A.

B. C.

Supplementary Figure 1. A. Amino acid sequence alignments for sorghum SbMATE and homologs from Arabidopsis [At1g51340, percent amino-acid identity (I) = 51%; expected value (E) = 1.1e-121] and rice (Os01g69010, I = 63%; E = 1.1e-152). B. Structure of the SbMATE protein showing predicted transmembrane domains and hydrophilic loops linking specific transmembrane domains. C. Intron-exon structure of the SbMATE gene.

NHCOOH

Cytoplasm

3

Supplementary Figure 2. Electrophysiological characterization of SbMATE expressed in X. laevis oocytes. A. Representative currents recorded from oocytes microinjected with SbMATE cRNA (left panel) and water injected control oocytes (right panel) in ND96Al solution. The holding potential was set to 0 mV and voltage pulses were stepped between +40 and -160 mV (in 10 mV increments). B. Mean current to voltage relationships from control (open circles) and SbMATE-expressing oocytes (closed circles) obtained from recordings like those shown in A. Values represent the average of measurements from at least 10 different cells. Error bars denote the standard error and are not shown when smaller than the symbol.

TX430 GGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTATACTCCTACAGA CTATTAAAGTTG

BR007 GGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTATACTCCTACAGACTATTAAAGTTG

BR012 GGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTATACTCCTACAGACTATTAAAGTTG

SC283 GGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTAGCTAGGAGTACTCCTACAGACTATTAAAGTTGGGCCTTGTTTAGTTCCAAATAATTTTGCAAAATAGGAATAGTAGCATTTTCGTTTGTATTTGACAAATATTGTCCAATCATGAACTAATTAGACTCAAAAGATTCGTCTCGTTAATTTCGACCAAACTGTGAAATTAGTTTTTATTTTCGTCTATATTTAATACTTCATGCATGCGTCTAAAGATTTGATGTGACGGAGAATCTAAAAAATTTTGCAAAACTTTTTGGGAACTAAACAAGGCCTTGGTTGGTGCGATGATGTTGGATCCAGTGAGCTACCGGTGAAGGTGCTCGTTATGCGTTTAAACATTGTTCCGTCCGGCGGCATCTATACTCCTACAGACTATTAAAGTTG

Supplementary Figure 3. Nucleotide sequence of the MITE-containing region in the promoter of SbMATE for the four sorghum accessions presented in Figure 3 of the manuscript as representative of the four size classes for this region. As depicted in

Figure 3 of the manuscript, the MITE-containing regions contain 3 repeating elements: a 100-bp element highlighted in yellow and labeled as (a) in Figure 3; the 243-bp MITE insertion highlighted in blue and labeled (b) in Figure 3; and a 21-bp element following the MITE insertion highlighted in pink and labeled (c) in Figure 3 of the manuscript. Each of the MITE-containing regions ends with an imperfect (a) element that contains either an 8-bp deletion, highlighted in grey, or a 12-bp deletion, highlighted in green. Note that as the MITE-containing region increases in size, the number of a-b-c repeats increases from 1 in TX430, to 3, 4 and 5 in BR007, BR012, and SC283, respectively.

Supplementary Figure 4. Al tolerance as measured by % relative root growth (%RRG). %RRG values were calculated from root growth measured over 24 hrs in +Al solution divided by root growth measured over 24 hrs in control (-Al) solution x 100 for the Al sensitive wheat cultivar, Bobwhite, and 4 T1 transgenic Bobwhite families expressing SbMATE under the constitutive maize ubiquitin promoter. Wheat plants were grown in 0.2 mM CaCl2 solution with or without 5 µM AlCl3 (pH 4.5). The data are means ± S.D. (n = 19 for Bobwhite; 11 for 6001A; 11 for 6001D; 8 for 6001F; and 9 for 6053).

1

SUPPLEMENTARY METHODS Phenotypic Analysis of Sorghum Al Tolerance

Sorghum seeds were surface-sterilized with 0.5% (w/v) NaOCl for 15 min,

rinsed with ultra-pure water and allowed to germinate for 4 days at 26oC on wet filter

paper. Seedlings were then transplanted to 8 L tubs containing complete Magnavaca

nutrient solution31 lacking Al at pH 4.0. After 24 hrs, the solution was changed to either

control nutrient solution (-Al) or nutrient solution containing {27} µM Al3+ for the

subsequent measurement of root growth.

Relative root growth (RRG) values were determined as follows. After a 4-day

germination period, seedlings were allowed to acclimate in control nutrient solution

lacking Al for 24 hr, at which time the initial length of each seedling’s root in control

solution (ilc) was measured. Final lengths in control solution (flc) were recorded 24 hrs

later, followed by replacement of the control nutrient solution with a solution of

identical composition but containing Al. Final root lengths under Al treatment (flAl)

were obtained after 5 days of exposure to Al. The degree of root growth inhibition

caused by Al over the 5-day exposure period relative to the control root growth was

calculated as RRG (% relative root growth) = [(flAl – flc)5d / (flc – ilc)1d x 5] x 100.

AltSB genotypes were assigned based on RRG values and by scoring visual root

damage caused by Al as described in Magalhaes et al.5, using 14 individuals per

BR007xSC283 RIL family. For high resolution mapping, genotypes for F2

recombinants were assigned by progeny testing 21 individuals per F2:3-derived family

in each of two separate experiments. The AltSB genotypes of true breeding Al tolerant

RILs or F2:3 progeny were considered homozygous tolerant whereas those of true

breeding Al sensitive families were considered homozygous sensitive. Segregating

families were considered heterozygous for AltSB.

2

Relative net root growth (RNRG) means for the sorghum lines BR012, BR007,

IS8577, SC549, 3DX, SC175, 9DX, CMS225, SC283, SC566, which were used for the

correlation analyses with SbMATE expression, citrate release and size of the MITE

insertion region, are those shown in Table S4 from Caniato et al.10. RNRG values were

determined based on growing 21 individuals per inbred line in either control (-Al)

nutrient solution or the same nutrient solution plus Al. After 5 days of root growth in the

+/-Al solutions, root growth over that 5-day period was determined and RNRG

calculated as (root growth in Al) / (control root growth) x100, thus being expressed as a

per cent of control root growth.

Positional Cloning of AltSB

A recombinant inbred line (RIL) map of AltSB was developed with 354 RILs

derived from a cross of highly Al tolerant SC283, and Al sensitive BR007. The

subsequent high resolution map was constructed by screening 2085 F2 individuals

from a BR007xSC283 cross and identifying individuals with single recombination

events between the markers CTG29 and M181. Recombinant F2 individuals were self-

pollinated and Al tolerance was assessed in F2:3 families as described in the previous

session.

Genomic DNA isolation and genotyping of the two sequenced-tagged site

markers CTG29 and M181 were carried out as described in Caniato et al.10. See

Supplementary Table 2 for the primer sequences for these markers. Genetic distances

in the RIL population were determined from recombination frequencies using the

Kosambi function32 with LOD=3 and maximum recombination frequency of � =0.4.

Our initial mapping study indicated that the RFLP markers isu68 and isu52.2

were tightly linked to AltSB5, and isu68 is also located on rice chromosome 1 near

position 157 cM. Sequence-tagged site (STS) markers were developed both from rice

ESTs nearby or via sample sequencing of syntenic sorghum BACs in this region of

3

chromosome 333, and two of these STS markers, CTG29 and M181, mapped at 0.2 and

0 cM from AltSB, respectively (Fig 1A). High resolution mapping of AltSB involved the

screening 4170 gametes from a SC283 x BR007 (an Al sensitive standard) F2

population which identified 27 individuals showing single recombination events in the

CTG29-M181 interval. Of those, 6 individuals showed recombination events between

AltSB and M181, indicating that CTG29 and M181 flanked the AltSB locus. The STS

marker T755 was developed for the leftmost end of sorghum BAC 55D12 and was

completely linked to AltSB in the high resolution map; thus, BAC181g10, which

spanned T755, was selected for shotgun sequencing. A G/A SNP next to open reading

frame (ORF) 2 within 181g10 (Figure 1B) was subsequently placed onto the high

resolution map, and for the 21 individuals with recombination events between CTG29

and AltSB, only 4 were recombinant for the G/A SNP-AltSB interval, indicating that ORF2

was significantly closer to AltSB than CTG29 and confirming that BAC 181g10 spanned

the AltSB locus. Two additional markers 24.6 Kb apart within 181g10 (in red in Figure

1B) each identified a single recombination event with AltSB, yielding an average

recombination ratio of ~513 Kb cM-1 across the region, and allowed us to identify three

predicted candidate ORFs (ORFs 7, 8 and 9) for AltSB (Figure 1B).

Isolation of the Full-Length cDNA for SbMATE

The 5’ and 3’ end sequences of SbMATE transcripts were identified by 5’/3’-

rapid amplification of cDNA ends (RACE) using a GeneRacer kit (Invitrogen)

according to the manufacturer’s instructions. The sequence information for the primers

used for the 5’ and 3’ RACE experiment can be found in Supplementary Table 2. First-

strand cDNA was reverse transcribed from 1 µg of total RNA from roots of the Al-

tolerant NIL (ATF10B) with the GeneRacer oligo dT primer. The PCR of 5’- cDNA ends

was carried out with the GeneRacer kit using a 5’ primer specific to the adaptor ligated

to cDNA ends and JL56, a gene specific primer (GSP) corresponding to the SbMATE

4

cDNA. Nested PCR was carried out with GeneRacer 5’ nested primer and JL54, the

nested GSP. The PCR of 3’- cDNA ends was carried out with the GeneRacer kit using

GeneRacer 3’ primer, a 3’ primer specific to the oligo dT primer ligated to 3’-cDNA

ends and JL55, a GSP. Nested PCR was carried out with GeneRacer 3’ Nested

primer and JL48, a nested GSP. Cycling parameters used in this 3’/5’-RACE protocol

were 34 cycles at 94oC for 30 s, at 58oC for 40s and 72oC for 1 min. The PCR products

were subcloned into a pCR4-TOPO vector (Invitrogen, Carlsbad, CA, USA) and

sequenced with a DNA sequencer. According to the 5’- and 3’-end sequences, the

full-length cDNA of SbMATE was amplified with PCR primers JL96 and JL97. The

PCR products were subcloned into a pCR4-TOPO vector and sequenced.

Determination of Gene Expression via Semi-quantitative Reverse Transcription

(RT) PCR

Sorghum seedlings were grown as described above and in Caniato et al.10 in

nutrient solution with {27} µM Al3+ or without Al (control solution) for 3 days. Each

experimental unit consisted of 10 root apices (1 cm) and 3 replications were used for

each treatment. Root apices were collected, frozen in liquid nitrogen, and total RNA

was extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). First strand

cDNA was synthesized using 300 ng of DNase I-treated RNA, 500 ng of anchored

oligo(dT) 12-18 mers and 100 �moles of each dNTP in a total volume of 12 �L. Samples

were heated at 65oC for 5 minutes and transferred to ice. Subsequently, 4 µL of 5x

reaction buffer (Invitrogen, Carslbad CA), 2 �l DTT 0.1 M and 1 µL DEPC water were

added to the mix. The mix was incubated at 42oC for 2 min followed by addition of 1 µL

of SuperScript II RT (Invitrogen) and final incubation at 42oC for 90 minutes. First

strand cDNA samples were then subjected to 70oC for 15 min and stored at -20oC. A

800-bp cDNA fragment, spanning the 3rd to 5th exons of the SbMATE transcript, was

amplified from the 1st-strand cDNA samples using the primers JL57 and JL58. A ~500

5

bp fragment of the Actin gene amplified with primers ActF and ActR was used as an

internal control. See Supplementary Table 2 for sequence information for these

primers. PCR reactions were run with 2 µL of the cDNA mix; 20 mM Tris-HCl (pH 8.4);

50 mM KCl; 2 mM MgCl2; 0.125 mM dNTP; 10 pmoles of each primer and 0.5 U of Taq

DNA polimerase (Invitrogen) in a 20 µl reaction volume. PCR reactions were

undertaken with an initial denaturation step at 94oC for 1 min followed by 30 cycles at

94oC for 30s, 60oC for 40s and 72oC for 90s, with a final step at 72oC for 5 min. The

PCR reactions for SbMATE and the Actin gene were found to be in the linear phase at

30 PCR cycles, which was the number of cycles chosen for the semi-quantitative RT-

PCR assays.

Determination of Gene Expression via Quantitative Real-Time Reverse

Transcription (RT) PCR

Sorghum seedlings were grown as described above and in Caniato et al.10 in

nutrient solution with {27} µM Al3+ or without Al (control solution). Root and shoot

tissues were collected 1, 3, and 6 days after +/-Al treatment. Three replications were

employed for each treatment.

Total RNA was extracted from individual tissue samples using the RNasy Plant

Mini Kit (Qiagen, Valencia, CA). First-strand cDNA was synthesized using 7.5 µg of

total RNA with the High-Capacity Archive Kit (Applied Biosystems).

SbMATE transcripts were quantified using the ABI Prism 7900 Sequence

Detection System (Applied Biosystems). A series of cDNA dilutions were used for

making standard curves both for SbMATE transcripts and for 18S RNA which was

used as the internal reference. Then, the selected dilution for specific cDNA samples

(100ng for SbMATE transcripts, 1ng for 18S RNA) were used as real-time PCR

templates to quantify relative transcript levels following the conditions recommended

by the manufacturer. The forward and reverse primers, as well as the probe specific to

6

SbMATE, are ORF7-F, ORF7-R and ORF7-Probe, respectively. See Supplementary

Table 2 for sequence information for these primers. Levels of endogenous 18S RNAs

were determined using Taqman Ribosomal RNA Control Reagents (Applied

Biosystems). Distilled water or products of room temperature reactions without reverse

transcriptase were used as negative controls. The levels of the SbMATE transcripts

were normalized to endogenous 18S RNA. Each set of experiments was replicated

three times.

Subcellular Localization of SbMATE

The membrane localization of SbMATE was determined by examining the

transient expression of the SbMATE coding region tagged with GFP in onion (Allium

cepa) epidermal cells. 35S::SbMATE:GFP constructs were generated by amplification

of the SbMATE coding region using adaptor primers that incorporated SpeI restriction

site linkers for sub-cloning into the plant transformation vector, pCAMBIA 1302. The

latter contains a 35S promoter driving the expression of an mGFP5 gene. Insertion of

SbMATE between the 35S promoter and the GFP coding region of the opened

pCAMBIA vector generated the translational in-frame fusion of the SbMATE::GFP

chimera driven by the 35S promoter. The resulting construct was fully sequenced and

checked for sequence accuracy. Transient expression of the SbMATE::GFP chimera

was achieved by particle bombardment of onion epidermal cells. Briefly, M10 tungsten

particles (1.1 �m) were coated with one microgram of the SbMATE::GFP (or empty

pCAMBIA 1302 vector as a negative control) plasmid DNA in 2.5 M CaCl2 and 1 M

spermidine (Sigma, USA). Onion epidermal peels were bombarded at a helium

pressure of 27 MPa (rupture disks 1300 p.s.i; Biolistic PDS-2000/He BioRad

Laboratories, Hercules, CA, USA) with the DNA-coated particles, and the tissue was

incubated on filter paper immersed in tap water in the dark at room temperature for 24

7

h. Imaging of GFP fluorescence was carried out using confocal microscopy (Leica TCS

SP2 system, Leica, Germany).

Determination of Al-activated Root Citrate Exudation in Sorghum Near-Isogenic

Lines (NILs)

Seeds for the Al-tolerant (ATF10B) and Al-sensitive (ATF8B) NILs were surface-

sterilized with 0.5 (w/v) NaOCl for 15 min, rinsed with ultrapure water and allowed to

germinate for 3 days at 26oC. Seedlings were then transplanted to 8 L tubs containing

complete nutrient solution lacking Al30 at pH 4.0. After 24 hrs the solution was changed

to either control nutrient solution (-Al) or nutrient solution containing {27} µM Al3+ and

root exudates were collected after 1, 3 and 6 days of treatment with {0} or {27} µM Al3+.

Six seedlings were used for each determination of organic acids in the root exudates;

root exudate collection began by transferring the six seedlings to a 50 ml plastic

centrifuge tube containing 4.3 mM CaCl2.6H20 with or without Al added as AlCl3.6H2O

with the same free Al3+ activity of {27} µM Al3+ (pH 4.5) as was used when seedlings

were grown in the Magnavaca nutrient solution30 prior to collection of root exudates.

Each root exudate determination was replicated four times. Root exudates were

collected for 6 hrs, and then the exudate solution was passed through anionic and

cationic chromatography columns to remove Al and inorganic anions that interfere with

the determination of organic acids. Subsequently, 1 mL sub-samples were lyophilized

and resuspended in 0.2 mL of ultrapure water. Analysis of organic acids in root

exudates was performed using a capillary electrophoresis system as described in

Piñeros et al.28.

Expression of Sorghum SbMATE in Transgenic Arabidopsis Seedlings and

Analysis of Arabidopsis Al Tolerance and Root Organic Acid Exudation

A cDNA fragment spanning from the start codon to the termination codon of

SbMATE was amplified by PCR with the primers JL115 and JL116 from the Al-tolerant

8

NIL, ATF10B. See Supplementary Table 2 for primer sequences. Restriction-digested

PCR products were cloned into the pBAR2 vector between the corresponding

restriction sites, which are located after the 35S promoter.

Both the empty vector and the vector carrying the SbMATE construct were

individually electroporated into the Agrobacterium tumefaciens strain GV3101

(Invitrogen) and used for Arabidopsis thaliana transformation (in both the Columbia-0

and AtALMT knockout backgrounds). The presence of the transgene was confirmed by

Basta herbicide resistance of the transgenic plants and by PCR confirmation of T-DNA

insertions.

About 60 independent T2 lines transformed with the 35S::SbMATE construct

were tested for root growth in hydroponic solution as described by Hoekenga et al.18

Three T2 lines transformed with the empty vector in each of the wild-type (Col-0) and

AtALMT knockout backgrounds were also included as controls for the root growth

experiments.

Individual T2 lines with enhanced root growth rate in the presence of Al

indicating increased Al tolerance as compared to corresponding controls were selfed

and the segregating T3 progeny analyzed to identify transgenic and non-transgenic

homozygous T3 progenies, which were confirmed by progeny testing. Corresponding

transgenic and non-transgenic homozygous T3 lines were then used for determination

of Al tolerance (root growth) as described in Hoekenga et al.18.

For determination of root organic acid exudation, ~2-3 mg of surface sterilized

and stratified seeds for each Arabidopsis transgenic and non-transgenic line were

germinated in Magenta boxes containing sterile hydroponic growth solution for six

days18. Subsequently, seedlings of individual transgenic lines were transferred to 20

mL of the filter-sterilized exudation solution (pH 4.2) with or without a total Al

concentration of 13.6 µM AlCl3 (Al3+ activity of 1.5 µM) in a sterile petri dish for 2 days18.

9

The exudation solutions were collected at the end of the second-day of treatment, and

passed through anionic and cationic chromatography columns to remove Al3+ and

inorganic anions that interfere with the determination of organic acid anions.

Subsequently, 1 mL sub-samples were analyzed for organic acids in the root exudates

using the capillary electrophoresis system as described in Piñeros et al.28.

Expression of Sorghum SbMATE in Transgenic Wheat Seedlings and Analysis of

Al Tolerance

A cDNA fragment spanning from the start codon to the termination codon of

SbMATE was amplified by PCR with the primers, M1F and M1R. See Supplementary

Table 2 for primer sequence information. Restriction-digested PCR products were

cloned into a modified pCAMBIA p3301 vector (Canberra, ACT 2601

Australia) between the corresponding restriction sites, which are located after the

maize ubiquitin promoter. Transformation into the Al sensitive wheat line Bobwhite

was undertaken as described in Anand et al. (2003)34. Briefly, this involves biolistic

transformation of immature embryogenic wheat calli and subsequent regeneration of

T0 plants. The presence of the transgene in specific T0 plants was verified via PCR.

For the Al tolerance assay, T1 seedlings were grown on 0.2 mM CaCl2 (pH 4.5)

solution for 2 days, and root growth was determined in the –Al solution. Then

seedlings were transferred to the same solution containing 5 µM AlCl3 and root growth

determined for a subsequent 2 day period. Percent relative root growth (%RRG) was

calculated by dividing root growth values in Al by root growth values in control (-Al)

media and multiplying these numbers by 100.

Comparative Analysis with the Rice Genome

Comparative sequence analysis revealed a high degree of gene conservation

between the sorghum BAC 181g10 where AltSB resides and the rice BAC AP003437

located at position ~159 cM on rice chromosome 1; this syntenic rice BAC harbors a

10

MATE gene that was the best hit found using SbMATE as query. Furthermore, a major

rice Al tolerance QTL detected on chromosome 1 is linked to the marker Xwg11026 at

position ~158 cM, in a rice BAC contiguous to AP003437. The BAC positions for the

RLFP marker Xwg110 (Gene Bank accession AA231665) and isu68 in rice were

determined by sequence similarity analysis. The genetic positions for the BACs on rice

chromosome 1 can be found at http://rgp.dna.affrc.go.jp.

Expression of SbMATE in Xenopus oocytes

cRNA was prepared using the RNA Capping Kit (Stratagene, La Jolla, CA,

USA) from SmaI-digested T7TS plasmid DNA, which contained the SbMATE coding

region, flanked by the 3'- and 5'-untranslated regions of a Xenopus β-globin gene.

Harvested and defolliculated stage V to VI Xenopus laevis oocytes were maintained in

standard ND96 solution (supplemented with 50 µg/mL gentamycin) at 18°C overnight

prior to injections. Oocytes were injected with 48 nL of water containing 20 ng of cRNA

encoding SbMATE (or 48 nL of water as a control). Whole-cell currents were recorded

as described in Hoekenga et al.18. All recordings were performed in ND96AL solution

consisting of (in mM): 96 NaCl, 1 KCl, 0.8 CaCl2, 0.1 LaCl3, and 0.1 AlCl3 (pH 4.5). The

holding potential was set to 0 mV and voltage test pulses (500 ms in duration) were

stepped from +40 mV to -160 mV (in 10 mV increments), with a 10 s resting phase at 0

mV between each voltage pulse. The current-voltage (I/V) relationships measured

under different conditions were constructed by measuring the current amplitude at the

end of the test pulses.

Literature Cited

31. Magavaca, R., Gardner, C.O. & Clark, R.B. in Genetic Aspects of Plant Mineral

Nutrition. (eds Gableman, H. W., Loughman, B. C.) 101-121 (Martinus Nijhoff

Publishers, Dordrecht, 1987).

11

32. Kosambi, D. D. The estimation of map distances from recombination values. Ann.

Eugen. 12, 172-175 (1944).

33. Klein, P. E., Klein, R. B., Vrebalov, J. & Mullet J. E. Sequence-based alignment of

sorghum chromosome 3 and rice chromosome 1 reveals extensive conservation of

gene order and one major chromosomal rearrangement. Plant J. 34, 605-621 (2003).

34. Anand, A. et al. Greenhouse and field testing of transgenic wheat plants stably

expressing genes for thaumatin-like protein, chitinase and glucanase against

Fusarium graminearum. J. Exp. Bot. 54, 1101-111 (2003).

SUPPLEMENTARY NOTE

Funding Acknowledgement The work was supported by Generation Challenge Program Grant # IC69, USDA-NRI

Competitive Grant # 2006-35301-16884, a McKnight Foundation Collaborative Crop

Research Program grant, and a FAPEMIG – Brazil grant.

Supplementary Table 1. Disruption via T-DNA insertion of the six most closely related SbMATE homologues in Arabidopsis has no effect on Al tolerance. For comparison, the Al tolerance determined via measurement of per cent relative net root growth (% RNRG) for Col-0 wild type plants assayed in the same experiment was 64.4 ± 4.14%.

Gene Name Total T-DNA Lines

Tested Al Tolerance

(% RNRG) P-Value* At1g51340 1 63.27 ± 2.20 0.1133 At2g21340 1 64.00 ± 3.16 0.4881 At2g38330 3 65.45 ± 3.78 0.6338

63.70 ± 4,22 0.4484 63.43 ± 3.10 0.2792

At3g08040 1 63.36 ± 4.61 0.3556 At4g38380 2 63.56 ± 3.32 0.3200

63.70 ± 4.22 0.4485 At4g39030 2 63.43 ± 3.32 0.3201

63.80 ± 2.30 0.3110 Ten Arabidopsis T-DNA insertion lines were acquired from the Arabidopsis Biological Resource Center (ABRC), where the six Arabidopsis genes that are most closely related to SbMATE 30 were disrupted by T-DNA insertion. The T-DNA insertions in each of the lines were confirmed by PCR with primers specific to individual genes and to the left border of the T-DNA insertions. Homozygous progeny were generated for each of the T-DNA lines and they were used for determination of Al tolerance (root growth). *The P-values were determined via t-tests for %RNRG values between the wild-type (Col-0) and individual T-DNA lines. References Cited 30. Rogers, E. E. & Guerinot, M. L. FRD3, a member of the multidrug and toxin efflux family, controls iron deficiency responses in Arabidopsis. Plant Cell 14, 1787-1799 (2002).

Supplementary Table 2 Primer/Probe

Name Primer/Probe Sequence Application

AltSB-F 5’-GTG CTG GAT CCG ATC CTG AT-3’

forward primer for RT-PCR

AltSB-R 5’-CAC TGC CGA AGA AAC TTC CA-3’

reverse primer for RT-PCR

Actin-F 5’-TGA TGA AGA TTC TCA CTG AG-3’

forward primer for RT-PCR, internal control

Actin-R 5’-GAT CCA CAT CTG TTG GAA CG-3’

reverse primer for RT-PCR, internal control

CTG29F 5´-HEX-ATG CAG TAT CTG CAG TAT CAT TT-3’

forward primer for marker CTG29

CTG29R 5’-AAT CCG TCA GGT CAG CAA TC-3’

reverse primer for marker CTG29

M181F 5´-6FAM- AAG GCA ACA ACT GAG GCA CT-3’

forward primer for marker M181

M181R 5’-TCG CTA GAG TGG TGC AAG AA-3’

reverse primer for marker M181

GeneRacer oligo dT primer

5’-GCT GTC AAC GAT ACG CTA CGT AAC GGC ATG ACA GTG (T)24-3’

5', 3' RACE

GeneRacer 5' adaptor primer

5’-CGA CTG GAG CAC GAG GAC ACT GA-3’

5’ primer specific to the adaptor ligated to cDNA ends; for 5', 3' RACE

JL56 5’-ATC CGA GGA AGC GCC GGA AT-3’

gene specific primer (GSP) for SbMATE, used for 5' 3' RACE

GeneRacer 5’ nested primer

5’-GGA CAC TGA CAT GGA CTG AAG GAG TA-3’

5' nested primer for 5', 3' RACE

JL54 5’-CCT TGA ACC CAC GGA AGA CT-3’

nested GSP for SbMATE or 5', 3' RACE

GeneRacer 3’ primer

5’-GCT GTC AAC GAT ACG CTA CGT AAC G-3’

3’ primer specific to the oligo dT primer ligated to 3’-cDNA ends; For 5', 3' RACE

JL55 5’-GCC CGC GCT GCG CTA CCT GA-3’

GSP for SbMATE; For 5', 3' RACE

Primer/Probe Name

Primer/Probe Sequence Application

GeneRacer 3’ Nested primer

5’-CGC TAC GTA ACG GCA TGA CAG TG-3’

3' nested primer for 5', 3' RACE

JL48 5’-ACG CTG ATA ATG CTG AGC AAG CTG-3’

GSP for SbMATE; For 5', 3' RACE

JL96 5’-GTA CGA TCG ACA CGA GAC TGT ACG TA-3’

forward primer for amplification of full-length SbMATE cDNA

JL97 5’-TGC TTG CAA GGT TTG TAG CTA GGC CGA-3’

reverse primer for amplification of full-length SbMATE cDNA

JL57 5’-GTG CTG GAT CCG ATC CTG AT-3’

forward primer for semi-quantitative RT-PCR

JL58 5’-CAC TGC CGA AGA AAC TTC CA-3’

reverse primer for semi-quantitative RT-PCR

ActF 5’-TGA TGA AGA TTC TCA CTG AG-3’

forward primer for the Actin gene

ActR 5’-GAT CCA CAT CTG TTG GAA CG-3’

reverse primer for the Actin gene

ORF7-F 5'-CAG CCA TTG CCC ATG TTC TTT-3'

forward primer for Real-Time PCR

ORF7-R 5'-ACC AGC TTG CTC AGC ATT ATC A-3'

reverse primer for Real-Time PCR

ORF7-Probe 6FAM-CCC AGT ACC TGA TAA CGC- TAMRA

probe for Real-Time PCR

JL115 5'-AAT ATC TAG ACG ATC GAC ACG AGA CTG TAC GT-3'

forward primer for constructing SbMATE over-expression vector for Arabidopsis transformation (the underlined bases denote the XbaI site)

JL116 5'-AAT ACC CGG GAA GGT TTG TAG CTA GGC CGA-3'

reverse primer for constructing SbMATE over-expression vector for Arabidopsis transformation (the underlined bases denote the XmaI site)

M1F 5'-AAT AGG ATC CAT GGA GGA ACA CCG GTC AC-3'

forward primer for constructing SbMATE over-expression vector for wheat transformation (underlined bases denote BamH I sites)

Primer/Probe

Name Primer/Probe Sequence Application

M1R 5'-AAT AGG ATC CTC ACT GCC GAA GAA ACT TCC-3'

reverse primer for constructing SbMATE over-expression vector for wheat transformation (underlined bases denote BamH I sites)