Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis...

20
Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu, a,b,1 Wanqi Liang, a,1 Changsong Yin, a Xiao Cui, a Jie Zong, a Xing Wang, a Jianping Hu, c and Dabing Zhang a,b,2 a School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China b Bio-X Research Center, Key Laboratory of Genetics and Development and Neuropsychiatric Diseases, Ministry of Education, Shanghai Jiao Tong University, Shanghai 200240, China c Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824 The rice (Oryza sativa) floral homeotic C-class gene, MADS3, was previously shown to be required for stamen identity determination during early flower development. Here, we describe a role for MADS3 in regulating late anther development and pollen formation. Consistent with this role, MADS3 is highly expressed in the tapetum and microspores during late anther development, and a newly identified MADS3 mutant allele, mads3-4, displays defective anther walls, aborted microspores, and complete male sterility. During late anther development, mads3-4 exhibits oxidative stress-related phenotypes. Microarray analysis revealed expression level changes in many genes in mads3-4 anthers. Some of these genes encode proteins involved in reactive oxygen species (ROS) homeostasis; among them is MT-1-4b, which encodes a type 1 small Cys-rich and metal binding protein. In vivo and in vitro assays showed that MADS3 is associated with the promoter of MT-1-4b, and recombinant MT-1-4b has superoxide anion and hydroxyl radical scavenging activity. Reducing the expression of MT-1-4b causes decreased pollen fertility and an increased level of superoxide anion in transgenic plants. Our findings suggest that MADS3 is a key transcriptional regulator that functions in rice male reproductive development, at least in part, by modulating ROS levels through MT-1-4b. INTRODUCTION In higher plants, the formation of the stamen, the male repro- ductive organ consisting of anther and filament, is a complex biological process that includes stamen specification and de- velopment of specialized anther tissues in which the haploid microspores/pollens are generated (McCormick, 1993). Genetic and functional analyses of homeotic mutants with changed floral organ identities in the model dicot plants Arabidopsis thaliana and Antirrhinum majus led to the ABC model, which explains how the stamen is specified by the combinatorial action of class B, C, and E genes (Coen and Meyerowitz, 1991; Ditta et al., 2004). Most of the A, B, C, and E genes encode MIKC-type MADS box transcription factors, which contain a 60–amino acid MADS box domain in the N-terminal region, a less conserved intervening region of ;30 amino acids, a moderately conserved ;70–amino acid keratin-like domain involved in protein–protein interaction, and a highly variable C-terminal region of variable length (Cho et al., 1999; Egea-Cortines et al., 1999; Yang et al., 2003). Recent studies suggest that the ABC model can also partially explain how stamen determination is specified in the monocot plant rice (Oryza sativa; Nagasawa et al., 2003; Yamaguchi et al., 2006; Dreni et al., 2007; Ohmori et al., 2009; Zhang and Wilson, 2009; Li et al., 2010a). For example, a rice B-class gene, SUPERWOMEN1 (SPW1 or MADS16), which is orthologous to the Arabidopsis APETALA3 gene, has been shown to be crucial for stamen specification (Nagasawa et al., 2003). spw1 mutants show homeotic conversions of stamens to carpels and lodicules to palea/lemma-like structures. In Arabidopsis, the C-class gene AGAMOUS (AG) acts to specify stamen and carpel identities and floral meristem determinacy (Yanofsky et al., 1990; Bowman et al., 1991). Studies in rice identified two C-class MADS box genes, MADS3 and MADS58, that may have distinct functions in specifying stamen identity, with MADS3 playing a more impor- tant role (Yamaguchi et al., 2006). Other MADS box genes, such as SPOROCYTELESS/NOZZLE (SPL/NZZ) (Yang et al., 1999; Schiefthaler et al., 1999) and AG (Ito et al., 2004, 2007) from Arabidopsis and MADS2 from maize (Zea mays; Schreiber et al., 2004), have been implicated in regulating anther development. SPL/NZZ regulates the formation of anther walls and pollen mother cells, as the primary sporogenous cells cannot form pollen mother cells in spl anthers, thereby blocking early cell differentiation (Yang et al., 1999). AG has been shown to activate the expression of SPL/NZZ, suggesting that this gene is necessary for early stamen development (Ito et al., 2004). During later developmental stages, AG continues its expression in the anther and regulates anther dehiscence by directly regulating the expression of the gene that encodes a jasmonic acid (JA) 1 These authors contributed equally to this work. 2 Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Dabing Zhang ([email protected]). W Online version contains Web-only data. OA Open Access articles can be viewed online without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.110.074369 The Plant Cell, Vol. 23: 515–533, February 2011, www.plantcell.org ã 2011 American Society of Plant Biologists

Transcript of Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis...

Page 1: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

Rice MADS3 Regulates ROS Homeostasis during LateAnther Development W OA

Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a Xiao Cui,a Jie Zong,a Xing Wang,a Jianping Hu,c

and Dabing Zhanga,b,2

a School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, Chinab Bio-X Research Center, Key Laboratory of Genetics and Development and Neuropsychiatric Diseases, Ministry of Education,

Shanghai Jiao Tong University, Shanghai 200240, Chinac Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824

The rice (Oryza sativa) floral homeotic C-class gene, MADS3, was previously shown to be required for stamen identity

determination during early flower development. Here, we describe a role for MADS3 in regulating late anther development

and pollen formation. Consistent with this role, MADS3 is highly expressed in the tapetum and microspores during late

anther development, and a newly identified MADS3 mutant allele, mads3-4, displays defective anther walls, aborted

microspores, and complete male sterility. During late anther development, mads3-4 exhibits oxidative stress-related

phenotypes. Microarray analysis revealed expression level changes in many genes in mads3-4 anthers. Some of these

genes encode proteins involved in reactive oxygen species (ROS) homeostasis; among them is MT-1-4b, which encodes a

type 1 small Cys-rich and metal binding protein. In vivo and in vitro assays showed that MADS3 is associated with the

promoter of MT-1-4b, and recombinant MT-1-4b has superoxide anion and hydroxyl radical scavenging activity. Reducing

the expression of MT-1-4b causes decreased pollen fertility and an increased level of superoxide anion in transgenic plants.

Our findings suggest that MADS3 is a key transcriptional regulator that functions in rice male reproductive development, at

least in part, by modulating ROS levels through MT-1-4b.

INTRODUCTION

In higher plants, the formation of the stamen, the male repro-

ductive organ consisting of anther and filament, is a complex

biological process that includes stamen specification and de-

velopment of specialized anther tissues in which the haploid

microspores/pollens are generated (McCormick, 1993). Genetic

and functional analyses of homeotic mutants with changed floral

organ identities in the model dicot plants Arabidopsis thaliana

andAntirrhinummajus led to the ABCmodel, which explains how

the stamen is specified by the combinatorial action of class B, C,

and E genes (Coen and Meyerowitz, 1991; Ditta et al., 2004).

Most of the A, B, C, and E genes encode MIKC-type MADS box

transcription factors, which contain a 60–amino acid MADS box

domain in the N-terminal region, a less conserved intervening

region of;30 amino acids, amoderately conserved;70–amino

acid keratin-like domain involved in protein–protein interaction,

and a highly variable C-terminal region of variable length (Cho

et al., 1999; Egea-Cortines et al., 1999; Yang et al., 2003).

Recent studies suggest that the ABC model can also partially

explain how stamen determination is specified in the monocot

plant rice (Oryza sativa; Nagasawa et al., 2003; Yamaguchi et al.,

2006; Dreni et al., 2007; Ohmori et al., 2009; Zhang and Wilson,

2009; Li et al., 2010a). For example, a rice B-class gene,

SUPERWOMEN1 (SPW1 or MADS16), which is orthologous to

the Arabidopsis APETALA3 gene, has been shown to be crucial

for stamen specification (Nagasawa et al., 2003). spw1 mutants

show homeotic conversions of stamens to carpels and lodicules

to palea/lemma-like structures. In Arabidopsis, the C-class gene

AGAMOUS (AG) acts to specify stamen and carpel identities and

floral meristem determinacy (Yanofsky et al., 1990; Bowman

et al., 1991). Studies in rice identified two C-class MADS box

genes,MADS3 andMADS58, that may have distinct functions in

specifying stamen identity, with MADS3 playing a more impor-

tant role (Yamaguchi et al., 2006).

Other MADS box genes, such as SPOROCYTELESS/NOZZLE

(SPL/NZZ) (Yang et al., 1999; Schiefthaler et al., 1999) andAG (Ito

et al., 2004, 2007) from Arabidopsis andMADS2 frommaize (Zea

mays; Schreiber et al., 2004), have been implicated in regulating

anther development. SPL/NZZ regulates the formation of anther

walls and pollen mother cells, as the primary sporogenous cells

cannot form pollen mother cells in spl anthers, thereby blocking

early cell differentiation (Yang et al., 1999).AG has been shown to

activate the expression of SPL/NZZ, suggesting that this gene is

necessary for early stamen development (Ito et al., 2004). During

later developmental stages, AG continues its expression in the

anther and regulates anther dehiscence by directly regulating

the expression of the gene that encodes a jasmonic acid (JA)

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: Dabing Zhang([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.110.074369

The Plant Cell, Vol. 23: 515–533, February 2011, www.plantcell.org ã 2011 American Society of Plant Biologists

Page 2: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

synthetic enzyme, DEFECTIVE IN ANTHER DEHISCENCE1 (Ito

et al., 2007). MaizeMADS2 is required for anther dehiscence and

pollen maturation, and knockdown of MADS2 resulted in abor-

tion of anthers and defective pollen development (Schreiber

et al., 2004).

Reactive oxygen species (ROS) are generated in plants when

molecular dioxygen (O2) is used as a terminal electron acceptor,

creating molecules such as superoxide anion (O22), hydroper-

oxide radicals (OHd

), and hydrogen peroxide (H2O2). Most of

these molecules are toxic by-products of aerobic metabolism in

plants subjected to abiotic stresses, such as drought, salinity,

flooding, heat, and cold (Miller and Mittler, 2006). Plants have

evolved diverse protective mechanisms, including various en-

zymes and nonenzymatic systems, to adjust ROS levels (Mittler

et al., 2004). The ROS-scavenging enzyme system contains

superoxide dismutase, catalase, and peroxidase (Apel and Hirt,

2004). Superoxide dismutase is able to rapidly convert super-

oxide radicals to hydrogen peroxide and dioxygen, and the

generated hydrogen peroxide is then converted to water and

dioxygen by peroxidase and catalase (Gechev et al., 2006). The

so-called nonenzyme system includes low molecular mass

antioxidants, such as ascorbate, glutathione, carotenoids, and

metallothioneins (MTs), which are known to remove hydroxyl

radicals and singlet oxygen (Gechev et al., 2006). MTs are low

molecular weight, Cys-rich proteins; plant MTs are grouped into

four types (Robinson et al., 1993; Cobbett and Goldsbrough,

2002). Some rice MTs have been shown to be ROS scavengers.

For instance, recombinant MT2b protein has superoxide- and

hydroxyl radical-scavenging activities (Wong et al., 2004), and

reduction in MT2b expression caused accumulation of ROS,

which triggers epidermal cell death in rice roots (Steffens and

Sauter, 2009).

ROS can also serve as important signaling molecules that

participate in a diverse range of plant processes, such as root

hair development and elongation, leaf expansion, apical domi-

nance, tracheary element maturation, trichome development,

senescence, and response to biotic and abiotic stress (Rodrıguez

et al., 2002; Foreman et al., 2003; Overmyer et al., 2003;

Sagi et al., 2004; Gapper and Dolan, 2006; Gechev et al., 2006;

Miller et al., 2008). Emerging evidence indicates that some ROS

species, such as the superoxide anion radical and hydrogen

peroxide, are central regulators of cell death in plants (Moeder

et al., 2002; Overmyer et al., 2003; Bouchez et al., 2007). The

cellular level of ROS is thus tightly regulated by an efficient and

elaborate system, which modulates the production and scav-

enging of ROS. However, how plants regulate ROS levels

according to cellular needs at different developmental stages

and within different cell types and organs remains poorly under-

stood.

In this study, we show that MADS3 has a critical role in

regulating rice late anther development via modulating ROS

homeostasis. MADS3 is expressed during late anther develop-

ment, in the tapetum and microspores. The rice mads3-4 mu-

tant is male sterile, contains aborted anther walls, and shows

disrupted pollen development due to oxidative stress. The ab-

normal increase in ROS level, peroxisome-like organelles, su-

peroxide dismutase, and peroxidase activities in the mads3-4

mutant during later anther development is likely the result of the

altered expression of genes involved in maintaining ROS level.

Moreover, using chromatin immunoprecipitation (ChIP) and the

electrophoretic mobility shift assay (EMSA), we demonstrated

that MADS3 is able to bind to the promoter region of a metal-

lothionein gene, MT-1-4b, which encodes a small Cys-rich and

metal binding protein in a ROS-scavenging pathway. These

findings together provide insight into the role of MADS3 in reg-

ulating male reproductive development and show that it acts,

at least in part, through regulating ROS homeostasis.

RESULTS

Isolation of amads3 Allele with Complete Male Sterility

We identified a complete male sterile mutant from a rice mutant

library, which had been generated by treating seeds of rice

cultivar 9522 (Oryza sativa ssp Japonica) with 60Co g-ray radia-

tion (Chen et al., 2006; Li et al., 2006, 2010b). Thismutant exhibits

normal vegetative and panicle development (Figures 1A and 1B),

yet its flowers have ectopic lodicules in whorl 2 and homeotic

transformation of stamens into lodicule-like organs in whorl 3

(Figures 1C to 1F; see Supplemental Table 1 online). The mutant

anthers seem to form a normal tetrad at stage 8 of anther

development (see Supplemental Figures 1A and 1B online) and

have a yellow appearance with bicellular pollen grains at stage

11, just like wild-type flowers (Figures 1C and 1E; see Supple-

mental Figures 1B and 1E online). However, the anthers start to

turn brown at stage 12 (Figures 1D and 1F). At stage 12, the

mutant contains degenerated pollen grains that are unable to

enter or complete the second mitosis (Figures 1G and 1H; see

Supplemental Figures 1C and 1F online). Transverse section

analysis of the mutant anthers revealed a brown color that is

caused by degenerated tapetum and pollen grains (Figures 1I

and 1J). Although its flowers occasionally develop more carpels

in whorl 4 (see Supplemental Table 1 online), themutant is female

fertile, as shown by reciprocal cross analysis. When the mutant

was backcrossed with a wild-type plant, all F1 plants were fertile

and F2 plants showed a phenotype segregation of 3:1 (fertility:

sterility = 239:81), suggesting that this phenotype is most likely

caused by a single recessive mutation.

A map-based cloning approach was used to clone the gene

responsible for the mutant phenotype. First, we mapped the

mutation roughly to a genetic distance between two markers

OS103 (25.4 centimorgans) and ZH104 (28.4 centimorgans) on

chromosome 1 (Figure 2A). Subsequently, by screening 1183

individuals of the F2 mapping population with six pairs of PCR

primers (see Supplemental Table 2 online), the mutation was

narrowed down to a region of 18 kb between two markers,

FQ120 and FQ121 (Figure 2A). Through repeated sequencing of

the 18-kb region in the mutant, we confirmed a deletion of two

sequential nucleotides (TA) in the fifth exon of MADS3, which

causes a frame shift and premature translational termination in

the 137th amino acid within the K domain (Figure 2B). Expression

of the full-length cDNA fragment of MADS3 under the control of

the cauliflower mosaic virus (CaMV) 35S promoter restored

fertility to the homozygous mutant plants (see Supplemental

Figure 2 online), confirming that MADS3 is responsible for the

516 The Plant Cell

Page 3: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

mutant phenotype. This mutant was thus termed mads3-4 be-

cause three alleles of MADS3 had been identified previously

(Yamaguchi et al., 2006). Similar to the previously described

strong allele mads3-3, mads3-4 has altered floral organ identi-

ties, but overall the defects seem weaker than those inmads3-3

(Yamaguchi et al., 2006).

MADS3 Is Expressed Strongly in the Tapetum during Late

Anther Development

Yamaguchi et al. (2006) showed that the expression ofMADS3 is

detectable in stamen primordia, when the lemma and palea

primordia initiate, but disappears soon after the appearance of

the stamen primordia. However,mads3-4 seems to show strong

defects in late anther development (Figures 1C to 1F). Thus, we

performed RT-PCR analysis, a b-glucuronidase (GUS) assay,

and in situ hybridization to test whether MADS3 is expressed

during late anther development. RT-PCR detected no obvious

expression of MADS3 in vegetative organs, nonreproductive

floral organs (i.e., glumes, lemma, and palea) (Figure 3A). By

contrast, an increase in MADS3 expression was detected in

anthers starting from stage 9, when young microspores form.

The expression of MADS3 peaked at stage 11, when the bicel-

lular pollen forms, and decreased at stage 12 (Figure 3A). In

addition, theMADS3 transcript was weakly detected in the pistil

at the heading stage (Figure 3A). Consistent with the RT-PCR

data, transgenic rice plants expressing the GUS gene driven by

the MADS3 promoter (;3.1 kb) exhibited GUS activity from

stage 9 to 12 during anther development (Figure 3B). GUS

activity was also observed in the stigma of the pistil (Figure 3C).

Furthermore, transverse anther sections showed GUS expres-

sion in tapetal cells and microspores (Figures 3D and 3E). Using

in situ hybridization analysis, we confirmed the expression of

MADS3 in the tapetum and microspores from stage 9 to stage

12, with stronger expression in the tapetum (Figures 3F to 3H).

Only background-level signals were observed in the control

(Figure 3I). Taken together, these results suggested thatMADS3

likely plays a role in the late stages of anther development.

mads3-4 Has Defects in Late Anther Development

To further characterize the role ofMADS3 in anther development,

we performed a detailed anther morphological analysis. The

mads3-4 mutant undergoes normal meiosis, as revealed by

4’,6-diamidino-2-phenylindole (DAPI) staining (see Supplemen-

tal Figures 1A and 1D online). Semithin section analysis revealed

no obvious phenotypic alterations inmads3-4 at stage 9 (Figures

4A and 4E). At stage 10, wild-type anthers develop condensed

and deeply stained tapetal cells and form vacuolated micro-

spores (Figure 4B). Although mads3-4 had vacuolated micro-

spores, the tapetum seemed less condensed andweakly stained

(Figure 4F). At stage 11, wild-type anthers formed falcate-

shaped pollen with degenerated tapetal cells (Figure 4C),

whereas the mads3-4 anther wall layers, including the tapetum,

became disordered, enlarged, and broken, and microspores

appeared degraded and irregularly shaped (Figure 4G). At stage

12, thewild-type anther epidermiswas collapsed, and themiddle

layer and endothecium were mostly degraded (Figure 4D). The

mature pollen grains were deeply stained, indicating that starch,

lipids, and other nutrient materials were stored (Figure 4D). By

contrast, the mads3-4 anther still had the anther wall layers,

including the endothecium, degenerated tapetal cells, microspores,

Figure 1. Phenotypic Analysis of mads3-4.

(A) Plant after bolting.

(B) Panicle at the heading stage.

(C) to (F) Flowers at stage 11 ([C] and [E]) and stage 12 ([D] and [F]) (Zhang and Wilson, 2009).

(G) and (H) Pollen grains at stage 12 stained by I2-KI.

(I) and (J) Transverse sections of fresh anthers prepared from flowers like those in (D) and (F) without the staining treatment.

MP, mature pollen; Msp, microspore; T, tapetum; WT, wild type. Bars = 5 mm in (C) to (F), 50 mm in (G) and (H), and 10 mm in (I) and (J).

MADS3 Regulates Anther Development 517

Page 4: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

and abundant granular objects in the microspores and at the

edge of the anther locule (Figure 4H). These results suggested

that MADS3 is required for the proper development of anthers.

mads3-4 Anthers Display Features of Cells Undergoing

Oxidative Stress

Transmission electron microscopy (TEM) analysis detected no

obvious differences between wild-type and mads3-4 anthers at

stage 9. Both types of plants contained tapetal layers with band-

like structures, abundant plastids, endoplasmic reticulum, and

mitochondria, a number of Ubisch bodies at the interface be-

tween tapetal cells and the microspore surface, and a nearly

completed pollen exine configuration (see Supplemental Figure

3 online). At stage 10, wild-type tapetal cells contained vacuoles

and plastids and mitochondria (Figures 5A and 5E). By contrast,

mads3-4 tapetal cells had a greatly increased number of single-

membrane, round-shaped structures filled with an electron

dense granular matrix, which are characteristic of peroxisomes

(Antonenkov et al., 2010), whereas plastids and mitochondria

were barely found (Figures 5B, 5F, and 5f). At stage 11, wild-type

tapetal cells were more condensed, forming a band-like struc-

ture (Figures 5C and 5G), andmicrospores were full of cytoplasm

and numerous plastid-like organelles and mitochondria (Figure

5I). By contrast, mads3-4 tapetal cells continued their abnormal

development, containing dark and less condensed cytoplasm

with peroxisome-like organelles (Figures 5D and 5H). Micro-

spores also seemed to be damaged, with numerous round and

deeply stained structures (Figure 5J). At stage 12 in wild-type

anthers, tapetal cells were hardly visible (Figure 5K), pollen grains

were deeply stained with a lot of starch, and a well-organized

intine configuration had developed (Figure 5M). By contrast, the

mads3-4 pollen was shrunken, irregularly shaped, and contained

abundant electron-dense compounds instead of starch grains

(Figures 5L and 5N). These cellular defects seemed to be char-

acteristic of cells subjected to oxidative stress.

Oxidative stress is frequently caused by higher levels of ROS,

which cause oxidative damage to cellular structures and mole-

cules (Gechev et al., 2006). ROS is believed to be a signal that

promotes programmed cell death (PCD) in plants and animals

(Wiseman, 2006; Doyle et al., 2010). Tapetal PCD is initiated from

stage 8 of anther development in rice (Li et al., 2006), but whether

ROS plays a role in tapetal development remains unknown. To

examine whether changes in ROS levels are associated with the

mutant phenotype inmads3-4, we measured ROS levels in both

wild-type and mads3-4 anthers using 2’,7’-dichlorofluorescin

(DCFH), which can be oxidized by ROS to the highly fluorescent

2’,7’-dichlorofluorescein (Simontacchi et al., 1993; Schopfer

et al., 2001). Low ROS levels were observed in both wild-type

and mads3-4 anthers before stage 8, while fluorescent levels

increased in wild-type and mads3-4 anthers at stage 8 (Figure

6A) and reached a higher level at stage 9. From stage 10 to stage

11, the ROS level decreased to a very low level in wild-type

anthers but remained high in mads3-4 anthers. At stage 12,

mads3-4 anthers still showed a relatively higher level of ROS

compared with the wild type (Figure 6A).

Superoxide anion, hydrogen peroxide, and hydroxyl radicals

are the major ROS species in plants, and superoxide anion and

hydrogen peroxide are known to play central roles in plant cell

death (Moeder et al., 2002; Overmyer et al., 2003; Bouchez et al.,

2007). Given that hydroxyl radicals are unstable and difficult to

be detected directly in biological samples (Babbs et al., 1989;

Tiedemann, 1997), we performed an analysis of superoxide

anion and hydrogen peroxide in wild-type andmads3-4 anthers.

The presence of superoxide anion was quantified using WST, a

water-soluble tetrazolium salt reagent, which can be efficiently

reduced by superoxide to a stable water-soluble formazan dye

with high molar absorptivity (Peskin and Winterbourn, 2000; Tan

and Berridge, 2000; Schopfer et al., 2001). In both wild-type and

mads3-4 anthers, the level of superoxide anionwas relatively low

before stage 8, notably increased from stage 8 to stage 9, and

reached the highest level at stage 9 (Figure 6B). From stage 10 to

Figure 2. Molecular Identification of Rice MADS3.

(A) Fine mapping of the MADS3 gene to an 18-kb region on chromosome 1.

(B) Positions of the mutations in the fourmads3 alleles. Boxes represent exons, thick lines are introns, and numbers indicate lengths in base pairs of the

exons.

518 The Plant Cell

Page 5: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

stage 11, superoxide anion content, like that of total ROS,

decreased in the wild-type anthers but remained high in

mads3-4 (Figure 6B). Furthermore, we analyzed the cellular

localization of superoxide anion in rice anthers using nitroblue

tetrazolium (NBT), which can be oxidized by superoxide anion

to form a dark blue formazan precipitation (Liszkay et al.,

2004). This approach obtained results consistent with WST

measurements with respect to changes in superoxide anion

levels in both wild-type and mads3-4 anthers (Figures 6C and

6D). Finally, transverse section analysis revealed the super-

oxide anion staining signals to be localized within the tapetal

cells and microspores (Figures 6E and 6F). These results

suggested that the dynamics of superoxide anion levels in rice

anther development may play an essential role in proper

microspore development and that the mads3-4 mutation

may have disturbed the equilibrium between production and

scavenging of superoxide molecules, thereby causing defec-

tive late anther development.

Measurement of hydrogen peroxide and staining of these

molecules with 3,5,3,59-tetramethylbenzidine (TMB) showed no

obvious differences in changes in the level of hydrogen peroxide

between the wild type and mads3-4 (see Supplemental Figure 4

online), suggesting that themads3-4mutation has little effect on

the hydrogen peroxide content in rice anther development.

Peroxisomes are subcellular organelles that not only partici-

pate in the production of ROS, leading to deleterious cellular

effects, cell death, and diverse pathological conditions, but also

house numerous ROS scavenging enzymes in both plants and

animals (Lopez-Huertas et al., 2000; del Rıo et al., 2002). These

organelles contain antioxidantmolecules, such as ascorbate and

glutathione, as well as a battery of antioxidant enzymes, includ-

ing catalase, superoxide dismutase, peroxidase, glutathione

Figure 3. Rice MADS3 Expression Pattern Analysis.

(A) Spatial and temporal expression analyses of MADS3 by qualitative (top) and quantitative (bottom) RT-PCR. The quantitative data are given as

means 6 SE (n = 3).

(B) to (E) GUS staining analysis of various tissues and developmental stages in theMADS3pro:GUS transgenic line. In (B), BS8, anthers before stage 8;

S8-S12, anthers in stage 8 to 12.

(C) A pistil.

(D) Tissue section of an anther at stage 10, showing the tapetum and microspores.

(E) Microspores at stage 11.

(F) to (I) In situ analyses of MADS3 expression at stages 9 (F), 10 (G), and 11 (H), with (I) as a control showing stage 10 detected with the sense probe.

Msp, microspore; St, stigma; T, tapetum. Bars = 5 mm in (E) to (I), 1 mm in (B), and 0.5 mm in (C).

MADS3 Regulates Anther Development 519

Page 6: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

reductase, and monohydroascorbate reductase (Schrader and

Fahimi, 2006). Higher ROS levels usually induce increases in the

number of peroxisomes (Schrader and Fahimi, 2006). Consistent

with the occurrence of numerous peroxisome-like structures in

mads3-4, higher activities of superoxide dismutase and perox-

idase were also detected in the mutant anthers from stage 10 to

stage 12 (see Supplemental Figure 5 online).

Transcriptome Analysis of Wild-Type andmads3-4 Anthers

To identify downstream targets of MADS3, we compared

genome-wide mRNA levels in wild-type and mads3-4 anthers

at stage 9, using microarray analysis with an Agilent 4344K–

oligonucleotide DNA chip. To assess the reproducibility of

microarray signals, we performed two biological replicates for

both the wild type and mads3-4. The empirical Bayes method

was employed to analyze the data (Smyth, 2004). A false dis-

covery (FDR) cutoff of 0.5% was used for the initial filtering of

candidate genes, followed by a secondary selection of at least

2-fold changes in gene expression. The predicted functions of

genes were obtained from the Institute for Genomic Research

(http://tigrblast.tigr.org/euk-blast). A total of 1728 genes were

found to show at least 2-fold expression changes in mads3-4

anthers compared with thewild type (see Supplemental Data Set

1 online). Among them, 1217 genes were previously found to be

expressed in rice microspore/pollen and tapetum by laser mi-

crodissection (LM) and microarray analyses (Suwabe et al.,

2008). Out of the 1217 genes, 630 were downregulated and

587 were upregulated (see Supplemental Data Set 2 online).

The 1728 genes with altered expression levels in mads3-4

were functionally grouped into four categories: (1) information

storage and processing (53 down- and 62 upregulated), (2)

cellular processes and signaling (166 down- and 176 upregu-

lated), (3) metabolism (288 down- and 201 upregulated), and (4)

unknown functions (388 down- and 394 upregulated) (see Sup-

plemental Table 3 online). To further characterize these genes,

we employed the Web-based DAVID 6.7 program (http://david.

abcc.ncifcrf.gov/) to search for differential distributions of gene

ontology (GO) and putative biological functions of these genes

with altered expression in the mads3-4 mutant (Huang et al.,

2009). A high proportion of the GO terms were associated with

the polysaccharide metabolic/catabolic process, oxidation re-

duction/stress response, lipid transport/localization, sexual re-

production, cell wall organization/modification/macromolecule

metabolic process, and carbohydrate catabolic process (see

Supplemental Table 4 online).

Besides MADS3, which was downregulated 5.66-fold, 95

genes that encode putative transcriptional factors also showed

altered gene expression. These include MADS box proteins

MADS47, MADS68, and MADS98, a basic helix-loop-helix

protein (Os04g0599300), and MYB proteins Os05g0490600,

Os01g0709000, Os08g0433400, and Os04g0470600 (see Sup-

plemental Table 5 online). Os01g0709000, which was down-

regulated 2.87-fold in themutant, is a close homolog to the Pinus

taeda gene,MYB1, which encodes a putative transcription factor

involved in the regulation of phenylpropanoid metabolism and

secondary cell wall biogenesis (Bomal et al., 2008).

Phytohormones, such as gibberellic acid (GA), jasmonic acid,

and auxin, play crucial roles in many development processes,

including anther and pollen development (Ishiguro et al., 2001;

Feng et al., 2006; Aya et al., 2009). In mads3-4 anthers, a group

of genes involved in phytohormone signaling and metabolism

exhibited altered expression. Among these were genes puta-

tively encoding the gibberellin receptor GID1L2, gibberellin

2-oxidase, gibberellin 3-b-dioxygenase 2-2, auxin-induced pro-

tein 5NG4, auxin response factor (Os04g0664400), and an auxin

efflux carrier component (Os01g0643300) (see Supplemental

Table 6 online).

The expression of genes known to be associated with early

rice anther development, such as MULTIPLE SPOROCYTE1

(MSP1) (Nonomura et al., 2003; Wang et al., 2006), HOMOLO-

GOUS PAIRING ABERRATION IN RICE MEIOSIS2 (PAIR2)

Figure 4. Transverse Section Analysis of Wild-Type and mads3-4 Anthers.

Anthers were obtained from stages 9 ([A] and [E]), 10 ([B] and [F]), 11 ([C] and [G]), and 12 ([D] and [H]). (A) to (D) are the wild type, and (E) to (H) are

mads3-4. E, epidermis; En, endothecium; MP, mature pollen; Msp, microspore; T, tapetum. Bars = 10 mm.

520 The Plant Cell

Page 7: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

(Nonomura et al., 2004), GAMYB (Kaneko et al., 2004; Aya et al.,

2009; Liu et al., 2010),Wax-deficient anther1 (Wda1) (Jung et al.,

2006), CP1 (Lee et al., 2004), and RAFITIN (Wang et al., 2003),

was not significantly changed in mads3-4 (see Supplemental

Table 7 online), suggesting that these genes may act earlier than

MADS3 in regulating anther development. Conversely, several

genes related to late anther development, such as MST8, which

encodes a monosaccharide transporter crucial for the sugar

unloading pathway, and INV4, which encodes a cell wall inver-

tase required for hexose production and starch synthesis for

pollen maturation (Oliver et al., 2005, 2007), were significantly

downregulated (8.88- and 9.99-fold, respectively; see Supple-

mental Table 7 online). A reduction in the expression ofMST8 and

INV4was shown to be related to male sterility in rice (Oliver et al.,

2005, 2007). In mads3-4 anthers, we also observed a 2.36-fold

upregulation of Tapetum Degeneration Retardation (TDR), which

is required for tapetal development and degeneration in rice, with

its mutants showing delayed tapetal PCD (Li et al., 2006).

Moreover, the expression of C6, which encodes a small mobile

lipid transfer protein required for postmeiotic anther develop-

ment in rice (Zhang et al., 2010a), was upregulated 2.36-fold

in mads3-4 (see Supplemental Table 7 online). The NADPH-

dependent HC-toxin reductase gene, Os07g0602000, showed a

17.9-fold reduction in expression. This protein likely plays a role

in increasing the level of the NAD(P)H pool and preventing the

host from induced cell death (Hayashi et al., 2005). Furthermore,

the expression of Os08g0515900, which encodes a putative

dihydroflavonol 4-reductase essential for male fertility in Arabi-

dopsis (Tang et al., 2009), was upregulated 21.93-fold inmads3-4.

Finally, four male sterility–related genes also had changed ex-

pression, including, Os04g0354600 (7.47-fold downregulated),

Os04g0353600 (5.16-fold downregulated), Os09g0567500 (3.29-

fold upregulated), and Os03g0167600 (4.46-fold upregulated).

These results together support the view that MADS3 plays a key

role in late anther development.

Plants have a large number of P450 proteins with diverse

functions in biosynthetic and detoxication pathways (Schuler

and Werck-Reichhart, 2003). For example, many P450s are

involved in the synthesis of lignins, UV protectants, pigments,

defense compounds, fatty acids, hormones, and signaling mol-

ecules, as well as in the breakdown of compounds, including

those that are toxic (Schuler and Werck-Reichhart, 2003). In

mads3-4 anthers, we detected changes in gene expression for

42 genes encoding P450s: 29 were downregulated and 13 were

upregulated (see Supplemental Table 8 online). Among them,

CYP704B2, a gene essential to rice postmeiotic anther devel-

opment, was upregulated 4.29-fold. The CYP704B2 gene is

expressed in the tapetum and microspores, its loss-of-function

mutant, cyp704B2, is male sterile, and recombinant CYP704B2

expressed in yeast is able to catalyze the production of

v-hydroxylated fatty acids with 16 and 18 carbon chains (Li

et al., 2010b). Similarly, another rice male sterile gene,CYP703A3

(Aya et al., 2009), was upregulated 9.58-fold inmads3-4. Further-

more, Os03g0371000, which has a homolog in Catharanthus

roseus encoding a putative cytochrome P450 (CYP72A1) with the

ability to cleave the oxidative ring to convert loganin into secolo-

ganin (Irmler et al., 2000), was downregulated 7.41-fold.

We verified the microarray expression data by quantitative

RT-PCR (qRT-PCR), using independently generated RNA

samples and focusing on nine differentially expressed genes.

Genes subjected to validation were MADS3, MT-like pro-

tein (Os12g0568500), two peroxidases (Os05g0499300 and

Os11g0112400), three transcription factors (Os03g0186600,

Os03g0671800, and Os01g0972800), and two cytochrome

P450s (Os02g0596300 and Os04g0469800) (see Supplemental

Figure 6 online). Proteins encoded by most of these genes have

Figure 5. Transmission Electron Micrographs of Wild-Type and mads3-4

Anthers from Stages 10 to 12.

(A) to (D) Anthers were obtained from stage 10 ([A] and [B]) and 11 ([C]

and [D]) plants. (A) and (C) are the wild type, and (B) and (D) are from the

mutant.

(E) The wild-type tapetum at stage 10.

(F) Themads3-4 tapetum at stage 10 with abundant peroxisomes. (f) is a

magnified picture of a peroxisome-like structure from the mutant.

(G) and (H) The wild-type (G) and mads3-4 (H) tapetum at stage 11.

(I) and (J) The wild-type (I) and mads3-4 (J) microspore at stage 11.

(K) and (L) Stage 12 anthers from wild-type (K) and mads3-4 (L) plants.

(M) The wild-type mature pollen at stage 12, showing lots of starch

granules.

(N) The mads3-4 microspore at stage 12, showing the accumulation of

dark, electron-dense compounds.

E, epidermis; En, endothecium; Ex, exine; In, intine; Mp, Mature pollen,

Msp, microspore; Mt, mitochondrion; Pe, peroxisome; Pl, plastid; St,

starch; T, tapetum; V, vacuole; Ub, Ubisch body. Bars = 2 mm in (A) to

(D), 1 mm in (E) to (H) and (K) to (N), 10 mm in (I) and (J), and 0.5 mm in (f).

MADS3 Regulates Anther Development 521

Page 8: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

been shown or predicted to be involved in ROS homeostasis.

Close correlations were found between changes detected by the

microarray analysis and those obtained with qRT-PCR, confirm-

ing the reliability of the microarray results.

ExpressionChanges inGenes Involved inROSHomeostasis

inmads3-4 Anthers

In agreement with the abnormally high levels of ROS in mads3-4

anthers, we observed changes in the expression of a number of

genes related to ROS homeostasis in the mutant. In plant cells,

ROS are mainly produced by enhancing the enzymatic activity

of plasma membrane–bound NADPH oxidases, pH-dependent

cell wall–bound peroxidases, and amine oxidases in the apo-

plast (Mittler et al., 2004). Genes involved in the ROS-producing

pathway (e.g., Os01g0360200, Os12g0541300, Os01g0835500,

Os09g0438000, Os01g0734200, and Os05g0528000), which en-

code putative respiratory burst oxidases (Mittler et al., 2004;

Gechev et al., 2006), hadminimal expression changes inmads3-4

anthers. On the other hand, genes encoding putative peroxidases

(20 upregulated; seven downregulated) and amine oxidases

(downregulated, such as Os09g0368500; upregulated, such as

Os04g0671300) showed at least a 2-fold change in expression in

mads3-4 anthers (Table 1). Because no detailed annotation of

peroxidases is available in the rice genome, we could not distin-

guish cell wall–bound peroxidases involved in ROS production

from other peroxidases required for ROS removal.

The balance between the activity of superoxide dismutase,

catalase, and/or peroxidase and sequestering of metal ions by

metal binding proteins is thought to be crucial in preventing the

formation of the highly toxic ROS (Mittler et al., 2004). Notably, a

total of 49 genes (27 upregulated; 22 downregulated) among the

1728 genes with altered expression levels in mads3-4 were

annotated as being involved in ROS scavenging. The gene prod-

ucts include glutathione S-transferase (four downregulated and

five upregulated), metallothionein (five downregulated), monode-

hydroascorbate reductase (two downregulated and one upregu-

lated), glutaredoxin (Os02g0512400,upregulated;Os01g0235900,

downregulated), peroxiredoxin (Os07g0638300, downregulated),

and thioredoxin (Os01g0168200, downregulated) (Table 1). These

data suggested that themads3-4mutation affects the expression

of genes whose protein products may be components in the

network of ROS homeostasis during anther development.

MADS3 Is Associated with the Promoter of a

ROS-Scavenging Gene

MADS box proteins have been shown to regulate gene expres-

sion by binding to a consensus core element called the CArG-box

[59-CC(A/T)4NNGG-39] (Riechmann et al., 1996). To further reveal

the regulatory role of MADS3 during rice anther development,

putative MADS binding sequences (CArG-box) were identified

using the tools described in plant CARE (http://bioinformatics.

psb.ugent.be/webtools/plantcare/html/; Thijs et al., 2002). A

total of 189 of the 895 downregulated genes and 169 of the

833 upregulated genes (see Supplemental Data Set 1 online)

were found to contain at least one CArG-box within the 1-kb

upstream region (see Supplemental Data Sets 3 and 4 online),

suggesting thatMADS3may directly regulate a number of target

genes during late anther development in rice. These genes

encode factors with putative roles in signal transduction, hor-

mone metabolism, and stress response; notably, four of the 189

genes (two MTs and two peroxidases), and 10 of the 169 genes

(six peroxidases, three glutathione S-transferases, and one

thioredoxin) are related to the ROS-scavenging system (Table 1).

To confirm the binding ability of MADS3 to these promoters,

we selected three genes that are downregulated in mads3-4:

Os12g0571100 (encoding a metallothionein, MT-1-4b, 7.37-fold

downregulation), Os12g0150200 (encoding P450, 7.37-fold

Figure 6. Analyses of ROS and Superoxide Anion levels in Wild-Type

and mads3-4 Anthers.

(A) and (B) ROS (A) and superoxide anion (O2�; [B]) levels. Data are

presented as means 6 SE (n = 3).

(C) and (D) NBT staining analysis of O2� production from early stages

8 to 12 showing the shallow to dark-blue colors.

(E) and (F) O2� localization in the tapetum and microspores in tissue

sections of anthers at stage 9. Bars = 5 mm.

Msp, microspore; T, tapetum; WT, wild type. BS8, anthers before stage

8; S8-S12, anthers at stage 8 to 12.

522 The Plant Cell

Page 9: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

Table 1. Genes Involved in ROS Homeostasis with Expression Changes of at Least 2-Fold (mads3-4/Wild Type) in the mads3-4 Anther and Their

Putative CArG-Boxes within the 1-kb Upstream Region

Locus Annotation Fold Change Direction

Position

(Relation to TSS) Box Sequence

ROS-producing (2)

Os09g0368500 Amine oxidase �2.09

Os04g0671300 Amine oxidase 2.04

ROS-scavenging (23)

Downregulated (15)

Os03g0288000 MT, putative, expressed �8.83 � �138 to 129 CCATTTCTGG

Os03g0288000 MT, putative, expressed �8.83 � �425 to 416 CCTTTTCTGG

Os12g0568500 MT, putative, expressed �4.86

Os12g0571100 MT, putative, expressed �7.37 + �879 to 870 CCTTTTGGGG

Os12g0571000 MT, putative, expressed �4.58

Os09g0454500 Monodehydroascorbate reductase �4.33

Os07g0638300 Peroxiredoxin, putative �3.19

Os08g0423500 Monodehydroascorbate reductase �3.04

Os01g0353400 Glutathione S-transferase, putative,

expressed

�2.46

Os10g0525500 Glutathione S-transferase, N-terminal

domain-containing protein, expressed

�2.41

Os12g0567800 MT, putative, expressed �2.34

Os01g0149800 MT, putative, expressed �2.34

Os01g0235900 Glutaredoxin, putative, expressed �2.32

Os10g0525400 Glutathione S-transferase, putative, expressed �2.08

Os01g0369700 Glutathione S-transferase, putative, expressed �2.03

Upregulated (8)

Os02g0533300 Monodehydroascorbate reductase 2.14

Os01g0370200 Glutathione S-transferase, putative, expressed 2.25

Os01g0168200 Thioredoxin, putative, expressed 2.36 + �653 to 644 CCTTTTGAGG

Os10g0395400 Glutathione S-transferase, putative, expressed 2.42

Os02g0512400 Glutaredoxin subgroup III 2.49

Os03g0134900 Glutathione S-transferase, putative, expressed 2.86

Os10g0543800 Glutathione S-transferase, putative, expressed 3.01 + �519 to 510 CCAATACGGG

Os08g0550500 Glutathione S-transferase, C-terminal

domain-containing protein, expressed

3.58 � �456 to 447 CCAATAGAGG

Peroxidase (ROS-producing and ROS-scavenging) (26)

Downregulated (7)

Os05g0499300 Peroxidase precursor, putative, expressed �15.45

Os01g0327100 Peroxidase precursor, putative, expressed �12.54 � �336 to 327 CCTATTTGGG

Os12g0112000 Peroxidase precursor, putative, expressed �7.61

Os11g0112400 Peroxidase precursor, putative, expressed �6.41

Os07g0639400 Peroxidase precursor, putative, expressed �2.97

Os06g0681600 Peroxidase precursor, putative, expressed �2.56

Os01g0327400 Peroxidase precursor, putative, expressed �2.13 + �621 to 612 CCTATTAGGG

Os01g0327400 Peroxidase precursor, putative, expressed �2.13 � �622 to 613 CCTAATAGGG

Upregulated (19)

Os03g0368000 Peroxidase precursor, putative, expressed 2.02

Os03g0368300 Peroxidase precursor, putative, expressed 2.03 + �814 to 805 CCTATTAAGG

Os02g0236600 Peroxidase precursor, putative, expressed 2.35

Os03g0235000 Peroxidase precursor, putative, expressed 2.37 � �186 to 177 CCATAACTGG

Os10g0536700 Peroxidase precursor, putative, expressed 2.38 � �13-4 CCAATTGGGG

Os10g0536700 Peroxidase precursor, putative, expressed 2.38 � �172 to 163 CCATTTGTGG

Os03g0368900 Peroxidase precursor, putative, expressed 2.44 + �239 to 230 CCTAAATAGG

Os01g0205900 Peroxidase precursor, putative, expressed 2.66

Os01g0326000 Peroxidase precursor, putative, expressed 2.67

Os04g0465100 Peroxidase precursor, putative, expressed 2.73 �Os11g0210100 Peroxidase precursor, putative, expressed 2.82

Os12g0191500 Peroxidase precursor, putative, expressed 2.96

Os04g0689000 Peroxidase precursor, putative, expressed 3.05

(Continued)

MADS3 Regulates Anther Development 523

Page 10: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

downregulation), and Os01g0327100 (encoding a peroxidase,

12.54-fold downregulation), for ChIP experiments. First, we

developed rabbit polyclonal antibodies against a bacterially

expressed recombinant protein that contains a unique region

at the C terminus of rice MADS3 (see Methods). Specificity of

the antibody was confirmed using immunoblot analysis. We

detected a band of ;35 kD, which is slightly larger than the

expected size, in protein isolated from the wild type, implying

possible protein modification of MADS3 in vivo. By contrast, no

obvious signal was observed in themads3-4mutant because the

C-terminal region, against which the antibody was raised, was

absent in the truncated MADS3 protein in the mutant (see

Supplemental Figure 7 online). ChIP-qPCR analysis showed

specific enrichment of the affinity-purified MADS3 antibody on

the promoter region of MT-1-4b, which contains the CArG-box,

but not on the promoters of Os12g0150200 and Os01g0327100,

both of which also contain the predicted CArG-box (Figure 7A).

To further confirm the ChIP-qPCR data, we performed EMSA

using the recombinant MADS3 protein and a DNA fragment

containing the MT-1-4b promoter region (2980 to 2837 bp

upstream of ATG). A supershifted band was observed when the

MT-1-4b promoter region was incubated with MADS3 (Figure

7B, lane 2), while no supershifted signal was observed in the

control sample containing purified bacterial protein expressed

from the empty pET-32a plasmid (Figure 7B, lane 1). Moreover,

we observed decreased signals when a 25-, 50-, or 100-fold

molar excess of unlabeled MT-1-4b probe was added to the

EMSA reaction as competitors (Figure 7B, lanes 3 to 5). This

result suggested that MADS3 directly regulates MT-1-4b ex-

pression. Consistent with the ChIP-qPCR and mobility shift

results, qRT-PCR analysis revealed strong expression of MT-1-

4b in wild-type anthers from stage 9 to stage 12 (Figure 7C), and

MT-1-4b transcripts were previously found in rice microspore/

pollen and tapetum using LM samples and microarray analysis

(Suwabe et al., 2008). In addition,MT-1-4b is highly expressed in

roots (see Supplemental Figure 8 online), which is in agreement

with the previous report by Zhou et al. (2006).

A previous study demonstrated superoxide- and hydroxyl

radical-scavenging activities for the recombinant rice MT2b

protein, a homolog of MT-1-4b (Wong et al., 2004). To test

whether MT-1-4b has similar activities, we determined the hy-

droxyl radical-scavenging activity of recombinant MT-1-4b in

vitro. In this method, a reaction between salicylate and hydroxyl

radicals leads to the formation of dihydroxybenzoic acid, which

can be suppressed by a scavenger in a dose-dependent manner

(Smirnoff and Cumbes, 1989). Addition of recombinant MT-1-4b

at concentrations of 10 to 50 mM effectively protected salicylate

from attack by hydroxyl radicals (Figure 7D), suggesting that MT-

1-4b is a potent scavenger of hydroxyl radicals. In addition,

recombinant MT-1-4b was shown to have superoxide scaveng-

ing activity, whereby it was able to scavenge superoxide anion by

inhibiting the oxidation of WST in vitro (Figure 7E).

Silencing ofMT-1-4bCausesDefective PollenDevelopment

To understand the biological role of MT-1-4b in rice anther

development, we adopted an artificial microRNA (amiRNA) ap-

proach to reduce the expression of the MT-1-4b gene. Two 21-

nucleotide amiRNA sequences, which target aMT-1-4b–specific

region, were designed by the customized version of the Web

MicroRNA Designer platform and used to replace the sequence

of endogenous microRNA and microRNA* of osa-MIR528

(Schwab et al., 2006; Ossowski et al., 2008; Warthmann et al.,

2008). The final construct was transformed into calli induced

from wild-type rice mature seeds, and a total of 11 T0 trans-

formant lines expressingMT-1-4b-amiRNAwere obtained. None

of the transgenic plants displayed any obvious growth pheno-

types during vegetative or floral organ development. However,

using I2-KI staining, seven lines were found to contain reduced

mature pollen grain formation, suggesting defective anther de-

velopment and pollen formation in these transgenic plants. Three

of the seven lines (lines 1, 3, and 4; called MT-1-4b-amiRNA-S

lines) showed a relatively strong phenotype by having a greatly

reduced number of deeply stained pollen grains (Figures 8A and

8B), whereas the other four lines (called MT-1-4b-amiRNA-W)

showed relatively weak phenotypes (Figures 8A and 8C). In

addition, qRT-PCR analysis of stage 9 anthers revealed an

overall correlation between the level of MT-1-4b expression

and the severity of the phenotype in pollen maturation (Fig-

ure 8D).

To further determine whether MT-1-4b functions as a ROS

scavenger in vivo, we examined ROS levels in the MT-1-4b-

amiRNA lines. At stages 10 and 11, theMT-1-4b-amiRNA-S lines

displayed relatively high levels of ROS compared with the wild-

type anthers, whereas at stage 12, the wild-type and amiRNA

anthers showed equally high levels of ROS, suggesting thatMT-

1-4bmay be crucial for ROS homeostasis (Figure 8E). Moreover,

abnormal accumulation of superoxide anion was observed in the

anthers ofMT-1-4b-amiRNA-S lines at stages 10 and 11 (Figures

8F and 8G). Additional cytological analysis showed the abnormal

Table 1. (continued).

Locus Annotation Fold Change Direction

Position

(Relation to TSS) Box Sequence

Os06g0274800 Peroxidase precursor, putative, expressed 3.07

Os03g0339300 Peroxidase precursor, putative, expressed 3.79

Os04g0688100 Peroxidase precursor, putative, expressed 3.97

Os12g0530100 Peroxidase precursor, putative, expressed 5.14

Os07g0677600 Peroxidase precursor, putative, expressed 5.36 + �818 to 809 CCAAAACAGG

Os08g0302000 Peroxidase precursor, putative, expressed 7.13

Os05g0231900 Peroxidase precursor, putative 14.73 + �518 to 509 CCATTTCTGG

524 The Plant Cell

Page 11: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

accumulation of superoxide anion signals in the tapetal layer and

microspores of MT-1-4b-amiRNA-S lines (Figures 8H and 8I).

DISCUSSION

RiceMADS3 Regulates Late Anther Development and

Pollen Formation

Previous investigations showed that the rice floral homeotic

C-class gene, MADS3, is expressed in stamen and carpel

primordia during early floral development, and that the protein

it encodes is crucial for stamen specification (Kang et al., 1998;

Yamaguchi et al., 2006). In this study, we characterized a mutant

allele of MADS3 (mads3-4), further analyzed the expression

pattern of this gene, and revealed an important role for MADS3 in

regulating postmeiotic anther development in rice.

Of the previously reported MADS3 alleles, both mads3-1 and

mads3-2 are in the Nipponbare (a japonica cultivar) background

and contain Tos17 retrotransposon insertions in the gene.

mads3-1 is a weak allele carrying an insertion in the C-terminal

region of the gene and showing no defective floral organs,

whereasmads3-2 is an intermediate allele with a mutation at the

C terminus of the K-domain and reduced expression of the gene,

which resulted in mild transformation of stamens into lodicules

(Yamaguchi et al., 2006).mads3-3 is a strong allele in the Dongjin

(a japonica cultivar) background. It contains a T-DNA insertion in

the second intron of the gene, which leads to no detectable

MADS3 transcript and homeotic transformation of nearly all

stamens in whorl 3 into lodicule-like organs (Yamaguchi et al.,

2006). mads3-4, which results in a mutation in the middle region

of the K-domain of MADS3, seems to be another intermediate

mutant with reduced expression of MADS3 (see Supplemental

Figure 6 online). In contrast withmads3-3, the allele introduced in

this study is in the 9522 (a japonica cultivar) background and is

male sterile. These phenotypic differences between the mads3

allelesmay be caused by differences in genetic background and/

or the nature of the mutations in MADS3. Moreover, we cannot

Figure 7. Assays to Show the Transcriptional Regulation of MT-1-4b by Rice MADS3 and Enzymatic Activity of MT-1-4b.

(A) Fold enrichment calculations from qPCR on three independent ChIP experiments; the data are presented as means 6 SE (n = 3). The predicted

MADS box binding sites CC(A/T)4NNGG are indicated in the 1-kb promoter regions of Os12g0571100, Os12g0150200, and Os01g0327100, and the

PCR amplicons containing the CArG-box are underlined. The gray lines represent the amplified fragments containing the CArG-box by ChIP-PCR using

primers listed in Supplemental Table 2 online, the numbers below the line indicate the positions of PCR primers relative to ATG, and the numbers on the

line indicate the position of CArG-box relative to ATG.

(B) EMSA to show the binding ability of recombinant MADS3 purified from bacteria to the promoter of MT-1-4b. The probe for P-MT-1-4b is 144 bp in

length (�837 to �980). MADS3 was mixed with digoxigenin-labeled P-MT-1-4b, and a 25-, 50- and 100-fold molar excess of unlabeled P-MT-1-4b

probe (lanes 2 to 4) was added as competitor to the EMSA reaction. Protein translated from the pET32a plasmid served as a control (Cont, lane 1).

(C) qRT-PCR analysis of theMT-1-4b expression pattern in wild-type (WT) andmads3-4mutant anthers at various stages of anther development. BS8,

anthers before stage 8; S8-S12, anthers from stage 8 to stage 12.

(D) Comparison of hydroxyl radical scavenging activity between pET-32a and pET-32a-MT-1-4b, measured as the ability to inhibit hydroxyl radical-

mediated salicylate hydroxylation.

(E) Comparison of superoxide anion scavenging activity between pET-32a and pET-32a-MT-1-4b, measured as the ability to inhibit WST oxidation.

Values in (C) to (E) are the mean from three individual experiments.

MADS3 Regulates Anther Development 525

Page 12: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

exclude the possibility that mads3-2 also has defective anther

development because in the previous report on mads3-2, the

authors did not perform a detailed observation of anther devel-

opment (Yamaguchi et al., 2006).

The finding that MADS3 is important for postmeiotic anther

development in rice is consistent with the expression pattern of

MADS3 in the tapetum andmicrospores from stage 9 to stage 12

(Figure 9). Furthermore, transcriptome analyses demonstrated

thatMADS3 affects the expression of 1728 genes,many of which

are associated with cellular processes and signaling, carbohy-

drate transport and metabolism, secondary metabolite bio-

synthesis, detoxification, and transcriptional regulation. Among

these 1728 genes, some genes, such as TDR (Li et al., 2006;

Zhang et al., 2008), CYP703A3 (Aya et al., 2009), CYP704B2 (Li

et al., 2010b), C6 (Zhang et al., 2010a), MST8, and INV4 (Oliver

et al., 2005, 2007), have been shown to be involved in anther

development, suggesting that MADS3 may regulate anther de-

velopment by affecting the expression of these genes. Unlike

MADS3, the Arabidopsis C-class gene AG is expressed from

anther initiation to anther maturation in sporogenous cells, the

connective, anther walls and the filament, and no AG expression

was detected in pollen grains (Bowman et al., 1991). The distinct

spatial and temporal expression patterns of AG and MADS3

suggest that these two proteins have diversified functions in

anther development. AG plays a key role in regulating early

anther development by activating the expression of the MADS

box gene SPL/NZZ (Ito et al., 2004) and in late anther develop-

ment and anther dehiscence by regulating JA synthesis (Ito et al.,

2007) (Figure 9). BesidesMADS3 and AG, there are other MADS

box genes reported to be expressed in anther development in

rice (Hobo et al., 2008), maize (Schreiber et al., 2004), Arabidop-

sis (Alvarez-Buylla et al., 2000), and Antirrhinum (Zachgo et al.,

1997), indicating important roles for MADS box genes in plant

anther development and pollen formation.

MADS3 Regulates ROS Homeostasis during

Anther Development

Cell death is involved in various plant development events, from

seed setting to gametophyte development. The tapetum, the

innermost sporophytic layer within the anther wall, undergoes

cell degeneration to supply nutrients for pollen development

Figure 8. Analyses of the MT-1-4b-amiRNA Lines.

(A) to (C) I2-KI–stained pollen grains of the wild type (WT) andMT-1-4b-amiRNA lines at stage 12. The percentage of deeply stained pollen grains among

400 pollen grains from the wild type was 98.8%6 2.44%, that for the threeMT-1-4b-amiRNA-S lines (MT-1-4b-amiRNA-1, 3, and 4) was 51.52, 57.81,

and 51.23%, respectively (average percentage = 53.50% 6 3.73%), and that from the four MT-1-4b-amiRNA-W lines (lines 2, 5, 6, and 7) was 72.5,

77.8, 67.03, and 71.4%, respectively (average percentage = 72.18% 6 4.43%) (n = 3). Pollen grains from the wild type (A), line 1 (B), and line 2 (C) are

shown.

(D) qRT-PCR analysis ofMT-1-4b transcripts inMT-1-4b-amiRNA lines (lines 1 to 7, indicated by 1 to 7, respectively) at stage 9 of anther development.

Data are given as means 6 SE (n = 3).

(E) and (F) Comparison of ROS (E) and O2� (F) levels in wild-type andMT-1-4b-amiRNA lines. WST could be reduced by O2

� to a stable water-soluble

formazan with high absorbance at 450 nm. Data are means 6 SE (n = 3 for the wild type and MT-1-4b-amiRNA-S; n = 4 for MT-1-4b-amiRNA-W).

(G) Comparison of O2� production in wild-type and MT-1-4b-amiRNA anthers from stage 9 to 12 using NBT staining.

(H) and (I) O2� signals detected in the tapetum and microspores in stage 10 wild-type and MT-1-4b-amiRNA anthers.

T, tapetum; Msp, microspore; BS8, anthers before stage 8; S8-S12, anthers at stage 8 to 12. Bars = 20 mm in (A) to (C), 50 mm in (G), and 10 mm in (H)

and (I).

526 The Plant Cell

Page 13: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

(Papini et al., 1999; Varnier et al., 2005; Li et al., 2006). Tapetal

cell differentiation and disintegration coincide with the postmei-

otic developmental program, and premature or delayed degra-

dation of the tapetum is associated with male sterility. Several

rice genes encoding putative transcription factors, such as

GAMYB (Kaneko et al., 2004; Tsuji et al., 2006; Aya et al.,

2009; Liu et al., 2010),UNDEVELOPED TAPETUM1 (UDT1) (Jung

et al., 2005), and TDR (Li et al., 2006), have been reported to be

associated with tapetal function and degeneration. GAMYB is

regulated by GA, and gamyb-2 tapetal cells appear swollen and

have defects in PCD (Aya et al., 2009). UDT1 and TDR encode

basic/helix-loop-helix transcription factors, andmutants of these

genes aremale sterile. TDR acts as a positive regulator of tapetal

PCD in rice, with itsmutant showing delayed tapetal nuclear DNA

fragmentation and abortion of microspores (Li et al., 2006).

ROS is known to act as a signal to promote PCD in both plants

and animals (Wiseman, 2006; Doyle et al., 2010), but how ROS

regulates tapetal development remains unknown in plants. During

anther development in wild-type rice, tapetal PCD is first detect-

able at stage 8, and strong PCD signals are present at stage 9 (Li

et al., 2006; Zhang and Wilson, 2009). Our study showed that

ROS level is detectable at stage 8, increases at stage 9, and is

much reduced at stage 10 (Figure 6), suggesting that the timely

production of ROS, superoxide anion in particular, is associated

with the initiation of tapetal PCD during anther development.

The steady state ROS level, which is important for normal

development processes, is determined by the interplay between

ROS-producing and ROS-scavenging mechanisms (Gapper and

Dolan, 2006; Gechev et al., 2006; Miller et al., 2008). In Arabi-

dopsis, this appears to require a large gene network composed

of at least 152 genes (Mittler et al., 2004). Investigations in

Arabidopsis suggested that the coordination between different

components of theROSscavenging network in plants is complex

(Karpinski et al., 1999; Rizhsky et al., 2003). For instance, even

though most ROS produced during light stress are thought to be

generated in chloroplasts or peroxisomes (Asada, 1999), light

stress to Arabidopsis causes the induction of cytosolic but not

chloroplastic defense enzymes (Karpinski et al., 1997, 1999;

Pnueli et al., 2003). Moreover, to date, there are at least three

different enzymes of plant ROS-removal pathways found to be

dual targeted to chloroplasts andmitochondria, implying the close

coordination of these two typesoforganelles in defense responses

(Creissen et al., 1995; Obara et al., 2002; Chew et al., 2003).

In rice, however, functional aspects of ROS-related genes

remain less understood. We observed expression changes in 51

genes with putative functions in the ROS network in mads3-4

anthers (Table 1), suggesting that MADS3 may play a role in

adjusting ROS homeostasis during anther development. Con-

sistent with the gene expression profile analysis, mads3-4 has

high ROS accumulation in the anther, starting from stage 9, when

young microspores form. Furthermore, mad3-4 tapetal cells at

stages 10 to 12 display features characteristic of those associ-

ated with oxidative stress, including less cytoplasmic conden-

sation, fewermitochondria, andmore peroxisome-like organelles.

Increased ROSmight be associated with the decreased expres-

sion of ROS-scavenging genes, including those encoding MTs

and peroxidase. The increased number of peroxisomes in

mads3-4 tapetal cells and elevated peroxisome-associated en-

zyme activities, such as superoxide dismutase and peroxidase,

may in part result from higher ROS levels during late anther

development. Our in vivo and in vitro analyses suggested that

MADS3 may directly regulate a ROS-removal gene, MT-1-4b.

Consistent with these results, an LM-mediated microarray anal-

ysis revealed the expression of MT-1-4b in the tapetum and

microspores during late anther development (Hobo et al., 2008),

an expression pattern that is similar to that of MADS3 in rice

anther development. The biological importance of the regulation

of MT-1-4b by MADS3 is further supported by the biochemical

activity of the recombinant MT-1-4b protein in removing the

superoxide anion and hydroxyl radical (Figure 7). Moreover,

knockdown of MT-1-4b led to reduced pollen fertility and ab-

normal accumulation of superoxide anion in the transgenic lines,

which is similar to the phenotypes in mads3-4 during late anther

development. Lastly, we found changes in the expression of a

few other genes encoding proteins putatively involved in ROS

removal in mads3-4, suggesting that these genes may also

encode components of a ROS-scavenging network that modu-

lates late anther development.

In conclusion, we have demonstrated a role of the floral

homeotic C-class gene MADS3 in regulating late anther devel-

opment and pollen formation. We have shown that this protein

functions, at least in part, by regulating ROS homeostasis in

anthers through the ROS-removal protein MT-1-4b. Although it

remains to be seen whether MADS3 functions in late anther

development through other cellular targets, our study sheds light

on the molecular mechanisms underlying the genetic control of

postmeiotic tapetum degeneration and development.

METHODS

Mutant Material and Growth Conditions

The F2 mapping population was generated from a cross between

mads3-4 (japonica) and GuangLuAi4 (indica) rice (Oryza sativa). In the

Figure 9. Comparison of the functions of Rice MADS3 and Arabidopsis

AG during Flower Organ Determination and Anther Development.

Timelines for the functions of the two proteins are shown. The spikelet

development stages refer to Itoh et al. (2005). Ito et al. (2007) was used as

a reference for the function of AG. See Discussion for details of the

comparison. (A) shows the function of rice MADS3, and (B) indicates the

role of Arabidopsis AG.

MADS3 Regulates Anther Development 527

Page 14: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

F2 population, male sterile plants were selected for gene mapping. All

plants were grown in the paddy field of Shanghai Jiao Tong University.

Characterization of Mutant Phenotypes

Plants were photographed with a Nikon E995 digital camera and a Motic

K400 dissecting microscope. Observation of anther development by

semithin sections and TEM was performed as described by Li et al.

(2006). DAPI staining was performed according to Han et al. (2006).

Histochemical GUS Assays

Histochemical GUS staining was performed as described by Jefferson

et al. (1987) and Dai et al. (1996). Freshly collected samples from

transgenic plants expressing the promoter-GUS fusion were put in the

staining solution under vacuum conditions for 15 min, followed by

incubation at 378C overnight. After staining, samples were rinsed in

70% ethanol for 1 h before a photo was taken with a Nikon E995 digital

camera. Staining solution contained 0.02 M 5-bromo-4-chloro-3-indolyl-

b-D-glucuronide, 0.1 M NaH2PO4, 0.25 M EDTA, 5 mM potassium

ferricyanide, 5 mM potassium ferrocyanide, and 1.0% (v/v) Triton X-100,

pH 7.0.

RT-PCR and qRT-PCR

Total RNA was isolated from rice tissues (e.g., root, culm, leaf, and

lemma/palea) at stage 9 of anther development as well as anthers at

different stages from the wild type and mads3-4. The stages of anthers

were classified according to spikelet length (Feng et al., 2001; Zhang and

Wilson, 2009). First-strand cDNA was synthesized using the ReverTra

Ace-a-First Strand cDNA synthesis kit (Ferment). qRT-PCR analysis was

performed using SYBR Premix EX Taq (Takara) on a Rotor-Gene

RG3000A detection system (Corbett Research) (Li et al., 2010b).

Complementation of themads3-4Mutant

For functional complementation of the ricemads3-4mutant, the MADS3

fragment was amplified with primers MADS3-F and MADS3-R from the

cDNA clone (RGRC-NIAS, Japan; http://www.rgrc.dna.affrc.go.jp/stock.

html) (see Supplemental Table 2 online) and inserted into a binary vector

pHB (kindly provided byHongquan Yang), which contains a double CaMV

35S promoter and a hygromycin resistance marker, to make pHB-

MADS3. Calli induced from homogenous mads3-4 young panicles were

used for transformation with Agrobacterium tumefaciens EHA105 carry-

ing the pHB-MADS3 plasmid, as described by Hiei et al. (1997).

Map-Based Cloning

Bulked segregant analysis used to identify markers linked to mads3-4,

and fine mapping was performed as described by Li et al. (2006) using

mapping markers listed in Supplemental Table 2 online.

Microarray Analysis

Agilent 4*44K rice oligoarrays (Agilent Technologies) containing 44,000

features were employed for two-color oligoarrays in this study. Two

biological replicates of total RNA were prepared frommads3-4 and wild-

type anthers at stage 9 using TRIzol reagent (Invitrogen), and the mRNA

was isolated from total RNAs using the RNAeasy mini kit (Qiagen).

Developing antherswere classified according tomicroscopy analysis and

spikelet length (Feng et al., 2001; Zhang and Wilson, 2009). Microarray

hybridization was carried out in a Hybridization Chamber (Agilent,

G2534A) according to procedures provided by the company. The micro-

arrays were scanned with an Agilent instrument (G2565BA), and the

quality of the chip data was analyzed with R statistical language and the

limma package of the Bioconductor project (http://www.bioconductor.

org/) (Smyth, 2005). Acquired signals were normalized internally and

across all arrays as described by Smyth and Speed (2003), and the

average signals of replica were used for analysis. Meanwhile, linear

models and empirical Bayes methods were applied to find the differen-

tially expressed genes (Smyth, 2004).

In Situ Hybridization

Agene-specific region at the 39 end untranslated region ofMADS3 (796 to

1209 bp), which shares little sequence similarity with other genes

according to the results of a BLASTn analysis, was amplified by PCR

from the cDNA clone, digested withBamHI andHindIII, and transcribed in

vitro under T7 and SP6 promoters with RNA polymerase, using the DIG

RNA labeling kit (Roche). In situ hybridizationwas performed according to

Kouchi and Hata (1993) and Li et al. (2006).

Rice MADS3 Polyclonal Antibody Preparation and

Specificity Analysis

For production of the MADS3-specific polyclonal antibody, a MADS3-

specific fragment (643 to 828) was amplified from the cDNA clone and

with the primer pair MADS3-anti-F1 and MADS3-anti-R (see Supple-

mental Table 2 online). The PCR product, called MADS3-1, was cloned

into the BamHI and SalI sites of pET-32a (Novagen) to produce p32-

MADS3-1. Then, the primer pairMADS3-anti-F2 andMADS3-anti-R (see

Supplemental Table 2 online) was used to amplify the same MADS3-

specific fragment (643 to 828) for insertion at the 59 end of MADS3-1 at

HindIII and XhoI sites in p32-MADS3-1 to obtain p32-MADS3. The fused

protein tagged by 63His at the N terminus was expressed and purified

according to Novagen instructions for pET-32a, and the antibody was

prepared as described by Huang et al. (2003). Nuclear extracts used for

immunoblot analysis to test the specificity of the MADS3 antibody were

isolated following the protocol used for ChIP experiments, except that the

tissue was not fixed (Zhang et al., 2010b).

ChIP-qPCR Analysis

Rice spikeletes at stage 10 were treated and sonicated with an Ultrasonic

Crasher Noise Isolating Chamber (Scientz). The procedure for ChIP of

MADS3-DNA complexes in wild-type andmads3-4 anthers was modified

from Haring et al. (2007).

For qPCR analysis, primers are listed in Supplemental Table 2 online,

and reactions were performed on a Rotor-Gene RG3000A detection

system (Corbett Research) using SYBR Green I. For PCR reactions, 0.5

mL of recovered DNA from ChIP or controls, or 1 mL of 50-fold diluted

input DNA,was added as template, and each reactionwas repeated three

times. The normalized mean cycle threshold (Ct) of each gene was

calculated and used for fold change calculations using the method

described by Rotor-Gene version 6.0 (Build 38) software and Zhang et al.

(2010b). The difference between the Ct of the wild type andmads3-4was

calculated to obtain the relative enrichment of the upstream fragment.

Determination of the Levels of ROS, Superoxide Anion, and

Hydrogen Peroxide, and Activities of Superoxide Dismutase

and Peroxidases

Measurement of ROS and superoxide anions was performed as de-

scribed by Schopfer et al. (2001), with DCFH-diacetate from Boehringer

Mannheim and 50 mg freshly detached anthers in each reaction. For

superoxide anion measurement, 50 mg freshly detached anthers were

incubated in 1mLK-phosphate buffer (20mm, pH 6.0) containing 500mM

WST (Dojindo) for 8 h at 258C in the darkness on a shaker. Blanks without

528 The Plant Cell

Page 15: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

plant material were run in parallel and used as the control. Hydrogen

peroxide wasmeasured with a commercial kit (Biyuntian Laboratories), in

which ferrous ions (Fe2+) are oxidized to ferric ions (Fe3+) by hydrogen

peroxide, and the Fe3+ then forms a complex with an indicator dye,

xylenol orange (3,3-bis[N,N-di(carboxymethyl)-aminomethyl]-o-cresol-

sulfone-phthalein (Deiana et al., 1999).

Protein extractions used for superoxide dismutase and peroxidase

activity assays were prepared as described by Chen et al. (2004).

Superoxide dismutase activities were measured with a Superoxide

Dismutase Assay Kit-WST (Dojindo Laboratories).

Histochemical Assays of the Production of Superoxide Anion and

Hydrogen Peroxide

In vivo hydrogen peroxide staining was performed as described by

Barcelo (1998), using TMB. Freshly collected anthers were put in the

staining solution (0.1mgmL21 TMB solution in Tris-acetate, pH 5.0) under

vacuumconditions for 15min and then incubated at 258Cuntil a blue color

appeared. Production of superoxide anion was visualized by incubating

intact anthers in 10 mM K-citrate buffer, pH 6.0, containing 0.5 mM NBT

(Liszkay et al., 2004).

MT-1-4b-amiRNA Construct

The online tool WMD2 (http://wmd2.weigelworld.org) was used to select

the suitableMT-1-4b-amiRNA sequences (Schwab et al., 2006; Ossowski

et al., 2008). The recommended 59-TATGGATTCAGTCGCACTCAC-39

asMT-1-4b-amiRNAsequenceand59-GTGAGAGCGTCTGAATCCATA-39

as MT-1-4b-amiRNA* sequence were employed to replace the natural

osa-MIR528 precursor as described byWarthmann et al. (2008). The osa-

MIR528-MT-1-4b-amiRNA sequence synthesized by the Jie Rui Biotech

Company (Shanghai) was then inserted into the BamHI and XbaI sites of

the binary vector pHB, which has the double CaMV 35S promoter, for rice

transformation.

EMSA

The MADS3 fragment, which contains 702 bp of the putative full-length

coding region, was amplified with MADS3-PF and MADS3-PR (see

Supplemental Table 2 online) and cloned into pET32a at BamHI and

XhoI sites before the construct was transformed into BL21 (DE3) pLysS.

The transformed cells were cultured at 378C until the OD600 was 0.6 and

then induced with 1 mM isopropyl b-D-thiogalactopyranoside for 36 h at

158C. For purification of the fusion protein, the cells were lysed using a

high-pressure cell crusher, and the His-tagged fusion proteins were

purified with Ni-NTA resin (Qiagen) according to the manufacturer’s

instructions. The P-MT-1-4b DNA fragment containing the CCTTTTGGGG

boxwas amplified usingprimersMT-1-4b-EMSA-F andMT-1-4b-EMSA-R

(see Supplemental Table 2 online). The DNA fragments were cloned into

pMD18-T (TaKaRa) for sequence confirmation and then labeled with the

DIG labeling kit (DDLK-010). The DNA binding reactions were performed

according to Wang et al. (2002).

Production of Recombinant MT-1-4b and Assays to Test Its Activity

A fragment containing the entire open reading frame (237 bp) ofMT-1-4b

was amplified using primers MT-1-4b-F and MT-1-4b-R (see Supple-

mental Table 2 online), cloned into the EcoRI and XhoI sites of pET-32a

(Novagen) to produce a pET-32a-MT-1-4b expression vector, and then

transformed intoBL21 (DE3) pLysS. The transformed cells were cultured at

378C until the OD600 reached 0.6 and then induced with 1 mM isopropyl

b-D-thiogalactopyranoside for 4 h at 378C. Purification of the native

ET-32a-MT-1-4b fusion protein (32 kD) was performed following the

protocol used for MADS3 recombinant protein purification in the EMSA

experiment (see above).

For hydroxyl radical scavenging assays, antioxidant-mediated com-

petitive inhibition of the salicylate hydroxylation by hydroxyl radicals was

performed as described previously (Smirnoff and Cumbes, 1989) using

protein expressed from pET-32a as a control. The measurement was

repeated three times. For superoxide anion scavenging assays, the

inhibition of WST reduction by an antioxidant was performed as de-

scribed by Wong et al. (2004).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis

Genome Initiative or GenBank/EMBL databases under the follow-

ing accession numbers: MADS3 (Os01g0201700), cDNA of MADS3

(AK108568), MT2b (Os05g0111300), MADS47 (Os03g0186600),

MADS68 (Os11g0658700), MADS98 (Os01g0913900), GID1L2

(Os07g0643000), gibberellin 2-oxidase (Os05g0158600), gibberellin

3-b-dioxygenase 2-2 (Os05g0178100), auxin-induced protein 5NG4

(Os02g0103600), MSP1 (Os01g0917500), PAIR2 (Os09g0506800),

GAMYB (Os01g0812000),Wda1 (Os10g0471100),CP1 (Os04g0670500),

RAFITIN (Os08g0496800),MST8 (Os01g0567500), INV4 (Os04g0413200),

TDR (Os02g0120500), C6 (Os11g0582500), CYP704B2 (Os03g0168600),

CYP703A3 (Os08g0131100), and UDT1 (Os07g0549600).

Supplemental Data

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

Supplemental Figure 1. DAPI Staining Analysis of Wild-Type and

mads3-4 Microspores.

Supplemental Figure 2. Complementation of the mads3-4 Mutant.

Supplemental Figure 3. Transmission Electron Micrographs of An-

thers in Wild-Type and mads3-4 Plants at Stage 9.

Supplemental Figure 4. Higher Superoxide Dismutase and Peroxi-

dase Activities in mads3-4 Anthers from Stage 10 to Stage 12.

Supplemental Figure 5. Analysis of Hydrogen Peroxide in Wild-Type

and mads3-4 Anthers.

Supplemental Figure 6. qRT-PCR Analysis of Selected Genes

Showing Changed Expression in mads3-4 Anthers.

Supplemental Figure 7. Analysis of the Specificity of Polyclonal

Antibodies against MADS3.

Supplemental Figure 8. Analysis of Spatial and Temporal Expression

of MT-1-4b by Qualitative and Quantitative RT-PCR.

Supplemental Table 1. Summary of Floral Organ Defects in mads3-4.

Supplemental Table 2. Primers Used in This Study.

Supplemental Table 3. Functional Classification by Cluster of

Orthologous Groups of Proteins (COG) Analysis of Genes That Are

Upregulated or Downregulated (at Least 2-Fold Change in Expres-

sion) in Anthers, as Identified by Bayes Analysis (<0.5% FDR).

Supplemental Table 4. GO Term Enrichments (P Value < 0.05) for

Genes with Expression Changes of at Least 2-Fold (mads3-4/Wild

Type), as Identified by Bayes Analysis (<0.5% FDR).

Supplemental Table 5. Genes Encoding Transcriptional Factors with

Expression Changes of at Least 2-Fold (mads3-4/Wild Type) in

mads3-4 Anthers.

Supplemental Table 6. Genes with Putative Functions in Phytohor-

mone Biosynthesis, Metabolism and Signaling That Have Expression

Changes of at Least 2-Fold (mads3-4/Wild Type) in mads3-4

Anthers.

MADS3 Regulates Anther Development 529

Page 16: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

Supplemental Table 7. Changes in Expression of Genes Required for

Rice Anther Development in mads3-4.

Supplemental Table 8. Forty-Two Cytochrome P450 Genes That

Have Expression Changes of at Least 2-Fold (mads3-4/Wild Type) in

mads3-4 Anthers and Putative CArG-Boxes within the 1-kb Upstream

Region in These Genes.

Supplemental Data Set 1. Genes Whose Expression Differs in

Stage-9 mads3-4 Anthers from the Wild Type (by at Least 2-Fold),

as Identified by Limma Analysis (FDR > 0.05%).

Supplemental Data Set 2. Genes with an Expression Change of at

Least 2-Fold in mads3-4 and Found to Be Expressed in Tapetal Cells

and/or Microspores.

Supplemental Data Set 3. The 189 Downregulated Genes with

Expression Changes of at Least 2-Fold inmads3-4 Anthers and Putative

CArG-Boxes within the 1-kb Upstream Regions of These Genes.

Supplemental Data Set 4. The 169 Upregulated Genes with Expres-

sion Changes of at Least 2-Fold in mads3-4 Anthers and Putative

CArG-Boxes within the 1-kb Upstream Region of These Genes.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Sheila McCormick and the anon-

ymous reviewers for comments on the manuscript, Changjun Liu for

discussions, and the Rice Genome Resource Center for providing the

MADS3 cDNA clone. We thank Zhijing Luo and Mingjiao Chen for

mutant screening and generation of F2 populations for gene mapping,

Xiaoyan Gao and Ping Zhang for making plastic sections and TEM

observations, and Hongquan Yang for providing the binary vector pHB.

This work was supported by funds from the National Key Basic

Research Developments Program, Ministry of Science and Technology,

P.R. China (2009CB941500 and 2006CB101700), National “863” High-

Tech Project (2006AA10A102), National Natural Science Foundation of

China (30725022 and 30600347), Shanghai Leading Academic Disci-

pline Project (B205), and the National Transgenic Major Program

(2011ZX08009-003-003).

Received January 30, 2010; revised January 6, 2011; accepted January

19, 2011; published February 4, 2011.

REFERENCES

Alvarez-Buylla, E.R., Liljegren, S.J., Pelaz, S., Gold, S.E., Burgeff, C.,

Ditta, G.S., Vergara-Silva, F., and Yanofsky, M.F. (2000). MADS-

box gene evolution beyond flowers: Expression in pollen, endosperm,

guard cells, roots and trichomes. Plant J. 24: 457–466.

Antonenkov, V.D., Grunau, S., Ohlmeier, S., and Hiltunen, J.K.

(2010). Peroxisomes are oxidative organelles. Antioxid. Redox Signal.

13: 525–537.

Apel, K., and Hirt, H. (2004). Reactive oxygen species: Metabolism,

oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55:

373–399.

Asada, K. (1999). The water-water cycle in chloroplasts: Scavenging of

active oxygens and dissipation of excess photons. Annu. Rev. Plant

Physiol. Plant Mol. Biol. 50: 601–639.

Aya, K., Ueguchi-Tanaka, M., Kondo, M., Hamada, K., Yano, K.,

Nishimura, M., and Matsuoka, M. (2009). Gibberellin modulates

anther development in rice via the transcriptional regulation of

GAMYB. Plant Cell 21: 1453–1472.

Babbs, C.F., Pham, J.A., and Coolbaugh, R.C. (1989). Lethal hydroxyl

radical production in paraquat-treated plants. Plant Physiol. 90: 1267–

1270.

Barcelo, A.R. (1998). Hydrogen peroxide production is a general

property of the lignifying xylem from vascular plants. Ann. Bot.

(Lond.) 82: 97–103.

Bomal, C., et al. (2008). Involvement of Pinus taeda MYB1 and MYB8 in

phenylpropanoid metabolism and secondary cell wall biogenesis: A

comparative in planta analysis. J. Exp. Bot. 59: 3925–3939.

Bouchez, O., Huard, C., Lorrain, S., Roby, D., and Balague, C. (2007).

Ethylene is one of the key elements for cell death and defense

response control in the Arabidopsis lesion mimic mutant vad1. Plant

Physiol. 145: 465–477.

Bowman, J.L., Drews, G.N., and Meyerowitz, E.M. (1991). Expression

of the Arabidopsis floral homeotic gene AGAMOUS is restricted to

specific cell types late in flower development. Plant Cell 3: 749–758.

Chen, J., Song, L., Dai, J., Gan, N., and Liu, Z. (2004). Effects of

microcystins on the growth and the activity of superoxide dismutase

and peroxidase of rape (Brassica napus L.) and rice (Oryza sativa L.).

Toxicon 43: 393–400.

Chen, L., Chu, H.W., Yuan, Z., Pan, A.H., Liang, W.Q., Huang, H.,

Shen, M.S., Zhang, D.B., and Chen, L. (2006). Isolation and genetic

analysis for rice mutants treated with 60 Co g-Ray. J. Xiamen Uni. 45:

82–85.

Chew, O., Rudhe, C., Glaser, E., and Whelan, J. (2003). Character-

ization of the targeting signal of dual-targeted pea glutathione reduc-

tase. Plant Mol. Biol. 53: 341–356.

Cho, S., Jang, S., Chae, S., Chung, K.M., Moon, Y.H., An, G., and

Jang, S.K. (1999). Analysis of the C-terminal region of Arabidopsis

thaliana APETALA1 as a transcription activation domain. Plant Mol.

Biol. 40: 419–429.

Cobbett, C., and Goldsbrough, P. (2002). Phytochelatins and metal-

lothioneins: Roles in heavy metal detoxification and homeostasis.

Annu. Rev. Plant Biol. 53: 159–182.

Coen, E.S., and Meyerowitz, E.M. (1991). The war of the whorls: Ge-

netic interactions controlling flower development. Nature 353: 31–37.

Creissen, G., Reynolds, H., Xue, Y., and Mullineaux, P. (1995).

Simultaneous targeting of pea glutathione reductase and of a bacte-

rial fusion protein to chloroplasts and mitochondria in transgenic

tobacco. Plant J. 8: 167–175.

Dai, Z., Gao, J., An, K., Lee, J.M., Edwards, G.E., and An, G. (1996).

Promoter elements controlling developmental and environmental reg-

ulation of a tobacco ribosomal protein gene L34. Plant Mol. Biol. 32:

1055–1065.

Deiana, L., Carru, C., Pes, G., and Tadolini, B. (1999). Spectropho-

tometric measurement of hydroperoxides at increased sensitivity by

oxidation of Fe2+ in the presence of xylenol orange. Free Radic. Res.

31: 237–244.

del Rıo, L.A., Corpas, F.J., Sandalio, L.M., Palma, J.M., Gomez, M.,

and Barroso, J.B. (2002). Reactive oxygen species, antioxidant

systems and nitric oxide in peroxisomes. J. Exp. Bot. 53: 1255–1272.

Ditta, G., Pinyopich, A., Robles, P., Pelaz, S., and Yanofsky, M.F.

(2004). The SEP4 gene of Arabidopsis thaliana functions in floral organ

and meristem identity. Curr. Biol. 14: 1935–1940.

Doyle, S.M., Diamond, M., and McCabe, P.F. (2010). Chloroplast

and reactive oxygen species involvement in apoptotic-like program-

med cell death in Arabidopsis suspension cultures. J. Exp. Bot. 61:

473–482.

Dreni, L., Jacchia, S., Fornara, F., Fornari, M., Ouwerkerk, P.B., An,

G., Colombo, L., and Kater, M.M. (2007). The D-lineage MADS-box

gene OsMADS13 controls ovule identity in rice. Plant J. 52: 690–699.

Egea-Cortines, M., Saedler, H., and Sommer, H. (1999). Ternary

complex formation between the MADS-box proteins SQUAMOSA,

530 The Plant Cell

Page 17: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

DEFICIENS and GLOBOSA is involved in the control of floral archi-

tecture in Antirrhinum majus. EMBO J. 18: 5370–5379.

Feng, J.H., Lu, Y.G., Liu, X.D., and Xu, X.B. (2001). Pollen development

and its stages in rice (Oryza sativa L.). Chinese J. Rice Sci. 15: 21–28.

Feng, X.L., Ni, W.M., Elge, S., Mueller-Roeber, B., Xu, Z.H., and Xue,

H.W. (2006). Auxin flow in anther filaments is critical for pollen grain

development through regulating pollen mitosis. Plant Mol. Biol. 61:

215–226.

Foreman, J., Demidchik, V., Bothwell, J.H., Mylona, P., Miedema, H.,

Torres, M.A., Linstead, P., Costa, S., Brownlee, C., Jones, J.D.,

Davies, J.M., and Dolan, L. (2003). Reactive oxygen species pro-

duced by NADPH oxidase regulate plant cell growth. Nature 422:

442–446.

Gapper, C., and Dolan, L. (2006). Control of plant development by

reactive oxygen species. Plant Physiol. 141: 341–345.

Gechev, T.S., Van Breusegem, F., Stone, J.M., Denev, I., and Laloi,

C. (2006). Reactive oxygen species as signals that modulate plant

stress responses and programmed cell death. Bioessays 28: 1091–

1101.

Han, M.J., Jung, K.H., Yi, G., Lee, D.Y., and An, G. (2006). Rice

Immature Pollen 1 (RIP1) is a regulator of late pollen development.

Plant Cell Physiol. 47: 1457–1472.

Haring, M., Offermann, S., Danker, T., Horst, I., Peterhansel, C., and

Stam, M. (2007). Chromatin immunoprecipitation: Optimization, quan-

titative analysis and data normalization. Plant Methods 3: 11.

Hayashi, M., Takahashi, H., Tamura, K., Huang, J., Yu, L.H., Kawai-

Yamada, M., Tezuka, T., and Uchimiya, H. (2005). Enhanced

dihydroflavonol-4-reductase activity and NAD homeostasis leading

to cell death tolerance in transgenic rice. Proc. Natl. Acad. Sci. USA

102: 7020–7025.

Hiei, Y., Komari, T., and Kubo, T. (1997). Transformation of

rice mediated by Agrobacterium tumefaciens. Plant Mol. Biol. 35:

205–218.

Hobo, T., et al. (2008). Various spatiotemporal expression profiles of

anther-expressed genes in rice. Plant Cell Physiol. 49: 1417–1428.

Huang, W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and

integrative analysis of large gene lists using DAVID bioinformatics

resources. Nat. Protoc. 4: 44–57.

Huang, Y., Liang, W., Pan, A., Zhou, Z., Huang, C., Chen, J., and

Zhang, D. (2003). Production of FaeG, the major subunit of K88

fimbriae, in transgenic tobacco plants and its immunogenicity in mice.

Infect. Immun. 71: 5436–5439.

Irmler, S., Schroder, G., St-Pierre, B., Crouch, N.P., Hotze, M.,

Schmidt, J., Strack, D., Matern, U., and Schroder, J. (2000). Indole

alkaloid biosynthesis in Catharanthus roseus: New enzyme activities

and identification of cytochrome P450 CYP72A1 as secologanin

synthase. Plant J. 24: 797–804.

Ishiguro, S., Kawai-Oda, A., Ueda, J., Nishida, I., and Okada, K.

(2001). The DEFECTIVE IN ANTHER DEHISCIENCE gene encodes a

novel phospholipase A1 catalyzing the initial step of jasmonic acid

biosynthesis, which synchronizes pollen maturation, anther dehis-

cence, and flower opening in Arabidopsis. Plant Cell 13: 2191–2209.

Itoh, J., Nonomura, K., Ikeda, K., Yamaki, S., Inukai, Y., Yamagishi,

H., Kitano, H., and Nagato, Y. (2005). Rice plant development: From

zygote to spikelet. Plant Cell Physiol. 46: 23–47.

Ito, T., Ng, K.H., Lim, T.S., Yu, H., and Meyerowitz, E.M. (2007). The

homeotic protein AGAMOUS controls late stamen development by

regulating a jasmonate biosynthetic gene in Arabidopsis. Plant Cell

19: 3516–3529.

Ito, T., Wellmer, F., Yu, H., Das, P., Ito, N., Alves-Ferreira, M.,

Riechmann, J.L., and Meyerowitz, E.M. (2004). The homeotic pro-

tein AGAMOUS controls microsporogenesis by regulation of SPOR-

OCYTELESS. Nature 430: 356–360.

Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUS

fusions: Beta-glucuronidase as a sensitive and versatile gene fusion

marker in higher plants. EMBO J. 6: 3901–3907.

Jung, K.H., Han, M.J., Lee, D.Y., Lee, Y.S., Schreiber, L., Franke, R.,

Faust, A., Yephremov, A., Saedler, H., Kim, Y.W., Hwang, I., and

An, G. (2006). Wax-deficient anther1 is involved in cuticle and wax

production in rice anther walls and is required for pollen development.

Plant Cell 18: 3015–3032.

Jung, K.H., Han, M.J., Lee, Y.S., Kim, Y.W., Hwang, I., Kim, M.J.,

Kim, Y.K., Nahm, B.H., and An, G. (2005). Rice Undeveloped

Tapetum1 is a major regulator of early tapetum development. Plant

Cell 17: 2705–2722.

Kaneko, M., Inukai, Y., Ueguchi-Tanaka, M., Itoh, H., Izawa, T.,

Kobayashi, Y., Hattori, T., Miyao, A., Hirochika, H., Ashikari, M.,

and Matsuoka, M. (2004). Loss-of-function mutations of the rice

GAMYB gene impair alpha-amylase expression in aleurone and flower

development. Plant Cell 16: 33–44.

Kang, H.G., Jeon, J.S., Lee, S., and An, G. (1998). Identification of

class B and class C floral organ identity genes from rice plants. Plant

Mol. Biol. 38: 1021–1029.

Karpinski, S., Escobar, C., Karpinska, B., Creissen, G., and

Mullineaux, P.M. (1997). Photosynthetic electron transport regulates

the expression of cytosolic ascorbate peroxidase genes in Arabidop-

sis during excess light stress. Plant Cell 9: 627–640.

Karpinski, S., Reynolds, H., Karpinska, B., Wingsle, G., Creissen, G.,

and Mullineaux, P. (1999). Systemic signaling and acclimation in

response to excess excitation energy in Arabidopsis. Science 284:

654–657.

Kouchi, H., and Hata, S. (1993). Isolation and characterization of novel

nodulin cDNAs representing genes expressed at early stages of

soybean nodule development. Mol. Gen. Genet. 238: 106–119.

Lee, S., Jung, K.H., An, G., and Chung, Y.Y. (2004). Isolation

and characterization of a rice cysteine protease gene, OsCP1, using

T-DNA gene-trap system. Plant Mol. Biol. 54: 755–765.

Li, H., Pinot, F., Sauveplane, V., Werck-Reichhart, D., Diehl, P.,

Schreiber, L., Franke, R., Zhang, P., Chen, L., Gao, Y.W., Liang,

W.Q., and Zhang, D.B. (2010b). Cytochrome P450 family member

CYP704B2 catalyzes the omega-hydroxylation of fatty acids and is

required for anther cutin biosynthesis and pollen exine formation in

rice. Plant Cell 22: 173–190.

Li, H.F., Liang, W.Q., Jia, R.D., Yin, C.S., Zong, J., Kong, H.Z., and

Zhang, D.B. (2010a). The AGL6-like gene OsMADS6 regulates floral

organ and meristem identities in rice. Cell Res. 20: 299–313.

Li, N., et al. (2006). The rice tapetum degeneration retardation gene is

required for tapetum degradation and anther development. Plant Cell

18: 2999–3014.

Liu, Z.H., Bao, W.J., Liang, W.Q., Yin, J.Y., and Zhang, D.B. (2010).

Identification of gamyb-4 and analysis of the regulatory role of

GAMYB in rice anther development. J. Integr. Plant Biol. 52: 670–678.

Liszkay, A., van der Zalm, E., and Schopfer, P. (2004). Production of

reactive oxygen intermediates (O(2)(.-), H(2)O(2), and (.)OH) by maize

roots and their role in wall loosening and elongation growth. Plant

Physiol. 136: 3114–3123, discussion 3001.

Lopez-Huertas, E., Charlton, W.L., Johnson, B., Graham, I.A., and

Baker, A. (2000). Stress induces peroxisome biogenesis genes.

EMBO J. 19: 6770–6777.

McCormick, S. (1993). Male gametophyte development. Plant Cell 5:

1265–1275.

Miller, G., and Mittler, R. (2006). Could heat shock transcription factors

function as hydrogen peroxide sensors in plants? Ann. Bot. (Lond.)

98: 279–288.

Miller, G., Shulaev, V., and Mittler, R. (2008). Reactive oxygen signal-

ing and abiotic stress. Physiol. Plant. 133: 481–489.

MADS3 Regulates Anther Development 531

Page 18: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

Mittler, R., Vanderauwera, S., Gollery, M., and Van Breusegem, F.

(2004). Reactive oxygen gene network of plants. Trends Plant Sci. 9:

490–498.

Moeder, W., Barry, C.S., Tauriainen, A.A., Betz, C., Tuomainen, J.,

Utriainen, M., Grierson, D., Sandermann, H., Langebartels, C., and

Kangasjarvi, J. (2002). Ethylene synthesis regulated by biphasic

induction of 1-aminocyclopropane-1-carboxylic acid synthase and

1-aminocyclopropane-1-carboxylic acid oxidase genes is required for

hydrogen peroxide accumulation and cell death in ozone-exposed

tomato. Plant Physiol. 130: 1918–1926.

Nagasawa, N., Miyoshi, M., Sano, Y., Satoh, H., Hirano, H., Sakai, H.,

and Nagato, Y. (2003). SUPERWOMAN1 and DROOPING LEAF

genes control floral organ identity in rice. Development 130: 705–718.

Nonomura, K., Miyoshi, K., Eiguchi, M., Suzuki, T., Miyao, A.,

Hirochika, H., and Kurata, N. (2003). The MSP1 gene is necessary

to restrict the number of cells entering into male and female sporo-

genesis and to initiate anther wall formation in rice. Plant Cell 15:

1728–1739.

Nonomura, K.I., Nakano, M., Murata, K., Miyoshi, K., Eiguchi, M.,

Miyao, A., Hirochika, H., and Kurata, N. (2004). An insertional

mutation in the rice PAIR2 gene, the ortholog of Arabidopsis ASY1,

results in a defect in homologous chromosome pairing during meiosis.

Mol. Genet. Genomics 271: 121–129.

Obara, K., Sumi, K., and Fukuda, H. (2002). The use of multiple

transcription starts causes the dual targeting of Arabidopsis putative

monodehydroascorbate reductase to both mitochondria and chloro-

plasts. Plant Cell Physiol. 43: 697–705.

Ohmori, S., Kimizu, M., Sugita, M., Miyao, A., Hirochika, H., Uchida,

E., Nagato, Y., and Yoshida, H. (2009). MOSAIC FLORAL OR-

GANS1, an AGL6-like MADS box gene, regulates floral organ identity

and meristem fate in rice. Plant Cell 21: 3008–3025.

Oliver, S.N., Dennis, E.S., and Dolferus, R. (2007). ABA regulates

apoplastic sugar transport and is a potential signal for cold-induced

pollen sterility in rice. Plant Cell Physiol. 48: 1319–1330.

Oliver, S.N., Van Dongen, J.T., Alfred, S.C., Mamun, E.A., Zhao, X.,

Saini, H.S., Fernandes, S.F., Blanchard, C.L., Sutton, B.G.,

Geigenberger, P., Dennis, E.S., and Dolferus, R. (2005). Cold-

induced repression of the rice anther-specific cell wall invertase gene

OSINV4 is correlated with sucrose accumulation and pollen sterility.

Plant Cell Environ. 28: 1534–1551.

Ossowski, S., Schwab, R., and Weigel, D. (2008). Gene silencing in

plants using artificial microRNAs and other small RNAs. Plant J. 53:

674–690.

Overmyer, K., Brosche, M., and Kangasjarvi, J. (2003). Reactive

oxygen species and hormonal control of cell death. Trends Plant Sci.

8: 335–342.

Papini, A., Mosti, S., and Brighigna, L. (1999). Programmed-cell-death

events during tapetum development of angiosperms. Protoplasma

207: 213–221.

Peskin, A.V., and Winterbourn, C.C. (2000). A microtiter plate assay for

superoxide dismutase using a water-soluble tetrazolium salt (WST-1).

Clin. Chim. Acta 293: 157–166.

Pnueli, L., Liang, H., Rozenberg, M., and Mittler, R. (2003). Growth

suppression, altered stomatal responses, and augmented induction

of heat shock proteins in cytosolic ascorbate peroxidase (Apx1)-

deficient Arabidopsis plants. Plant J. 34: 187–203.

Riechmann, J.L., Wang, M., and Meyerowitz, E.M. (1996). DNA-

binding properties of Arabidopsis MADS domain homeotic proteins

APETALA1, APETALA3, PISTILLATA and AGAMOUS. Nucleic Acids

Res. 24: 3134–3141.

Rizhsky, L., Liang, H., and Mittler, R. (2003). The water-water cycle is

essential for chloroplast protection in the absence of stress. J. Biol.

Chem. 278: 38921–38925.

Robinson, N.J., Tommey, A.M., Kuske, C., and Jackson, P.J. (1993).

Plant metallothioneins. Biochem. J. 295: 1–10.

Rodrıguez, A.A., Grunberg, K.A., and Taleisnik, E.L. (2002). Reactive

oxygen species in the elongation zone of maize leaves are necessary

for leaf extension. Plant Physiol. 129: 1627–1632.

Sagi, M., Davydov, O., Orazova, S., Yesbergenova, Z., Ophir, R.,

Stratmann, J.W., and Fluhr, R. (2004). Plant respiratory burst oxi-

dase homologs impinge on wound responsiveness and development

in Lycopersicon esculentum. Plant Cell 16: 616–628.

Schiefthaler, U., Balasubramanian, S., Sieber, P., Chevalier, D.,

Wisman, E., and Schneitz, K. (1999). Molecular analysis of NOZZLE,

a gene involved in pattern formation and early sporogenesis during

sex organ development in Arabidopsis thaliana. Proc. Natl. Acad. Sci.

USA 96: 11664–11669.

Schopfer, P., Plachy, C., and Frahry, G. (2001). Release of reactive

oxygen intermediates (superoxide radicals, hydrogen peroxide, and

hydroxyl radicals) and peroxidase in germinating radish seeds controlled

by light, gibberellin, and abscisic acid. Plant Physiol. 125: 1591–1602.

Schrader, M., and Fahimi, H.D. (2006). Peroxisomes and oxidative

stress. Biochim. Biophys. Acta 1763: 1755–1766.

Schreiber, D.N., Bantin, J., and Dresselhaus, T. (2004). The MADS

box transcription factor ZmMADS2 is required for anther and pollen

maturation in maize and accumulates in apoptotic bodies during

anther dehiscence. Plant Physiol. 134: 1069–1079.

Schuler, M.A., and Werck-Reichhart, D. (2003). Functional genomics

of P450s. Annu. Rev. Plant Biol. 54: 629–667.

Schwab, R., Ossowski, S., Riester, M., Warthmann, N., and Weigel,

D. (2006). Highly specific gene silencing by artificial microRNAs in

Arabidopsis. Plant Cell 18: 1121–1133.

Simontacchi, M., Caro, A., Fraga, C.G., and Puntarulo, S. (1993).

Oxidative stress affects [alpha]-tocopherol content in soybean em-

bryonic axes upon imbibition and following germination. Plant Physiol.

103: 949–953.

Smirnoff, N., and Cumbes, Q.J. (1989). Hydroxyl radical scavenging

activity of compatible solutes. Phytochemistry 28: 1057–1060.

Smyth, G.K. (2004). Linear models and empirical bayes methods for

assessing differential expression in microarray experiments. Stat.

Appl. Genet. Mol. Biol. 3: Article3.

Smyth, G.K. (2005). Limma: Linear models for microarray data. In

Bioinformatics and Computational Biology Solutions Using R and

Bioconductor, R. Gentleman, V. Carey, S. Dudoit, R. Irizarry, and W.

Huber, eds (New York: Springer), pp. 397–420.

Smyth, G.K., and Speed, T.P. (2003). Normalization of cDNA micro-

array data. Methods 31: 265–273.

Steffens, B., and Sauter, M. (2009). Epidermal cell death in rice is

confined to cells with a distinct molecular identity and is mediated by

ethylene and H2O2 through an autoamplified signal pathway. Plant

Cell 21: 184–196.

Suwabe, K., et al. (2008). Separated transcriptomes of male gameto-

phyte and tapetum in rice: Validity of a laser microdissection (LM)

microarray. Plant Cell Physiol. 49: 1407–1416.

Tan, A.S., and Berridge, M.V. (2000). Superoxide produced by acti-

vated neutrophils efficiently reduces the tetrazolium salt, WST-1 to

produce a soluble formazan: A simple colorimetric assay for measur-

ing respiratory burst activation and for screening anti-inflammatory

agents. J. Immunol. Methods 238: 59–68.

Tang, L.K., Chu, H., Yip, W.K., Yeung, E.C., and Lo, C. (2009). An

anther-specific dihydroflavonol 4-reductase-like gene (DRL1) is es-

sential for male fertility in Arabidopsis. New Phytol. 181: 576–587.

Thijs, G., Marchal, K., Lescot, M., Rombauts, S., De Moor, B.,

Rouze, P., and Moreau, Y. (2002). A Gibbs sampling method to

detect overrepresented motifs in the upstream regions of coex-

pressed genes. J. Comput. Biol. 9: 447–464.

532 The Plant Cell

Page 19: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

Tiedemann, A.V. (1997). Evidence for a primary role of active oxygen

species in induction of host cell death during infection of bean leaves

with Botrytis cinerea. Physiol. Mol. Plant Pathol. 50: 151–166.

Tsuji, H., Aya, K., Ueguchi-Tanaka, M., Shimada, Y., Nakazono, M.,

Watanabe, R., Nishizawa, N.K., Gomi, K., Shimada, A., Kitano, H.,

Ashikari, M., and Matsuoka, M. (2006). GAMYB controls different

sets of genes and is differentially regulated by microRNA in aleurone

cells and anthers. Plant J. 47: 427–444.

Varnier, A.L., Mazeyrat-Gourbeyre, F., Sangwan, R.S., and Clement,

C. (2005). Programmed cell death progressively models the develop-

ment of anther sporophytic tissues from the tapetum and is triggered

in pollen grains during maturation. J. Struct. Biol. 152: 118–128.

Wang, A., Xia, Q., Xie, W., Datla, R., and Selvaraj, G. (2003). The

classical Ubisch bodies carry a sporophytically produced structural

protein (RAFTIN) that is essential for pollen development. Proc. Natl.

Acad. Sci. USA 100: 14487–14492.

Wang, H., Tang, W., Zhu, C., and Perry, S.E. (2002). A chromatin

immunoprecipitation (ChIP) approach to isolate genes regulated by

AGL15, a MADS domain protein that preferentially accumulates in

embryos. Plant J. 32: 831–843.

Wang, Y., Wang, Y.F., and Zhang, D.B. (2006). Identification of the rice

(Oryza sativa L.) mutant msp1-4 and expression analysis of its UDT1

and GAMYB genes. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue

Bao 32: 527–534.

Warthmann, N., Chen, H., Ossowski, S., Weigel, D., and Herve, P.

(2008). Highly specific gene silencing by artificial miRNAs in rice. PLoS

ONE 3: e1829.

Wiseman, A. (2006). p53 protein or BID protein select the route to either

apoptosis (programmed cell death) or to cell cycle arrest opposing

carcinogenesis after DNA damage by ROS. Med. Hypotheses 67:

296–299.

Wong, H.L., Sakamoto, T., Kawasaki, T., Umemura, K., and

Shimamoto, K. (2004). Down-regulation of metallothionein, a reactive

oxygen scavenger, by the small GTPase OsRac1 in rice. Plant Physiol.

135: 1447–1456.

Yamaguchi, T., Lee, D.Y., Miyao, A., Hirochika, H., An, G., and

Hirano, H.Y. (2006). Functional diversification of the two C-class

MADS box genes OSMADS3 and OSMADS58 in Oryza sativa. Plant

Cell 18: 15–28.

Yang, W.C., Ye, D., Xu, J., and Sundaresan, V. (1999). The SPORO-

CYTELESS gene of Arabidopsis is required for initiation of spo-

rogenesis and encodes a novel nuclear protein. Genes Dev. 13:

2108–2117.

Yang, Y., Fanning, L., and Jack, T. (2003). The K domain mediates

heterodimerization of the Arabidopsis floral organ identity proteins,

APETALA3 and PISTILLATA. Plant J. 33: 47–59.

Yanofsky, M.F., Ma, H., Bowman, J.L., Drews, G.N., Feldmann, K.A.,

and Meyerowitz, E.M. (1990). The protein encoded by the Arabidop-

sis homeotic gene agamous resembles transcription factors. Nature

346: 35–39.

Zachgo, S., Saedler, H., and Schwarz-Sommer, Z. (1997). Pollen-

specific expression of DEFH125, a MADS-box transcription factor in

Antirrhinum with unusual features. Plant J. 11: 1043–1050.

Zhang, D.B., and Wilson, Z.A. (2009). Stamen specification and anther

development in rice. Chin. Sci. Bull. 54: 1–12.

Zhang, D.S., Liang, W.Q., Yin, C.S., Zong, J., Gu, F.W., and Zhang,

D.B. (2010a). OsC6, encoding a lipid transfer protein, is required

for postmeiotic anther development in rice. Plant Physiol. 154:

149–162.

Zhang, D.S., Liang, W.Q., Yuan, Z., Li, N., Shi, J., Wang, J., Liu, Y.M.,

Yu, W.J., and Zhang, D.B. (2008). Tapetum degeneration retardation

is critical for aliphatic metabolism and gene regulation during rice

pollen development. Mol. Plant 1: 599–610.

Zhang, H., Liang, W.Q., Yang, X.J., Luo, X., Jiang, N., Ma, H., and

Zhang, D.B. (2010b). Carbon starved anther encodes a MYB domain

protein that regulates sugar partitioning required for rice pollen

development. Plant Cell 22: 672–689.

Zhou, G.K., Xu, Y.F., Li, J., Yang, L.Y., and Liu, J.Y. (2006). Molecular

analyses of the metallothionein gene family in rice (Oryza sativa L.).

J. Biochem. Mol. Biol. 39: 595–606.

MADS3 Regulates Anther Development 533

Page 20: Rice MADS3 Regulates ROS Homeostasis during Late Anther ... · Rice MADS3 Regulates ROS Homeostasis during Late Anther Development W OA Lifang Hu,a,b,1 Wanqi Liang,a,1 Changsong Yin,a

DOI 10.1105/tpc.110.074369; originally published online February 4, 2011; 2011;23;515-533Plant Cell

ZhangLifang Hu, Wanqi Liang, Changsong Yin, Xiao Cui, Jie Zong, Xing Wang, Jianping Hu and Dabing

Rice MADS3 Regulates ROS Homeostasis during Late Anther Development

 This information is current as of December 8, 2020

 

Supplemental Data /content/suppl/2011/02/04/tpc.110.074369.DC1.html

References /content/23/2/515.full.html#ref-list-1

This article cites 112 articles, 40 of which can be accessed free at:

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists