplification for microarray
RNA was isolated from specific cell-types of A. thaliana enhancer trap line
KC464 as per Roy et al. (2008) with the following modifications. Cellular sap from 3
individual mesophyll cells sampled with 3 different capillaries or 30 epidermal cells
sampled with a single capillary was expelled into 10 U of RNaseOUT (Invitrogen) in
3 μL of DEPC-treated water and amplified using the TargetAMP 2-round aRNA
amplification kit (EpiCentre Biotechnologies). RNA was quantified with a nanodrop
ND-1000 (Thermo Scientific) and 100 ng was reverse-transcribed with random
hexamers using Superscript® II reverse transcriptase (Invitrogen). RT-PCR was used
to screen for genomic DNA and cellular contamination: epidermal contamination was
screened by the use of mGFP5, GAL4 AtCUT1 and AtLTP1 and mesophyll
contamination was screened by the use of AtRBCS-3b and AtCA1 (see Supplemental
Table 2 for list of primers) (Inada & Wildermuth, 2005). RNA size and quality was
assessed after RNA amplification using an Agilent Bioanalyzer 2100 RNA picochip
(Agilent Technologies), with only samples possessing an average cRNA size greater
than 500 nt passing to the next round of analysis. aRNA was labelled with either Cy3
or Cy5 using the Kreatech ULS™ aRNA labelling kit, according to manufacturer
instructions for Agilent arrays (Kreatech Diagnostics). The degree of labelling (DoL)
was calculated using a Nanodrop 1000 prior to fragmentation. Bioanalyzer analysis
was performed on pre- and post-fragmented, labelled aRNA to confirm correct size
profiles.
Custom 4 x 44 k Agilent DNA microarrays designed using eArray v.5.3.1
(Agilent Technologies), incorporating the entire Arabidopsis 3 oligo gene list, in
addition to user defined probes for both mGFP5 and GAL4-VP16 and duplicates of
known leaf-expressed ion transporters (Design ID: 017700), were used in this study.
Eight hundred and twenty-five nanograms of Cy3 and Cy5 labelled aRNA was
hybridised to each array, washed and scanned as per manufacturer’s instructions for
two-colour arrays and imaged on an Agilent Microaray Scanner in XDR mode. Image
files were processed and within-slide normalization was carried out using Agilent's
"Feature Extraction Software" (V. 9.5.3.1, Agilent Technologies). Additional
between-slide normalization was not undertaken because this would be compromised
by the expected stochastic related fluctuations in gene expression within individual
cells (Elowitz et al., 2002). Instead, a list of putatively differently expressed genes
was prepared by splitting transcripts into groups that were consistently expressed at
very high levels (log2(expression) > 13.3) in one cell-type while being consistently
expressed at below background levels (log2(expression) < 8.0) in the other tissue.
Hybridised arrays were omitted from analysis if they contained control genes from
both an epidermal and mesophyll origin (see Supplemental Table 2 for list of control
genes), or they did not pass standard quality control tests (Bioanalyzer median aRNA
product size > 500 nt and sufficient fluorescence degree of labelling).
References
Elowitz, M.B., Levine, A.J., Siggia, E.D., and Swain, P.S. (2002). Stochastic gene
expression in a single cell. Science 297: 1183-1186.
Inada N, Wildermuth MC. 2005. Novel tissue preparation method and cell-specific
marker for laser microdissection of Arabidopsis mature leaf. Planta 221: 9-16.
Roy SJ, Gilliham M, Berger B, Essah P, Cheffings C, Miller AJ, Widdowson L,
Richardson P, Leigh RA, Tester M. 2008. Investigating glutamate-like receptor
gene co-expression in Arabidopsis thaliana. Plant, Cell and Environment 31: 861-71.
Table S1 List of Arabidopsis T-DNA insertion lines investigated in the course of this study
MRS2 Loci
AtMRS2-1
AtMRS2-5
AtMRS2-10
SALK ID
Line name
Primers for confirming insertion site (5’ → 3’)
T-DNA Insertion
site
SALK_006797
SALK_038768
SALK_139989
SALK_105475
SALK_115410
SALK_100361
SALK_006528
mrs2-1a (2-1a)
mrs2-1b (2-1b)
mrs2-1c
mrs2-5a (2-5)
mrs2-5b
mrs2-10a (2-10)
mrs2-10b
LP: tctgtttgggctaattggttg; RP: gataaaagcttgagcattgcg
LP: catagctgaagcaggtcttgg; RP: agatgctccaccggttaagtc
n.d.
LP: ccagtcggaactgtgtaaacg; RP: tgattctgcacattccccagc
n.d.
LP: cagtaccagagctcatggacg; RP: tcccacaaaatcagatggatc
n.d.
3rd Intron/4th Exon
Promoter
4th Exon
4th Exon
5‘ UTR
4th Exon
5‘ UTR
Loss of
functional
transcript
+
+
+
+
-
Lines with a verified loss of transcription (+, see Fig. S1A) are highlighted and were used for phenotype screening. In three cases homozygous
T-DNA insertions did not lead to a loss of intact mRNAs (-) and so were excluded from this assay (Fig. S1). Primers used to confirm T-DNA
insertion site (LP, and RP) calculated from Signal SALK website (http://signal.salk.edu/tdnaprimers.2.html). Results presented in Gebert et al.
(2010)
were
confirmed
in
our
study.
n.d.
not
determined.
Table S2 Recipes for hydroponics growth solutions utilised in this study
Final conc (mM)
LCS
2
Macronutrients
Stock Conc. (M)
NH4NO3
1
BNS
2
KNO3
1
3
5
3
CaCl2•2H20
KCl
Ca(NO3)2•4H20
1
1
0.4
0.1
2
2
0.05
0
0
0.9
2
2.2
MgSO4•7H20
0.4
2
4.5
3.5
KH2PO4
0.1
0.6
6
0.6
NaCl
NaNO3
Mg(NO3)2•6H20
MgCl2
K2SO4
1
1
0.4
0.5
0.1
0.4
1.2
0.5
0
0
1.7
0
0
14.5
0.2
Micronutrients
NaFe(III)EDTA
H3BO3
1.5
0
0
0
0
NaOH to pH 5.6
Stock Conc. (mM)
50
50
50
50
Final conc (μM)
50
50
HMS
2.3
50
50
MnCl2•4H20
5
10
10
10
ZnSO4•7H20
10
150
150
150
CuSO4•5H20
0.5
1
1
1
Na2MoO3
0.1
0.5
0.5
0.5
Table S3 Primers used for qPCR analysis. Amplification efficiency (E) of each primer pair calculated across 5 serial log dilutions, with
Ct values plotted against log10 (concentration) using qGene software
Gene
AGI
Forward Primer (5’→3’)
Reverse Primer (5’→3’)
AtEF-1α
Atβ-Tubulin5
AtActin2
AtGAPDH-A
AtCUT1
AtLTP1
GAL4
mGFP5
AtCA1
AtRBCS-3b
AtMRS2-1
AtMRS2-4
AtMRS2-5
AtMRS2-10
AtMRS2-11
AtMHX1
AtCAX1
At1g07940
At1g20010
At3g18780
At3g26650
At1g68530
At2g38450
N/A
N/A
At3g01500
At5g38410
At1g16010
At3g58970
At2g03620
At1g80900
At5g22830
At2g47600
At2g38170
gacaggcgttctggtaaggag
cgtgaaatccagcgtttgtga
tgagcaaagaaatcacagcact
tggttgatctcgttgtgcaggtctc
cattcacgcaggaggcagag
atagccaagacgaccccaga
cggaggagagcagcaacaag
tgtccttttaccagacaaccatta
gtgaaagggaggcggtgaa
tgactaccttctccgcaacaa
gatctttgggatgaactttgag
cattgacgacacagaagactacg
tccgacgactttccagtatgt
tcagaagacgcaaccgaga
tgaggaagaagaggaggaaatc
atcgtggtgcttgtgttgag
catcatcgtggcgtggatt
gcggaaagagttttgatgttca
tcgtccattccttctcctgtg
cctggacctgcctcatcatac
gtcagccaagtcaacaactctctg
ccacacggcagagttacacttg
tagctcatcacctcacggttt
attccaagggcatcggtaaac
agctgttacaaactcaagaagga
atcacagtcaaaggcacattacaa
gcattcttcaacttccttcaacac
gtctactttctggggagcaca
tggtttgtaagagagagggagat
tgcgtcttcttcttcttctctaac
accccgaagatggctaca
cccgttgttagccagaatg
ttttccttttgttgcttcttct
gcattttgtttctggggaagt
Product
Length
(bp)
275
171
166
263
229
179
265
103
288
181
181
290
138
218
282
167
207
Amplification
Efficiency
(E)
1.98
1.98
1.99
1.97
1.96
1.97
1.95
1.96
1.95
1.99
1.99
1.95
1.98
1.96
1.98
1.97
2.00
The slope of this standard curve was used in the following equation to calculate E; E = 10(-1/slope), with each qPCR performed in triplicate.
Table S4 Primers for amplification of the coding sequence of MRS2s for generating
YFP fusions and for complementation experiments as described in Materials and
Methods
Gene
Forward Primer (5’→3’)
Reverse Primer (5’→3’)
AtMRS2-1
AtMRS2-5
AtMRS2-10
atgtctgagctaaaagagcg
atgggagaacaactagatcca
atgtctgaactcaaagagcgtttg
ttatagaggcatgagtcttctgtac
tcagagagggaatactttcttg
tcacagaggcatgagtcttctacg
Reverse Primer minus stop
(5’→3’)
tagaggcatgagtcttctgtacttg
gagagggaatactttcttgtgc
cagaggcatgagtcttctacgtttg
Table S5 Leaf water content and soluble sugar concentration of Col-0, and all mrs2
T-DNA insertion lines utilised in this study, grown in both BNS and LCS hydroponics
conditions
Leaf Water Content (WCOM)
Soluble Sugar Conc. (mg.g-FW-1)
Line
Col-0
mrs2-1a
mrs2-1b
mrs2-5
mrs2-10
cax1/cax3
BNS
LCS
BNS
LCS
89.99 ± 0.33
90.05 ± 0.39
89.84 ± 0.67
89.36 ± 0.55
89.93 ± 0.41
89.30 ± 0.82
89.50 ± 0.59
90.04 ± 0.92
89.35 ± 0.80
90.01 ± 0.71
89.57 ± 0.38
90.14 ± 0.66
21.41 ± 0.67
21.03 ± 0.56
21.29 ± 0.64
21.13 ± 0.67
21.31 ± 0.88
26.31 ± 0.88
22.25 ± 0.48
22.07 ± 0.51
22.09 ± 0.64
22.12 ± 0.46
22.20 ± 0.19
20.80 ± 0.39
Shoots of 6-wk-old plants grown for one week in BNS or LCS were excised and
weighed (fresh weight, FW), dried at 42oC for 48 h (dry weight, DW) (mean ± SEM.;
n = 6 plants). Water content presented as mass of water (FW-DW) per mass of
organic matter (WCOM). Soluble sugar concentration calculated from 3 independent
plants under each treatment with data shown as mean ± SEM.
Fig. S1 Confirmation of homozygosity for T-DNA insertion lines of MRS2 genes
used in this study and location of T-DNA inserts. (a) PCR used to amplify the entire
coding region of AtMRS2-1, AtMRS2-5 and AtMRS2-10 from each SALK T-DNA
insertion line and the Col-0 (wild-type) background. Underlined are the lines lacking
corresponding gene transcript. (b) Image showing insertion site of all loss of transcript
lines and naming conformity referred to in (a). 2,500 bp of genomic sequence (sense
orientation) incorporating the gene is shown to scale with exons (shaded boxes),
introns (unshaded boxes), 5’ and 3’ non-coding regions (unfilled lines) and the
insertion site(s) and direction (arrow) of T-DNAs.
Fig. S2 Standard curves used to quantify magnesium concentration by both (a) single
cell sampling and analysis (SiCSA), or (b) cryogenic scanning electron microscopy
(cryo-SEM) in combination with X-ray microanalysis (XRMA). For (a), equal
volumes (≈20 pl) of internal standard (250mM rubidium fluoride (RbF) in 250mM
Mannitol) and a dilution series from 1.25-200 mM MgCl2 was spotted onto 1% (w/v)
pioloform-coated copper SEM grids, respectively. Each concentration was spotted in
quintuplicate and grids were prepared and analysed as for other SiCSA samples. For
(b), dilution of calibration solution was mixed with 5% (w/v) colloidal
graphite/carbon that had been ballmilled and passed through a 50 mm sieve (Treeby et
al., 1987). Solutions were placed in a brass stub and prepared identically to plant
material analysed by cryo-SEM as per described methods. Data was analysed with
XRMAplot and plotted to form standard curves for each element. Filled circle
indicates detection limit for magnesium for each method, defined as the first point
distinguishable from 0 mM Mg, which is 15 mM for SiCSA and 35 mM for cryoSEM.
Fig. S3 Validation of SiCSA-based RNA sampling and amplification for qPCR and
microarray study using the enhancer trap line, KC464. qPCR check of cell-specific
gene expression and contamination of palisade mesophyll samples by RNA from
adaxial epidermal cells. RNA was amplified from samples taken from 30 adaxial
epidermal cells and 3 palisade mesophyll cell of leaf 8 of 6-wk-old KC464 plants.
Data are normalised against AtEF1α (At1g07940), Atβ-Tubulin5 (At1g20010) and
AtActin2 (At3g18780). Three independent amplifications were performed, with data
presented as mean normalised expression levels ± SEM (n = 3 PCR technical repeats).
Cell-specific genes showed the expected pattern for epidermis, AtCUT1, AtLTP1 and
mesophyll RBCS-3b, AtCA1 (Brandt et al., 2002; Inada and Wildermuth, 2005). GFP
and GAL4 transcripts were also found only within the epidermal RNA samples as
expected from GFP fluorescence profile. Equally amplified transcripts AtEF1a and
Atβ-Tubulin5 were normalised against AtActin2, known to be expressed in both cell
types (Laval et al., 2002). Primers listed in Supplemental Material Table S2.
Fig. S4 AtMRS2-1 and AtMHX expression are positively correlated with leaf [Mg] and
inversely correlated with leaf [Ca] in Arabidopsis thaliana ecotypes. Correlation of
inductively-coupled plasma (ICP) spectroscopy and microarray data across 23
Arabidopsis ecotypes, reinforcing the importance of mesophyll enriched MRS2s in
leaf Mg accumulation. Normalised microarray data for of (a-b) AtMRS2-1, (c-d)
AtMRS2-4, (e-f) AtMRS2-10, (g-h) AtMRS2-11, and (i-j) AtMHX
obtained from
Affymetrix Arabidopsis ATH1 Genechip® arrays performed by Lempe et al. (2005)
and ICP data obtained from Purdue ionomics information management system
(PiiMS) database (Baxter et al., 2007)(www.ionomicshub.org) on leaves of plants
grown in soil with the same fertilization and light regimens. Weight normalised
values (in ppm) were utilised and presented relative to Columbia-0 to facilitate interexperimental normalization of ICP data for (a,c,e,g,i) magnesium and (b,d,f,h,j)
calcium (Mean ± SEM). Linear regression line shown in red, r2 values calculated
using PRISM ver. 5.1 (GraphPad software). Correlations with AtMRS2-5 were not
possible given there was no probe sets for this transcript on the ATH1 array.
Figure S5. Subcellular localisation of N-terminal and C-terminal YFP fusions with
the PM-localised AtMRS2-10 showing (a) diffuse cytosolic localisation as an N-
terminal fusion, yet (b) discrete plasma membrane targeting as a C-terminal fusion.
Cytosolic fluorescence signal for both (c) AtMRS2-1 and (d) AtMRS2-5 as Nterminal YFP fusions. Cells were imaged by confocal fluorescence microscopy as per
methods. (e) Predicted cleavage site at the N-terminus (aa16-19) of AtMRS2-10 using
the SignalP 3.0 server (Bendtsen et al., 2004).
Fig. S6 Perturbations in mesophyll vacuolar Mg accumulation in T-DNA insertion
lines under serpentine conditions only confirmed for AtMRS2-1 and AtMRS2-5. Cellspecific vacuolar [Mg] determined by SiCSA/XRMA on adaxial epidermis and
palisade mesophyll of 6-wk-old Arabidopsis plants grown in hydroponics solutions:
(a) basal nutrient solution or (b) high magnesium solution after 4 d (Mean + S.E.M; n
= 25 cells of each type, from 5 independent plants). Asterisk indicates significant
difference determined by one-way ANOVA (P < 0.05).
Fig. S7 Elemental (Mg, K, Ca) responses of Arabidopsis Col-0 mesophyll over 7 d
growth in LCS as determined by SiCSA/XRMA (Mean ± SEM.; n = 50 mesophyll
samples, from 10 independent plants, experiment performed in duplicate).
Fig. S8 Correlation between growth rate and (a) chlorophyll content, or (b) leaf
osmolality in Col-0, mrs2-1a, mrs2-1b, mrs2-5, mrs2-10 and cax1/cax3 lines grown in
both BNS- and LCS-hydroponics.