Cytological Analysis and Genetic Control of Rice Anther Development

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Journal of Genetics and Genomics 38 (2011) 379e390www.jgenetgenomics.org

Cytological analysis and genetic control of rice anther development

Dabing Zhang a,b,*, Xue Luo a, Lu Zhu a

a Institute of Plant Science, State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology,

Shanghai Jiao Tong University, Shanghai 200240, ChinabBio-X Research Center, Shanghai Jiao Tong University, Shanghai 200240, China

Received 11 May 2011; revised 29 July 2011; accepted 1 August 2011

Abstract

Microsporogenesis and male gametogenesis are essential for the alternating life cycle of flowering plants between diploid sporophyte andhaploid gametophyte generations. Rice (Oryza sativa) is the world’s major staple food, and manipulation of pollen fertility is particularlyimportant for the demands to increase rice grain yield. Towards a better understanding of the mechanisms controlling rice male reproductivedevelopment, we describe here the cytological changes of anther development through 14 stages, including cell division, differentiation anddegeneration of somatic tissues consisting of four concentric cell layers surrounding and supporting reproductive cells as they form maturepollen grains through meiosis and mitosis. Furthermore, we compare the morphological difference of anthers and pollen grains in both monocotrice and eudicot Arabidopsis thaliana. Additionally, we describe the key genes identified to date critical for rice anther development and pollenformation.

Keywords: Rice (Oryza Sativa); Anther; Developmental stages; Cellular morphology; Arabidopsis thaliana

1. Introduction

Rice is one of the most important agricultural crops. Hybridrice exhibits heterosis, or hybrid vigor, which is indicated bymore rapid growth and considerablely higher yields thanproduced by the parental lines. Rice male sterility is frequentlycaused by environmental effects or genetic mutations, leadingto defective anther development and pollen fertility. Since thefemale reproductive development remains normal in somemale sterile lines, these lines can be fertilized by the pollengrains of other rice cultivars, greatly contributing to theproduction of hybrid seeds (Wilson and Zhang, 2009; Ouyang,et al., 2009, 2010). Furthermore, because of its small genomeand high efficiency of transformation, rice also has been usedas a model monocot plant for comparative studies with othermodel plants such as Arabidopsis thaliana (IRGSP, 2005; Junget al., 2008).

* Corresponding author. Tel: þ86 21 34205073, fax: þ86 21 34204869.

E-mail address: [email protected] (D. Zhang).

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Published by Elsevier Limited and Science Press. All rights reserved.

doi:10.1016/j.jgg.2011.08.001

Rice and other agriculturally important cereals includingbarley (Hordeum vulgare) and maize (Zea mays) belong to thegrass family (Poaceae), which is one of the largest families inflowering plants (Linder and Rudall, 2005). As knowledge ofits development can be extrapolated to other monocot crops,rice is useful not only as an excellent model plant for bio-logical studies, but also as a model crop for agronomicalimprovement.

Evolutionary adaptations in the organization and structureof grass inflorescence (or panicle) have resulted in theirdistinct morphologies from those of core eudicots and non-grass monocots (Grass Phylogeny Working Group, 2001;Zanis, 2007). The rice inflorescence or panicle has a centralstem that terminates after the generation of several primaryand secondary branches (Fig. 1A). Spikelets are directlyformed on primary and secondary branches that are attachedon the main axis called the rachis (Itoh et al., 2005). Each ricespikelet contains a flower with one pistil, six stamens and twolodicules subtended by an inner bract or prophyll, called thepalea, and an outer bract called the lemma (Fig. 1B) (Yuanet al., 2009b). Each stamen consists of a filament and an

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Fig. 1. Morphology of rice panicle, spikelet organs and anther section. A: a mature inflorescence of rice at the heading stage. B: a mature spikelet of rice with

formation of ovule and pollen grains. C: a mature anther of rice. D: section of anther at stage 9 showing the anther wall layers, microspores in the locule as well as

vascular tissues and connective tissues. Rachis, inflorescence axis; pib ¼ primary inflorescence branch; sib ¼ secondary inflorescence branch; sp ¼ spikelet;

pi ¼ pistil; st ¼ stamens; sl ¼ sterile lemma; pa ¼ palea; le ¼ lemma; fi ¼ filament; lo ¼ lodicule; E ¼ epeidermis; En ¼ endothecium; T ¼ tapetum; C ¼ cavity

for dehiscence; V ¼ vascular bundle.

380 D. Zhang et al. / Journal of Genetics and Genomics 38 (2011) 379e390

anther with four lobes linked to the filament by connectivetissues (Fig. 1B). The rice anther includes two thecae linkedby the connective tissue, and each theca contains two locules,one is longer at the base and the other is shorter. The twolocules are connected by a septum and stomium (consisting ofsmall epidermal cells), which are crucial for anther dehiscence(Fig. 1C) (Matsui et al., 1999).

Successful male reproductive development within the maleorgan, the anther, includes a number of critical developmentalevents such as meristem specification, cell differentiation, cell-to-cell communication, meiosis and mitosis (Scott et al., 2004,McCormick, 2004, Ma, 2005; Wilson and Zhang, 2009). Thenumber of total protein-coding genes in rice has been esti-mated at 52,214 [RAP2 (Rice Annotation Project 2008) http://rapdb.dna.affrc.go.jp/] or 56,797 [MSU release 6.1 (Ouyanget al., 2007) http://rice.plantbiology.msu.edu/]. Transcriptomeanalyses in rice using staged anthers and pollen grains (ordissected tapetal cells/microspores) identified approximately29,000 unique transcripts in the anther and male gametophyte(Hobo et al., 2008; Suwabe et al., 2008; Huang et al., 2009;Jiao et al., 2009; Fujita et al., 2010; Tang et al., 2010; Wanget al., 2010; Wei et al., 2010). This observation clearly indi-cates that variations in gene expression occur during antherdevelopment and pollen formation.

Several reports published on the developmental staging ofrice anther contribute to our understanding of the whole malereproductive developmental process (Feng et al., 2001; Itohet al., 2005; Zhang and Wilson, 2009). Feng et al. (2001)observed anther morphology in semi-thin sections anddivided the anther developmental process into eight stages:microspore mother cell formation stage, microspore mother

cell meiosis stage, early microspore stage, middle microsporestage, late microspore stage, early bicellular pollen stage, latebicellular pollen stage and mature pollen stage. Itoh et al.(2005) also described the rice anther development in eightstages, from An1 of archesporial cells (ACs) differentiation toAn8 of mature pollen grain production, and meiosis in 12stages. In Arabidopsis, anther development has been dividedinto 14 stages based on morphological features (Sanders et al.,1999; Ma, 2005). Recently, we analyzed the cellular changes ofthe rice anther (O. sativa ssp. japonica cv. 9522 and Zhonghua11) using light microscopic observations of transverse sectionsand divided the rice anther developmental course into 14 stages(Zhang and Wilson, 2009), which was meant to be consistentwith that of Arabidopsis (Sanders et al., 1999; Ma, 2005). Here,we further characterize the cellular changes during the 14anther developmental stages by detailed analysis of semi-thinsections of O. sativa ssp. japonica cv. 9522, according tomethods described by Li et al. (2006a). The recently reportedkey genes for rice anther development identified by mutantcharacterization are also discussed. In addition, we comparethe divergent morphology of rice anther development with thatof Arabidopsis.

2. Cytological analysis of rice anther development

Table 1 summarizes the key events that occur at all 14stages of anther development. The key genes expressed in riceare listed as well as their homologous genes in Arabidopsis.

Stage 1: The stamen primordium is formed after cell divi-sions and differentiation of floral meristem. The anther primor-dium contains three layers, L1, L2, and L3 (Fig. 2, stage 1).

http://rapdb.dna.affrc.go.jp/

http://rapdb.dna.affrc.go.jp/

http://rice.plantbiology.msu.edu/

Table 1

Major cytological events during rice anther development.

Stage Event Anther

length (mm)

Expressed genes in rice Homologous genes in Arabidopsis

1 Floral meristem differentiates into round-shape stamen

primordia containing L1, L2 and L3 cellular layers.

0.05e0.1

2 The stamen meristem form the anther primordia with

four corners, and the L2 layer forms archesporial cells

in each corner.

0.1e0.15

3 Archesporial cells divide and differentiate into primary

parietal cells.

0.15e0.2

4 Cell divisions further generate the secondary parietal

layers and sporogenous cells.

0.2e0.25 MSP1 (Nonomura et al., 2003;

Wang et al., 2006)

OsTDL1A (Zhao et al., 2008)

EMS1/EXS (Zhao et al., 2002;

Canales et al., 2002)

TPD1 (Yang et al., 2003a, 2003b;

Jia et al., 2008)

5 The primary sporogenous cells divide and form the

secondary sporogenous cells. The outer secondary

parietal layer forms the endothecium layer and the

middle layer, and the inner secondary parietal layer

develops into the tapetum.

0.25e0.3 GAMYB (Kaneko et al., 2004;

Aya et al., 2009; Liu et al., 2010)

MYB33/MYB65

(Millar and Gubler, 2005)

6 Development of microspore mother cells (MMCs)

surrounded by four-layered anther walls.

0.3e0.4 UDT1 (Jung et al., 2005) DYT1 (Zhang et al., 2006)

7 Meiocytes start meiotic division, and meiocytes are

associated with the tapetal layer. The middle layer

becomes a less visible band-like structure.

0.4e0.45 PAIR1/PAIR2/PAIR3

(Nonomura et al., 2004; 2006;

Yuan et al., 2009a, 2009b)

ZEP1 (Wang et al., 2010)

MEL1 (Nonomura et al., 2007)

PSS1 (Zhou et al., 2011),

API5 (Li et al., 2011a, 2011b, 2011c)

ASY1 (Caryl et al., 2000;

Armstrong et al., 2002)

ZYP1(Higgins et al., 2005)

8a Formation of dyads; at the end of meiosis I, one

meiocyte forms two nuclei separated by a cell plate

(Chen et al., 2005). The cytoplasm of tapetal cells

become condensed, and programmed cell death is

initiated as indicated by the nuclear DNA fragmentation

(Li et al., 2006a, 2006b).

0.45e0.8 TDR (Li et al., 2006a, 2006b) AMS (Sorensen et al., 2003;

Xu et al., 2010)

8b After meiosis II, the tetrad containing microspores

enclosed by the callose wall is formed. The tapetum

becomes more condensed and vacuolated. Primexines

are formed by microspores outside their surface.

0.8e1.1

9 Free haploid microspores, spherical in shape and

forming thin exines, are released from the tetrads. Wild-

type tapetal cells become condensed and form obvious

characteristic orbicules/Ubisch bodies on the inner

surface facing the microspores.

1.1e1.7 WDA1 (Jung et al., 2006)

PTC1 (Li et al., 2011a, 2011b,

2011c)

CYP703A3 (Morant et al., 2007)

CYP704B2 (Li et al., 2010)

DPW (Shi et al., 2011)

MS1 (Vizcay-Barrena and Wilson,

2006)

CYP703A2 (Aya et al., 2009)

CYP704B1 (Dobritsa et al., 2009)

10 Tapetal cells become more degenerated and produce

abundant electron-density Ubisch bodies. The

microspore becomes more vacuolated with a round

shape.

1.7e2.2 OsC6 (Zhang et al., 2010a, 2010b)

RIP1 (Han et al., 2006),

API5 (Li et al., 2011a, 2011b, 2011c)

11 The microspore undergoes the first mitotic division,

generating a generative cell and a vegetative cell. The

tapetum cells almost completely degenerate into cellular

debris and Ubisch bodies on the internal surface.

2.2 MADS3 (Hu et al., 2011)

12 The generative cell undergoes the second mitosis and

generates the mature pollen grain containing three

invisible nuclei. The tapetum completely disappears.

2.2 CSA (Zhang et al., 2010a, 2010b)

13 The flower opens, pollen sacs become connected and

anther dehiscence occurs.

2.2

14 The anther continues releasing mature pollen grains. 2.2 RIP1 (Han et al., 2006)

AID1(Zhu et al., 2004)

MSP1 ¼ MULTIPLE SPOROCYTES1; EMS1/EXS ¼ EXTRA SPOROGENOUS CELLS 1/EXCESS MICROSPOROCYTES; TPD1 ¼ TAPETUM DETER-

MINANT1; UDT1 ¼ RICE UNDEVELOPED TAPETUM1; DYT1 ¼ DYSFUNCTIONAL TAPETUM1; PAIR1/PAIR2/PAIR ¼ PAIRING ABERRATION IN

RICE MEIOSIS 1/2/3; MEL1 ¼ MEIOSIS ARRESTED AT LEPTOTENE1; PHS1 ¼ POOR HOMOLOGOUS SYNAPSIS 1; PSS1 ¼ POLLEN SEMI-

STERLITY 1; TDR ¼ TAPETUM DEGENERATION RETARDATION; AMS ¼ ABORTED MICROSPORES; PTC1 ¼ PERSISTENT TAPETAL CELL 1;

MS1 ¼ MALE STERILITY1; OSCP1 ¼ CYSTEINE PROTEASE 1;RIP1 ¼ RICE IMMATURE POLLEN 1; API5 ¼ APOPTOSIS INHIBITOR5;

CSA ¼ CARBON STARVED ANTHER; DAD1 ¼ DEFECTIVE IN ANTHER DEHISCENCE1; AID1 ¼ ANTHER INDEHISCENCE1.

381D. Zhang et al. / Journal of Genetics and Genomics 38 (2011) 379e390

Fig. 2. Cytological observation and diagrams of rice anther development and

pollen formation at 14 stages. L1, L2, and L3, the three cell layers in stamen

primordia; Ar ¼ archesporial cell; C ¼ connective layer; V ¼ vascular bundle;

1�P ¼ primary parietal layer; 2�P ¼ secondary parietal cell layer;


Eventually, the L1 layer cells divide to form the epidermis andthe stomium, which play roles in protecting microspore devel-opment and anther dehiscence.

Stage 2: The transverse section analyses indicate that theoutermost layer is the epidermis, which stems from anticlinalcell division of the L1 layer. The anther primordium isquadrangular in shape, with the palea and lemma closetogether. Stem cells in the L2 layer form archosporial cells viathe rapid mitotic division of cells at the four corners (Fig. 2,stage 2). The archesporial cells appear slightly larger thanother cells. During the later stages, the L3 layer cells divideand form connective cells, vascular tissues and circular cellclusters close to the stomium. The connective cells lie betweenmicrosporangia and vascular bundles and degenerate duringlater anther developmental stages.

Stage 3: Periclinal divisions of archesporial cells generatedistinct primary parietal cells (Fig. 2, stage 3).

Stage 4: Archesporial cells undergo periclinal divisions andgenerate primary sporogenous cells. The primary parietal cellsform two secondary parietal layers. The developing anther atthis stage has characteristic locules, a three-layer anther wall,and connective and vascular tissues (Fig. 2, stage 4).

Stage 5: The primary sporogenous cells divide and form thesporogenous cells, and the outer secondary parietal layerfurther generates the endothecium layer and the middle layer.The inner secondary parietal layer develops into the tapetallayer. The anther at this stage has four concentric somaticlayers that surround the sporgenous cells (from the surface tointerior): the epidermis, the endothecium, the middle layer andthe tapetum (Fig. 2, stage 5).

Stage 6: The secondary sporogenous cells generate micro-spore mother cells (MMCs, also called pollen mother cells,PMCs) within the locule (Fig. 2, stage 6).

Stage 7: Meiocytes initiate meiotic division and contact thetapetal layer. The middle layer becomes a less visible band-like structure (Fig. 2, stage 7).

Stage 8a: Meiocytes continue the process of meiosis andbecome slightly separated, forming ellipsoidal shaped dyads.In rice, one meiocyte forms two nuclei separated by the cellplate at the end of meiosis I (Chen et al., 2005), whereas inArabidopsis, at the end of meiosis I a band of numerousorganelles is formed at the equatorial level of the dyad (Ma,2005). Tapetal cells become vacuolated and shrunken withdarkly stained cytoplasm, and initiation of programmed celldeath in tapetal cells occurs with the nuclear DNA fragmen-tation (Li et al., 2006a, 2011a) (Fig. 2, stage 8a).

Stage 8b: Meiocytes undergo meiosis II, and four newlygenerated haploid microspores in one tetrad are enclosed bythe callose wall deposited on the primexine of the microspore(Zhang et al., 2008; Li et al., 2010; Li and Zhang, 2010).The tapetal layer continues to degenerate (Fig. 2, stage 8b) (Liet al., 2006a, 2006b, 2010, 2011a).

Sp ¼ sporogenous cell; StR ¼ stomium region; T ¼ tapetum; Tds ¼ tetrads.

Dy ¼ dyad cell; E ¼ epidermis; En ¼ endothecium; MC ¼ meiotic cell;

ML ¼ middle layer; MMC ¼ microspore mother cell; MP ¼ mature pollen;

Msp ¼ microspore parietal cell; BP ¼ bicellular pollen; bars ¼ 35 mm.


Stage 9: Free haploid microspores are released from thetetrads as the callose wall is degraded by callase secreted fromthe tapetal cells. Early in this stage, microspores are sphericalwith thin exines (Li and Zhang, 2010). Wild-type tapetal cellsreabsorb their vacuoles, and the cytoplasm becomescondensed (Fig. 2, stage 9). The secretory rice tapeta producecharacteristic orbicules/Ubisch bodies, which are assumed toexport tapetum-produced sporopollenin precursors across thehydrophilic cell wall to the locule (Fig. 5) (Huysmans et al.,1998, Li et al., 2010, 2011a). The middle layer becomesinvisible.

Stage 10: Tapetal cells become more degenerated with hill-like structures and form more electron-dense Ubisch bodiesalong the tangential surface of tapetal cells. Thicker exine withnexine and sexine layers are formed on the outer surface of themicrospores (Li and Zhang, 2010). The microspore vacuolateswith an increase of volume, resulting in a round-shapedmicrospore (Fig. 2, stage 10).

Stage 11: The vacuolated microspore undergoes the firstmitotic division with asymmetric cell division, generatinga much smaller generative cell and a larger vegetative cell. Atthe beginning of the stage, microspores are falcate shaped andthen become enlarged. As the starch accumulates inside themicrospore, the vacuole diminishes gradually. Subsequently,the generative cells separate from the pollen wall and moveclose to the vegetative nucleus. The tapetum cells almostcompletely degenerate into cellular debris and Ubisch bodieson the internal surface (Fig. 2, stage 11) (Feng et al., 2001; Liet al., 2010).

Stage 12: The generative cell in the microspore undergoesthe second mitosis and divides into two sperm cells, and themature pollen grain contains three nuclei, which cannot beseen because of the accumulation of starch and lipidic mate-rials (Feng et al., 2001; Zhang et al., 2010a). The epidermisand the endothecium degenerate further, and the tapetumcompletely disappears. At this stage, the anther developmentand pollen maturation are nearly complete, with the sphericalmicrospores full of reserve substances (Fig. 2, stage 12)(Zhang et al., 2010a).

Stage 13: Pollen grains become more spherical in shape,the lemma and palea interlocking structure is opened and thefilament elongates. The two adjacent pollen sacs becomelinked, and anther dehiscence occurs, leaving only theepidermis and endothecium layers (Fig. 2, stage 13).

Stage 14: The septum breaks, and the anther continues therelease of mature pollen grains (Fig. 2, stage 14).

3. Key genes required for anther development in rice

Recent forward and reverse genetic investigations greatlyfacilitated our knowledge of the molecular mechanisms ofmale reproductive development in plants, particularly inArabidopsis, the model eudicot plant that offers excellentadvantages for developmental studies. As a monocot, rice isbecoming another model crop for developmental biology dueto the release of the rice genome sequence and a large numberof available tools for the analysis of gene function, such as

collections of T-DNA insertion mutants, full-length cDNAsand a highly efficient transformation system (Jung et al.,2008). Recent genetic and biochemical investigations identi-fied several key regulators in rice male reproductive devel-opment (Zhang and Wilson, 2009). We had previouslydescribed the molecular regulatory networks controllinganther and pollen development in Arabidopsis and rice,including such genes as those determining anther cell divisionand differentiation, meiosis, pollen development and antherdehiscence (Wilson and Zhang, 2009; Zhang and Wilson,2009). Here, we briefly introduce key regulators for antherdevelopment in rice.

3.1. Morphological difference of anthers and pollengrains between rice and Arabidopsis

Overall, the developmental events are highly similarbetween rice and Arabidopsis, although there are severalmorphological differences in the appearance of the rice anthercompared with that of wild-type Arabidopsis anther (Fig. 3).The Arabidopsis anther lobes seem to be highly fused witha less obvious independent pollen sac, and the anther cuticlehas a longitudinal striped pattern (Fig. 3A and C). However,rice anthers have obvious lobe boundaries and reticulate anthercuticles (Fig. 3B and D). This observation may reflect thedifferences in anther cuticle development in monocots anddicots. The anther cuticle is identified as a thin hydrophobiclayer continuously coating the outermost surface of the anthersand provides a protective role for microspore development.The cutin matrix and waxes are the major components of theanther cuticle. The cutin is an insoluble polymer composed ofhydroxylated and epoxy C16 and C18 fatty acids, and waxescontain different substances such as alkanes, alcohols, ketonesand wax esters (Li et al., 2010).

Although both rice and Arabidopsis have secretory tapeta(Huysmans et al., 1998; Furness and Rudall, 1998), the tapetaof rice and other cereals display characteristic orbicules/Ubisch bodies, which are thought to export tapetum-producedsporopollenin precursors across the hydrophilic cell wall to thelocule (Fig. 4B, D and E). Orbicules have not been observed inthe Brassicaceae family including Arabidopsis, which containunique secretory tapeta with specialized organelles such aselaioplasts and tapetosomes (Fig. 4A and C) (Wu et al., 1997;Furness and Rudall, 1998; Huysmans et al., 1998).

After anther dehiscence, the exine, forming the outer wallof the pollen, offers another major protective barrier for spermcells and provides high resistance to environmental stresses(Blackmore et al., 2007; Li et al., 2010; Li and Zhang, 2010;Shi et al., 2011). The biochemical nature of the pollen exineremains elusive because of the technical limitation of puri-fying and obtaining a large quantity of materials for analysis.In addition, sporopollenin, the major component of the pollen/spore exine, is highly insoluble, resistant to degradation andexceptionally stable (Ahlers et al., 2003). Emerging evidenceindicate that lipidic pollen exine is made of sporopollenin, anextremely resilient material derived from polymerization offatty acid metabolites and phenolic acid (Piffanelli et al., 1998;

Fig. 3. Outer surface observation of Arabidopsis and rice anthers by SEM. A: Arabidopsis anther at stage 12, bar ¼ 100 mm. B: rice anther at stage 12,

bar ¼ 100 mm. C: enlargement of (A) showing the anther cuticle, bar ¼ 10 mm. D: enlargement of (B) showing the anther cuticle, bar ¼ 10 mm.


Morant et al., 2007). Morphological analysis also reveals thatrice pollen wall ontology is distinct from that of Arabidopsis(Li and Zhang, 2010). The outer surface of Arabidopsis pollengrains display elegant reticulate cavities with an abundantpollen coat (tryphine) deposited inside the pollen exine(Fig. 5A, C and E). Meanwhile, rice pollen have a smooth andparticulate exine patterning, which has a wider inter-layerspace between the nexine (foot layer) and sexine comparedwith that of Arabidopsis (Fig. 5B, D and F) (Li et al., 2010,2011a; Li and Zhang, 2010). This difference may be associ-ated with the different methods of pollination between the twoplants. Arabidopsis belongs to insect-pollinated (entomophi-lous) plants, while rice is wind-pollinated (anemophilous)

(Ariizumi and Toriyama, 2011). In addition, Arabidopsispollen exine consists of two layers, the tectum and the footlayer, and the baculum forms between the two layers.Compared with that of Arabidopsis, rice mature pollen exinecontains a larger space between the two layers (Fig. 5) (Li andZhang, 2010).

3.2. Molecular control of early anther development

MSP1 (MULTIPLE SPOROCYTE) transcripts, encodinga Leu-rich repeat receptor-like protein kinase in rice, areobserved in young panicles at stage 4. MSP1 is responsible forlimiting the number of cells entering into male and female

Fig. 4. Tapetal cells of Arabidopsis and rice. A: tapetal cells containing tapetosomes in Arabidopsis at stage 12, bar ¼ 1 mm. B: tapetal cells containing Ubisch

bodies in rice at stage 12, bar ¼ 1 mm. C: enlargement of (A) showing the tapetosome, bar ¼ 0.5 mm. D: enlargement of (B) showing Ubisch bodies, bar ¼ 0.5 mm.

E: outer surface of inner side of rice tapetal layer facing the microspores observed by SEM, bar ¼ 1 mm. Ne ¼ nexine; Te ¼ texine; U ¼ ubisch bodies;

t ¼ tapetosome.


sporogenesis and triggers the differentiation of anther wallformation in rice. The msp1 mutant produces an excessivenumber of both male and female sporocytes, and its antherfails to produce the tapetum layer (Nonomura et al., 2003;Wang et al., 2006). OsTDL1A, co-expressed with MSP1 inboth anthers and the ovule, encodes a small peptide that wasshown to bind to MSP1. However, knock-down of OsTDL1Amimics the msp1 phenotype in the ovule but not in the anther(Zhao et al., 2008). The general importance of the MSP1/

Fig. 5. Pollen wall morphology analysis of rice and Arabidopsis at stage 12. A: poll

bar ¼ 5 mm. C: pollen wall section of Arabidopsis by TEM, bar ¼ 1 mm. D: pollen

pollen exine structure. F: proposed model of rice pollen exine structure. In ¼ i

PC ¼ pollen coat; PM ¼ plasma membrane; M ¼ microspore; Cy ¼ cytoplasm.

OsTDL1A1-dependent pathway(s) in reproductive develop-ment, particularly precursors for male sporocytes into thetapetum is the discovery of their counterparts in A. thaliana(Nonomura et al., 2003; Ma, 2005; Wang et al., 2006; Zhaoet al., 2008). In Arabidopsis, the EXCESS MALE SPOR-OCYTES1 (EMS1) (also called EXTRA SPOROGENOUSCELLS, EXS ) EMS1/EXS gene encodes a LRR-RLK. Muta-tions of EMS1/EXS or its putative ligand TPD1 (TAPETUMDETERMINANT1) cause male sterility due to defective

en grain of Arabidopsis by SEM, bar ¼ 5 mm. B: pollen grain of rice by SEM,

wall section of rice by TEM, bar ¼ 1 mm. E: proposed model of Arabidopsis

ntine; Ne ¼ nexine; Ex ¼ exine; Se ¼ sexine; Te ¼ tectum; Ba ¼ bacula;


anther cell differentiation with excess male sporocytes andlack of tapetum (Canales et al., 2002; Zhao et al., 2002; Yanget al., 2003a, 2003b, 2005; Jia et al., 2008; Feng andDickinson, 2010). EMS1/EXS is initially expressed in theprecursors of sporogenous cells and the tapetum and thenbecomes more strongly expressed in the tapetum than in themale sporocytes (Zhao et al., 2002). Conversely, TPD1 isexpressed in sporogenous cells and tapetum precursors andlater is more highly expressed in the male sporocytes than thetapetum (Yang et al., 2003a, 2003b).

Rice GAMYB (OsGAMYB) is a transcriptional factor thatpositively regulates gibberellin (GA)-dependent a-amylaseexpression. GAMYB is highly expressed in stamen primordiaand tapetal cells, moderately in the endothecium and themiddle layer, but has no expression in MMCs. The gamybmutants display signs of defective anther development, such asdelayed degeneration of tapetal cells and abnormal meiosisprocess (Kaneko et al., 2004; Aya et al., 2009; Liu et al.,2010). MYB33 and MYB65 are closely related genes ofGAMYB in Arabidopsis, and a myb33 myb65 double mutantdisplays defective anther development with hypertrophictapetal cells and premeiotic abortion of pollen development(Millar and Gubler, 2005).

UNDEVELOPED TAPETUM1 (UDT1) encodes a basichelix-loop-helix (bHLH)econtaining protein and plays a crucialrole in the differentiation of secondary parietal cells to maturetapetal cells. The expression of UDT1 is detectable in tapetalcells and microspores from late stage 6 to stage 8, and in thetapetum, connective tissue and vascular bundles but not inyoung microspores after stage 8 (Jung et al., 2005). The Ara-bdopsis ortholog of UDT1 is DYSFUNCTIONAL TAPETUM1(DYT1), which is also required for proper cell differentiation inthe anther (Zhang et al., 2006). Moreover, the expression levelof UDT1 is down-regulated in msp1 mutants (Wang et al.,2006), and DYT1 expression is decreased in ems1/exs mutants(Zhang et al., 2006), suggesting the conserved regulatorypathway of MSP1eUDT1 in both rice and Arabidopsis.

3.3. Genes involved in meiosis

Meiosis plays a central role in the life cycles of all sexuallyreproducing organisms and is a highly conserved process ineukaryotes. Several key genes have been identified to beessential for meiosis during rice male reproductive develop-ment, such as PAIR1 (HOMOLOGOUS PAIRING ABERRA-TION IN RICE MEIOSIS1) (Nonomura et al., 2004), PAIR2(Nonomura et al., 2006), PAIR3 (Yuan et al., 2009b),MEIOSISARRESTED AT LEPTOTENE1 (MEL1) (Nonomura et al.,2007), OsRAD21-4 (Zhang et al., 2006), ZEP1 (Wang et al.,2010) and PSS1 (Pollen Semi-sterility1) (Zhou et al., 2011).PAIR1 encodes a novel putative coiled-coil protein that isrequired for homologous chromosome pairing in male andfemale meiocytes in rice (Nonomura et al., 2004). PAIR2 isrequired for homologous synapsis during meiosis I in rice(Nonomura et al., 2006). PAIR2 contains a HORMA-domainand is homologous to the Arabidopsis ASY1 (Caryl et al.,2000; Armstrong et al., 2002). A novel meiosis-related gene,

PAIR3, encodes a protein containing putative coiled-coil motifsand has no close homologs in other organisms. This gene wasdemonstrated to play a key role in homologous chromosomepairing and synapsis in rice male and female meicytes (Yuanet al., 2009a). MEL1 belongs to the ARGONAUTE (AGO)gene family, and the mel1 mutant aborts chromosomes duringearly meiotic stages, causing defective male and femalefertility (Nonomura et al., 2007). ZEP1 encodes a transversefilament protein homologous to Arabidopsis ZYP1. ZEP1 mayact as a central element of the synaptonemal complex, and zep1mutants show no formation of the synaptonemal complex inearly prophase I, equal chromosomal segregation in anaphase Ias well as increased numbers of crossovers (Wang et al., 2010).PSS1 encodes a kinesin-1-like protein with microtubule-stimulated ATPase activity, and pss1 mutants display laggingchromosomes and chromosomal bridges at anaphase I andanaphase II of male meiosis, causing reduced male fertility(Zhou et al., 2011).

3.4. Molecular control of postmeiotic male reproductivedevelopment

Tapetum Degeneration Retardation (TDR), the ortholog ofthe Arabidopsis AMS gene encoding a bHLH transcriptionfactor (Sorensen et al., 2003; Xu et al., 2010), plays a key rolein tapetal programmed cell death (PCD) in rice. The tdrmutant displays delayed tapetal degeneration and nuclearDNA fragmentation, and abortion of microspores after therelease from the tetrad. Moreover, TDR is able to directlyregulate the expression of a cysteine protease gene OsCP1in vivo and in vitro (Li et al., 2006a, 2006b). Furthermore,TDR also plays a role in pollen exine formation, as the tdrmicrospore does not have sporopollenin precursors in its pri-mexine, as well as regulates the expression of genes such asDefective Pollen Wall and OsC6 that are related to lipidicsynthesis and transport during anther development (Zhanget al., 2008; 2010a; Shi et al., 2011). ABORTED MICRO-SPORE (AMS ) encodes a post-meiotic, tapetally expressedbHLH protein (Sorensen et al., 2003), which also has a puta-tive ortholog in rice, TDR (Li et al., 2006a, 2006b; Zhanget al., 2008). The ams mutant displays an expanded tapetallayer and aborted microspores (Sorensen et al., 2003; Xu et al.,2010). The key functional role of AMS in anther and micro-spore development is the regulation of the expression ofa number of genes involved in various biological activities,particularly those associated with metabolism and depositionof the pollen wall. In addition, 13 genes involved in tapetaldevelopment and pollen wall formation were shown to bedirect regulatory targets of AMS (Xu et al., 2010). Thefunctional importance of this pathway is highlighted by thedemonstration that mutants of one of these downstream AMStargets, an ATP Binding Cassette (ABC) transporter, WhiteeBrown Complex homolog protein 27 (WBC27), displaydefective pollen development and male sterility (Xu et al.,2010).

Tapetal cell development and differentiation are criticalfor the early male reproduction; however, during late pollen


development, tapetal degeneration, promoted by an apoptosis-like process, is also crucial for viable pollen formation(Li et al., 2006a; Aya et al., 2009). Arabidopsis MALESTERILITY1 (MS1) encodes a putative PHD-finger (PlantHomeo Domain) protein (Wilson et al., 2001; Ito andShinozaki, 2002; Ito et al., 2007; Yang et al., 2005), andms1 mutants show altered tapetal development without normalPCD and abnormal tapetal degeneration associated with largeautophagic vacuoles and mitochondrial swelling (Vizcay-Barrena and Wilson, 2006). We recently identified a keyregulator, PERSISTENT TAPETAL CELL 1 (PTC1), which isthe ortholog of MS1. It controls programmed tapetal devel-opment and degradation during rice anther development.Mutation of PTC1 causes uncontrolled tapetal cell prolifera-tion and swelling, delayed DNA fragmentation, abnormalUbisch bodies/orbicules and pollen wall development, causingcomplete male sterility (Li et al., 2011a). PTC1 is brieflyexpressed in tapetal cells and microspores. Expression analysissuggests that PTC1 regulates the expression of genes associatedwith tapetal function and pollen exine formation. More inter-estingly, the conserved and crucial role of PTC1 is indicated byits ability to complement its Arabidopsis ortholog mutant, ms1.These observations present new insights into programmed malereproductive development in both dicots and monocots.

APOPTOSIS INHIBITOR5 (API5) is another newly iden-tified tapetal PCD positive regulator in rice and is critical forpost-meiotic anther development and proper formation of malegametophytes (Li et al., 2011b). API5 encodes a nuclearprotein, which is homologous to the anti-apoptosis proteinApi5 in animals. The osapi5 mutant displays delayed tapetaldegeneration, causing aborted pollen development. OsAPI5 isable to interact with two DEAD-box ATP-dependent RNAhelicases, API5-INTERACTING PROTEIN1 (AIP1) andAIP2, and regulates the expression of OsCP1, a rice cysteineprotease gene, providing a new understanding of tapetal PCDcontrol in rice (Li et al., 2011b).

Normal tapetal differentiation and degeneration are requiredfor successful male reproductive development and fertility inhigher plants (Ma, 2005; Wilson and Zhang, 2009). Timelydegeneration of tapetal cells is thought to contribute to therelease of wall materials, including carbohydrates, lipidicmolecules and other nutrients from the tapetal cells to thedeveloping microspores. Genetic investigations have identifiedseveral genes required for Arabidopsis pollen wall develop-ment, such as MS1, MS2, NEF1 (NO EXINE FORMATION 1)(Ariizumi et al., 2004), DEX1 (DEFECTIVE in EXINEPATTERN FORMATION) (Paxson-Sowders et al., 2001), FLP1(FACELESSPOLLEN1) (Ariizumi et al., 2003), CYP703A2(Morant et al., 2007), ACOS5 (Acyl-CoA Synthetase 5) (Souzaet al., 2009) and CYP704B1 (Dobritsa et al., 2009). Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE 5(ACOS5) can be condensed with malonyl-CoA by POLYKE-TIDE SYNTHASE A/LAP6 and B/LAP5 (PKSA/B) toproduce a-pyronepolyketides (Souza et al., 2009; Dobritsaet al., 2009; Kim et al., 2010), which can be reduced byTETRAKETIDE a-PYRONE REDUCTASE1 and 2 (TKPR1/2)(also called DIHYDROFLAVONOL 4-REDUCTASE LIKE1,

DRL1) to form hydroxylated a-pyrone compounds for sporo-pollenin biosynthesis (Tang et al., 2009; Grienenberger et al.,2010). In rice, cytochrome P450 family members,CYP703A3 and CYP704B2, were shown to be essential forpollen exine development (Li and Zhang, 2010). The riceortholog of CYP703A2, CYP703A3, which is directly regulatedby GAMYB, encodes a fatty acid hydroxylase, and the loss ofCYP703A3 function causes defective ubicsh body and pollenexine development (Aya et al., 2009). The rice ortholog ofCYP704B1, CYP704B2, which is conserved among terrestrialplants, is preferentially expressed in tapetal cells, and therecombinant CYP704B2 protein has the ability to catalyze thehydroxylation of palmitic acid and unsaturated C18 fatty acidsin the u position of the carbon chain. The cyp704B2 mutantshows defective anther epidermal cuticle and aborted pollengrains without obvious exines (Li et al., 2010), indicating thatCYP704B2 controls a conserved biosynthetic pathway for thebiopolymers sporopollenin and cutin. Furthermore, rice Wax-deficient anther1 (WDA1) is homologous to ArabidopsisECERIFERUM1 (CER1), which is related to anther cuticle andpollen exine development (Jung et al., 2006).

As a non-photosynthetic male reproductive organ, theanther requires the supply of photosynthetic assimilates fromsource organs to support normal pollen development andmaturation (Goetz et al., 2001). Within the anther, the devel-oping pollen is immersed in locular fluid containing nutrients,including sugars and lipids from the sporophytic (somatic)tissue tapetum (Pacini et al., 2006). Recently, we identifieda key regulator gene in rice, Carbon Starved Anther (CSA),which is a putative R2R3 MYB-type transcription factorinvolved in regulating sugar partitioning during male repro-ductive development. The csa mutant displays reduced levelsof carbohydrates in later anthers and is male sterile. The CSAgene is mainly expressed in the vascular tissue and the tapetumof the anther, as well as in other sinks, and the CSA protein isable to directly regulate the expression of MST8, whichencodes a monosaccharide transporter (Zhang et al., 2010b).This finding provides the first example for transcriptioncontrol of carbon partitioning in plants.

The Arabidopsis flower C-class gene, AGAMOUS (AG),plays a key role in specifying stamen, carpel identities, andfloral meristem determinacy as well as male reproductivedevelopment (Yanofsky et al., 1990; Bowman et al., 1991;Yang et al., 1999; Schiefthaler et al., 1999; Ito et al., 2004,2007). Studies in rice identified two C-class MADS boxgenes, OsMADS3 and OsMADS58, and OsMADS3 was shownto be required for specifying stamen, ovule and floral meristemidentities (Yamaguchi et al., 2006; Li et al., 2011c). Recently,we revealed a role for OsMADS3 in regulating late antherdevelopment and pollen formation. Consistent with thisfunction, OsMADS3 is highly expressed in the tapetum andmicrospores during late anther development, and a new allele,osmads3-4, displays defective anther walls, aborted micro-spores, leading to complete male sterility (Hu et al., 2011).The molecular mechanism of OsMADS3 involvement in ricemale reproductive development is modulation of reactiveoxygen species (ROS) levels through MT-1-4b. MT-1-4b


encodes a type 1 small Cys-rich and metal binding protein,and recombinant MT-1-4b has superoxide anion and hydroxylradical scavenging activity. Accordingly, the osmads3-4mutant exhibits oxidative stress-related phenotypes (Hu et al.,2011).

Additionally, Rice Immature Pollen 1 (RIP1) and ANTHERINDEHISCENCE1 are also required for late pollen maturationand anther development in rice (Zhu et al., 2004; Han et al.,2006). RIP1 encodes a nuclear-localized protein, which ishighly homologous to proteins containing five WD40 repeatsequences. RIP1 plays an essential role in the late stage ofpollen formation and germination. Pollen of the rip1 mutantdisplays delayed development, including defective formationof starch granules and the intine layer as well as the organ-elles. AID1 encodes a novel protein of 426 amino acids witha single MYB domain that is closely related to that of thetelomere-binding proteins of human, mouse and Arabidopsis,and of single MYB domain transcriptional regulators in plantssuch as PcMYB1 and ZmIBP1. The AID1 transcripts wereobserved in both the leaves and panicles of wild-type plants(Zhu et al., 2004), while aid1 mutants display partial tocomplete male sterility due to defective anther dehiscence,a consequence of failure to accumulate starch in pollen grains.

4. Conclusion

In this article, we describe the cytological analysis of riceanther development and pollen formation at 14 developmentalstages, showing the distinct aspects of rice male reproductivedevelopment. Furthermore, the identified regulators of properanther development and pollen formation are also brieflysummarized. We hope these descriptions are useful for bothmale reproductive developmental studies and the manipulationof male fertility in crops.

Acknowledgements

This work was supported by the funds from the NationalBasic Research Program of China (Nos. 2009CB941500 and2007CB108700), the National Natural Science Foundation ofChina (No. 30725022), the Chinese Transgenic Project (No.2009ZX08009-108B), and the National 863 High-Tech Project(No. 2011AA10A101).

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Cytological Analysis and Genetic Control of Rice Anther Development