DWARF27, an Iron-Containing Protein Required for the ... · auxin cannot directly enter the axillary buds and that a second messenger is required to inhibit the outgrowth of axillary

DWARF27, an Iron-Containing Protein Required for theBiosynthesis of Strigolactones, Regulates Rice TillerBud Outgrowth W OA

Hao Lin,a,1 Renxiao Wang,a,1,2 Qian Qian,b,1 Meixian Yan,b Xiangbing Meng,a,c Zhiming Fu,a,c Cunyu Yan,c

Biao Jiang,d Zhen Su,e Jiayang Li,a,c and Yonghong Wanga,c,3

a State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,

Beijing 100101, Chinab State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences,

Hangzhou 310006, Chinac National Center for Plant Gene Research (Beijing), Beijing 100101, Chinad Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, Chinae China Agricultural University, Beijing 100094, China

Tillering in rice (Oryza sativa) is one of the most important agronomic traits that determine grain yields. Previous studies on

rice tillering mutants have shown that the outgrowth of tiller buds in rice is regulated by a carotenoid-derived MAX/RMS/D

(more axillary branching) pathway, which may be conserved in higher plants. Strigolactones, a group of terpenoid lactones,

have been recently identified as products of the MAX/RMS/D pathway that inhibits axillary bud outgrowth. We report here

the molecular genetic characterization of d27, a classic rice mutant exhibiting increased tillers and reduced plant height.

D27 encodes a novel iron-containing protein that localizes in chloroplasts and is expressed mainly in vascular cells of

shoots and roots. The phenotype of d27 is correlated with enhanced polar auxin transport. The phenotypes of the d27 d10

double mutant are similar to those of d10, a mutant defective in the ortholog of MAX4/RMS1 in rice. In addition, 29-epi-5-deoxystrigol, an identified strigolactone in root exudates of rice seedlings, was undetectable in d27, and the phenotypes of

d27 could be rescued by supplementation with GR24, a synthetic strigolactone analog. Our results demonstrate that D27 is

involved in the MAX/RMS/D pathway, in which D27 acts as a newmember participating in the biosynthesis of strigolactones.

INTRODUCTION

Shoot branching plays an important role in determining the

diversity of plant architectures. In higher plants, branches are

derived from shoot apical meristems (SAMs). The primary SAM

provides the main axis of the plant body, while the secondary

SAMs in the axils of leaves generate axillary meristems (AMs)

(McSteen and Leyser, 2005). Formation of branches generally

comprises two distinct steps: the formation of AMs in the leaf

axils and the outgrowth of axillary buds (Shimizu-Sato and Mori,

2001). However, after initiation, an AM can arrest its growth and

form a dormant bud, which will be released in response to

particular environmental and/or developmental signals (Wang

and Li, 2008).

In some plant species, the outgrowth of axillary buds may be

inhibited by the primary shoot, a phenomenon known as apical

dominance (Sachs and Thimann, 1964; Cline, 1991). The plant

hormone auxin has long been implicated in participating in this

process (Thimann and Skoog, 1934; Cline, 1991; Leyser, 2003).

Indole-3-acetic acid (IAA) is the most abundant natural plant

auxin and is synthesized mainly in the shoot apex and young

leaves. It is transported along the shoot-root axis from cell to cell

in a polar manner, which is essential for inhibiting the outgrowth

of axillary buds (Ljung et al., 2001; Leyser, 2003; Sieberer and

Leyser, 2006). However, a large body of evidence suggests that

auxin cannot directly enter the axillary buds and that a second

messenger is required to inhibit the outgrowth of axillary buds

(Shelagh and John, 1975;Morris, 1977; Pilate et al., 1989; Prasad

et al., 1993; Booker et al., 2003). Cytokinin is the first reported

second messenger candidate, which is synthesized in roots and

transported acropetally in the xylem to promote directly the

outgrowth of axillary buds (Van Dijck et al., 1988; Cline, 1991;

Eklof et al., 1997; Kapchina-Toteva et al., 2000; Nordstrom et al.,

2004). Exogenous application of cytokinin to axillary buds pro-

motes their outgrowth (Sachs and Thimann, 1964; Cline, 1991).

Increased cytokinin levels lead to reduced apical dominance in

Arabidopsis thaliana (Tantikanjana et al., 2001; Jung et al., 2005).

It is plausible that auxin suppresses the outgrowth of axillary

buds by influencing the supply of cytokinin to axillary buds (Eklof

1 These authors contributed equally to this work.2 Current address: Institutes of Biology II, Albert-Ludwigs-Universitat ofFreiburg, Schanzlestrasse 1, D-79104 Freiburg, Germany.3 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: Yonghong Wang([email protected]).WOnline version contains Web-only data.OAOpen access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.109.065987

The Plant Cell, Vol. 21: 1512–1525, May 2009, www.plantcell.org ã 2009 American Society of Plant Biologists

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Figure 1. Morphological Comparison between Wild-Type and d27 Plants.

(A) The phenotype of wild-type (Shiokari) (left) and d27 seedling (right). Arrow indicates the first tiller in d27, which is absent in wild type at this stage.

Bar = 1 cm.

(B) Developmental process of tiller buds at the axil of the first leaves in the wild type (top panel) and d27 (bottom panel), showing the accelerated tiller

D27, a New Member of the MAX Pathway 1513

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et al., 2000; Li and Bangerth, 2003; Nordstrom et al., 2004;

Tanaka et al., 2006).

Recent studies on a series of branching mutants,more axillary

growth (max) of Arabidopsis, ramosus (rms) mutants of pea

(Pisum sativum), decreased apical dominance (dad) mutants of

petunia (Petunia hybrida), have revealed an additional carotenoid-

derived hormone as a second messenger of auxin action on the

regulation of AM outgrowth (Beveridge et al., 1994, 1996, 2000;

Napoli, 1996; Stirnberg et al., 2002; Sorefan et al., 2003; Booker

et al., 2004, 2005; Simons et al., 2007). Genetic analysis and

grafting experiments on these mutants have shown that this kind

of carotenoid derivative, synthesized in the root and transported

acropetally or synthesized locally, represses branch outgrowth

(Beveridge et al., 1997; Morris et al., 2001; Turnbull et al., 2002;

Sorefan et al., 2003). In Arabidopsis, four MAX loci, MAX1 to

MAX4, have been identified and proved to be involved in a

common signaling pathway, known as theMAXpathway (Booker

et al., 2005). MAX1, a cytochrome P450 family member, acts

downstream of MAX3 and MAX4, two carotenoid cleavage

deoxygenase (CCD) family proteins, CCD7 and CCD8, in the

biosynthesis of the signal (Sorefan et al., 2003; Booker et al.,

2004, 2005; Lazar and Goodman, 2006). By contrast, MAX2

encodes an F-box protein, which is responsible for perceiving

and transducing the signal (Stirnberg et al., 2002, 2007). In pea,

RMS4, RMS5, and RMS1 are orthologous of MAX2, MAX3, and

MAX4, respectively (Sorefan et al., 2003; Johnson et al., 2006).

Although the outgrowth behaviors between dicotyledonous

and monocotyledonous AMs are apparently different, the fact

that orthologs ofMAX2 toMAX4 have also been identified in rice

(Oryza sativa) suggests that monocots and dicots share a con-

served MAX-involved carotenoid-derived branching signal path-

way (Wang and Li, 2008). Rice plants defective inD3,HTD1/D17,

and D10, which correspond to Arabidopsis MAX2, MAX3, and

MAX4, respectively, give rise to more tillers and reduced plant

height (Ishikawa et al., 2005; Zou et al., 2006; Arite et al., 2007),

indicating their similar functions in suppressing the branch

development in monocotyledonous plants. Thereafter, this

carotenoid-derived branching inhibiting signal pathway was

generally known as the MAX/RMS/D pathway.

Recently, two groups have independently reported that the

MAX/RMS/D pathway is involved in the production and signaling

of strigolactones (Gomez-Roldan et al., 2008; Umehara et al.,

2008). Strigolactones, synthesized from carotenoids, are a group

of terpenoid lactones that have been found in root exudates of

diverse plant species (Cook et al., 1972; Bouwmeester et al.,

2003; Matusova et al., 2005; Humphrey and Beale, 2006; Lopez-

Raez et al., 2008). Mutations of CCD7 or CCD8 in pea, rice, and

Arabidopsis results in reduced strigolactones production. By

contrast, the signaling mutant d3, which is defective in the

ortholog of Arabidopsis MAX2, accumulates higher levels of

strigolactones. Furthermore, application of strigolactones in-

hibits shoot branching in the ccd mutants of pea, rice, and

Arabidopsis, but it has no effect on rms4/max2/d3 signaling

mutants (Gomez-Roldan et al., 2008; Umehara et al., 2008).

These data strongly suggest that strigolactones act as a new

class of phytohormones involved in regulating plant branching.

Strigolactones have been identified previously as seed germi-

nation stimulants of root parasitic plants (Cook et al., 1972;

Bouwmeester et al., 2003; Humphrey and Beale, 2006) and

hyphal branching signals when plants interact with mycorrhizal

fungi (Akiyama et al., 2005). However, the strigolactone biosyn-

thesis and signaling pathways still remain to be elucidated.

Identification of new branching mutants and isolation of their

genes in multiple plant systems will facilitate the elucidation of

the biosynthesis and signaling pathways of this new type of

hormone in plants.

Tillering in rice is one of the most important agronomic traits

that determine grain yields and a model system for elucidating

molecular mechanisms that regulate axillary buds (Wang and Li,

2005). In this study, we characterize a rice dwarf 27 (d27) mutant

that is defective in the outgrowth of axillary buds. Map-based

cloning and in-depth analysis of D27 revealed that it encodes a

novel chloroplast-located iron-containing protein. Our results

demonstrate that D27 regulates tiller bud outgrowth through the

MAX/RMS/D pathway and participates in the biosynthesis of

strigolactones.

RESULTS

Phenotypes of the Rice Tillering Dwarf Mutant d27

The rice d27mutant is a classic rice mutant described previously

(Ishikawa et al., 2005; Arite et al., 2007). To elucidate the

molecular mechanism that determines rice tiller number, we

further characterized d27 in depth. At the seedling stage, d27

Figure 1. (continued).

bud elongation in d27. The pictures were photographed when the third leaf was 0, 2, 4, and 6 cm in length. Arrows indicate the examined tiller buds.

Bars = 1 mm.

(C) Phenotype of wild-type (left) and d27 (right) plants at the heading stage. Bar = 10 cm.

(D) Kinetic analyses of the elongation of tiller buds at the axil of the first leaves indicated in (B). Each value represents the mean 6 SE of 15 replicates.

(E) Kinetic comparison of tiller numbers between wild-type and d27 plants at different developmental stages. Each value represents themean6 SE of 15

replicates.

(F) The types of tillers at the heading stage. Pt, primary tillers; St, secondary tillers; Tt, tertiary tillers; Qt, quaternary tillers. Each value represents the

mean 6 SE of 15 replicates.

(G) to (J) Cross sections of the wild type ([G] and [I]) and d27 ([H] and [J]) culms. Bars = 100 mm.

(I) and (J) are magnifications of indicated regions in (G) and (H), respectively. Bars = 50 mm.

(K) Morphological comparison of flag leaves between the wild type (left) and d27 (right). Bar = 1 cm.

(L) and (M) Micrographs of cleared flag leaves from the wild type (L) and d27 (M). The regions indicated in (K) are shown. Bars = 100 mm.

1514 The Plant Cell

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exhibits accelerated tiller production (Figure 1A). Kinetic analysis

of the tiller bud development demonstrated that the increased

tiller number is ascribed to accelerated tiller bud outgrowth

rather than to more tiller bud formation (Figures 1B and 1D; see

Supplemental Figure 1 online). At the mature stage, the d27

mutant plant showed a high tillering and severe dwarf phenotype

(Figure 1C). Kinetic analysis showed that the final tiller number of

d27 is three times that of the wild type (Figure 1E), which results

from the outgrowth of higher-order tillers (Figure 1F). Further

histological analysis suggested that the cell number and size are

both reduced in d27mutant culms and leaves, leading to severe

dwarfism in the mutant plant (Figures 1G to 1M).

Cloning and Characterization of D27

To isolate theD27gene, we took amap-based cloning approach.

D27 was primarily delimited in an interval of;3.0 centimorgans

between the two molecular markers C189 and RM206 on the

long arm of chromosome 11 (Figure 2A). To fine-map the D27

locus, we generated a large F2 mapping population derived

from a cross between d27-ZF802 and its isogenetic lines ZF802.

Of 21,000 F2 plants, 5200 mutant plants were used for fine-

mapping, and the D27 locus was located between the two

cleaved amplified polymorphic sequence (CAPS) markers P1

and P7 (Figure 2B). Screening with newly developed molecular

markers P1 to P7 (see Supplemental Table 1 online), D27 was

further placed within an 18-kb DNA region between the P3 and

P6 markers and cosegregated with the P5 marker (Figure 2C).

Within this region, there are two open reading frames (ORFs).

Sequencing of these two ORFs of d27-ZF802 revealed a

4-bp deletion at the fourth exon of a putative gene, ORF

LOC_Os11g37650, and this deletion results in a frame shift and

generates a premature translation termination product (see

Supplemental Figure 2 online).

The identity of D27 was further confirmed by a genetic com-

plementation test. The plasmid pD27C, containing a 9.25-kb

genomic DNA fragment consisting of a 2236-bp upstream se-

quence, the entire D27 gene including seven exons and six

introns, and a 2044-bp downstream region (Figure 2D), was

introduced into a d27-Nipponbare mutant. All four transgenic

lines of pD27C complement the d27 phenotype (Figure 2E).

Therefore, ORF LOC_Os11g37650 is the rice D27 gene, and its

4-bp deletion is responsible for the altered phenotype of d27.

D27 Encodes a Novel Iron-Containing Protein

Sequence analysis of 59- and 39-rapid amplification of cDNA

ends (RACE) products indicated that the full length of D27 cDNA

is 1254-bp long, with anORF of 837 bp, a 217-bp 59-untranslatedregion, and a 200-bp 39-untranslated region (see Supplemental

Figure 2 online). Sequence comparison between genomic DNA

and cDNAs revealed that D27 is composed of seven exons that

encodes a 278–amino acid polypeptide (Figure 2C; see Supple-

mental Figure 2 online). The 4-bp deletion in d27 results in a

premature translational product (Figure 3A; see Supplemental

Figure 2 online). The BLASTP (Altschul et al., 1997) analysis

revealed that D27 shares no homology with any functionally

identified protein and contains no conserved domain. However,

analysis of multiple alignment against the National Center for

Biotechnology Information database and The Institute for Ge-

nomic Research (TIGR) plant transcript assemblies showed that

D27 has homologies in many plant species, from lower plants to

higher plants (see Supplemental Figure 3 online), suggesting that

D27 may play a basic role in plants.

Interestingly, when we tried to express and purify recombinant

D27, we found that the bacterial cells expressing the maltose

Figure 2. Map-Based Cloning of D27.

(A) The D27 locus was mapped on the long arm of chromosome 11

between markers C189 and RM206.

(B) A BAC contig spanning the D27 locus. The numerals indicate the

number of recombinants identified from 5300 F2 mutant plants. BAC1,

AC137588; BAC2, AC104847; BAC3, AC136148; BAC4, AC146334.

(C) Fine-mapping of D27 with markers developed based on the

AC136148 sequence. The D27 gene was narrowed to an 18-kb genomic

DNA region between CAPS markers P3 and P6 and cosegregated with

marker P5. LOC_Os11g37650 is the candidate for D27.

(D) The complementation plasmid containing the entire D27 (pD27C).

(E) Phenotypic comparison among Nipponbare, d27-Nipponbare, and

the transgenic line harboring pD27C. Bar = 5 cm.


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binding protein (MBP)-D27 fusion protein were strikingly brown

in color, as was the purified MBP-D27 fusion protein (Figure 3B).

This result suggested that D27 is very likely to have a cofactor. To

explore this possibility, we analyzed the recombinant D27 with

inductively coupled plasma mass spectrometry (ICP-MS) and

found that the recombinant MBP-D27 protein contains ;1.7

mole of iron permole of protein, in contrast with an extremely low

level of iron bound to the C-terminal truncated polypeptide,

MBP-D271-187 (Figure 3C, Table 1), which is equivalent to the

mutated form of D27. The binding of iron to D27 was further

confirmed by characterizing the absorbtion spectrum of the

MBP-D27 fusion protein, which showed a specific peak at 420

nm, a characteristic for the presence of iron (Figure 3D). Fur-

thermore, when the recombinant MBP-D27 protein was treated

with the reducing agent dithionite, the peak at 420 nm exhibited a

dramatic decrease (Figure 3D), indicating that D27 is indeed an

iron-containing protein. Moreover, the purified recombinant

D27 protein contained no significant amount of other metals

(Table 1), suggesting that the binding of iron to D27 is specific.

Taken together, all these results indicate that D27 is an authentic

iron-containing protein in plants.

Expression Patterns of D27 and Subcellular Localization of

the D27 Protein

Real-time PCR analysis revealed that the D27 expression level is

high in axillary buds and young panicles, medium in shoot bases

and culms, and low in roots, sheaths, and leaves (Figure 4A). The

tissue-specific expression pattern of D27 was further examined

using mRNA in situ hybridization. D27 was predominantly ex-

pressed in young leaves (Figure 4B), axillary buds (Figure 4C),

inflorescence promodia (Figure 4D), lateral roots (Figure 4E), and

crown roots (Figure 4H). Furthermore, D27 expression was

detected in vascular cells at the shoot apex of the main stem

and young leaves (Figures 4F and 4I), in the nodal vascular

anastomosis (Figure 4G), and in large and small vascular bundles

of the internodes (Figure 4J).

To determine the subcellular localization of the D27 protein, we

performed a transient expression experiment of D27 in rice leaf

protoplasts. The C terminus of D27 was fused with green

fluorescent protein (GFP) under the control of cauliflower mosaic

virus (CaMV) 35S promoter, and the construct was transferred

into rice leaf protoplasts by the polyethylene glycol–mediated

method. In contrast with the control, which was ubiquitous in

protoplast cells, the D27-GFP fusion protein was predominantly

localized in chloroplasts (Figure 4K).

Enhanced Polar Auxin Transport in d27

Shoot branching has been reported to be correlated to polar

auxin transport (PAT) in Arabidopsis and pea (Morris, 1977;

Beveridge et al., 2000; Bennett et al., 2006; Dai et al., 2006). We

therefore investigated whether D27 is involved in PAT in rice. By

comparing the basipetal and acropetal IAA transport in upper-

most internodes between thewild-type and d27 plants, we found

that basipetal PAT in d27 was significantly elevated, whereas

acropetal PAT of 3H-IAA and basipetal transport of 3H-IAA

treated with the PAT inhibitor N-1-naphthylphtalamic acid

(NPA) showed no significant difference between the wild-type

and mutant plants (Figure 5A).

To investigate whether the increased auxin transport is related

to the d27 mutant phenotype, we further examined the effect of

NPA on d27 seedlings in hydroponic culture. Two-week-old

seedlings were treated with various concentrations of NPA. As

shown in Figures 5B and 5C, the tiller number of d27 mutant

plants was largely rescued when grown in the presence of 1.5

mM NPA for 5 weeks. Furthermore, when 2-week-old wild-type

and d27 seedlings were treated with as low as 0.5 mM NPA, the

Figure 3. Characterization of the D27 Protein.

(A) Schematic representation of D27 and D271-187 (premature D27).

(B) Escherichia coli strains (top panel) expressing MBP-D27 and

MBP-D271-187 and purified MBP-D27 and MBP-D271-187 proteins

(bottom panel), showing the brown color of D27 proteins.

(C) Iron contents bound to MBP, MBP-D27, and MBP- D271-187. Values

are means 6 SE of three independent experiments.

(D) UV visible absorbance spectra of purified MBP-D27 and MBP-

D271-187 proteins before (blue and red) and after (purple and green)

reduction with dithionite.

Table 1. Metal Contents of MBP-D27 Protein Determined by

ICP-MS Analysis

Metal

Moles of Metal per Mole of Protein

MBP MBP-D27 MBP-D271-187

Iron 0.01 6 0.01 1.74 6 0.04 0.17 6 0.01

Calcium 0.55 6 0.03 0.46 6 0.01 0.49 6 0.01

Chromium 0.01 6 0.01 0.03 6 0.01 0.02 6 0.01

Copper 0.01 6 0.01 0.02 6 0.01 0.02 6 0.01

Nickel 0.01 6 0.01 0.01 6 0.01 0.01 6 0.01

Magnesium <0.001 <0.001 <0.001

Manganese <0.001 <0.001 <0.001

Zinc <0.001 <0.001 <0.001

Values are means 6 SE of three independent experiments.

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Figure 4. Expression Patterns of D27 and Subcellular Localization of the D27 Protein.


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outgrowth of tiller buds of the wild type was significantly pro-

moted, which is consistent with the long-established concept

that too little auxin transport also leads to an increased shoot

branching (Chatfield et al., 2000). By contrast, the treatment of

NPA showed a remarkable inhibition to the tiller outgrowth of the

d27 seedlings, suggesting that the tillering phenotype of d27may

be correlated with an enhanced PAT.

D27 May Function through the MAX/RMS/D Pathway

In rice, one class of tillering dwarf mutants with an increased tiller

number and reduced plant height has been previously reported

(Kinoshita and Takahashi, 1991). Among them, d3,d17/htd1, and

d10 are found to function as their orthologs of MAX2/RMS4,

MAX3/RMS5, and MAX4/RMS1/DAD1 in Arabidopsis, pea, and

Petunia, respectively (Stirnberg et al., 2002; Sorefan et al., 2003;

Booker et al., 2004; Foo et al., 2005; Ishikawa et al., 2005;

Snowden et al., 2005; Johnson et al., 2006; Zou et al., 2006; Arite

et al., 2007). The phenotype of d27 and the involvement ofD27 in

PAT prompted us to test whether D27 is a new member of the

MAX/RMS/D pathway in rice. We therefore generated a d27 d10

double mutant and compared the phenotypes of single and

double mutants of d27, d10, and d27 d10. As shown in Figure 6,

d10 exhibits similar phenotype to d27, but has more tillers and a

more severe dwarf stature than d27 (Figures 6A and 6B). Phe-

notypic analysis revealed that the d27 d10 double mutant

showed similar tiller number and plant height to d10 (Figures

6A to 6D). Further investigation on the responses of d27, d10,

and d27 d10 to the treatment of different NPA concentrations

indicated that the d27 d10 double mutant has a similar response

to d10 (Figures 6E and 6F). These results strongly suggested that

D27 participates in the MAX/RMS/D pathway.

D27 Participates in the Biosynthesis of Strigolactones

Recent studies have shown that the proposed novel hormones

that inhibit plant branching and are derived from theMAX/RMS/D

pathway are strigolactones or their downstream metabolites.

MAX1, MAX3/RMS5/D17, and MAX4/RMS1/D10 are involved in

the biosynthesis of strigolactones, while MAX2/RMS4/D3 is

involved in strigolactone signaling (Gomez-Roldan et al., 2008;

Umehara et al., 2008). To further understand the role ofD27 in the

MAX/RMS/D pathway in rice, we investigated whether d27 is

deficient in strigolactone production or signaling. By applying 1.0

mM GR24, a synthetic strigolactone analog that acts as native

strigolactones, to wild-type and d27 seedlings in a hydroponic

culture, we found that the exogenous supplement of GR24 was

able to fully inhibit tiller bud outgrowth of 3-week-old d27

seedlings (Figure 7A), and continuous treatment for 7 weeks

fully restored the tillering dwarf phenotype of d27 (Figure 7B).

We further analyzed and compared the strigolactones pro-

duced in the root exudates of wild-type and d27 seedlings by

liquid chromatography–quadruple/time-of-flight tandem mass

spectrometry (LC/MS-MS). Our results clearly showed that

29-epi-5-deoxystrigol (epi-5DS), an identified strigolactone in

the hydroponic culture media of rice seedlings, was produced in

the wild-type cultivar Shiokari root exudates but was undetect-

able in d27 (Figure 7C). Moreover, we performed a highly

sensitive germination assay using Orobanche minor seeds to

estimate the strigolactone production in d27 root exudates. In

agreement with LC/MS-MS data, the germination-stimulating

activity of d27 root exudates dramatically decreased in compar-

ison with the wild type (Figure 7D; see Supplemental Figure 4

online). These results indicate that D27 is required for the

production of strigolactones in rice.

DISCUSSION

In higher plants, the degree and pattern of branching are major

determinants of plant architecture. Although there has been

significant progress in the characterization of branching mutants

and the understanding of corresponding regulatory genes has

been achieved recently, the molecular mechanisms underlying

plant branching remain to be elucidated. A rice tiller is a spe-

cialized grain-bearing branch that grows independently of the

main culmby its own adventitious roots. Tillering in rice is not only

an important agronomic trait for grain yield, but also an ideal

system for studying branching in higher plants, especially for

monocotyledonous species. In this article, we reported the

cloning of D27 and functional characterization of its mechanism

underlying the axillary tiller development, showing that D27

suppresses the outgrowth of tiller buds by producing strigolac-

tones.

D27 Is a Novel Iron binding Protein That Localizes

in Chloroplasts

Although the bioinformatic analysis shows that D27 encodes a

novel protein with no homology to any functionally known

Figure 4. (continued).

(A) D27 expression levels revealed by real-time PCR in various organs, including roots (R), culms (C), sheaths (Sh), leaves (L), panicles (P), shoot bases

(SB), and axillary buds (AB). Values are means 6 SE of three independent experiments.

(B) to (J) D27 expression patterns revealed by mRNA in situ hybridization. The cross sections of the vegetative shoot apexes show the expression of

D27 in young leaves (B) and axillary buds (C). The longitudinal section of an inflorescence meristem at the secondary branch differentiation stage shows

the expression of D27 in the inflorescence promodia (D). The cross sections of roots show the D27 expression in lateral roots (E) and the steles of crown

roots (H). The cross sections of the unelongated stem internodes ([F] and [I]), nodes (G), and internodes (J) of elongated culms indicate the expression

of D27 in vasculature tissues. (I) is the magnification image of the squared region in (F). Arrows indicate the expression sites of D27. YL, young leaf; LS,

leaf shealth; AB, axillary bud; IF, inflorescence; SA, shoot apex; NV, nodal vascular anastomosis; LR, lateral root; LB, large vascular bundle; SB, small

vascular bundle; ST, stele. Bars = 100 mm in (B) to (E) and (H) and 200 mm in (F), (G), (I), and (J).

(K) Subcellular localization of 35S:GFP (top panel) and 35S:D27-GFP (bottom panel) in rice protoplast cells. Bars = 5 mm.

1518 The Plant Cell

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proteins, D27 homologs are found from algae (cyanophyta) to

higher plants, but not in animals or fungi (see Supplemental

Figure 3 online), suggesting that D27 may be a plant-specific

protein. Analysis of the transient expression in rice protoplasts

demonstrates that the D27 protein is localized in chloroplasts,

similar to MAX3 and MAX4/D10 (Booker et al., 2004; Auldridge

et al., 2006; Arite et al., 2007).

Our data also showed that D27 is an iron-containing protein

and that the truncated D27 loses the ability to bind iron (Figure

3C). Bacterial cells expressing truncated MBP-D271-187 are

colorless, and the purified protein does not have a 420-nm

peak. By contrast, the full-length MBP-D27 fusion protein is

Figure 5. Comparison of PAT and Tillering Responses upon NPA

Treatments between Wild-Type and d27 Plants.

(A) Comparison of PAT between the wild type (Shiokari) and d27 in the

uppermost internodes. The acropetal auxin transport measurement is

used as a negative control. Values are means 6 SE of three independent

experiments. The asterisk represents significance difference between

the wild type (Shiokari) and d27 determined by the Student9s test at

P < 0.05.

(B) The phenotypic comparison between wild-type (ZF802) and d27

plants upon 5-week 1.5 mM NPA treatment. Bar = 5 cm.

(C) Comparison of tillering upon 5-week NPA treatment between wild-

type (ZF802) and d27 seedlings. Each value represents the mean6 SE of

15 seedlings.

Figure 6. Phenotypic Analyses of the d27 d10 Double Mutant.

(A) and (B) Phenotypes of wild-type, d27, d10, and d27 d10 at the

seeding stage (A) and at the heading stage (B). Red, white, and blue

arrows indicate first, second, and third tillers, respectively. Bars = 2 cm in

(A) and 10 cm in (B).

(C) Kinetic tillering analyses of d27, d10, and d27 d10 plants at different

developmental stages. DAG, days after germination. Each value repre-

sents the mean 6 SE of 15 seedlings.

(D) Dwarf phenotype of d27, d10, and d27 d10 plants. Internode length

was measured after harvest. P, panicle; I to IV, nodes numbered from top

to bottom. Each value represents the mean 6 SE of 15 seedlings.

(E) and (F) Comparison of tiller number (E) and plant height (F) of

13-week-old plants in response to NPA treatment at various concentra-

tions. Each value represents the mean 6 SE of 15 seedlings.


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brown and has the characteristic absorbance at 420 nm (Figures

3B and 3D), suggesting that the C terminus of D27 may contain

an iron binding domain.

D27 Suppresses the Outgrowth of Rice Tiller Buds through

the MAX Pathway

The development of shoot branching occurs in two steps, the

initiation of the AM and the outgrowth of axillary buds. Unlike

the elusivemolecular mechanism that regulates AM initiation, the

outgrowth of axillary buds is well understood due to the recent

breakthrough in theMAX/RMS/D pathway. Studies on a number

of mutants that display excess axillary branches, max in Arabi-

dopsis (Stirnberg et al., 2002, 2007; Sorefan et al., 2003; Booker

et al., 2004), rms in pea (Beveridge et al., 1994, 1996, 2000; Foo

et al., 2001, 2005; Morris et al., 2001; Sorefan et al., 2003), and

dad in petunia (Napoli, 1996; Snowden et al., 2005; Simons et al.,

2007), have revealed the existence of a carotenoid-derived AM

outgrowth regulating pathway. Although the outgrowth behav-

iors between dicotyledonous and monocotyledonous axillary

buds are different (for reviews, see McSteen and Leyser, 2005;

Wang and Li, 2008), they appear to share a conserved branching

signal pathway because orthologs of MAX2/RMS4, MAX3/

RMS5, and MAX4/RMS1 have also been identified in rice; they

are D3, HTD/D17, and D10, respectively (Ishikawa et al., 2005;

Zou et al., 2006; Arite et al., 2007). Rice plants harboring

individual loss-of-function mutations in these genes lead to

more tillers and reduced plant height, a similar phenotype to

those in Arabidopsis and pea, indicating their conserved func-

tions in suppressing branch development in monocotyledonous

plants.

The more tillers phenotype of d27 is ascribed to the extensive

outgrowth of tiller buds, especially to the higher-order tiller buds,

which are dormant in the wild-type plants (Figures 1A to 1F). The

comparable morphology of d27 to that of the rice tillering dwarf

mutant d3, htd1/d17, or d10 prompted us to test the hypothesis

that D27 is also involved in the MAX/RMS/D pathway. The

analysis of the double mutant d27 d10 confirms the hypothesis.

In the phenotypes tested, including tillering behavior, plant

height, and response to NPA treatment, d27 d10 resembles

d10 (Figure 6), suggesting that D27 may function the same as

D10 in the MAX/RMS/D pathway. In agreement with this, D27 is

expressed in roots and shoots, especially in the vasculature

tissue of the plants (Figures 4B to 4J), an expression pattern

similar to those of D10 and HTD1/D17. These results are con-

sistent with a role in the biosynthesis of strigolactones. Further

determination of strigolactone-related products in d27 and d10

will facilitate the understanding of the genetic relationship be-

tween D27 and D10.

D27 Is Required for the Biosynthesis of Strigolactones

The MAX/RMS/D pathway has been proven to interact with

classic plant hormones auxin and cytokinin, but all the evidence

obtained so far has demonstrated that the MAX/RMS/D

-dependent branching signals are not attributed to any known

hormones. Recent studies uncover the role of strigolactones

or their metabolites acting as a new class of branching hor-

mones, the signal derived from the MAX/RMS/D pathway

Figure 7. Analysis of Strigolactones in Wild-type and d27 Plants.

(A) Three-week-old wild-type (Shiokari) and d27 seedlings treated with

(bottom panel) or without (top panel) 1.0 mMGR24. Red and white arrows

indicate first and second tillers, respectively. Bars = 1.0 cm.

(B) Seven-week-old wild-type and d27 seedlings treated with (bottom

panel) or without 1.0 mM (top panel) GR24. Red and white arrows indicate

first and second tillers, respectively. Bars = 5.0 cm.

(C) LC-MS/MS chromatograms for the standard epi-5DS (top trace) and

root exudates from wild-type (middle trace) and d27 (bottom trace)

seedlings. Arrows indicate the detection of epi-5DS.

(D) Germination rate of O. minor seeds 5 d after treatments with water,

GR24, or extracts of wild-type or d27 root exudates. Each value

represents the average of three replicates 6 SE.

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(Gomez-Roldan et al., 2008; Umehara et al., 2008). Although

previous studies have shown that strigolactones are derived

from the carotenoid pathway and function as important signals

in establishing the interaction between plants and mycorrhizal

fungi (Akiyama et al., 2005; Matusova et al., 2005), the biosyn-

thetic and signaling pathways of strigolactones are poorly

understood. MAX1, MAX3/RMS5/D17, and MAX4/RMS1/D10

are essential components for the biosynthesis of strigolac-

tones, whereas MAX2/RMS4/D3 is involved in the perception of

the signal. Our studies provide direct evidence that D27 is a new

component of the MAX/RMS/D pathway and plays an essential

role in biosynthesizing strigolactones. First, the d27 phenotype

can be restored to the wild type upon supplemention with GR24

(Figures 7A and 7B). Second, the d27 root extract contains

undetectable strigolactone, which is normally produced in the

wild-type root extract (Figure 7C). Third, unlike thewild type, the

d27 root exudates failed to stimulate the seed germination ofO.

minor (Figure 7D; see Supplemental Figure 4 online). Based on

the findings that the D27 protein is localized in chloroplasts and

contains iron and the fact that the complex structure of

strigolactones should undergo a number of enzymatic reac-

tions, including hydroxylation, epoxydation, oxidation, etc., to

achieve its biosynthesis (Matusova et al., 2005), we hypothesize

that D27 may participate in a redox reaction involved in the

biosynthesis of strigolactones. Further biochemical experi-

ments are required to confirm this possibility in the future.

Roles of Strigolactones and Auxin in Regulating

Shoot Branching

The discovery of strigolactones as a product of the MAX/RMS/D

pathway provides an opportunity to elucidate mechanisms of

shoot branching in higher plants. Currently, how strigolactones

and auxin interact to regulate shoot branching is still vague, and

two models have been proposed based on different experimen-

tal systems. One is the PAT hypothesis, which proposes that the

MAX/RMS/D pathway acts by regulating PIN-dependent auxin

transport in the stem, which inhibits auxin transport from buds

(Bennett et al., 2006; Ongaro and Leyser, 2008; Leyser, 2009).

The other proposes that strigolactones function as secondary

messengers of auxin that repress the bud outgrowth directly

(Beveridge et al., 2000; Brewer et al., 2009; Ferguson and

Beveridge, 2009). Our work on the rice tillering mutant d27 has

revealed that the mutation in D27 leads to a deficiency in

strigolactone biosynthesis and an increase in PAT, which are

consistent with previous studies on Arabidopsis and pea

(Beveridge et al., 2000; Bennett et al., 2006). However, it is still

unclear whether the enhanced PAT is a direct consequence or a

feedback effect of the deficiency in strigolactones. Moreover, it

should be pointed out that, in contrastwith the complete rescue of

the mutant phenotypes by the treatment with GR24 (Figure 7), the

dwarf phenotype of d27 is completely recovered to the wild type

by treatment with NPA (Figures 5B and 5C). These results suggest

that the action of strigolactones or auxin may not be in a simple

linear pathway. A full elucidation of the actions of strigolactones

and their interactions with auxin and other branching signals

awaits the identification of more novel MAX/RMS/D-dependent

branching mutants and corresponding inhibiting signals.

METHODS

Plant Materials

The d27 mutant is in a Shiokari background. In this study, we also

generated d27-ZF802 and d27-Nipponbaremutants by backcrossing the

d27 mutant plants with indica cultivar ZF802 and japonica variety

Nipponbare. The d10 mutant also has a Shiokari background. The d27

and d10 mutants were provided by Takamure Itsuro of Hokkaido Uni-

versity. Rice (Orzya sativa) plants were cultivated in the experimental field

at the Institute of Genetics and Developmental Biology in Beijing in the

natural growing seasons. For NPA (at indicated concentrations) and 1mM

GR24 treatment, germinated seeds were grown on a nylon net floating on

hydroponic solution in the greenhouse.

Mapping of D27

To map the D27 locus, the d27-ZF802 mutant was crossed to the wild

type (ZF802), and the genomic DNA from 5200 F2 progeny with the

mutant phenotype was extracted with a modified CTAB method de-

scribed previously (Mou et al., 2000). To fine-mapD27, the CAPSmarkers

were generated based on single nucleotide polymorphisms identified in

the sequence. The molecular lesion of d27-ZF802 was identified by PCR

amplification of the D27 genomic region from the wild-type and d27-

ZF802 mutant plants and comparison of their sequences using ClustalW

within Lasergene version 5.0 software (DNASTAR). The primer sequences

are listed in Supplemental Table 1 online.

Complementation of d27

The BAC clone OSJNBa0029K08 was digested with BamHI and KpnI to

generate a 9.25-kb genomic DNA fragment. The DNA fragment was

ligated to the BamHI and KpnI digested pCAMBIA1300 vector (CAMBIA),

forming pD27C,which contains a 2236-bp upstream sequence, the entire

D27 gene, and a 2044-bp downstream region. The pD27C plasmid was

introduced into Agrobacterium tumefaciens EHA105 by electroporation,

and the rice d27-Nipponbare mutant was transformed according to a

published method (Hiei et al., 1994). The phenotype was scored in T1

transformants and T2 progeny.

RT-PCR, RACE-PCR, and Real-Time PCR Analyses

Total RNA was prepared using a TRIzol kit according to the user manual

(Invitrogen). One microgram of total RNA was treated with DNase I and

used for cDNA synthesis with an RT kit (Promega). The 59- or 39-RACE of

D27 was performed using a SMART RACE cDNA amplification kit

according to the manufacturer’s instructions (Clontech). Real-time PCR

experiments were performed using gene-specific primers in a total

volume of 10 mL with 1 mL of the RT reactions, 1 mM gene-specific

primers D27EF and D27ER, and 5 mL SYBR Green Master mix (Applied

Biosystems) on anABI 7900 real-timePCRmachine (Applied Biosystems)

according to themanufacturer’s instructions. The riceUbiquitin gene was

used as the internal control. The relative expression levels of D27 in

various organs were compared with that in the root, after normalization

with Ubiquitin transcript and averaged from three biological replicates.

The primer sequences used for the above studies are listed in Supple-

mental Table 2 online.

Histological Analysis and mRNA in Situ Hybridization

Tissues of rice were fixed with 4% (w/v) paraformaldehyde at 48C

overnight, followed by a series of dehydration and infiltration, and

embedded in paraffin (Paraplast Plus; Sigma-Aldrich). The tissues were

sliced into 8- to 10-mmsectionswith amicrotome (Leica RM2145), affixed


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to microscope slides, and stainedwith Safranin O and Fast Green (Fisher)

according to Ruzin (1999). Sections were observed under bright field

through a microscope (Leica DMR) and photographed using a Micro

Color CCD camera (Apogee Instruments).

To investigate the morphology of the leaf blade epidermal cells,

samples were cleared in benzyl-benzoate-four-and-half fluid as previ-

ously described (Herr, 1982).

RNA in situ hybridization was performed as described previously (Li

et al., 2007) with minor modification. Briefly, the 14- to 760-bp region of

the D27 gene was amplified by gene-specific primers D27IF and D27IR

with BamHI and KpnI adaptors (see Supplemental Table 2 online) and

subcloned into the BamHI- and KpnI-digested pBluescript II SK+ vector

(Stratagene). The construct was used as the template to generate sense

and antisense RNA probes. Digoxigenin-labeled RNA probes were pre-

pared using a DIG Northern Starter Kit (Roche) according to the manu-

facturer’s instructions. Slides were observed under bright field through a

microscope (Leica DMR) and photographed with a Micro Color CCD

camera (Apogee Instruments).

Subcellular Localization of D27

To generate CaMV35S-GFP, aHindIII-NotI fragment containingGFPwas

amplified by primers GFPF and GFPR (see Supplemental Table 2 online)

using CaMV35S-sGFP (S65T)-NOS-39 cassette vector (Niwa et al., 1999).

The resultant fragment was cloned into the pET28a (Novagen) vector to

generate pET28a-GFP. The b-glucuronidase fragment of the pBI221

vector (Clontech) was replaced by the BamHI-NotI fragment of pET28a-

GFP to generate theCaMV35S-GFP construct. ABamHI-HindIII fragment

containing the coding region of D27 amplified by the primers D27F and

D27R (see Supplemental Table 2 online) was subcloned into the BamHI

and HindIII sites of CaMV35S-GFP to generate CaMV35S:D27-GFP. The

plasmids CaMV35S-GFP and CaMV35S:D27-GFP were introduced into

rice leaf protoplasts as described (Bart et al., 2006). After overnight

incubation in the dark, the GFP signal and chlorophyll autofluorescence

were examined under a confocalmicroscope at excitationwavelengths of

488 and 647 nm, respectively (FluoView 1000; Olympus).

PAT Assay

The PATwas assayed according to themethod described previously with

some minor modifications (Okada et al., 1991; Li et al., 2007). Briefly, the

apical or basal ends of the 20-mm excised segments from the uppermost

internode at the early heading stage (for basipetal or acropetal transport

assays, respectively) were incubated in 10 mL of half-strength Murashige

and Skoog liquid medium containing 0.35% phytogel and 0.1 mM3H-labeled IAA (American Radiolabeled Chemicals) in 1.5-mL Eppendorf

tubes in the dark at room temperature. NPA (10 mM) was added to the

mediumas indicated to block IAA active transport so that the IAA diffusion

levels could be compared between wild-type and mutant plants. After a

3-h incubation, 5-mm sections from the nonsubmerged ends of seg-

ments were excised and transferred into Eppendorf tubes containing 2

mL of scintillation liquid. After an 18-h incubation in 2 mL of scintillation

liquid, the radioactivity of each tube was counted by a liquid scintillation

counter (1450 MicroBeta TriLux; Perkin-Elmer).

Expression and Purification of MBP-D27 Fusion Proteins in

Escherichia coli

The D27 cDNAs corresponding to full length and amino acids 1 to 187

were each amplified by the primer sets listed in Supplemental Table 2

online and cloned into the EcoRI andBamHI sites of the E. coli expression

vector pMAL-c2 (New England Biolabs). Expression of MBP, MBP-D27,

and MBP-D271-187 in BL21 Rosetta cells (Stratagene) was induced with

0.1 mM isopropyl-1-thio-D-galactopyranoside at 168C for 18 h. Fusion

proteins were purified using amylose-affinity chromatography (New

England Biolabs) according to the manufacturer’s protocols and quan-

tified by the Bio-Rad protein assay reagent.

Metal Quantitation

The purified recombinant MBP, MBP-D27, and MBP-D271-187 proteins

were digested with 40% nitric acid on a heating block, after cooling the

metal contents of the digests were determined using a Thermo ICP-MS

XII. The optical spectra of recombinant D27-MBP and MBP-D271-187(;10 to 20 mg/mL protein in 50 mM Tris, pH 8.0, and 50 mM NaCl) were

measured from the near UV to the near IR (200 to 800 nm) on a Beckman

Coulter DU800. Chemical reduction of D27-MBP and MBP-D271-187proteins was achieved by adding 2 mM dithionite to the protein solution.

LC/MS-MS Analysis of epi-5DS and Germination Assay of

Orobanche minor

The strigolactone epi-5DS measurement and the O. minor germination

assay were performed according to the method described by Yoneyama

et al. (2008). The hydroponic culture media were collected and extracted

twice with ethyl acetate. The ethyl acetate phase was washed with 0.2 M

K2HPO4, dried over anhydrous Na2SO4, and concentrated in vacuo. The

extracts were dissolved in 50% (v/v) acetonitrile and were subjected to

LC/MS-MS analysis using a system consisting of a triple quadruple

tandem mass spectrometer (Quattro Premier XE; Waters MS Technolo-

gies) and an Acquity Ultra Performance Liquid Chromatograph (Acquity

UPLC;Waters) equippedwith a reverse phase column (BEH-C18, 2.13 50

mm, 1.7 mm; Waters). The mobile phase was changed from 30% (v/v)

acetonitrile to 40% and 70% (v/v) linearly in 6 and 15 min after the

injection, respectively, at a flow rate of 0.4 mL min21. The column

temperature was set to 258C. MS parameters were set to the following

values: desolvation gas flow 800 L·h21, capillary voltage 3800 V, cone

voltage 30 V, desolvation temperature 3508C, source temperature 1208C,

collision energy 15 V, using MRM 331.16 > 216.10 transition for the epi-

5DS detection, and 5 pg/mL epi-5DS in 50% (v/v) acetonitrile was used as

reference for the qualification of epi-5DS in the root exudate sample.

To obtain strigolactone-containing exudates for germination assays,

the 1.5 liters of hydroponic culture media were concentrated using Oasis

HLB columns (Waters) and eluted with 5 mL acetone. The exudates for

each bioassay were prepared by mixing 100 mL of the concentrated

eluates in acetone and 900 mL of water and evaporating the acetone in a

vacuum centrifuge. Deionized water and GR24 were used as negative

and positive controls, respectively.

Accession Numbers

The GenBank accession number for the rice Dwarf27 sequence reported

in this article is FJ641055. Sequence data from this article can be found in

the GenBank database and TIGR plant transcript assemblies database

(boldfaced) under the following accession numbers or plant TA identifier.

GenBank identification numbers and TIGR numbers are as follows:

Acaryochloris marina (Am): YP_001515237.1; Arabidopsis thaliana

(At): NP_680560.1; NP_564838.1; NP_973748.1; NP_563673.1;

TA47796_3702; Chlamydomonas reinhardtii (Cr): XP_001702558.1;

XP_001697941.1; Fragaria vesca (Fv): DY667171; Glycine max (Gm):

BI470614; Lactuca perennis (Lp): DW093521; Manihot esculenta (Me):

TA7061_3983; Medicago truncatula (Mt): TA29020_3880; Ostreococcus

lucimarinus (Ol): XP_001420448.1; XP_001421321.1; XP_001419261.1;

XP_001420823.1; Oryza sativa (Os): NP_001060847.1 (Os08g0114100);

NP_001054553.1 (Os05g0131100); EAZ41303.1 (OsJ_25811); D27,

ABA94460.1; EEC68482.1; EAY81567.1 (OsI_36731); EEC78459.1

(OsI_18326); Ostreococcus tauri (Ot): CAL57302.1; CAL55718.1;

CAL57640.1; CAL54767.1; Physcomitrella patens (Pp): XP_001755220.1;

1522 The Plant Cell

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XP_001752784.1; XP_001763276.1; XP 001,763,362.1; XP_

001767010.1; Picea sitchensis (Ps): ABK22858.1; ABK23534.1;

Phaeodactylum tricornutum (Pt): EEC48282.1; EEC51126.1; Selaginella

moellendorffii (Sm): DN838054; Synechococcus sp PCC 7335 (Sy):

YP_002711663.1; Triticum aestivum (Ta): TA111626_4565; Thermosyne-

chococcus elongates (Te): NP_682732.1; Taraxacum officinale (To):

DY820710; TA1119_50225; Triphysaria versicolor (Tv): DR172918;

TA4072_64093; Vitis vinifera (Vv): CAO40130.1; CAO62908.1;

CAO22611.1; Zea mays (Zm): NP_001144840.1; ACG26781.1;

ACG28622.1; Zingiber officinale (Zo): TA7516_94328.

Supplemental Data

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

Supplemental Figure 1. Comparison of Tiller Bud Formation be-

tween the Wild Type and d27.

Supplemental Figure 2. D27 cDNA Sequence and Its Deduced

Amino Acid Sequence.

Supplemental Figure 3. Multiple Sequence Alignment of the De-

duced Amino Acid Sequence of D27 with Its Homologs.

Supplemental Figure 4. Germination of O. minor Seeds 5 d after

Treatment with Root Exudate Extracts of Shiokari, d27, d10, Water

(Negative Control), or GR24 (Positive Control).

Supplemental Table 1. List of PCR-Based Molecular Markers

Developed in This Study.

Supplemental Table 2. Primer Sequences Used for D27 Analyses.

ACKNOWLEDGMENTS

We thank Zhijie Liu and Neil Shaw (Institute of Biophysics, Chinese

Academy of Sciences) for advice on protein purification, Jindong Zhao

(Institute of Hydrobiology, Chinese Academy of Sciences, and Peking Uni-

versity) for suggestions on protein analysis, Takamure Itsuro (Hokkaido

University) for providing d27 and d10 mutants, and Dun Li (Stony Brook

University) for the improvement of the English language. We also thank

Koichi Yoneyama and Xiaonan Xie (Utsunomiya University) for sharing

information on strigolactone analysis and kindly providing GR24 and

Kohki Akiyama (Osaka Prefecture University) for providing epi-5DS. This

work was supported by grants from the Ministry of Science and

Technology of China (2006AA10A101) and the National Natural Science

Foundation of China (90817108 and 30830009).

Received January 28, 2009; revised April 30, 2009; accepted May 7,

2009; published May 26, 2009.

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DWARF27, an Iron-Containing Protein Required for the ... · auxin cannot directly enter the axillary buds and that a second messenger is required to inhibit the outgrowth of axillary