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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.170

NATURE PLANTS | www.nature.com/natureplants 1

A polarly localized transporter for efficient manganese uptake in rice Daisei Ueno1†, Akimasa Sasaki2†, Naoki Yamaji2†, Takaaki Miyaji3, Yumi Fujii1, Yuma Takemoto1,2, Sawako Moriyama4, Jing Che2, Yoshinori Moriyama3,4, Kozo Iwasaki1, and Jian Feng Ma2* 1Faculty of Agriculture, Kochi University, 200 Otsu, Monobe, Nankoku, Kochi 783-8502, Japan 2Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, 710- 0046, Japan 3Advanced Science Research Center, Okayama University, Okayama 700-8530, Japan 4Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan *Corresponding authors †These authors contributed equally to this work. Corresponding authors Jian Feng Ma Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, 710- 0046, Japan Tel/Fax: 81-86-434-1209; E-mail: [email protected]

http://dx.doi.org/10.1038/nplants.2015.170

2 NATURE PLANTS | www.nature.com/natureplants

SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.170

Supplementary Methods

Plant materials and growth conditions

Two T-DNA insertion mutants of MTP9 (1B-05023 and 2C-40574, refer to mtp9-1 and

mtp9-2 in the text) of Dongjin background RISD

(http://www.postech.ac.kr/life/pfg/risd/) were used in this study. Seeds were

germinated in tap water for 3 d at 30°C in the dark after surface sterilization with 0.5%

(v/v) NaClO for 1 h. After germination, seedlings were transferred to a net floated on

a 0.5 mM CaCl2 solution in 1.5-l pots for 5 d and then on a half-strength Kimura B

nutrient solution (pH 5.4) containing the macronutrients MgSO4 (0.28 mM), (NH4)2SO4

(0.18 mM), Ca(NO3)2 (0.18 mM), KNO3 (0.09 mM), and KH2PO4 (0.09 mM); and the

micronutrients Fe(II)SO4 (10 µM), H3BO3 (3 µM), MnCl2 (0.5 µM), CuSO4 (0.2 µM),

ZnSO4 (0.4 µM), and (NH4)6Mo7O24 (1 µM). The solutions were renewed every 2 d.

The seedlings were further grown in 1.2 or 3.5-l pots containing the nutrient solution

until use in the following experiments. Plants were grown in a greenhouse at 25°C to

30°C under natural sun-light for soil culture, or in growth chamber with a 14 h : 10 h,

light (30°C) : dark (25°C) photoperiod for hydroponic culture.

Cloning of full-length MTP9 cDNA

Total RNA was extracted from rice (Oryza sativa L. 'Dongjin') roots using an RNeasy

Plant Mini Kit (Qiagen) and converted to cDNA using SuperScript II reverse

transcriptase after treatment with DNase I (Life Technologies). Full-length MTP9

cDNA was amplified using the primers 5′-ACCAGCAGCAAGCAGGCCATG-3′ and

5′-TGAAGAGAAAATTTGCATAAATGAGA-3′, which were designed to target the

5′-UTR (untranslated region) with start codon, and the 3′-UTR according to the




sequence information for MTP9 that corresponded to LOC_Os01g03914 deposited in

the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/). The PCR

fragment was cloned into the pGEM-T Easy vector (Promega) and sequenced using a

Big-Dye sequencing kit (Applied Biosystems) on a sequence analyser (ABI 3130

Genetic Analyser; Applied Biosystems). Protein transmembrane domains were

predicted using the TMHMM server (version 2.0,

http://www.cbs.dtu.dk/services/TMHMM/).

Phylogenetic analysis

The phylogenetic tree was constructed using MEGA 6 software (version 6.06,

http://www.megasoftware.net/) after alignment using ClustalW

(http://clustalw.ddbj.nig.ac.jp/) with 1000 bootstrap trials.

Transient expression of a GFP:MTP9 fusion protein

An MTP9 cDNA fragment was amplified from the pGEM-T Easy-MTP9 plasmid using

the primers 5′-AGGTACCTTATGGGGAGCCG-3′ and

5′-TGGTACCTCAGACCTCGGCC-3′ and was cloned into the BsrGI site of GFP in

the pUC18 carrying the cauliflower mosaic virus (CaMV) 35S promoter and nopaline

synthase (NOS) terminator. Rice leaf protoplasts were prepared from leaves of

2-w-old seedlings grown hydroponically, and used for transformation by the

polyethylene glycol method as described by Chen et al1. GFP fluorescence was

observed by laser confocal microscopy (LSM700; Carl Zeiss).




Western blot analysis

Microsomal membrane was prepared from roots of plants treated with various

concentration (0, 0.5, 50, 200 µM) of Mn for 48 h. SDS-PAGE and detection of

chemiluminescence of the antigen-antibody complexes was performed according to

Chen et al2. Anti-H+-ATPase (Agrisera) polyclonal antibody was used for detecting

tonoplast. A HPR-conjugated goat anti-rabbit antibody (Promega) was used as a

secondary antibody, and an ECL Prime Western Blotting Detection System (GE

Healthcare) was used for detecting chemiluminescence of the antigen-antibody

complexes. The chemiluminescence was detected using LAS-1000 (Fujifilm).

Expression, purification, and reconstitution of MTP9

The expression, purification, and reconstitution of MTP9 were carried out as described

previously3. Escherichia coli C43 (DE3) cells were transformed with expression

vectors and grown in TB medium containing 20 µg mL-1 kanamycin sulphate at 37°C.

Escherichia coli cells were grown until A600 reached 0.6 – 0.8, and then

isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 1 mM and

culture was continued for 16 hours at 18°C. The cells were then harvested by

centrifugation and suspended in a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM

sucrose and 1 mM phenylmethylsulphonyl fluoride. The cell suspension was then

disrupted by sonication with a TOMY UD200 tip sonifier (OUTPUT4), and centrifuged

at 5,856 × g at 4°C for 10 minutes to remove large inclusion bodies and cell debris.

The resultant supernatant was carefully obtained and centrifuged again at 150,000 × g

for 1 hour at 4°C. The pellet was suspended in buffer containing 70 mM Tris-HCl (pH

8.0), 100 mM NaCl, 10 mM KCl, 15% glycerol, and 1 mM phenylmethylsulphonyl




fluoride, and the protein concentration was adjusted to 10 mg mL-1. Then, the

membranes were treated with 1.5% Fos-choline 14 (Affymetrix) and centrifuged at

150,000 × g at 4°C for 1 hour. The supernatant containing MTP9 was obtained,

diluted twofold with buffer containing 70 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10

mM KCl, 15% glycerol and 1 mM phenylmethylsulphonyl fluoride, and then applied to

a column containing 1 mL of cOmplete His-tag purification resin (Roche) equilibrated

with buffer containing 70 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM KCl, and 15%

glycerol. After incubation for 1 hour at 4°C, the column was washed with washing

buffer containing 70 mM Tris-HCl (pH 8.0), 10 mM imidazole, 100 mM NaCl, 10 mM

KCl, 20% glycerol, and 0.1% n-decyl-β-D-thiomaltopyranoside (DTM) (Affymetrix).

The MTP9 protein was eluted with buffer containing 20 mM Tris-HCl (pH 8.0), 300

mM imidazole, 100 mM NaCl, 10 mM KCl, 20% glycerol, and 0.1% DTM, and then

stored at –80°C, at which it was stable without loss of activity for at least several

months. Aliquots of 30 µg of purified MTP9 were mixed with 500 µg of liposomes

and frozen at –80°C for at least 10 minutes. The mixture was diluted 100-fold with

reconstitution buffer containing 40 mM MES-Tris (pH 5.6) and 0.1 M potassium acetate.

Reconstituted proteoliposomes were pelleted by centrifugation at 200,000 × g for 1 hour

at 4°C, and then suspended in 0.2 mL of reconstitution buffer. Asolectin liposomes

were prepared as described previously3.

Transport assay

Transport assays were carried out by the gel permeation procedure as described

previously3. Reaction mixtures (130 µL) containing 0.6 µg of protein incorporated

into proteoliposomes, 20 mM MOPS-Tris (pH 7.0) or 40 mM MES-Tris (pH 5.6), 0.1 M




potassium acetate, 10 mM KCl, and 1 µM 54MnCl2 (0.5 GBq µmol-1; PerkinElmer) were

incubated at 27°C. At the times indicated, transport was terminated by separating the

proteoliposomes from the external medium using centrifuge columns containing

Sephadex G-50 (fine). The radioactivity in the eluate was measured by Auto Well

Gamma System ARC-370M (Aloka).

Yeast assay

MTP9 cDNA was amplified from pGEM-T Easy-MTP9 using the primers

5′-AAAGCTTAAAAATGGGGAGCCGA-3′ and

5′-TGAATTCAGACCTCGGCCTTGT-3′ and cloned into HindIII and EcoRI sites of

pYES2 vector (Life Technology). The construct and pYES2 empty vector were

introduced to yeast (Saccharomyces cerevisiae) mutant strains pmr1 (Mat a; his3Δ1;

leu2Δ0; met15Δ0; ura3Δ0; PMR1::kanMX4), ccc1 (Mat a; his3Δ1; leu2Δ0; met15Δ0;

ura3Δ0; CCC1::kanMX4), ycf1 (Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0;

YCF1::kanMX4), and zrc1cot1 (Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0;

ZRC1::antMX3; COT1::kanMX4) which are hypersensitive to Mn, Fe, Cd, Zn/Co/Ni,

respectively. pmr1, ccc1 and ycf1 were purchased from Euroscarf

(http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html). zrc1cot1 was a kind

gift from David Eide. The cells were grown to the stationary phase in a synthetic

complete liquid medium consisting of yeast nitrogen base (Difco), amino acids without

uracil, and 2% glucose (SC-U/Glu). Cells were then incubated for 5 h to achieve

log-phase growth (OD600 approximately 0.8) and were harvested by centrifugation,

washed, and resuspended to OD600 0.5 with sterile water. Four 10-fold serial dilutions

were prepared in sterile water for each culture, and 5 µl of each dilution was spotted




onto SC-U plates containing 2% glucose or galactose (SC-U/Gal) supplemented with

0.15 and 0.3 mM MnCl2, 4 and 8 mM FeSO4, 20 and 40 µM CdSO4, 0.2 and 0.4 mM

ZnSO4, 0.25 and 0.5 mM CoCl2, or 0.75 and 1.5 mM NiCl2. After 1 to 2-d-growth, the

plates were photographed. To investigate expression level of MTP9 in yeast, the

parental wild type strain (BY4741) carrying the pYES2 empty vector or pYES2-MTP9

were grown to the stationary phase in SC-U/Glu (for 10 h) and SC-U/Gal (for 20 h)

medium. Total RNA was extracted using PureLink RNA Mini Kit (Life Technology)

after enzymatic disruption with Lyticase (Sigma), and the expression level was

determined with quantitative RT-PCR as described below.

Quantitative RT-PCR analyses

The expression of MTP9 and other putative Mn transporter genes in different organs

with different treatments in rice plants including T-DNA lines or yeast were determined

by quantitative real-time RT-PCR using a Thunderbird SYBR qPCR mix (Toyobo) on a

Prism 7300 Real-time PCR System (Applied Biosystems) or SsoFast EvaGreen

Supermix (Bio Rad) on real-time PCR machine CFX384 (Bio-Rad). The primers used

were 5′-AGGACCATTTCTTCGACGTG-3′ and 5′-TCCATCCACCATTTGTACCG-3′

for MTP9, 5′-CAGCAGCAGTAAGAGCAAGATG-3′ and

5′-GTGCTCAGGAAGTACATGTTGAT-3′ for Nramp5,

5′-ATTGAGCTTGGTCAGACTCG-3′ and 5′-AACCTGAACAACCACGCTAC-3′ for

IRT1, 5′-GACCACGGGCACTGTCAC-3′ and 5′-ATGACGCGATTCCTGAGAAG-3′

for IRT2, 5′-CACACAAATTCAAAAGCCATTTCTG-3′ and

5′-CGATAGACCGTTGTGGAGAAGC-3′ for Nramp3,

5′-GAGGGACAACGGTGTCATTGCTGGT-3′ and




5′-TGCAGAAAAGCCCTCGACGCCAAGA-3′ for YSL2,

5′-ATCTGAGGATATGCGCTTGG-3′ and 5′-AAAGCACGCTCAACTTCTGG-3′ for

MTP8, 5′-AAGGAGGCACATGCTATTGG-3′ and

5′-ATGTTGTGCTCTGGCTTGTG-3′ for MTP8.1,

5′-TCTGTTATGGCGACACTTGG-3′ and 5′-CAAGCATGATGTCCACAACC-3′ for

MTP11, and 5′-AAACGTCCACTCCTTGGTTG-3′ and

5′-TGACAGCCTTGTGATGGTTC-3′ for MTP11.1. Histone H3 was used as an

internal control in rice using the primers 5′-GGTCAACTTGTTGATTCCCCTCT-3′ and

5′-AACCGCAAAATCCAAAGAACG-3′. To investigate tissue-specific expression

and response to mineral deficiency, seedlings (4-w-old) were grown in the absence of

Zn, Fe, Mn, or Cu for 7 d, and then roots, basal region (0-1 cm from the root-to-shoot

junction) and shoots were sampled. To examine the effect of varying Mn supply on

MTP9 expression, seedlings (11-d-old) were treated with 0.05, 0.5, 200, 500, or 1000

µM Mn for 3 d. For spatial expression analysis in the roots, samples of apical (0–1

cm) and basal (1–2 and 2–3 cm) segments of the roots were taken and subjected to

qPCR. The absolute quantification method was used to determine OsMTP9 expression

in yeast. To generate standard curve for absolute quantification of MTP9 copy number,

8 points of 10-times dilution series (from 1×10-1 to 10-8 ng) of plasmid were subjected

to real-time PCR. Amplification efficiency was calculated to be 99.5%. CT values

for each sample were converted into absolute copy numbers using the standard curve.

The expression of MTP9 at different growth stage was also investigated as described

above. Rice (cv Nipponbare) was grown in a paddy field from mid June to the end of

September. Different tissues, including root, basal stem, lower leaf sheath, lower leaf




blade, flag leaf sheath, flag leaf blade, node III, node II, internode I, node I, peduncle,

rachis, spikelet, and husk, were sampled at different growth stages.

Phenotypic analysis

To investigate the effect of knockout on growth and metal accumulation, seedlings

14-d-old or 9-d-old) were exposed to a nutrient solution containing 0.5 µM Mn and 1

µM NiCl2, 1 µM CoCl2, 1 µM PbCl2, or 0.05 µM CdSO4 for 10 d. After the treatments,

shoots and roots were harvested and washed twice with deionized water, dried at 70°C

for 2 d, weighed, and then subjected to material analysis.

To compare the growth of knockout lines with wild-type rice in soil culture,

seedlings (16-d-old) were transferred to 3.5-l pots (3 lines per pot) filled with 3.5 kg of

soil. The soil used was taken from field of Okayama University as described

previously4. The plants were grown under upland condition in a controlled greenhouse.

After 4 months, the plants were washed with deionized water three times before harvest.

The plants were separated to brown rice, husk, empty seed, rachis node I-III, internode

I-III, flag leaf blade (leaf blade I), flag leaf sheath (leaf sheath I), lower leaf blade (leaf

blade II), lower leaf sheath (leaf sheath II), and the other parts. Hydroponic culture

was also performed similarly with the wild-type rice and two mutants using Kimura B

solution (pH 5.6) containing 50 µM Mn. The solution was changed every two days.

At harvest, the leaf blade, node, grain and other remaining tissues were sampled

separately. Each part was dried at 70°C and then weighed. Grain yield was also

recorded. Grain fertility (percentage of filled spikelets) was determined in a salt

solution with a gravity of 1.06.




Determination of chlorophyll fluorescence

To analyze PSII activity in photosynthesis, seedlings (14-d-old) were cultured under Mn

sufficient (0.5 µM) or deficient (0.05 µM) conditions for 14 days. The second

youngest leaf blade was used to determine chlorophyll fluoresce and Mn concentration.

The chlorophyll fluoresce was determined using a photosynthesis yield analyzer

(MINI-PAM; WALZ, Effeltrich, Germany). The quantum yield (Fv/Fm) was

automatically calculated on the MINI-PAM.

Short-term Mn uptake experiments

Seedlings (5-w-old) grown hydroponically were exposed to a 50 ml nutrient solution

containing 54Mn (10 kBq; 250 pM) in the presence of 0.5 µM cold Mn. At different

time points up to 180 min, the roots and shoots were sampled separately after wash and

the fresh weight was weighed. Radioactivity of each sample was measured

immediately by Auto Well Gamma System AccuFLEX γ7001 (Aloka). Mn uptake

and translocation was calculated based on the radioactivity of each part.

Determination of metal concentration

Dried samples were digested with concentrated HNO3 (60%) at 140°C. Concentration

of metals in the digest solution was determined with atomic absorption spectrometry

(AA-6800; Shimadzu, Japan) and ICP-MS (7700X; Agilent Technologies). Certified

rice (NCS ZC 73029) was used for checking the accuracy of the determination.




Supplementary Reference

1. Chen, S. et al. A highly efficient transient protoplast system for analyzing defence

gene expression and protein-protein interactions in rice. Mol. Plant. Pathol. 7, 417–

427 (2006).

2. Chen, Z. et al. Mn tolerance in rice is mediated by MTP8.1, a member of the cation

diffusion facilitator family. J. Exp. Bot. 64, 4375–4387 (2013).

3. Miyaji, T. et al. AtPHT4;4 is a chloroplast-localized ascorbate transporter in

Arabidopsis. Nat. Commun. 6, doi: 10.1038/ncomms6928 (2015).

4. Tamai, K., & Ma, J. F. Reexamination of silicon effects on rice growth and

production under field conditions using a low silicon mutant. Plant Soil 307, 21–27

(2008).




Supplementary Fig. 1: Phylogenetic tree of Mn-CDF (cation diffusion facilitator) family proteins. Phylogenetic relationship OsMTP8 (Phytozome: LOC_Os02g53490), OsMTP8.1 (Phytozome: LOC_Os03g12530), OsMTP9 (Phytozome: LOC_Os01g03914), OsMTP11 (Phytozome: LOC_Os01g62070), OsMTP11.1 (Phytozome: LOC_Os05g38670) from Oryza sativa, AtMTP8 (GenBank: At3g58060); AtMTP9 (GenBank: At1g79520), AtMTP10 (GenBank: At1g16310), AtMTP11 (GenBank: At2g39450) from Arabidopsis thaliana, ShMTP8 (GenBank: AAO38707) from Stylosanthes hamata, PtMTP11.1 (Phytozome: POPTR_0010s21810), PtMTP11.2 (Phytozome: POPTR_0008s04940) from Populus trichocarpa, CsMTP8 (GenBank: AFJ24703) from Cucumis sativus, and HvMTP8.1 (GenBank: AFP33387), HvMTP8.2 (GenBank: AFP33388) from Hordeum vulgare are shown. The tree was generated using MEGA6 software (version 6.06). The 0.05 scale shows substitution distance.

ShMTP8 CsMTP8

OsMTP8.1 HvMTP8.1

AtMTP8 OsMTP8

HvMTP8.2 AtMTP9

AtMTP10 OsMTP9

PtMTP11.1 PtMTP11.2 AtMTP11 OsMTP11

OsMTP11.1

1000

1000

1000999

1000999

892

1000

1000

990

792

708

0.05




0.0

0.2

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0.6

0.8

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Root Basal region Shoot

Rel

ativ

e m

RN

A le

vel Control

-Zn -Fe -Mn -Cu

0.0

0.5

1.0

1.5

2.0

0.05 0.5 200 500 1000

Rel

etiv

e m

RN

A le

vel

Mn supply (µM)

0.0

2.0

4.0

6.0

8.0

10.0

0-1 1-2 2-3

Rel

ativ

e m

RN

A le

vel

Distance from root apex (cm)

a

b c b b

a

abb

aba a

Supplementary Fig. 2: Expression pattern of MTP9. (a) Tissue-specific expression and response to mineral deficiency. Seedlings (4-w-old) were grown in the presence or absence of Zn, Fe, Mn or Cu for 7 days. Expression relative to control root is shown. (b) Response of MTP9 to different Mn concentrations. Seedlings (11-d-old) were exposed to different Mn concentrations (0.05, 0.5, 200, 500, and 1000 µM) for 3 d. Expression relative to 0.5 µM Mn is shown. (c) Spatial expression in different root segments. Expression relative to 0–1 cm is shown. Expression levels were determined using real-time RT-PCR. Histone H3 was used as an internal control for each experiment. Data in all figures represent the mean ± SD (n = 3). Different letters indicate a significant difference (P < 0.01) using Tukey’s test.




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Supplementary Fig. 3:Expression of MTP9 in different organs at different growth stage. Rice was grown in a paddy field until ripening and various organs were sampled. The expression level was determined by quantitative real-time RT-PCR. Expression relative to the root of 6 week is shown. Histone H3 and Actin were used as internal standards. Data represents the mean ± SD (n = 3). �




Supplementary Fig. 4: T-DNA insertion mutants of MTP9. (a) Positions of T-DNA insertion in two independent mutant alleles. Grey boxes, black boxes, and horizontal line indicate untranslated region, exon of coding region, and intron, respectively. T-DNAs are shown to be located on the first exon (for mtp9-1) and intron between the first and second exons (for mtp9-2) by triangles. Arrows indicate MTP9-specific primers used for screening of homozygous lines and semi-quantitative RT-PCR analysis. (b) Expression of MTP9 in the roots of wild-type (WT) rice and T-DNA lines. Histone H3 was used as an internal control. Data represent the mean ± SD (n = 4). Significant differences between wild-type and T-DNA lines were determined using Dunnett’s test are indicated by ***(P < 0.0001). (c) Western blot analysis of microsomal membrane prepared from roots. Arrow indicates predicted MTP9. The plasma membrane marker protein H+-ATPase detected using a specific antibody was used as an internal control.

mtp9-1

ATG TGA

a

MTP9

mtp9-2 500 bp

0.0

0.5

1.0

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Rel

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*** ***

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H+-ATPase

0.05 0.5 50 200Mn (µM)

MTP9

Supplementary Fig. 5: Accumulation of MTP9 at different Mn concentration. Microsome fractions were prepared from roots treated with various concentrations of Mn (0.05 (deficient), 0.5 (replete), 50, and 200 (excessive) µM) for 48 hours. The plasma membrane marker protein H+-ATPase was used as an internal control.




0

5000

10000

15000

20000

Vector MTP9 Vector MTP9

Cop

y nu

mbe

r ng-

1 RN

A

Glucose Galactose

Supplementary Fig. 6: Expression level of MTP9 in yeast (Saccharomyces cerevisiae). The yeast BY4741 strain carrying the pYES2 empty vector or pYES2-MTP9 was grown to the stationary phase in SC-U/Glucose (for 10 h) and SC-U/Galactose (for 20 h) medium. Data represent the mean ± SD (n = 3). Different letters indicate a significant difference (P < 0.05) using Tukey’s test.

ab

a

c




+MetalControl ++Metal

ccc1

zrc1cot1

Vector

MTP9pmr1

0.25 / 0.50 mM Co

4.0 / 8.0 mM Fe

0.20 / 0.40 mM Zn

0.75 / 1.50 mM Ni

ycf1 20 / 40 µM Cd

Supplementary Fig. 7: Transport assay of MTP9 in yeast (Saccharomyces cerevisiae). The yeast mutant strains pmr1, ccc1, ycf1, and zrc1cot1 carrying the pYES2 empty vector or pYES2-MTP9 were grown on SC-U medium containing glucose (left panel) or galactose (right panel) in the absence or presence of different concentration of MnCl2, FeSO4, CdSO4, ZnSO4, CoCl2, or NiCl2. Metal concentrations used are indicated on the right. Five µl (OD600 = 0.5) of serial dilutions (10-fold) were spotted. Plates were incubated for 24 – 48 h at 30°C in the dark.

0.15 / 0.30 mM Mn

Vector

MTP9

Vector

MTP9

Vector

MTP9

Vector

MTP9

Vector

MTP9

+MetalControl ++MetalGalactoseGlucose




Supplementary Fig. 8: Grain yield and fertility at the reproductive stage grown hydroponically. Wild-type rice (WT) and independent two mutant lines were grown in nutrient solution till ripening.Total shoot (straw + grains) dry weight (a), grain yields (b) and fertility (c, d). Data represent the mean ± SD (n = 3). Different letters indicate a significant difference (P < 0.05) using Tukey’s test.

c

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Mn

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. in

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(µg

g-1 D

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Mn treatment (µM)

WT mtp9-1 mtp9-2

0 100 200 300 400 500 600 700 800

0.05 0.5 Q

uant

um y

ield

of P

SII

Mn treatment (µM)

Supplementary Fig. 9: Mn concentration and photosynthesis activity in leaf blade. Concentration of Mn (a) and quantum yield of PSII (b) in the second youngest leaf blades of wild-type (WT) rice and two independent mutant lines are shown. Seedlings (14-d-old) were exposed to a nutrient solution for 14 d. Data represent the mean ± SD (n = 4). Different letters indicate a significant difference (P < 0.01) in individual treatment using Tukey’s test.

ab b

ab a b

ab b

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a b




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g-1 D

W)

Supplementary Fig. 10: Metal accumulation in hydroponic culture. Concentration of micronutrients (a) and macronutrients (b) in the shoots (upper) and roots (lower) of wild-type (WT) rice and two independent mutant lines. Seedlings (9-d-old) were exposed to a nutrient solution for 10 d. Data represent the mean ± SD (n = 4). Different letters indicate a significant difference (P < 0.01) in individual element using Tukey’s test.

0

5

10

15

20

25

K Mg Ca

Roo

t met

al c

onc.

(m

g g-

1 DW

)

a bab

b

0

20000

40000

60000




0.0

2.0

4.0

6.0

8.0

10.0

Sho

ot N

i con

c.

(µg

g-1 D

W)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

Sho

ot C

o co

nc.

(µg

g-1 D

W)

0.0

0.5

1.0

1.5

2.0

2.5

Sho

ot P

b co

nc.

(µg

g-1 D

W)

Supplementary Fig. 11: Accumulation of Ni, Co, Pb, and Cd in hydroponic culture. Concentration of Ni (a), Co (b), Pb (c), and Cd (d) in the shoots (upper) and roots (lower) of wild-type (WT) rice and two independent mutant lines. Seedlings were exposed to a nutrient solution containing Ni, Co, Pb, or Cd for 10 d. Data represent the mean ± SD (n = 3). Different letters indicate a significant difference (P < 0.01) using Tukey’s test.

a b c

0

50

100

150

200

250

Roo

t Ni c

onc.

(µ

g g-

1 DW

)

0 100 200 300 400 500 600 700

Roo

t Co

conc

. (µ

g g-

1 DW

)

0

500

1000

1500

2000

2500

Roo

t Pb

conc

. (µ

g g-

1 DW

)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

Sho

ot C

d co

nc.

(µg

g-1 D

W)

d

0

50

100

150

200

250

Roo

t Cd

conc

. (µ

g g-

1 DW

)

a

b b




0 500

1000 1500 2000 2500 3000 3500 4000 4500 5000

Brown rice Root Flag leaf Others

Mn

conc

. (µg

g-1

DW

)

WT mtp9-1 mtp9-2

Supplementary Fig. 12: Mn concentration in different organs of rice grown hydroponically. Wild-type (WT) and two independent mutants (mtp9-1 and mtp9-2) were grown in nutrient solution for 4 months. Data represent the mean ± SD (n = 3). Different letters indicate a significant difference (P < 0.01) in individual material using Tukey’s test.

a b b

a

b

ca

b b

a

b b

0 20 40 60 80

100

Brown rice

a

b b




0

50

100

150

200

250

Fe c

once

ntra

tion

(mg

kg-1

DW

)

0 20 40 60 80

100 120 140

Cu

conc

entra

tion

(mg

kg-1

DW

)

0

200

400

600

800

Zn c

once

ntra

tion

(m

g kg

-1 D

W) WT

mtp9-1 mtp9-2

a bab

a

b

a bab

c

a

b b ab b

ab b

ab b a

b b

Supplementary Fig. 13: Metal accumulation in soil culture. Concentration of Zn (a), Cu (b), and Fe (c) in different tissues of wild-type (WT) rice and two independent mutant lines. Plants were grown in soil till ripening. Data represent the mean ± SD (n = 3). Different letters indicate a significant difference (P < 0.01) in individual material using Tukey’s test.




0.0

0.5

1.0

1.5

2.0

2.5

Nramp5 IRT1 IRT2 Nramp3 YSL2 MTP8 MTP8.1 MTP11 MTP11.1

Rel

ativ

e m

RN

A le

vel

WT

mtp9-1

mtp9-2

Uptake Tissue distribution Subcellular trafficking and sequestration

a abb

Supplementary Fig. 14: Expression of putative Mn transporter genes in MTP9 mutants. Expression of transporter genes possibly involving uptake, tissue distribution, and subcellular trafficking and sequestration of Mn in the roots of wild-type (WT) rice and two independent mutants (mtp9-1 and mtp9-2) are shown. Histone H3 was used as an internal control. Data represent the mean ± SD (n = 4). Different letters indicate a significant difference (P < 0.05) using Tukey’s test.




0 20 40 60 80

100 120 140 160

Base Apex

Mn

conc

. (µg

g-1

FW

) WT mtp9-1 mtp9-2

a

Supplementary Fig. 15: Spatial distribution of Mn in roots. Mn concentration of basal roots (1–3 cm) and root apexes (0–1 cm). Seedlings (4-d-old) of the wild-type rice and two mutants were cultured in a solution containing 200 µM Mn for 40 h. Data represent the mean ± SD (n = 3). Different letters indicate a significant difference (P < 0.01) using Tukey’s test.

bb

Basal




Supplementary Fig. 16: Schematic representation of Mn uptake system in rice root. Nramp5 (blue color) is localized at the distal side of both exodermis and endodermis, while MTP9 (orange color) is localized at the proximal side of the same cell layers. Both Nramp5 and MTP9 are required to transport Mn across the Casparian strips (grey lines) and cooperation of Nramp5 and MTP9 leads to an unidirectional flux of Mn from the rhizosphere to the stele in rice roots.

AerenchymaEpidermis

Exodermis Endodermis

Casparian stripCasparian strip

Aerenchyma Stele

��

Stele


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