a bacterial-type abc transporter is involved in aluminum ...(a) molecular mapping of star1 using 716...

A Bacterial-Type ABC Transporter Is Involved in AluminumTolerance in Rice W

Chao Feng Huang,a,1 Naoki Yamaji,a,1 Namiki Mitani,a Masahiro Yano,b Yoshiaki Nagamura,c and Jian Feng Maa,2

a Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, JapanbQTL Genomics Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, JapancGenome Resource Center, Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences,

Tsukuba, Ibaraki 305-8602, Japan

Aluminum (Al) toxicity is a major factor limiting crop production in acidic soil, but the molecular mechanisms of Al tolerance

are poorly understood. Here, we report that two genes, STAR1 (for sensitive to Al rhizotoxicity1) and STAR2, are responsible

for Al tolerance in rice. STAR1 encodes a nucleotide binding domain, while STAR2 encodes a transmembrane domain, of a

bacterial-type ATP binding cassette (ABC) transporter. Disruption of either gene resulted in hypersensitivity to aluminum

toxicity. Both STAR1 and STAR2 are expressed mainly in the roots and are specifically induced by Al exposure. Expression

in onion epidermal cells, rice protoplasts, and yeast showed that STAR1 interacts with STAR2 to form a complex that

localizes to the vesicle membranes of all root cells, except for those in the epidermal layer of the mature zone. When

expressed together in Xenopus laevis oocytes, STAR1/2 shows efflux transport activity specific for UDP-glucose.

Furthermore, addition of exogenous UDP-glucose rescued root growth in the star1 mutant exposed to Al. These results

indicate that STAR1 and STAR2 form a complex that functions as an ABC transporter, which is required for detoxification of

Al in rice. The ABC transporter transports UDP-glucose, which may be used to modify the cell wall.

INTRODUCTION

Aluminum (Al) is the most abundant metal in the earth’s crust. It

occurs primarily in aluminosilicate minerals, most commonly as

feldspars in metamorphic and igneous rocks and as clay min-

erals in well-weathered soils (Driscoll and Schecher, 1990).

Metallic aluminum and aluminosilicate minerals are nontoxic.

However, under acidic conditions, aluminum is released into the

soil in the ionic form (Al3+), which is toxic to all living cells. Low

concentrations of ionic Al rapidly inhibit root elongation by

targeting multiple cellular sites, including the cell walls and

plasma membranes, and various cellular processes, such as

signal transduction pathways and Ca homeostasis (Ryan et al.,

2001; Barcelo and Poschenrieder, 2002; Rengel, 2004; Kochian

et al., 2005; Ma, 2008). Consequently, aluminum inhibits uptake

of water and nutrients and increases the plant’s sensitivity to

various stresses, especially drought. Therefore, aluminum tox-

icity has been recognized as a major factor limiting crop pro-

duction in acid soils, which comprise;30 to 40% of the world’s

arable soils (von Uexkull and Mutert, 1995).

There are large variations in the tolerance to aluminum toxicity

among plant species and cultivars within a species. Some plant

species and cultivars have evolved mechanisms to cope with Al

toxicity both externally and internally (Ma et al., 2001; Ryan et al.,

2001; Kochian et al., 2005; Ma, 2008). An example of a mech-

anism for external detoxification of Al is the release of organic

acid anions from roots (Ryan et al., 2001; Kochian et al., 2005;

Ma, 2008). Recently, genes encoding transporters for Al-induced

secretion of malate and citrate have been identified and charac-

terized in wheat (Triticum aestivum), barley (Hordeum vulgare),

and sorghum (Sorghum bicolor) (Sasaki et al., 2004; Furukawa

et al., 2007; Magalhaes et al., 2007). By contrast, internal

detoxification of Al is achieved by the formation of nontoxic Al

complexes with organic acids or other chelators and sequestra-

tion of these complexes in the vacuoles (Ma et al., 1997; Shen

et al., 2002, 2004a).

Rice (Oryza sativa) is themost Al-tolerant species among small

grain cereal crops, although genotypic variations in aluminum

tolerance also exist within this species (Ma et al., 2002). How-

ever, neither the physiological mechanisms nor the genes re-

sponsible for Al tolerance in rice have been identified. Unlike

other cereal species, such as rye (Secale cereale), wheat, and

barley, secretion of organic acid anions does not seem to be the

mechanism of Al tolerance in rice because the amount of organic

acid anions secreted is too small (Ma et al., 2002). Several

genetic studies have identified >10 quantitative trait loci (QTL) for

aluminum tolerance in rice, but the responsible genes have not

been cloned so far. In this study, we have identified two genes

that are responsible for aluminum tolerance in rice.We found that

proteins encoded by these two genes form a complex that

functions as a unique ATP binding cassette (ABC) transporter.

1 These authors contributed equally to this work.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Jian Feng Ma([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.108.064543

The Plant Cell, Vol. 21: 655–667, February 2009, www.plantcell.org ã 2009 American Society of Plant Biologists

RESULTS

Identification of an Aluminum-Tolerance Gene, STAR1,

Which Encodes a Nucleotide Binding Domain of an ATP

Binding Cassette Transporter

star1 (for sensitive to aluminum rhizotoxicity1, formerly known as

als1, for Al sensitive1) is a recessive rice mutant with hyper-

sensitivity to aluminum toxicity, which was isolated from an

aluminum-tolerant cultivar of rice irradiatedwith g-rays (Ma et al.,

2005). In the absence of Al, growth is similar in wild-type and

star1 mutant rice. Furthermore, the mutant is highly sensitive to

aluminum toxicity but not to other metals, including cadmium

and lanthanum (Ma et al., 2005). To clone the responsible gene,

we constructed an F2 mapping population derived from a cross

between star1 and Kasalath, an indica cultivar. We roughly

mapped the gene (STAR1) to the long arm of chromosome 6

between the InDel markers MaOs0615 andMaOs0619 (Ma et al.,

2005). For fine mapping of STAR1, we evaluated 716 star1

homozygotes. We developed three polymorphic InDel markers

between the two InDel markers mentioned above (i.e.,

MaOs0615 and MaOs0619) and mapped this gene between

MaOs0624 and MaOs0617 on the two overlapping PAC clones

(AP003770 and AP003771) (Figure 1A). We further developed

two new InDel markers and three CAPS (cleaved amplified

polymorphisms) markers on the two PACs and finally mapped

STAR1 to an 88-kb candidate region on the PACcloneAP003771

(Figure 1A). There are 14 predicated genes within this region

based on the rice genome annotation in The Institute for Ge-

nomic Research (TIGR) website (http://www.tigr.org), including

seven putative retrotransposon genes (Figure 1B). We se-

quenced the other seven candidate genes and found that,

relative to the wild type, a 15-bp deletion was present in the

second exon of one gene (Figure 1C), while there were no

mutations in other genes. This gene consists of four exons and

three introns, encoding a 291–amino acid protein (Figure 1C; see

Supplemental Figure 1 online). The gene (STAR1) is predicted to

encode a nucleotide binding domain (NBD) of a putative ABC

transporter protein and contains all the typical motifs conserved

in aNBD, namely, theWalker-A, Q-loop, ABC signature,Walker-B,

D-loop, and H-loop motifs (see Supplemental Figure 1 online).

According to new a nomenclature system (Sanchez-Fernandez

et al., 2001; Verrier et al., 2008), STAR1 was categorized into the

NO subgroup of subfamily I, which includes NBD proteins with

low similarity to each other and of unknown functions. There is no

homolog of STAR1 in the rice genome (Figure 1D). Database

searches led to the identification of one homolog of STAR1 in

maize (Zea mays), Arabidopsis thaliana, grape (Vitis vinifera), and

moss (Physcomitrella patens) (Figure 1D; see Supplemental Data

Set 1 online), but their function is unknown. STAR1 also showed

similarity to the NBD subunit of the bacterial phosphate trans-

porter, pstB, and the similarity to this transporter is higher than

any other plant ABC transporters (Figure 1D). The mutation in

star1 resulted in the deletion of five amino acids in the region

between the Walker-A and Q-loop motifs (C178 to V182; see

Supplemental Figure 1 online).

To test whether the mutation in STAR1 is responsible for the

observed phenotype, we performed a complementation test by

introducing a 5.4-kb clone containing the promoter and the

candidate gene into the star1 mutant. In the two independently

generated T1 transgenic lines carrying STAR1, the tolerance to

aluminum toxicity was increased to a similar level as seen in wild-

type rice in both hydroponic and acidic soil cultures (Figures 1E

and 1F). These results confirm that the mutation in STAR1 is

responsible for the aluminum sensitivity in the star1 mutant.

To investigate the localization of STAR1 in rice roots, we

generated transgenic rice plants carrying the translational fusion

between STAR1 and the green fluorescent protein (STAR1-GFP)

gene under the control of the STAR1 promoter (ProSTAR1:

STAR1-GFP). TheGFP fluorescence signal was observed in both

the main root and the lateral root and in all root cells except for

those in the epidermis of the mature region (Figures 2A and 2B).

We also examined STAR1 protein localization by immunostain-

ing. Similar to the result of STAR1-GFP fluorescence, the STAR1

antibody was detected in all cells of the root tip (Figures 2C and

2D). Furthermore, we found that STAR1 is present inside vesicle-

like granules in root cells (Figure 2D).

To investigate the subcellular localization of STAR1, we per-

formed protein gel blot analysis on root membrane fractions. We

found that STAR1 is present in the microsome fraction but not in

the soluble fraction in wild-type rice roots (Figure 2E). In the

mutant, no signal was found in either fraction, indicating the high

specificity of the antibody anti-STAR1. Surprisingly, fractionation

of the rootmicrosomalmembraneswith sucrose density gradients

showed that STAR1 was detected in the upper fraction (mainly in

the 20/30% sucrose boundary) (Figure 2F), which is different from

a marker protein of plasma membrane, Lsi1 (Ma et al., 2006),

detected in the lower fractions (at 40/50%). These results suggest

that STAR1 protein is not localized at the plasma membrane.

We also delivered STAR1-GFP into onion epidermal cells by

particle bombardment and examined whether STAR1-GFP is

colocalized with the mitochondria staining dye tetramethylrhod-

amine methyl ester or with the peroxisome-targeted red fluores-

cent protein, DsRed-PTS1. Our results showed that STAR1-GFP

did not colocalize with either tetramethylrhodamine methyl ester

or DsRed-PTS1 in onion epidermal cells (see Supplemental

Figure 2 online). Taken together, STAR1 is probably localized

at the membrane of some vesicles.

Isolation of STAR2, Which Encodes a Transmembrane

Domain Protein of an ABC Transporter

Although STAR1 contains only an NBD without transmembrane

domains (TMDs) (see Supplemental Figure 1 online), it was found

in the membrane fraction (Figure 2E). This inconsistency sug-

gests that STAR1 requires another factor to be localized on the

membrane. There are 10 genes encoding a NBD alone, including

STAR1, and three genes encoding only a TMD in the rice genome

(Garcia et al., 2004). InArabidopsis, a gene (als3) encoding only a

TMD has been implicated in aluminum tolerance (Larsen et al.,

2005). Therefore, there is a possibility that a homolog of ALS3 in

rice may interact with STAR1. We found a rice homolog of ALS3

and then cloned this gene, named STAR2, from rice root cDNA.

STAR2 consists of one exon, encoding a 285–amino acid protein

(Figure 3A), which is predicted to have seven TMDs (see Sup-

plemental Figure 3 online). In the rice genome, there is no close

656 The Plant Cell

homolog of this protein (Figure 3B; see Supplemental Data Set 2

online), but there is one homolog of STAR2 inmaize,Arabidopsis,

grape, and P. patens.

To investigate whether STAR2 is also involved in aluminum

tolerance in rice, we prepared star2 knockdown transgenic lines

by RNA interference (RNAi). In the RNAi transgenic lines, the

mRNA accumulation of STAR2 was reduced to <10% of that in

wild-type rice (Figure 3C). The tolerance to aluminum toxicitywas

significantly decreased in the RNAi transgenic lines compared

with that of the vector control plants (Figure 3D). These results

Figure 1. Map-Based Cloning and Complementation of STAR1.

(A)Molecular mapping of STAR1 using 716 F2 Al-sensitive mutants from a star1/Kasalath population. Molecular markers and their genetic distances in

a Rice Genome Research Program (RGP) genetic map are given above the vertical lines, and the numbers below the vertical lines represent

recombinants between each marker and the gene. The long horizontal line interrupted by two parallel lines represents the rice chromosome 6, and

overlapped horizontal lines indicate the PAC clones. STAR1 was finally mapped to an 88-kb candidate region on PAC clone AP003771.

(B) Candidate genes of STAR1. Fourteen genes were predicted to occur in the 88-kb candidate region based on the pseudomolecules annotation of the

rice genome. Black boxes represent retrotransposons, a white box indicates the mutated gene, and gray boxes represent other candidate genes that

lack mutations.

(C) Gene structure of STAR1. White boxes indicate exons, and black horizontal lines represent introns. STAR1 contains four exons and three introns.

The position of the deletion mutation in star1 caused by g-ray irradiation is indicated with a filled black triangle.

(D) Phylogenetic relationship of the STAR1 protein in maize, Arabidopsis, V. vinefera (grapevine), the moss P. patens, Thermoanaerobacter ethanolicus

(a thermophile), and Escherichia coli. The sequences used to generate the phylogeny are presented in Supplemental Data Set 1 online. Bootstrap values

from 1000 trials are indicated. The 0.1 scale shows substitution distance.

(E) and (F) Complementation test.

(E) Relative root elongation (root elongation with Al/root elongation without Alx100) of two independent transgenic lines transformed with STAR1

genomic fragment, the vector control, the wild type, and star1 grown in hydroponic conditions. The seedlings were exposed to an aluminum solution for

24 h. Data are means 6 SD of biological replicates (n = 10).

(F) Root growth on acidic soil (Andosol; pH 4.8). The seedlings were grown for 10 d.

A Rice Bacterial-Type ABC Transporter 657

clearly indicate that STAR2 is also required for aluminum toler-

ance in rice.

Immunostaining with STAR2 antibody showed that, like

STAR1, the STAR2 protein is localized in root cells (Figures 4A

and 4B). STAR2 is also present as vesicle-like granules in the

cells (Figure 4C).

Expression Pattern of STAR1 and STAR2

Both STAR1 and STAR2 were mainly expressed in the roots

(Figure 5A), and mRNA accumulation of STAR1 and STAR2 was

significantly enhanced when seedlings were exposed to alumi-

num. Accumulation of mRNA was greater in the mature zones (1

to 2 cm) of the roots than in the root tip (0 to 1 cm), both in the

presence and absence of aluminum (see Supplemental Figure 4

online). A time-course experiment showed that the expression of

STAR1 and STAR2 was similarly induced by aluminum as early

as 2 h after the exposure (Figure 5B). Furthermore, an aluminum

concentration as low as 5 mM triggered the expression of STAR1

and STAR2, and the expression level increased with increasing

external aluminum concentration (Figure 5C). The mRNA accu-

mulation of STAR1 and STAR2 did not respond to low pH (Figure

5C) and other metals, including cadmium and lanthanum (Figure

5D), indicating that STAR1 and STAR2 are specifically induced

by aluminum. These results suggest that STAR1 and STAR2 are

similarly regulated by aluminum.

Figure 2. Localization of STAR1.

(A) and (B) Fluorescence of GFP driven by the STAR1 promoter (ProSTAR1:GFP) in a transgenic plant root treated with 100 mMAl for 6 h: root tip (A) and

mature zone (B).

(C) Immunostaining of root cross section 5 mm from the tip using the anti-STAR1 polyclonal antibody.

(D) Close-up of immunostaining. Bars = 100 mm.

(E) and (F) Protein gel blot analysis of STAR1.

(E) Microsomal membrane fraction (M) and soluble protein fraction (S) were prepared from wild-type and star1 mutant roots treated with 20 mM Al

for 6 h.

(F) The membrane fraction of the wild-type root was separated by sucrose gradient centrifugation as indicated. Each fraction was subjected to SDS-

PAGE and protein gel blot analysis using anti-STAR1 antibody. Anti-Lsi1 antibody was used as a marker of the plasma membrane.

658 The Plant Cell

Interaction between STAR1 and STAR2

To examine the interaction between STAR1 and STAR2, we first

used the DUALmembrane system to detect protein–protein

interactions between an integral membrane protein and its

interaction partners (either integral membrane proteins or soluble

proteins) in the yeast S. cerevisiae (Johnsson and Varshavsky,

1994). When STAR1 was fused to the C-terminal half of ubiquitin

and STAR2 to the mutated N-terminal half of ubiquitin, yeast

growth was observed on the selective medium (Figure 6A),

indicating protein interaction between STAR1 and STAR2. By

contrast, when STAR1 was replaced by the mutant star1, the

growth of yeast was reduced to the same level as the negative

control (Figure 6A), suggesting that mutant star1 cannot interact

with STAR2.

To confirm protein interaction between STAR1 and STAR2, we

conducted bimolecular fluorescent complementation (BiFC)

analysis. In rice callus protoplasts expressing both EYFP-C-

STAR1 and EYFP-N-STAR2, complemented enhanced yellow

fluorescent protein (EYFP) fluorescence was observed around

the nuclei and in the internal region of the cytoplasm, but not in

the nuclei (see Supplemental Figure 5A online). In the protoplasts

expressing EYFP-C and EYFP-N, fluorescence was observed in

the cytoplasmand nuclei (see Supplemental Figure 5Donline), as

excess free N- and C-terminal proteins complement each other

to forma fluorescent complex.WhenEYFP-C-STAR1andEYFP-N

were expressed in protoplasts (see Supplemental Figure 5B

online), complemented YFP fluorescence was observed in the

cytoplasm and nuclei, as was seen for the EYFP-C and EYFP-N

combination, because EYFP-C-STAR1 alone is not targeted to

the membrane. By contrast, in protoplasts expressing EYFP-C

and EYFP-N-STAR2, fluorescence was not observed (see Sup-

plemental Figure 5C online). Taken together, these results sug-

gest that EYFP-C-STAR1 is able to interact with EYFP-N-STAR2

and that the complex is likely to be localized at some vesicles

inside the cytosol.

We also constructed fused genes betweenSTAR1 or star1 and

GFP (STAR1-GFP or star1-GFP) and between STAR2 and red

fluorescent protein DsRed-monomer (STAR2-DsRed) to further

examine the interaction between STAR1 and STAR2. When

STAR1-GFP alone was expressed in the epidermal cells of

onions, fluorescence signal in granule shape was observed

(Figure 6B). However, when STAR1-GFP and STAR2-DsRed

were introduced together into the epidermal cells of onions,

STAR1-derived green fluorescence (Figure 6D) and STAR2-

derived red fluorescence (Figure 6F) colocalized, producing a

merged yellow signal (Figure 6H). By contrast, when mutant

star1-GFP was coexpressed with STAR2-DsRed, star1-GFP did

not colocalize with STAR2-DsRed, but appeared as punctate

fluorescence signals (Figure 6E), similar to those seen in cells

Figure 3. Phylogenetic Relationship and Knockdown Assay of STAR2.

(A) Gene structure of STAR2. STAR2 contains one exon and no introns.

(B) Phylogenetic relationship of the STAR2 protein with homologs in maize, Arabidopsis, V. vinefera, P. patens, T. ethanolicus, and E. coli. The

sequences used to generate the phylogeny are presented in Supplemental Data Set 2 online. Bootstrap values from 1000 trials are indicated. The 0.1

scale shows substitution distance.

(C) Relative root transcript level of STAR2 in two independent transgenic RNAi lines, 52-4 and 97-1. Actin1 was used as an internal control, and values

are relative to the level of STAR2 in the vector control.

(D) Effect of STAR2 suppression by RNAi silencing on aluminum tolerance. The seedlings (four leaves stage) were exposed to a solution without

aluminum for 1 d and then to a solution with 100 mM aluminum for another day. The relative root elongation (root elongation with Al/root elongation

without Alx100) is shown. Data are means 6 SD of biological replicates (n = 6).


expressing STAR1-GFP alone (Figure 6B). Although the cellular

localization of these proteins in onion epidermal cells did not

reflect their localization in rice (Figures 2D and 4C), these results,

together with the split-ubiquitin system data and BiFC results,

indicate that STAR1 interacts with STAR2 by forming a complex.

The mutant star1 lacks five amino acids between the Walker A

and Q-loop motifs (see Supplemental Figure 1 online), and this

mutationmay inhibit its interactionwith STAR2. This is supported

by the structural analysis of a model bacterial ABC transporter,

BtuCD, which indicates that residues around the Q-loop are

involved in the interface with the TMD (Locher et al., 2002).

The STAR1-STAR2 Complex Transports UDP-Glucose

The results described above indicate that STAR1 and STAR2 are

translated to NBD and TMD, respectively, and then function as

an ABC transporter protein complex. To identify the substrate of

this putative transporter, we tested aluminum ion and phosphate

transport activity in both yeast and Xenopus laevis oocyte

expression systems. These two substrates were tested because

efflux of toxic aluminum from the cells is expected to enhance

tolerance to aluminum. STAR1 is similar to the bacterial phos-

phate transporter subunit PstB (Chan and Torriani, 1996) (Figure

1D), although there are no homologous genes of the bacterial

TMD proteins PstA and PstC in the rice genome. Phosphate is

also able to detoxify aluminum by forming a precipitate. How-

ever, the STAR1/STAR2 complex had no detectable aluminum

ion or phosphate transport activity in either of the heterologous

expression systems used.

We then compared the aluminum content in the roots of wild-

type rice and the star1 mutant. After a short-term (6 h) exposure

to aluminum, the roots and the cell walls in the segment 0 to 1 cm

from the root tip contained similar levels of aluminum in the wild

type and themutant (see Supplemental Figure 6 online), although

long-term (24 h) exposure to aluminum resulted in increased Al

accumulation in the root tip of themutant relative to thewild type,

due to severe inhibition of root growth (Ma et al., 2005). By

contrast, in the root segments 1 to 2 cm from the root tip, where

bothSTAR1 andSTAR2 are expressed at higher levels than in the

extreme tip, the aluminum content was higher in wild-type rice

than in the mutant. However, roots of the star1 mutant stained

with morin, an Al-specific fluorochrome, showed a fluorescent

signal intensity much higher than that of wild-type rice (see

Supplemental Figure 7 online).

We also performed amicroarray analysis to compare the effect

of aluminum exposure on the gene expression profile of the wild

type with that of the star1 mutant and found that several genes

encoding UDP-glucuronosyl/UDP-glucosyltransferase are sig-

nificantly upregulated by aluminum in the mutant roots (see

Supplemental Figure 8 online). Analysis with RT-PCR confirmed

this result (see Supplemental Figure 9 online). Furthermore, we

found that more soluble sugars accumulated in star1 mutant

roots exposed to aluminum than in those of the wild type (Figure

7A), with glucose being the predominant sugar (see Supplemen-

tal Figure 10 online). Based on all these findings, we speculated

that the STAR1-STAR2 complex transports UDP-glucose (UDP-

Glc) as a substrate. To examine the transport activity for UDP-

Glc, we coinjected Xenopus oocytes with cRNA encoding

STAR1 and STAR2, or with water as a control, and measured

the influx and efflux activity of 14C-labled UDP-Glc. STAR1 and

STAR2 showed an efflux activity for UDP-Glc (Figure 7B) but no

influx activity. Interestingly, the STAR1-STAR2 complex did not

transport UDP-glucuronic acid (UDP-GlcA, Figure 7B).

To further confirm this result, we also used an oocyte two-

electrode voltage clamp technique (Figure 7C; see Supplemental

Figures 11 to 13 online). When STAR1 was expressed alone, no

change in current was observed compared with the water-

injected control. However, when STAR1 and STAR2 were

coexpressed, a significant inward current was induced in the

UDP-Glc preloaded oocytes (see Supplemental Figure 11 online).

Figure 4. Localization of STAR2.

(A) to (C) Immunostaining of a root cross section 5 mm (A) or 15 mm

([B] and [C]) from the tip using anti-STAR2 polyclonal antibody. Bars =

100 mm.

(C) Close-up of immunostaining of STAR2.

660 The Plant Cell

Expression of STAR2 alone also resulted in current change, but it

lost the specificity for the preloaded substrates (UDP-Glc, UDP-

GlcA, and water) probably due to increased membrane perme-

ability for all substrates (see Supplemental Figure 12 online). By

contrast, when STAR1 and STAR2 were coexpressed, the com-

plex showed a high specificity for the substrates, transporting

only UDP-Glc, but not UDP-GlcA or UDP-galactose (Figure 7C).

External UDP-Glc did not affect the specific current for pre-

loaded UDP-Glc in oocytes expressing STAR1 and STAR2 (see

Supplemental Figure 13 online). These results indicate that the

STAR1-STAR2 complex has transport activity specific for UDP-

Glc and that the interaction between STAR1 and STAR2 is

necessary for the substrate-specific transport activity.

We also investigated the effect of exogenously applied UDP-

Glc on root elongation in both wild-type and star1 mutant rice.

UDP-Glc alone did not affect root growth of either thewild type or

themutant rice in the absence of aluminum (Figure 7D). However,

in the presence of aluminum, exogenous UDP-Glc significantly

alleviated the aluminum-induced inhibition of root growth in the

mutant (Figure 7D). Partial recovery of root elongation in the

mutant by the addition of UDP-Glu is probably due to lower

incorporation efficiency of exogenous UDP-Glu into the cell wall

than UDP-Glu secreted from endogenous site. Root growth of

the wild type was unaffected by UDP-Glc in the presence of

aluminum (Figure 7D).

DISCUSSION

ABC transporters represent a large family in plants with >120

members in both Arabidopsis and rice (Sugiyama et al., 2006;

Verrier et al., 2008). However, the function of most ABC trans-

porters is still unknown. In this study, we identified an Al-tolerant

gene, STAR1, from an Al-sensitive rice mutant using a map-

based cloning technique (Figure 1). This gene is unique because,

unlike most plant ABC transporters, which contain both NBDs

and TMDs, STAR1 only encodes a NBD (see Supplemental

Figure 1 online). Moreover, it shows higher similarity to subunit

PstB of the bacterial phosphorus transporter than any other plant

ABC transporters (Figure 1D).We further isolated a gene,STAR2,

which only encodes a TMD (see Supplemental Figure 3 online).

This gene is also involved in Al tolerance in rice because

knockdown of this gene resulted in increased Al sensitivity

(Figures 3C and 3D). Expression in onion epidermal cells, rice

protoplasts, and yeast showed that STAR1 interacts with STAR2

(Figure 6; see Supplemental Figure 5 online). Furthermore, the

Figure 5. Expression Analysis of STAR1 and STAR2.

(A) RT-PCR of STAR1, STAR2, and actin (internal standard) transcripts in the roots and shoots of wild-type rice treated with or without aluminum.

(B) and (C) Time-dependent (B) and aluminum concentration- and pH-dependent (C) expression of STAR1 and STAR2 in rice roots. One-week-old

seedlings of rice (cv Koshihikari) were treated with 50 mMAl, pH 4.5, for different time periods (B) or treated with various concentrations of Al at pH 4.5 or

with different pHs (4.5 and 5.6) in the absence of Al for 6 h (C). The root tips (0 to 10 mm from tip) were harvested.

(D) Response to different metals. Five-day-old seedlings were exposed to a 0.5 mM CaCl2 solution at pH 5.6 or to a 0.5 mM CaCl2 solution, pH 4.5,

containing 50 mM Al, 30 mM Cd, or 10 mM La for 6 h at 258C. Entire roots were harvested. Expression levels of STAR1, STAR2, and Actin as an internal

control were determined by quantitative RT-PCR. Relative value to STAR1 or STAR2 expression levels in plants without Al treatment was shown, and

values are means 6 SD of biological replicates (n = 3) in (B) to (D).


expression pattern is very similar between STAR1 and STAR2 in

terms of tissue specificity, dose and time response, and metal

specificity (Figure 5). All these results indicate that, like the

bacterial-type ABC transporter, STAR1 and STAR2 are trans-

lated to an NBD and TMD, respectively, and then function as an

ABC transporter protein complex, which is required for Al toler-

ance in rice.

STAR2 is a homolog of ALS3, which is involved in Al tolerance

in Arabidopsis (Larsen et al., 2005). However, STAR2 and ALS3

differ in expressionpattern andcellular localization. STAR2 is only

expressed in the roots (Figure 5A), whereas ALS3 is expressed in

all organs, including roots, leaves, stems, and flowers (Larsen

et al., 2005). The expression of both STAR2 and ALS3 is induced

by Al (Figure 5; Larsen et al., 2005). In the roots exposed to Al,

ALS3 is localized in the phloem and root tips. By contrast, STAR2

is localized in all cells excepting epidermal cells in themature root

zone (Figure 4). Although the partner of an equivalent of STAR1

has not been identified in Arabidopsis, the difference in the

expression and localization of ALS3 and STAR2 suggest that

Arabidopsis and rice have different mechanism for detoxifying Al.

Figure 6. Interaction between STAR1 and STAR2.

(A) DUALmembrane system. STAR1 and STAR2 were fused to the C-terminal half (Cub) and the mutated N-terminal half (NubG) of ubiquitin,

respectively. Yeast cells were cotransformed with various plasmid combinations as indicated at the right and were grown on SD medium without Leu

and Trp as a control (left panel) or on a selective medium for protein interaction (right panel) without Leu, Trp, His, and Ade and containing 10 mM

3-aminotriazole at different concentrations (10 times dilution from left side). All plates were grown for 3 d at 308C.

(B) to (I) Transient expression of fluorescent fusion proteins in onion epidermal cells by particle bombardment.

(B) Expression of STAR1-GFP alone.

(C) Expression of STAR2-DsRed alone.

(D), (F), and (H) Coexpression of STAR1-GFP and STAR2-DsRed observed with green channel for STAR1-GFP (D), red channel for STAR2-DsRed (F),

and merge of (D) and (F) in (H).

(E), (G), and (I), Coexpression of mutant star1-GFP and STAR2-DsRed observed with green channel for star1-GFP (E), red channel for STAR2-DsRed

(G), and merge of (E) and (G) in (I). Bars = 100 mm.

662 The Plant Cell

Recently, a eukaryotic half-type ABC transporter (ALS1),

which is involved in Al tolerance, was identified in Arabidopsis

(Larsen et al., 2007). This transporter is expressed in all organs,

including roots, leaves, stems, and flowers. In the roots, ALS1 is

localized at the vacuolar membrane of root cells, and it was

suggested to be important for sequestration of Al into the

vacuoles. However, this transporter does not have any similarity

to STAR1/2, suggesting that ALS1 and STAR1/2 have different

functions in Al tolerance.

Transport substrate experiments showed that the STAR1-

STAR2 complex had efflux transport activity for UDP-Glc (Fig-

ures 7B and 7C). Furthermore, the transporter is specific to this

substrate (Figures 7B and 7C; see Supplemental Figures 11 to 13

online). UDP-Glc is an activated form of glucose used as a

substrate for glycosyltransferases to synthesize various glyco-

sides (Ross et al., 2001). Although the link between the STAR1-

STAR2 complex and Al tolerance remains to be examined in

more detail in the future, there are two possibilities for the

involvement of UDP-Glc in Al tolerance. First, secreted UDP-Glc

may function as a chelator, like organic acid anions, and there-

fore detoxify Al by forming a nontoxic complex with it. However,

this possibility is unlikely because addition of exogenous UDP-

Glc did not alleviate Al-induced inhibition of root elongation in

wild-type rice (Figure 7D). Second, STAR1-STAR2 located on the

membrane may be responsible for transporting UDP-Glc from

the cytosol into the vesicles. UDP-Glc or glycoside derived from

UDP-Glc would then be released from the vesicles to the

apoplast by exocytosis and used to modify the cell walls to

mask the sites for aluminum binding, resulting in aluminum

tolerance in rice. This hypothesis is supported by the morin

staining result (see Supplemental Figure 7 online). Morin reacts

with aluminum to form a stable fluorescent complex by two

coordination sites (Jones et al., 2006). Aluminum has six coor-

dination sites. It has been reported that Al bound by the cell wall

could not be stained with morin (Eticha et al., 2005). Therefore,

the absence of morin staining in wild-type roots suggests that

aluminum has been masked by glycosides from UDP-Glc. This

hypothesis is also supported by the observations that glucose

accumulates in star1mutant roots (Figure 7A; see Supplemental

Figure 10 online) and that expression of UDP-glucosyltransferase

genes is upregulated in the star1 mutant (see Supplemental

Figures 8 and 9 online). The alleviating effect of exogenous UDP-

Glc further supports the hypothesis that transport of UDP-Glc by

STAR1-STAR2 is required for aluminum tolerance in rice.

A number of QTL for Al tolerance in rice have been detected

on various chromosomes (Ma and Furukawa, 2003; Xue et al.,

Figure 7. Physiological Changes in the star1 Mutant and Transport Activity of UDP-Glc.

(A) Soluble sugar concentration in the root. Roots exposed (+Al) or not (�Al) to 20 mM Al for 6 h were cut into a segment 0 to 2 cm from the tip. Soluble

sugar in the root cells was determined after freeze thawing. Data are means 6 SD of biological replicates (n = 3).

(B) Efflux transport activity for UDP-Glc. Oocytes that had been injected with mixed STAR1 and STAR2 or water were preloaded with 50 nL of 1 mM 14C-

labeled UDP-Glc (11.1 MBq mmol�1) or UDP-glucuronic acid (6.7 MBq mmol�1). Data are means 6 SD of biological replicates (n = 3 to 4).

(C) Substrate specificity. Oocyte two-electrode voltage clamp analysis was performed in oocytes expressing both STAR1 and STAR2. I/V relationships

were recorded in oocytes preloaded with water, UDP-Glc, UDP-glucoronic acid (UDP-GlcA), or UDP-galactose (UDP-Gal). Means 6 SD of biological

replicates (n = 12) are shown.

(D) Effect of exogenous UDP-Glc on the root growth. Seedlings were exposed to a solution containing 0 or 20 mM Al in the presence or absence of 500

mM UDP-Glc. Root elongation over a 24-h period was measured. Data are means 6 SD of biological replicates (n = 10).


2007). However, the position of STAR1 and STAR2 is different

from these reported QTL. Therefore, STAR1/2 may represent a

basal Al tolerance in rice. Based on the phylogenetic analysis

(Figures 1D and 3B; see Supplemental Data Sets 1 and 2 online),

genes similar to STAR1 and STAR2 are present in other species,

such as maize, Arabidopsis, grape, and, interestingly, also in P.

patens, a model organism of initial land plants (Figures 1D and

3B). This suggests that STAR1/2 is a universal Al tolerance

mechanism conserved in land plants, although it remains to be

investigated in plant species other than rice in future.

In conclusion, our results show that STAR1 interacts with

STAR2 to form a complex that functions as a bacterial-type ABC

transporter. The complex transports UDP-Glc and is required for

Al tolerance in rice.

METHODS

Plant Materials and Growth Conditions

The rice (Oryza sativa) mutant star1 (formerly als1) was previously isolated

from M3 seeds of Koshihikari mutagenized with g-ray irradiation (Ma

et al., 2005). Seeds of both wild-type rice and the star1 mutant were

soaked in deionized water overnight at 308C in the dark and then

transferred to a net floating on a 0.5 mM CaCl2 solution in a 1.5-liter

plastic container. Seedlings were grown for 4 to 7 d at 258C before being

used for various experiments. For root elongation measurement, the root

length of each seedling was measured with a ruler before and after the

treatments. Relative root elongation was calculated as follows: (root

elongation with Al treatment)/(root elongation without Al) 3 100.

Cloning of STAR1 and STAR2

STAR1 was cloned by the map-based cloning technique. We used an F2

population generated from a cross between star1 and Kasalath. We used

a total of 716 F2 plants with increased sensitivity to Al for mapping, which

were isolated from an F2 population based on relative root elongation.

Polymorphic markers were developed based on the rice InDel database

at http://shenghuan.shnu.edu.cn/genefunction/ricemarker.htm (Shen

et al., 2004b) and the comparison of the genomic sequence of Nippon-

bare with that of 93-11 by BLASTn searches in GenBank.

The coding sequences of the candidate genes predicted in the TIGR

website in the 88-kb candidate region were amplified in the star1 mutant

and in wild-type rice (cv Koshihikari) and sequenced using the ABI PRISM

310 Genetic Analyzer and the BigDye Terminators v3.1 cycle sequencing

kit (Applied Biosystems). The putative ABC transporter gene was inde-

pendently sequenced twice in the mutant.

STAR2 was amplified from rice root cDNA by PCR with primers

designed based on the rice genome database as described below.

Phylogenetic Analysis

Alignments were performed with ClustalW using default settings (http://

clustalw.ddbj.nig.ac.jp/), and phylogenetic trees were constructed using

the neighbor-joining algorithm with MEGA version 4 (Tamura et al., 2007)

with 1000 bootstrap trials.

Generation of Transgenic Rice

For the complementation test of STAR1, we obtained a BAC clone

(BAC3771) containing STAR1by screening a BAC library of Koshihikari.

After partial enzymatic cutting with Sau3AI, we selected a 5.4-kb clone

containing STAR1 and its promoter. This clone was inserted into the

pPZP2H-lac vector (Fuse et al., 2001) and then transformed into Agro-

bacterium tumefaciens (Strain EHA101). Calluses derived from the rice

mutant star1 were transformed by Agrobacterium-mediated transforma-

tion (Hiei et al., 1994). We obtained two independent transgenic lines and

a vector control line and thenmeasured root elongation over a 24-h period

in a 0.5 mM CaCl2 solution containing 0 or 20 mM Al, pH 4.5. We also

cultured these lines in acidic soil (Andosol; pH 4.8) for 10 d.

To generate the hairpin RNAi construct, we cloned a 411-bp fragment

(706 to 1116 bases from the transcriptional start) of two copies of STAR2

cDNA as inverted repeats into the pANDA vector under control of maize

ubiquitin1 promoter (Miki and Shimamoto, 2004) and subsequently

introduced this construct into calluses (cv Nipponbare) as described by

Toki et al. (2006). We measured the expression level of STAR2 using real-

time PCR as described below.We also evaluated the aluminum tolerance

in two independent RNAi transgenic and vector control lines by measur-

ing the root elongation before and after treatment in the absence or

presence of 100 mM aluminum.

RNA Isolation and RT-PCR

To examine the expression pattern of STAR1 and STAR2, we exposed

seedlings of the wild type to different aluminum concentrations (0 to 50

mM) for different periods of time, to different pH, and to a variety ofmetals,

namely Cd and La. Then, root tips (0 to 1 cm), basal roots (1 to 2 cm), or

whole roots and shoots were cut and frozen in liquid nitrogen within 5 min

of harvest. Total RNA was extracted using the RNeasy mini kit (Qiagen).

One microgram of total RNA was used for first-strand cDNA synthesis

using a SuperScript II kit (Invitrogen) and an oligo(dT)12-18 primer

(Invitrogen) following the manufacturer’s instructions. One-twentieth of

the reaction volume was used as template for the PCR amplification

of STAR1 and STAR2, and Actin1 was used as an internal control

(27 cycles). The primer sequences for RT-PCR of STAR1 were

59-TCGCATTGGCTCGCACCCT-39 and 59-TCGTCTTCTTCAG CCGCAC-

GAT-39, and the forward and reverse sequences used to amplify Actin1

were 59-GACTCTGGTGATGGTGTCAGC-39 and 59-GGCTGGAAGAG-

GACCTCAGG-39, respectively. For real-time RT-PCR analysis, 1 mL of

a 10-fold dilution of cDNA from each sample was used for the quantitative

analysis of gene expression using SYBR Premix Ex Taq (Takara). Histone

H3 (forward primer, 59-AGTTTGGTCGCTCTCGATTTCG-39; reverse

primer, 59-TCAACAAGTTGACCACGTCACG-39) was used as an internal

control in this experiment. Data were collected in accordance with the

7500 real time PCR system (Applied Biosystems).

Construction of Fluorescent Gene Fusion

For construction of a translational STAR1-GFP fusion with the STAR1

promoter, a 1.8-kb upstream region of STAR1 and the open reading

frame (ORF) of STAR1 except for the stop codon were amplified by PCR

from rice (cv Koshihikari) genomic DNA and root cDNA, respectively.

Primer pairs used for amplification and introduction of restriction sites

were 59-TTCTAGAGGATGCACTTTTTGTATGGTG -39 and 59-TTCTA-

GATCGCCGTGGTCGGTTTGGA-39 for the STAR1 promoter, and

59-GTTGGTGATCACTGTGTAGATCG-39 and 59-TTTCATGAGTCCGCT-

GAGCTCGAGGAAGCGC-39 for the STAR1 ORF. The promoter and

ORF were cloned GFP and the NOS terminator in pBluescript vector.

For construction of a translational fusion of STAR2 and DsRed, we

amplified the ORF of STAR2 andDsRed by PCR from rice (cv Koshihikari)

root cDNA and the pDsRed-Monomer vector (Takara Bio). Primer pairs

used for amplification and introduction of the restriction sites were

59-AAAGTCGACGCATGATGGCGAGCATGG-39 and 59-TTTCAT-

GACTCCGTCGGCGGCGAAGACGGC-39 for the STAR2 ORF and

59-TACCGGTCGCCACCATGGAC-39 and 59-TTTCTAGACTGGGA-

GCCGGAGTGGCG-39 for DsRed-Monomer. The STAR2 ORF and

664 The Plant Cell

DsRed-Monomer were cloned between the 35S promoter and NOS

terminator in the pBluescript vector.

BiFC Analysis of the Interaction between STAR1 and STAR2

Coding sequences of the N terminus and C terminus half of EYFP without

the stop codon, derived from nonstop EYFP-N in pUC19 and EYFP-C in

pUC19 (Abe et al., 2005), respectively, were introduced between the

cauliflower mosaic virus 35S promoter and the NOS terminator in the

pBluescript vector. ORFs of STAR1 and SATR2 were introduced after

the EYFP-C and EYFP-N sequences, respectively. These plasmids,

pEYFP-C-STAR1, pEYFP-N-STAR2, and control plasmids carrying 35S-

EYFP-C or 35S-EYFP-N alone, were transiently introduced into rice

callus protoplasts using the polyethylene glycol method according to

Kamiya et al. (2006). After overnight incubation, YFP fluorescence was

observed by a fluorescencemicroscope (Axio Imager with Apotome; Carl

Zeiss).

Protein Interaction Analysis in Yeast Using a

DUALmembrane System

Protein interaction between STAR1 and STAR2 was examined in yeast

using the DUALmembrane Kit 3, which takes advantage of a split-

ubiquitin system, according to the manufacturer’s protocol (Dualsystems

Biotech). Briefly, the ORF of the wild-type STAR1 or mutant star1 was

amplified by PCR using the following primers (forward 59-GGCCAT-

TACGGCCATGCGCATTGCCCGGATTCC-39 and reverse 59-GGC-

CGAGGCGGCCTTGCTGAGCTCGAGGAAGCGCC-39), and the PCR

product was cloned into the pGEM T-easy vector. The derived plasmids

were cleaved using the SfiI enzyme, and the generated fragment was

cloned in frame into the pBT3-STE vector (containing C-terminal half of

ubiquitin). The derived plasmid was verified by enzyme analysis and DNA

sequencing. STAR2 was cloned into the pPR3-STE vector (the mutated

N-terminal half of ubiquitin) by the same method. Forward and reverse

primers for amplification of STAR2 ORF were 59-GGCCATTACGGCCC-

GATGATGGCGAGCATGGCGGC-39 and 59-GGCCGAGGCGGCCGA-

GTCGGCGGCGAAGACGGCGT-39, respectively. The two constructs

and control vectors were cotransformed into the NMY51 yeast strain,

and the transformants were selected on SD-Trp-Leu media. Positive

clones were confirmed by PCR and cultured in SD-Trp-Leu liquid media

to the early log phase for growth assays. Ten microliters of transformed

yeast, with an OD value of 0.5, and 1:10 dilutions of this yeast, were

spotted on SD-Trp-Leu plates or selective plates (SD-Trp-Leu-His-Ade)

containing 10 mM 3-aminnotriazole and grown at 308C for 3 d.

Coexpression in Onion Epidermal Cells

We observed the subcellular localization of STAR1-GFP and STAR2-

DsRed and the protein interaction between STAR1-GFP and STAR2-

DsRed by transient expression in onion epidermal cells. Onion epidermal

cells were bombarded with 1-mmgold particles coated with plasmid DNA

carrying STAR1-GFP or star1-GFP and STAR2-DsRed and incubated in

the dark at 258C for 20 h. We observed fluorescence by confocal laser

scanning microscopy (LSM510; Carl Zeiss).

Localization of STAR1-GFP in Transgenic Rice

The construct carrying the STAR1 promoter, STAR1-GFP, and the NOS-

terminator were subcloned into the pPZP2H-lac binary vector (Fuse et al.,

2001) and subsequently introduced into rice calluses derived from wild-

type rice cv Nipponbare by means of Agrobacterium-mediated transfor-

mation, as described above. The GFP signal was observed by confocal

laser scanning microscopy (LSM510; Carl Zeiss).

Protein Gel Blot Analysis

Membrane proteins were prepared from the roots of both wild-type rice

and themutant star1, according to themethods of Sugiyama et al. (2007).

The samples (;20 g) were homogenized in 30 mL of ice-cold homog-

enization buffer consisting of 100 mM Tris-HCl, pH 8.0, 150 mM KCl,

0.5% (w/v) polyvinylpolypyrrolidone, 5 mM EDTA, 3.3 mM DTT, 1 mM

phenylmethylsulfonyl fluoride, and 10% (v/v) glycerol. After filtration, the

homogenates were centrifuged at 8000g for 10 min to yield the super-

natant and centrifuged again under the same conditions. The superna-

tants were then ultracentrifuged at 100,000g for 40 min. The pellets were

resuspended in a small volume of resuspension buffer containing 10 mM

Tris-HCl, pH 7.6, 10% (v/v) glycerol, 1 mM EDTA, and 1/100 protease

inhibitor cocktail and fractionated through a 20, 30, 40, 50, and 60% (w/v)

discontinuous sucrose gradient with 10 mM Tris-HCl, pH 7.6, 1 mM

EDTA, and 1 mM DTT by ultracentrifugation at 100,000g for 120 min. The

fractionated membranes were recovered by ultracentrifugation at

100,000g for 40 min. Each pellet was resuspended in resuspension

buffer supplemented with 1/100 volume of Protease Inhibitor Cocktail for

plant cell and tissue extracts (Sigma-Aldrich) and 1 mM DTT.

The synthetic peptide C-KIRVRGLTRRSEA (positions 111 to 123 of

STAR1) was used to immunize rabbits to obtain antibodies against

STAR1. The obtained antiserum was purified through the peptide affinity

column before use. Protein concentrations were measured by the

Bradford method (Bradford, 1976). Equal amounts of samples were

mixed with same volume of sample buffer containing 250 mM Tris-HCl,

pH 6.8, 8% (w/v) SDS, 40% (v/v) glycerol, 0.01% (w/v) bromophenol blue,

and 200 mM b-mercaptoethanol. The mixture was incubated at 378C for

60 min for Lsi1 and at 658C for 10 min for STAR1, and SDS-PAGE was

performed using 11% polyacrylamide gels containing 0.1% SDS. The

transfer to polyvinylidene difluoride membrane was performed with a

semidry blotting system, and the membrane was treated with purified

primary rabbit anti-STAR1 and anti-Lsi1 polyclonal antibodies diluted at

1:500 or 1:100, respectively. Anti-rabbit IgG HRP conjugate (1:10,000

dilution; GEHealthcare) was used as a secondary antibody, and ECL plus

(GE Healthcare) was used for detection via chemiluminescence.

Immunohistological Staining

For immunostaining, the anti-STAR2 antibody was used in addition

to the anti-STAR1 antibody described above. The synthetic peptide

C-AGQRARHVPRGKHI (positions 107 to 120 of STAR2) was used to

immunize rabbits to produce antibodies against STAR2. The roots of

1-week-oldseedlings (cvKoshihikari) treatedor notwithAl (20mM,6h)were

used for immunostaining as described previously (Yamaji and Ma, 2007).

Histological Staining of Aluminum

One-week-old wild-type (Koshihikari) and star1 mutant rice seedlings

were exposed to a 0.5 mM CaCl2 solution containing 30 mM aluminum.

After 9 h, the roots were sliced to 200 mm thickness with a razor blade in

distilled water and then stained in a 100 mM morin solution for 10 min at

room temperature. Stained sections were washed twice with distilled

water and observed by confocal laser scanning microscopy (LSM510;

Carl Zeiss).

Microarray Analysis

Wild-type rice (cv Koshihikari) and star1 mutant were grown in 0.5 mM

CaCl2 for 4 d. Root tips (0 to 1 cm) of 20 to 30 plants were sampled after

the roots were exposed to a solution containing 0 or 20 mM AlCl3 for 6 h,

and tips were then frozen in liquid nitrogen until used. Total RNA was

isolated using the RNeasy plant mini kit (Qiagen). Microarray analysis was

performed according to Agilent Oligo DNA Microarray Hybridization


protocols using the Rice Oligo DNA Microarray 44K RAP-DB (Agilent

Technologies) with three biological replicates. The microarrays were

scanned using a DNA microarray scanner (Agilent Technologies). Signal

intensities were detected by Feature Extraction software (Agilent Tech-

nologies).

Transport Activity in Xenopus laevisOocytes

We cloned the ORF of STAR1 and STAR2 individually into the pXBG-ev1

vector and performed in vitro transcription from them. We injected the

capped cRNA of STAR1, STAR2, mixed STAR1 and STAR2 (50 nL, 1 ng

nL21), or distilled water into Xenopus oocytes, selected according to size

and development stage (Ma et al., 2006). The efflux transport activity for

UDP-Glc or UDP-glucuronic acid was determined by directly injecting 50

nL of 1 mM 14C-labeled UDP-Glc (Perkin-Elmer; 11.1 MBq mmol21) or

UDP-glucuronic acid (Perkin-Elmer; 6.7 MBq mmol21) into the oocytes

(four oocytes per replicate). The oocytes were then washed with MBS

(modified Birth’s saline) to remove extracellular radioactivity and trans-

ferred to a 500-mL aliquot of fresh MBS at 188C. After 2 h, the incubation

medium was carefully sampled, and at the end of the experiment, the

oocytes were homogenized with 0.1 N HNO3. The radioactivity of the

incubation medium and the oocytes was determined using a liquid

scintillation counter (Liquid Scintillation System; Aloka).

For two-electrode voltage clamp analysis, whole-cell currents from

oocytes expressing STAR1, STAR2, or both STAR1 and STAR2 were

recorded with an Oocyte Clamp OC-725C (Warner Instruments) using

conventional two-electrode voltage clamp techniques. The output signal

was digitized and analyzed using Lab-Trax 4/16 and DataTrax software,

respectively (World Precision Instruments). Recording electrodes filled

with 3 M KCl had resistances between 1 and 4 MV. Data were recorded

from oocytes that were preloaded with water, UDP-Glc, UDP-glucoronic

acid, or UDP-galactose, by injecting 50 nL of water or 25 mM solutions

each, 1 to 3 h prior to recording. Measurements were performed in MBS

with or without 2 mM UDP-Glc at room temperature.

Measurement of Aluminum Concentration and Soluble Sugar

To compare the Al content in different compartments of the roots of wild-

type and star1 plants, seedlings were exposed to 20 mM Al for 6 h and

then 20 roots per replicate were excised into two segments (0 to 1 cm and

1 to 2 cm). The cell wall was extracted as described previously (Ma et al.,

2004). The Al concentration in the root segments and cell wall was

measured using a graphite furnace atomic absorption spectrophotome-

ter (model Z-8200; Hitachi) after the samples were dissolved in 2 M HCl.

Root symplast solution was obtained by centrifuging the frozen-

thawed root samples. Soluble sugar in the symplast solution was deter-

mined by the anthrone colorimetry method.

Effect of Exogenous UDP-Glc on Aluminum Tolerance

Seedlings (5 d old) of both wild-type rice and the star1 mutant were

exposed to a 0.5 mM CaCl2 solution containing 0 or 20 mM aluminum in

the presence or absence of 500 mMUDP-Glc for 1 d. Before and after the

treatments, the root length was measured with a ruler and the root

elongation was calculated.

Accession Numbers

Sequence data from this article can be found in the GenBank/DDBJ/

EMBL data libraries under accession numbers AB253626 (STAR1) and

AB379845 (STAR2).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. STAR1 Sequence, Conserved Motifs, and

star1 Mutation.

Supplemental Figure 2. STAR1-GFP Localization in Onion Epidermal

Cells.

Supplemental Figure 3. Alignment of STAR2 and ALS3.

Supplemental Figure 4. Expression of STAR1 and STAR2 in Different

Root Segments.

Supplemental Figure 5. BiFC Analysis of Interaction between STAR1

and STAR2.

Supplemental Figure 6. Al Content in the Root Segment and Cell

Wall.

Supplemental Figure 7. Morin Staining of the Wild-Type and star1

Root.

Supplemental Figure 8. Microarray Analysis of UDP-Glucosyltrans-

ferase Genes.

Supplemental Figure 9. RT-PCR of UDP-Glucosyltransferase Genes.

Supplemental Figure 10. Determination of Root Soluble Sugar

Components.

Supplemental Figures 11 to 13. Oocyte Two-Electrode Voltage

Clamp Analysis.

Supplemental Data Set 1. Sequences Used to Generate the Phy-

logeny Presented in Figure 1D.

Supplemental Data Set 2. Sequences Used to Generate the Phy-

logeny in Figure 3B.

ACKNOWLEDGMENTS

This research was supported by a grant from the Ministry of Agriculture,

Forestry, and Fisheries of Japan (Genomics for Agricultural Innovation

IPG-006 and QT3006 to J.F.M.) and by a Grant-in-Aid for Scientific

Research from the Ministry of Education, Culture, Sports, Science, and

Technology of Japan (18380052 to J.F.M.). We thank Ko Shimamoto for

providing pANDA vector, Tadashi Ishii and Naoki Sakurai for instruction

in cell wall sugar analysis, Fangjie Zhao for critical reading, Yasufumi

Daimon for kindly providing the plasmid vectors for BiFC analysis, and

Ritsuko Motoyama for assistance in microarray analysis.

Received November 21, 2008; revised January 6, 2009; accepted Feb-

ruary 12, 2009; published February 24, 2009.

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DOI 10.1105/tpc.108.064543; originally published online February 24, 2009; 2009;21;655-667Plant Cell

MaChao Feng Huang, Naoki Yamaji, Namiki Mitani, Masahiro Yano, Yoshiaki Nagamura and Jian Feng

A Bacterial-Type ABC Transporter Is Involved in Aluminum Tolerance in Rice

This information is current as of February 28, 2021

Supplemental Data /content/suppl/2009/02/12/tpc.108.064543.DC1.html

References /content/21/2/655.full.html#ref-list-1

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