regulatory role of a receptor-like kinase in specifying ...lrr-rlk, msp1 (multiple sporocyte1),...

Regulatory Role of a Receptor-Like Kinase in SpecifyingAnther Cell Identity1[OPEN]

Li Yang, Xiaoling Qian, Mingjiao Chen, Qili Fei, Blake C. Meyers, Wanqi Liang, and Dabing Zhang*

State Key Laboratory of Hybrid Rice, Shanghai Jiao Tong University and University of Adelaide Joint Centrefor Agriculture and Health, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University,Shanghai 200240, China (L.Y., X.Q., M.C., W.L., D.Z.); Department of Plant and Soil Sciences, DelawareBiotechnology Institute, University of Delaware, Newark, Delaware 19711 (Q.F., B.C.M.); Donald DanforthPlant Science Center, St. Louis, Missouri 63132 (B.C.M.); Key Laboratory of Crop Marker-Assisted Breeding ofHuaian Municipality, Jiangsu Collaborative Innovation Center of Regional Modern Agriculture andEnvironmental Protection, Huaian 223300, China (W.L., D.Z.); and School of Agriculture, Food, and Wine,University of Adelaide, South Australia 5064, Australia (D.Z.)

ORCID IDs: 0000-0002-0860-2175 (Q.F.); 0000-0003-3436-6097 (B.C.M.); 0000-0002-9938-5793 (W.L.); 0000-0002-1764-2929 (D.Z.).

In flowering plants, sequential formation of anther cell types is a highly ordered process that is essential for successful meiosisand sexual reproduction. Differentiation of meristematic cells and cell-cell communication are proposed to coordinate antherdevelopment. Among the proposed mechanisms of cell fate specification are cell surface-localized Leu-rich repeat receptor-likekinases (LRR-RLKs) and their putative ligands. Here, we present the genetic and biochemical evidence that a rice (Oryza sativa)LRR-RLK, MSP1 (MULTIPLE SPOROCYTE1), interacts with its ligand OsTDL1A (TPD1-like 1A), specifying the cell identity ofanther wall layers and microsporocytes. An in vitro assay indicates that the 21-amino acid peptide of OsTDL1A has a physicalinteraction with the LRR domain of MSP1. The ostdl1a msp1 double mutant showed the defect in lacking middle layers andtapetal cells and having an increased number of microsporocytes similar to the ostdl1a or msp1 single mutant, indicating the samepathway of OsTDL1A-MSP1 in regulating anther development. Genome-wide expression profiles showed the altered expressionof genes encoding transcription factors, particularly basic helix-loop-helix and basic leucine zipper domain transcription factorsin ostdl1a and msp1. Among these reduced expressed genes, one putatively encodes a TGA (TGACGTCA cis-element-bindingprotein) factor OsTGA10, and another one encodes a plant-specific CC-type glutaredoxin OsGrx_I1. OsTGA10 was shown tointeract with OsGrx_I1, suggesting that OsTDL1A-MSP1 signaling specifies anther cell fate directly or indirectly affecting redoxstatus. Collectively, these data point to a central role of the OsTDL1A-MSP1 signaling pathway in specifying somatic cell identityand suppressing overproliferation of archesporial cells in rice.

In flowering plants, germ cells originate from spo-rophytic tissues during the late developmental stage,which differs from most animals in which their germcells arise early in embryos (Walbot and Evans, 2003).The anther, the sporophytic male reproductive organ,produces male gametophytes via meiosis and subse-quent mitotic divisions to generate the sperm. Antherprimordia are generated by the floral meristem andusually contain three meristematic cell layers, L1 to L3.Within each of the anther lobes, the L1 generates theepidermis and the L2 forms both the sporogenous cells(microsporocytes) and three inner anther wall layers:the endothecium, the middle layer, and the tapetum(from outer to inner). The L3 produces the vasculatureand connective tissue at the center of each four-lobedanther (Poethig, 1987; McCormick, 1993; Ma, 2005;Wilson and Zhang, 2009; Zhang et al., 2011; Zhang andYang, 2014).

In the past decade, there have been models explain-ing the cell division and cell fate specification duringanther morphogenesis in Arabidopsis (Arabidopsisthaliana), rice (Oryza sativa), and maize (Zea mays; Ma,2005; Feng and Dickinson, 2010a, 2010b; Kelliher and

Walbot, 2011; Zhang and Yang, 2014). The lineagemodel proposes that a single hypodermal cell beneaththe anther epidermis undergoes sequential asymmetriccell divisions to form three concentric rings of somaticlayers and germ cells (Kiesselbach, 1999;Ma, 2005; Fengand Dickinson, 2010a, 2010b). Recent studies in maizedispute the existence of hypodermal cells (Kelliher andWalbot, 2011). By confocal microscopy immature an-ther lobes were shown to contain multiple L2-derived(L2-d) cells. Those cells with only L2-d neighbors re-spond to hypoxia by differentiating as archesporial cellsand subsequent sporogenous cells, while the peripheralL2-d cells differentiate into primary parietal cells(PPCs). The PPC then divides periclinally to form dis-tinctive morphological characteristics of two cell types:the endothecium and secondary parietal cell (SPC)enclosed by the epidermis. Then multipotent SPCscarry on a periclinal division generating daughter cellswith the same sizes that further differentiate into themiddle layer and the tapetum (Kelliher and Walbot,2011; Zhang and Yang, 2014).

Successful formation of male gametophytes andgametes requires the nutritive support of neighboring

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http://orcid.org/0000-0002-0860-2175http://orcid.org/0000-0003-3436-6097http://orcid.org/0000-0002-9938-5793http://orcid.org/0000-0002-1764-2929

somatic cells, especially the tapetal cell. Recently, acomplex and bidirectional interaction between antherwall layers andmicrosporocytes was uncovered to playcritical roles in determining the cell fate of somatic cellsand germ cells. For instance, maize OCL4 (OUTERCELL LAYER4) encodes a HD-ZIP IV transcriptionfactor and expresses exclusively in the epidermis. ocl4plants have anthers with an extra subepidermal celllayer with endothecial characteristics formed by anaberrant periclinal division in the endothecial cells(Vernoud et al., 2009). In Arabidopsis, the MADS-boxtranscription factor SPL/NZZ (SPOROCYTELESS/NOZZLE) regulates sporocyte development. Thespl/nzz mutant has no formation of microsporocytesand anther somatic wall layers; this AGAMOUS-regulated factor connects floral organ determinationwith the initiation of anther patterning (Schiefthaleret al., 1999; Yang et al., 1999). BAM1 (BARELY ANYMERISTEM1) and BAM2 encode CLAVATA1-relatedLRR-RLKs; BAM1/2 limit the expression domain ofSPL/NZZ, and SPL/NZZ promotes BAM1 expression inthe central sporogenous cells, contributing to the cor-rect differentiation of the endothecium, SPC, and theresulting middle layers and tapetal layers (DeYounget al., 2006; Hord et al., 2006; Sun et al., 2007). ERECTAfamily genes ER (ERECTA), ERL1 (ERECTA-LIKE1),and ERL2, encoding the LRR-RLKs, redundantly con-trol anther lobe formation and cell patterning (Toriiet al., 1996; Shpak et al., 2003; Shpak et al., 2004;Woodward et al., 2005). Furthermore, the signaling ofRPK2 (RECEPTOR-LIKE PROTEINKINASE2), another

LRR-RLK, controls the differentiation of the middlelayer andmaintenance of tapetal cell fate (Mizuno et al.,2007). Besides LRR-RLK proteins, two cytoplasmicmitogen-activated protein kinases (MAPKs), MPK3and MPK6, function redundantly in anther cell differ-entiation through a MAPK cascade (Hord et al., 2008).

In Arabidopsis, the cell fate decision of tapetalcells and microsporocytes involves a LRR-RLKcomplex including EMS1/EXS (EXCESS MALESPOROCYTES1/EXTRA SPOROGENOUS CELLS),TPD1 (TAPETUMDETERMINANT1), and SERK1/2(SOMATIC EMBRYO RECEPTOR KINASE1/2) pro-teins (Canales et al., 2002; Zhao et al., 2002; Yang et al.,2003; Albrecht et al., 2005; Colcombet et al., 2005; Yanget al., 2005). Mutants in EMS1/EXS and TPD1 displaythe same phenotypes of excess microsporocytes andimpaired tapetum and therefore are considered tofunction in the same pathway to regulate cell fate de-termination (Canales et al., 2002; Zhao et al., 2002; Yanget al., 2003; Feng and Dickinson, 2010a). TPD1, a pu-tative secreted ligand, might act as a ligand for theEMS1/EXS receptor kinase (Yang et al., 2003, 2005).Furthermore, the direct interaction between TPD1 andEMS1 has been confirmed in vitro and in vivo (Jia et al.,2008). A model explaining anther cell specification isthat the TPD1 signal is secreted from microsporocytesto surrounding tapetal cells and interacts with EMS1 topromote tapetal cell differentiation (Ma and Sundaresan,2010; Feng and Dickinson, 2010a). The serk1/serk2double mutant has a phenotype similar to ems1/exs andtpd1, a result suggesting that SERK1/2 and EMS1/EXSmay form heterodimeric receptors (Albrecht et al., 2005;Colcombet et al., 2005). The heterodimeric complexmay bind TPD1 to activate downstream targets to ac-quire anther cell fate; yet, the ability of TPD1 to bindheterodimers is unknown. Maize MAC1 (MULTIPLEARCHESPORIAL CELLS1), a homolog of ArabidopsisTPD1, suppresses archesporial cell proliferation andpromotes the periclinal division of subepidermal cells(Sheridan et al., 1996, 1999; Wang et al., 2012). TheMAC1 protein is accumulated preferentially in germcells, and it contains a predicted signal peptide thatleads to secretion (Wang et al., 2012). The mac1 mutantexhibits excess proliferation of archesporial cells notonly in anthers, but also in ovules (Sheridan et al., 1996,1999; Wang et al., 2012). This is different from Arabi-dopsis tpd1, which only exhibits defects in male tissues.

Rice OsTDL1A/MIL2 (MICROSPORELESS2) andMSP1 are orthologous to TPD1 (MAC1) and EMS1/EXS(Nonomura et al., 2003; Zhao et al., 2008; Hong et al.,2012a). Mutations in MSP1 give rise to a phenotypewith excessive sporogenous cells and failure of thesubepidermal cells to divide early. In addition, msp1also displays abnormal ovule development, whereas nofemale defects were reported in Arabidopsis (Canaleset al., 2002; Zhao et al., 2002; Nonomura et al., 2003).The expression of MSP1 is detectable mainly in neigh-boring cells surrounding male and female sporocytes(Nonomura et al., 2003; Hong et al., 2012a). Amutant ofOsTDL1A/MIL2 displays defects in early anther cell

1 This work was supported by funds from the National NaturalScience Foundation of China (31430009, 31230051, and 31271698);from the China Innovative Research Team, Ministry of Education,and the Program of Introducing Talents of Discipline to Universities(111 Project, B14016); from the National Key Basic Research Devel-opments Program, Ministry of Science and Technology, China(2013CB126902); from the Science and Technology Commission ofShanghai Municipality (grant no. 13JC1408200); and from the Lead-ing Scientist in Agriculture of ShanghaiMunicipality. Additional sup-port for research in the Meyers lab was provided by the U.S. NationalScience Foundation Plant Genome Research Program (award no.1339229). Collaborative research and travel between the lab of Zhangand Meyers at the University of Delaware and the Walbot lab atStanford University were supported by the British Council’s GlobalInnovation Initiative, which funds the Global Innovations Network(GII; for more details, see https://www.cpib.ac.uk/GII/).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Dabing Zhang ([email protected]).

W.L. and D.Z. conceived the original screening and research plans;L.Y. and W.L. designed the experiments and analyzed the data; X.Q.performed the map-based cloning of MSP1; M.C. and L.Y. con-structed the double mutant by crossing; L.Y., W.L., and Q.F. per-formed the transcriptomics assay; L.Y. wrote the article withcontributions of all the authors; D.Z. and B.C.M. supervised andcomplemented the writing.

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patterning, while the RNA interference (RNAi) lines ofOsTDL1A/MIL2 only show ovule defects (Zhao et al.,2008; Hong et al., 2012a). The OsTDL1A/MIL2mRNAsare present early in archesporal cells, later radically ininner somatic cells (Hong et al., 2012a). Even though ithas been hypothesized that OsTDL1A/MIL2 may actas a ligand of MSP1, and OsTDL1A-MSP1 signalingspecifies the early anther development (Zhang andYang, 2014), the mechanism underlying the OsTDL1A-MSP1 pathway remains largely unknown. Here, weprovide more evidence demonstrating that OsTDL1Ainteracts with MSP1 in regulating the specification ofcell fate in anthers, and their loss of function profoundlyalters expression of a set of genes.

RESULTS

OsTDL1A-MSP1 Genetic Pathway Specifies Anther CellIdentity and Impacts Ovule Development

To obtain further insights into the molecular mech-anism underlying rice sporogenesis, we identified threecompletely male-sterile mutants called ostdl1a, msp1-4,andmsp1-6, named as such because the mutations werein the OsTDL1A and MSP1 genes (see below). ostdl1a,allelic tomil2 (Hong et al., 2012a), was identified to havea 204-kb pair deletion betweenmarkers Y1213 andY1214on chromosome 12, which was genetically comple-mented by a 5.27-kb wild-type OsTDL1A (Os12g28750)genomic fragment (Supplemental Figs. S1A and S2).msp1-4was found to be caused by 10 bp deleted withinthe DNA sequence encoding the LRR domain ofMSP1, causing pretermination of MSP1 translation(Supplemental Fig. S1B; Wang et al., 2006). Latergene mapping and sequence analysis identified an-other novel allele msp1-6 with a single-base substi-tution (C to A) and a frameshift-causing insertion ofan A in the DNA sequence of the kinase domain(Supplemental Fig. S1B).To investigate whetherOsTDL1A andMSP1 function

in the same pathway, we built a double ostdl1a msp1-4mutant by crossing. Phenotypic analysis demonstratedthat this double mutant had small, white anthers lack-ing viable pollen grains (Fig. 1, A–F). Cytologicalanalysis indicated that ostdl1a msp1-4 had excessivearchesporial cells enclosed by a single layer of subepi-dermal PPCs (Fig. 2, A, B, E, and F). Subsequently,subepidermal cells could divide periclinally to forminner somatic layers in the wild type (Fig. 2, C and D).However, there was neither the middle layer nor thetapetum in the double mutant ostdl1a msp1-4 at stage5 (Fig. 2, G and H). At this stage, mutant anthers hadonly one continuous subepidermal layer, which sharesthe characteristics with endothecium, and some cellswith unknown identity were observed. However, thesporogenous cells showed overproliferation (Fig. 2, G–I).These observations indicate that the double mutantphenocopies both ostdl1a and msp1-4 single mutantsindicative of the two genes in the same pathway.

Microspore staining with 49,6-diamino-phenylindoleduring meiosis showed that despite an excess of mi-crosporocytes observed in ostdl1a and msp1-4, both hadnormal meiotic initiation until diakinesis, but failedafterward (Supplemental Fig. S3). This suggests that thesuccessful entry of microsporocytes into meiosis doesnot require the function of normal anther somatic tis-sues. Although the external appearance of carpels inostdl1a msp1-4 double mutant appeared normal (Fig. 1,G and H), few seeds were obtained when plants werepollinated with wild-type pollen grains (data notshown). These results suggest that ostdl1a and msp1-4mutations affect bothmale and female fertility, which isconsistent with prior results (Nonomura et al., 2003;Hong et al., 2012a). Examination of close transversesections demonstrated that ovules typically containedmultiple megaspores and a disorganized embryo sacsubdivided by cell walls in ostdl1a msp1-4 (Fig. 1J). Thiscontrasts with the wild type in which each ovule hasonly one megaspore in one embryo sac (Fig. 1I). Fromthe genetic and cytological analysis, we conclude thatOsTDL1A genetically interacts withMSP1 in specifyingthe early anther cell fate by promoting the transition ofparietal cells into the middle layer and tapetal layer, aswell as inhibiting the extra activity of forming repro-ductive cells of microsporocytes in rice. Here, wemainly focus on the specification of anther cell identityby the OsTDL1A-MSP1 pathway.

OsTDL1A Directly Binds to MSP1

Consistent with the fact that OsTDL1A geneticallyinteracts with MSP1, these two genes exhibit a distinctbut overlapping expression pattern (Supplemental Fig.S4A; Nonomura et al., 2003; Hong et al., 2012a). Tofurther investigate the regulatory relationship betweenOsTDL1A and MSP1, we performed qRT-PCR (quan-titative reverse transcription PCR) to analyze their

Figure 1. Phenotypic analysis of the ostdl1a msp1-4 double mutant. Aand B, Spikelets of the wild type and ostdl1a msp1-4 after removing thepalea and the lemma. C and D, Anthers of the wild type and ostdl1amsp1-4. E and F, The I2-KI staining pollen grains of the wild type andostdl1a msp1-4. G and H, Carpels of the wild type and ostdl1a msp1-4.I and J, Longitudinal sections of wild-type and ostdl1a msp1-4 ovules.gl, glume; lo, lodicule; pi, pistil; st, stamen. Bars = 1 mm (A and B),500 mm (C, D, G, and H), 5 mm (E and F), and 50 mm (I and J).

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expression and found that the expression level ofOsTDL1A and MSP1 did not show significant changesin msp1 and ostdl1a respectively (Supplemental Fig. S4,C and D). Therefore, OsTDL1A and MSP1 do not reg-ulate each other at transcriptional level. A proteinsubcellular localization assay showed that MSP1 waslocalized at plasmamembrane (Supplemental Fig. S4B).Given that MSP1 is a membrane kinase protein andOsTDL1A is a putative secretory protein containingan N-terminal signal peptide, it was proposed thatOsTDL1A is the ligand ofMSP1 (Zhao et al., 2008; Honget al., 2012a; Zhang and Yang 2014).

To test the feasibility of this hypothesis, we con-ducted yeast two-hybrid (Y2H) experiments. When theentire LRR domain of MSP1 was used as a bait, no in-teraction was detected for OsTDL1A (Fig. 3C). TheMSP1 protein has 28 LRRs with potential N-linkedglycosylation sites that may interfere with its proteininteraction as it is produced in yeast Saccharomycescerevisiae (van der Hoorn et al., 2005; Jia et al., 2008;Supplemental Fig. S5A). Therefore, we made a series ofshortened truncated cDNA fragments encoding theLRRs of MSP1 (Fig. 3A). We observed that only onefragment, named MSP1-LRR4 encoding the 20 to 28thLRRs, interacted with OsTDL1A (Fig. 3, A and C). TheOsTDL1A encodes a predicted protein of 226 aminoacids with a predicted cleavable signal peptide of 1 to35 amino acids. Multiple sequence alignments showedthat the C-terminal segment of OsTDL1A is well con-served in land plants (Supplemental Fig. S5B). To check

whether OsTDL1A encodes a small functional peptide,the full-length OsTDL1A and a C-terminal truncationencoding the peptide OsTDL1A-2 (110–226 aminoacids) were tested via Y2H assays, where both werefound to be capable of interacting with MSP1-LRR4. Incontrast, this was not a case for the N-terminal trunca-tion OsTDL1A-1 (1–107 amino acids; Fig. 3, B and D).Further assays confirmed that a 21-amino acid longpeptide of OsTDL1A-2 called OsTDL1A-4 (161–181amino acids) could interact with MSP1-LRR4 in vitro(Fig. 3, B and D). Surprisingly, this peptide was alsowell conserved in land plants, including multiplemonocot and eudicot species, a liverwort, and a moss(Supplemental Fig. S5B), implying that the conservedOsTDL1A may secrete a 21-amino acid peptide servingas a ligand to the membrane-bound LRR-RLK MSP1 inreproductive development.

A direct interaction between OsTDL1A and MSP1was further confirmed in vitro. AGST-OsTDL1A fusionprotein isolated from bacteria was able to pull downtransiently expressedMSP1 in tobacco (Nicotiana tabacum)leaves from the total soluble proteins (Fig. 3E).Thein vivo physical interaction between OsTDL1A andMSP1 was confirmed by coimmunoprecipitation intobacco leaves that transiently coexpressed MSP1-MycandOsTDL1A-FLAG fusion proteins.Membrane proteinextract from transgenic leaveswas immunoprecipitatedwith an anti-Myc antibody, and OsTDL1A -FLAG wasdetected by a western blot using an anti-FLAG anti-body (Fig. 3F). In the reciprocal coimmunoprecipitation

Figure 2. Comparison of anther development inthe wild type and ostdl1a msp1-4. A to D, Trans-verse sections of wild-type anthers. E to H,Transverse sections of ostdla1 msp1-4 anthers.Arrowheads in G and H represent cells with un-known characteristics. I, Diagram of cell lineagesduring early anther development in both the wildtype and ostdla1 msp1-4mutant. Ar, Archesporialcell; eAr, excess archesporial cell; E, epidermis;En, endothecium; eSp, excess sporogenous cell;L2-d, L2-derived cell; ML, middle layer; Sp, spo-rogenous cell; T, tapetum; uIC, unknown identitycell. Bars = 15 mm.


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experiment, we also detected MSP1-Myc from the anti-FLAG immunoprecipitates (Supplemental Fig. S6). Inconclusion, these results strongly suggest that OsT-DL1A directly interacts with the specific LRR extracel-lular region of MSP1 in planta.

A Wide Range of Genes Are Coregulated by OsTDL1A-MSP1 Signaling Pathway

To identify genes involved in the OsTDL1A-MSP1signaling, we collected spikelets from wild-type, ostdl1a,and msp1-4 plants with three biological replicates. Twokey stages were selected for analysis: stage 3 anthers inwhich L2-d cells have already differentiated into arche-sporial cells and primary parietal cells; and stage 5 inwhich middle layers and tapetal cells have been gener-ated by periclinal division of secondary parietal cells(Fig. 4A). Total RNA was extracted from these spikeletsand subject to RNA-seq analysis to identify differentiallyexpressed genes. In total, we identified 2,395 genes withsubstantial differential expression in ostdl1a and/ormsp1-4 compared with the wild type (SupplementalTable S1). Remarkably, a large number of genes weredown-regulated in both mutants: in ostdl1a, 793 at stage3 (1,122 in total) and 858 at stage 5 (1,267 in total); in

msp1-4, 613 at stage 3 (1,029 in total) and 885 at stage5 (1,122 in total; Fig. 4B, Supplemental Table S1). In ad-dition, ostdl1a and msp1-4 shared about 50% of the dif-ferentially expressed genes at stage 3, while a muchhigher ratio of altered genes was observed at stage 5:693 genes differentially coexpressed between ostdl1a andmsp1-4 at stage 3 and 833 genes at stage 5 (Fig. 4C).Among all commonly changed genes, 498 and 686 weredown-regulated relative to the wild type at stage 3 andstage 5, and 195 and 147 genes were up-regulated(Fig. 4C). These findings support the hypothesis thatOSTDL1A and MSP1 share a common molecularpathway for anther development, which is concomitantwith the above genetic and biochemical analysis. Al-though a large number of common genes was sharedbetween ostdl1a andmsp1-4, there were;400 and;300genes differentially expressed only in ostdl1a andmsp1-4, respectively (Fig. 4C). Given the diverse ex-pression patterns between OsTDL1A and MSP1, theymay also trigger signaling in different molecular path-ways during reproductive development.

Considering the great number of common geneswithdifferential expression shared between ostdl1a andmsp1-4, hierarchical clustering analysis of significantGene Ontology (GO) terms was performed to identify

Figure 3. Physical interaction between OsTDL1A and MSP1. A and B, Diagrams showing the structure and constructs of trun-cated MSP1 LRRs and OsTDL1A, respectively. C and D, Yeast two-hybrid analysis showing the interaction between truncatedMSP1 LRRs and OsTDL1A, and truncated OsTDL1A and MSP1-LRR4. E, In vitro pull-down assays showing the interaction be-tweenOsTDL1A andMSP1. The precipitatedMSP1was detected byanti-MSP1 antibody. F, In vivo coimmunoprecipitation assaysshowing the interaction between OsTDL1A and MSP1. Tobacco leaf extracts were immunoprecipitated by anti-Myc antibody.Molecular weight of proteins: GST = 26 kD, GST-OsTDL1A = 49 kD, MSP1 = 141 kD, MSP1-Myc = 159 kD, OsTDL1A-FLAG =26 kD. WB, Western blot; IP, immunoprecipitation.



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molecular processes that are affected by themutation ofOsTDL1A or MSP1 simultaneously (Fig. 4D). Theenriched processes were classified into four groups,namely, G1, G2, G3, and G4 (Fig. 4D). G1 cluster in-cluded enriched processes shared in ostdl1a and msp1-4at stage 3 and stage 5, such as hormone-mediatedsignaling pathways, multiorganism processes, DNA-templated transcription, cell death, and reactiveoxygen species (ROS) metabolic process, etc. Theidentification of hormone-mediated signaling path-ways is consistent with the fact that hormones playcritical roles in determining cell fate in anther devel-opment (Ye et al., 2010; Song et al., 2013); however, it isnot clear that whether they were directly or indirectlyaffected by OsTDL1A-MSP1 signaling. The enrichmentin DNA-templated transcription suggests a greatnumber of genes encoding transcription factors are in-volved in OsTDL1A-MSP1 signaling to regulate antherdevelopment. Noteworthy, ROS metabolic process wasobserved, supporting the recent evidence that redox

state specifies anther cell fate in plants (Kelliher andWalbot, 2012; Zhang and Yang, 2014). In the G2 clade,there is an enrichment of genes associated with sig-naling cascades, like signal transduction, cell commu-nication, and MAPK cascade at stage 5 in ostdl1a andmsp1-4, which is consistent with the biological functionof kinase protein MSP1. Nucleobase-containing com-pound metabolic process and photosynthesis wereenriched in clade 3. Furthermore, differentiallyexpressed genes in the G4 clade associated with celldivision were specifically observed in ostdl1a, possiblydue to its role in cell proliferation (Fig. 4D).

Previous transcriptome data sets generated usingmicroarray analysis in Arabidopsis ems1/exs and maizemac1 showed a wide range of changes in gene regula-tory networks (Wijeratne et al., 2007; Zhang et al., 2014).Given the conserved function of OsTDL1A-MSP1,MAC1, and EMS1 in male organ formation, a compar-ison of these three transcriptome sets was performedhere (Supplemental Fig. S7A). Of the differentially

Figure 4. Differential gene expression profiles of ostdl1a andmsp1-4. A, Developmental progression of an anther lobe for RNA-seqfrom the establishment the archesporial cells to post cell fate specification of four anther wall layers. B, Heat map for the expressionprofiles of differentially expressed genes in ostdl1a and msp1-4 compared with the wild type. C, Differentially expressed genesshared between ostdl1a and msp1-4 at stage 3 and stage 5. Red arrows represent genes up-regulated, and green arrows representgenes down-regulated. D, GO analysis for differentially expressed genes shared between ostdl1a andmsp1-4. Venn diagrams aboveeach set of columns are shaded to represent differentially expressed genes shared between the two mutants (from left to right):differentially expressed genes in ostdl1a only, in ostdl1a and msp1-4, and in msp1-4 only. G, group; S3, stage 3; S5, stage 5.


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expressed genes in Arabidopsis ems1 mutant, 10%overlapped with their corresponding rice orthologs inostdl1a and msp1-4 mutants, while a higher proportion(21%) was observed between maize and rice(Supplemental Fig. S7A and Supplemental Table S2).Further GO analysis revealed that these commonlyexpressed genes in rice, maize, and Arabidopsis areinvolved in similar processes, including transcriptionfactor activity, oxidoreductase activity, and phospha-tase activity, etc. (Supplemental Fig. S7B). This suggeststhat OsTDL1A (or MSP1) and its orthologs are ableto regulate similar biological processes during antherdevelopment.

Altered Expression of Genes EncodingTranscription Factors

To identify early signatures of transcriptional regu-lation in specifying anther cell fate, we analyzed theexpression of transcription factors in ostdl1a andmsp1-4(Supplemental Table S3). In total, there are 25 familiesof transcription factors, such as APETALA2, bHLH,MYB, and bZIP, enriched in the two mutants analyzed(Supplemental Fig. S8A). Overall, ostdl1a and msp1-4had similar numbers of differentially expressed tran-scription factors during anther development. Amongthese, 70% were down-regulated in the two mutants,such as bHLH, MYB, NAC, HB (homeobox domain),and bZIP families. Only some genes encodingmembersof the MADS, B3 (ABI3VP1 protein), OFP (ovate familyprotein), and HMG (high mobility group protein)families were up-regulated (Supplemental Fig. S8B).The DBB (double B-box) and ARR-B (the type-Bphospho-accepting response regulator) families weredown-regulated at stage 3 and up-regulated at stage5 specifically, while the opposite trend was observedfor GRAS (Supplemental Fig. S8B). This suggests thatthese transcription factors show common regulatoryfunction in the OsTDL1A-MSP1 signal pathway. In-terestingly, some bHLH members and bZIP membersshowed a dramatic reduction in the two mutants atstages 3 and 5 (Supplemental Fig. S8B).Among the 21 bHLH members with the altered

expression, three bHLH genes, OsbHLH164 (UDT1,UNDEVELOPED TAPETUM1), OsbHLH142 (TIP2,TDR INTERACTING PROTEIN2), and OsbHLH005(TDR, TAPETUM DEGENERATION RETARDATION)with known functions in anther development, weredown-regulated in both mutants at stage 5 (Fig. 5A).The expression of other members, such as OsbHLH035,OsbHLH036, OsbHLH010, and OsbHLH122, showedstrong reduction. Conversely, six other bHLHs wereslightly up-regulated in ostdl1a and/or msp1-4, includ-ing OsbHLH167, OsbHLH004, OsbHLH044, and an in-florescence architecture determinant OsbHLH167(LAX1, LAX PANICLE1; Fig. 5A). To further confirmwhether these bHLH genes have high expression levelsin rice anther, we examined their expression patternsusing a public microarray data set (GSE13988), where

the probes forOsbHLH167 andOsbHLH042were absent(Fig. 5B). OsbHLH006 and OsbHLH148, which are spe-cifically expressed at early stages of anther develop-ment, were identified in the same cluster with TIP2 andTDR. In theUDT1 cluster,OsbHLH035 andOsbHLH063showed relatively higher expressions at premeiosisstages, while the expression of OsbHLH010was high atearly meiosis stages (Fig. 5B). qRT-PCR analysis furtherconfirmed that OsbHLH6, OsbHLH148, and OsbHLH10were down-regulated in the two single mutants and thedouble mutant at stage 3 and stage 5, whereas TIP2showed reduced expression at stage 5 (Fig. 5, D–G). Inaddition, the expression of OsbHLH063 decreased onlyin ostdl1a msp1-4 at stage 5, while OsbHLH035 andOsbHLH036 did not show significant changes in thesingle mutants (Fig. 5H). These observations suggestthat the bHLHs regulated by OsTDL1A-MSP1 signal-ing play a diverse function in regulating cellular eventsduring anther development.

Furthermore, several bZIP transcriptional factors,especially one TGAmember,OsTGA10, showed down-regulation in ostdl1a and msp1-4, while the expressionof another OsTGA2 increased only in ostdl1a at stage5 (Fig. 5C). TGA proteins belong to a distinct subcladein the bZIP family, with 10 members in Arabidopsisand 14 members in rice. Among 14 OsTGA genes,OsTGA10, OsTGA11, and OsTGA12 were specifi-cally expressed in anthers before meiosis stages(Supplemental Figure S9A). Interestingly, the expres-sion of OsTGA10 was decreased more than 5-fold inthe two single mutants and the double mutant relativeto the wild type (Fig. 5I), while the expression ofOsTGA11 andOsTGA12was unchanged in the mutants(Supplemental Fig. S9, B and C). Surprisingly, similarchanges of the OsTGA10 homolog in maize were ob-served in the mac1 microarray data (SupplementalTable S2), consistent with a role conserved in maize andrice in anther development.

Genes for Redox Modulation Are Impacted by OsTDL1A-MSP1 Pathway

Redox regulation has recently emerged as a crucialmechanism for managing significant stages duringplant male reproduction (Kelliher and Walbot, 2012;Zhang and Yang, 2014). Among the genes with alteredexpression in ostdl1a and msp1-4, we observed numer-ous genes associated with redox modulation (Fig. 6A;Supplemental Table S4), and a great overlap was ob-served between ostdl1a and msp1-4 (Fig. 6A, red dottedline). These differentially expressed genes encodeROS-related enzymes and proteins, including putativeperoxidases, oxidoreductases, cytochrome P450s, thio-redoxins, and glutaredoxins, which are involved inthe ROS-producing and ROS-scavenging pathway.In agreement with the expression change of genesassociated with redox status, the result of 3,39-diaminobenzidine (DAB) staining analysis for thepresence of hydrogen peroxide showed the production



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of hydrogen peroxide only in the middle layer of wild-type anthers after the differentiation of the four cell walllayers at stage 5 (Fig. 6, C–F). No staining was observedin ostdla and msp1-4, suggesting that ostdl1a and msp1-4have defects in the production of hydrogen peroxideduring the anther development probably because theylack amiddle layer (Fig. 6, G–J). In support of this, therewere two differentially expressed genes encoding

plant-specific CC-type glutaredoxins, which function assmall oxidoreductases of the thioredoxin family pro-teins. Glutaredoxin OsGrx_I1 was significantly down-regulated in the two single mutants and the doublemutant at stage 3 and stage 5 (Fig. 6B). In addition,glutaredoxin MIL1 (MICROSPORELESS1), which waspreviously reported to control the meiosis initiation inmicrosporocytes and differentiation of the inner

Figure 5. Differential expressed genes encoding transcription factors in ostdl1a andmsp1-4. A, Expression profiles for individualmembers of the OsbHLH family. Each column represents average expression differences in a single mutant. B, Expression patternfor individual members of OsbHLH family in anther development. Expression signatures across wild-type anther libraries wereused to cluster genes with dynamic expression during anther development. C, Expression profiles for individual members of bZIPfamily. D to I, qRT-PCR analysis of the expression of transcription factors in ostdl1a, msp1-4, and ostdl1a msp1-4 at stage 3 andstage 5. FC, fold change.


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secondary parietal cells into the middle layer andtapetal cells (Hong et al., 2012b), decreased in themutants at stage 5 (Fig. 6B). These observations sug-gest that tightly regulated redox status plays an im-portant role in the process of anther development. Inaddition, ROS accumulation is reduced in mutantsdefective in formation of the middle layer, suggestingthat the middle layer cells might be the source of ROSproduction.Glutaredoxins are important for redox control in

normal anther development and are confirmed to act byposttranslationally modifying TGA transcription fac-tors through a direct biochemical interaction. Thephysical interaction between OsTGA10 and OsGrx_I1was detected by Y2H analysis (Fig. 7A). To gain furtherinsights into the role of OsTGA10 andOsGrx_I1 in earlyanther development, RNA in situ hybridization wasperformed. OsTGA10 transcripts were detectable in the

primary parietal cells at early anther developmentstage, and subsequently OsTGA10 expression wasstrongly detectable in the middle layer and tapetal cells,whereas its signal was almost invisible during meiosis(Fig. 7, B–G). Overall, the expression pattern ofOsTGA10 was similar to that of MSP1, which specifi-cally expressed in anther inner somatic layers, implyingthat OsTDL1A-MSP1 may specify anther cell fate bydirectly or indirectly affecting the expression ofOsTGA10. Supportively, the signal from OsTGA10 wasbarely observed in ostdl1a and msp1-4 (Fig. 7, H–M).Given the previous discovery that glutaredoxins act asinteracting partners of TGA transcription factors(Murmu et al., 2010; Hong et al., 2012b), we speculatethat the CC-type glutaredoxin OsGrx_I1 may modifythe conserved Cys residues within OsTGA10, therebyaffecting its transcriptional activity. Interestingly,OsTGA11 andOsTGA12were found to be expressed in

Figure 6. Analyses of redox status in ostdl1a and msp1-4 anthers. A, Venn diagrams of redox related differentially expressedgenes between ostdl1a andmsp1-4. The numbers of differentially expressedmembers are indicated in each set of column. B, qRT-PCR analysis of OsGrx_I1 and MIL1 showing changed expression in mutants. C to J, DAB staining analysis hydrogen peroxideproduction of anthers in the wild type from stages 3, 4, 5, and 7 (C–F), ostdl1a (G and H), and msp1-4 (I and J) at stage 3 andstage 5. ML, middle layer; T, tapetum. Bars = 15 mm.



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the inner parietal layer and archesporial cells at stage3 and strongly in the middle layer, tapetal cells, andsporogenous cells at later stage 5 (Supplemental Fig.S9, D–K). Expression pattern analysis showed thatthe mRNA levels ofOsTGA11 andOsTGA12were highat premeiosis stages, and they are homologousto Arabidopsis TGA9/10. Although the mutations ofOsTDL1A and MSP1 did not affect the expression ofOsTGA11 and OsTGA12, the expression of these twogenes partially overlap with that of OsTGA10. It ispossible that TGA members may function redundantlyin anther development.

OSTDL1A-MSP1 Pathway Affects Signal Transduction forCell Fate Specification

RLKs are associated with signal perception andtransduction in organisms, and several LRR-RLKswerereported to play major roles during anther develop-ment. Therefore, we were interested in whether therewere additional genes encoding LRR-RLK or RLKs,whose expression was affected by the ostdl1a and/ormsp1-4. In total, we observed 25 RLKs with changedexpression levels, including LRR-RLKs (8), S-locus do-main RLKs (6), WAK (wall-associated kinase)-RLKs (4),lectin-like RLKs (3), and other RLKs (4) (SupplementalTable S5). Among all the LRR-RLKs genes, MSP1 de-creased only in the msp1-4 mutant, not in the ostdl1a.Moreover, three LRR-RLK genes (Os05g51070,Os01g42294, and Os01g51400) belonging to the samesubfamily of MSP1 were up-regulated only in msp1-4.One LRR-V subfamily member (Os12g43640) wasdown-regulated in ostdl1a and msp1-4. Besides, onegene (Os06g35850) in the lectin-like RLK group showed2- to 3-fold increase in the two mutants at stage 3 andstage 5. In addition to RLKs, six genes encoding MEKkinases (MAPKKKs) and one MAPKK were down-regulated in ostd1la and/or msp1-4 (SupplementalTable S5). qRT-PCR analysis further confirmed that theexpression of OsMAPKKK55 and OsMAPKKK62 de-creased in the mutants at stage 3 and stage 5, whileOsMAPKKK63 decreased at stage 5 (Supplemental Fig.S10, B–D). Furthermore, one MAPK OsMAPK3, theortholog of Arabidopsis MPK3 functioning in earlyanther development, showed reduction in ostdl1a andmsp1-4 at stage 5 (Supplemental Table S5). Previousreports indicated that OsMAPK3 and OsMAPK6 haveroles in rice immunity (Kim et al., 2012), and whetherthey have similar functions in anther development asArabidopsis MAPK3/6 needs further study. Interest-ingly, the corresponding MAPKKK homologs frommaize were down-regulated in mac1 microarray data(Supplemental Table S2). These results suggest thatperturbations in OsTDL1A-MSP1 signaling affect theprotein kinase signaling pathways during antherdevelopment.

DISCUSSION

OsTDL1A-MSP1 Represents a Conserved and DiversifiedSignaling Pathway in Specifying Cell Fate duringPlant Reproduction

Establishment of new cell types is essential for theformation of organized and functional organs. Devel-opment of male organs and reproductive cells requiresthe initiation of the anther primordium, cell division,and differentiation, all of which when properly exe-cuted result in the development of concentric rings ofsomatic layers and germinal cells. In this study, ourdata support that the small secretory peptide OsTDL1Ais able to bind a LRR-RLK receptor, MSP1, andOsTDL1A-MSP1 signaling pathway specifies the early

Figure 7. Expression pattern of OsTGA10 and its interaction withOsGrx_I1. A, The interaction betweenOsTGA10 andOsGrx_I1 in yeasttwo hybrid assays. B to G, In situ analyses of OsTGA10 in wild-typeanthers from stage 2 to stage 7. OsTGA10 is transcribed mainly in pri-mary parietal at early development stage and later strongly in themiddle layer and tapetal cells. H to J, ostdl1a anthers hybridized withOsTGA10 antisense probe from stage 3 to stage 5. K to M, msp1-4anthers hybridized with OsTGA10 probe from stage 3 to stage 5. Noobvious expression of OsTGA10 in ostdl1a and msp1-4 anthers. ML,middle layer; T, tapetum. Bars = 15 mm.


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anther cell fate by triggering the formation of the mid-dle layer and tapetal layer from meristematic parietalcells and proper number of reproductive microsporo-cytes in rice.Our genetic and cytological analysis revealed that

ostdl1a msp1-4 double mutant shows defects includingthe lack in the middle layer and tapetal cells and anincreased number of microsporocytes and partiallyaborted ovule development similar to the single mutantostdl1a or msp1-4 (Figs. 1 and 2). This result indicatesthat OsTDL1A functions in the same signaling pathwaywith MSP1 in specifying the anther cell fate. Bio-chemically, the OsTDL1A-MSP1 interaction was con-firmed by coimmunoprecipitation assays (Fig. 3), whichis consistent with previous observation that OsTDL1Abinds to the LRR domain of MSP1 by bimolecular flu-orescence complementation in onion cells; however,OsTDL1B, the paralog of OsTDL1A, has no physicalinteraction with MSP1 (Zhao et al., 2008).OsTDL1A mRNA accumulates in both inner somatic

cells and sporogenous cells, while MSP1 mRNA wasdetectable early in parietal cells and later in middlelayers and tapetal cells. OsTDL1A/MIL2 proteinwas previously seen to localize on the margin of thetapetum and middle layer (Hong et al., 2012a). It islikely that OsTDL1A acts as a developmental signalsecreted from sporogenous cells and parietal cells. The

membrane-bound MSP1 perceives this signal and pro-motes the periclinal division of parietal cells to form theendothecium, SPC, and subsequent the middle layerand the tapetum, simultaneously suppressing the pro-liferation of reproductive cells (Fig. 8). In agreementwith the placement of OsTDL1A and MSP1 in the samepathway, our transcriptional analysis revealed thatOsTDL1A and MSP1 shared a variety of commondownstream genes with altered expression levels inostdl1a and msp1-4, even though there are some genesdisplaying differential expression changes betweenostdl1a and msp1-4 (Fig. 4). With the respect to thedifferentially expressed genes involved in signaltransduction enriched in msp1-4 at stage 5, in ostdl1a atstage 3 (Fig. 4D), we speculate that MSP1 may have anadditional partner(s) except for OsTDL1A, and viceversa. Consistently, more up-regulated LRR-RLKswere preferentially detected in msp1-4 (SupplementalTable S5).

To date, a few LRR-RLKs have been identified inperceiving signals such as steroids, peptides, and se-creted proteins (Larsson et al., 2007). Phytosulfokine is asulfated peptide five amino acids in length that servesas a ligand of Arabidopsis phytosulfokine receptor inpromoting cellular growth (Matsubayashi et al., 2002,2006). The 22-amino acid flagellin-derived peptide flg22binds to Arabidopsis FLAGELLIN SENSITIVE2 and

Figure 8. Proposed model of OsTDL1A-MSP1signaling in specifying early anther development.The small secreted peptide OsTDL1A serves as aligand that interacts with the LRR domain of MSP1localized on the outer surface of inner somaticlayers. Rice OsTDL1A-MSP1 signaling promotesthe division and differentiation of primary parietalcells for the formation of middle layer and tape-tum, and limits the proliferation of reproductivesporogenous cells. MAPK cascades and putativeRLKs may act downstream of OsTDL1A-MSP1complex to transduce the anther developmentalsignal. OsTDL1A-MSP1 signaling may indirectlyaffect the expression of the TGA transcriptionfactorOsTGA10 and glutaredoxinsOsGrx_I1 andMIL1. Moreover, the expression of genes encod-ing transcription factors such as OsbHLH mem-bers is affected by OsTDL1A-MSP1. Positive andnegative regulatory actions are indicated by ar-rows and lines with bars. Lines ending with dia-monds represent interactions. Ar, archesporialcell; KD, kinase domain; LRR, leu-rich repeat;ML,middle layer; SP, signal peptide; Sp, sporogenouscell; T, tapetum; TM, transmembrane domain.



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regulates defense response (Gómez-Gómez and Boller,2000). The small secreted EPIDERMAL PATTERNINGFACTOR-LIKE proteins are 45 to 76 amino acids in sizeand can bind to ER receptor kinases in Arabidopsis toregulate stomatal development (Kondo et al., 2010;Sugano et al., 2010). CLV3 (CLAVATA3) is a secretedpeptide producing the 12-amino acid hydroxylatedpeptide as an in vivo ligand for the CLV1-CLV2 re-ceptor kinase complex, with a role in regulation of thesize of the shoot apical meristem (Fletcher et al., 1999;Kondo et al., 2006). Here, we showed that a 21-aminoacid peptide of OsTDL1A interacts with MSP1 in vitro(Fig. 3). Moreover, the 21-amino acid peptide sequenceof OsTDL1A is evolutionarily conserved from lowerplants (such as liverwort and moss) to seed plants. It ishighly possible that OsTDL1A may be secreted andcleaved into a small functional peptide that acts as theligand of MSP1, but the exact size and sequence of thefunctional peptide remains to be elucidated.

Morphologically, monocots (including rice andmaize) and eudicots (including Arabidopsis) havesimilar and distinct aspects of anther development andpollen formation (Wilson and Zhang, 2009; Kelliher andWalbot, 2011; Zhang et al., 2011), suggesting that bothconserved and divergent mechanisms underlie plantmale reproduction. In the grasses, the related smallsecreted ligands OsTLD1A (and its receptor MSP1) andMAC1 are required for early anther development.Mutations of OsTLD1A or MAC1 cause the defectivesomatic patterning. Furthermore, MAC1 has beenshown to have a function in male and female devel-opment similar to that of OsTDL1A (Wang et al.,2012), suggesting that the functional conservation ofOsTDL1A-MSP1 in maize, even though the MSP1 ho-molog in maize has not been characterized. In contrast,in the absence of the ortholog TPD1 or EMS1/EXS inArabidopsis, the PPC could perform a periclinal divi-sion to establish a bilayer (the endothecium and SPC),although tapetal layers cannot be formed because of thefailure of subsequent symmetric cell divisions of SPCs(Canales et al., 2002; Zhao et al., 2002; Yang et al., 2003).It is possible that a related (but not yet defined) orthologor other factors are essential for differentiation of thePPC in Arabidopsis.

Role of Redox Status in Specifying Anther Cell Fate

It is well known that cellular redox states criticallycontrol the fate of anther cells (Kelliher and Walbot,2012; Zhang and Yang, 2014). In this work, transcrip-tional analysis revealed that many genes involved inredox process are differentially expressed in both mu-tants at stages 3 and 5, confirming the role of redoxstatus in controlling male gametogenesis (Fig. 6A).Previously, we showed that ROS molecules are highlydetectable in wild-type anthers at stages 8 to 9, invisibleat stages 10 and 11, then observed again at stage 12,which corresponds the initiation of tapetal pro-grammed cell death and then degeneration (Hu et al.,

2011). In this work, DAB staining analysis revealed thepresence of hydrogen peroxide in the middle layer (inwild-type anthers) after the differentiation of four an-ther wall layers (Fig. 6, C–F). The function of hydrogenperoxide in the middle layer is not clear, but ROS(hydrogen peroxide) could be a marker for the middlelayer. It is possible that ROS has a critical role in pro-moting the differentiation from parietal cells to inneranther wall layers. During the process of anther de-velopment, we can infer that spatiotemporal produc-tion of ROS is triggered by developmental signal,accompanying the temporal expression of redox-related factors. Thus, the redox status is a significantdeterminant of cell fate in early anther development.

Glutaredoxins (GRXs) have long been known assmall oxidoreductases that are involved in variouscellular processes and responses to oxidative stress,such as Arabidopsis ROXY1 and ROXY2 (Xing andZachgo, 2008; Murmu et al., 2010), maize MSCA1(MALE STERILE CONVERTED ANTHER1; Chaubalet al., 2003), and rice MIL1 (Hong et al., 2012b). In thiswork, we showed that two CC-type glutaredoxinsOsGrx_I1 andMIL1 had down-regulation in ostdl1a andmsp1-4 (Fig. 6B). Glutaredoxins have been recentlyrevealed to posttranslationally modify TGA transcrip-tion factors, thereby affecting their transcriptional ac-tivity (Li, 2014). In Arabidopsis, CC-type glutaredoxinsROXY1/2 play a redundant role in regulating earlyanther lobe formation and anther wall differentiationby interacting with TGA9/10 (Xing and Zachgo, 2008;Murmu et al., 2010).MSCA1, the ROXY1/2 homologs inmaize, plays a role in interpreting hypoxia to triggerarchesporial cell fate acquisition. msca1 has no pro-gression of archesporial cell differentiation and formsvasculature instead (Chaubal et al., 2003; Kelliher andWalbot, 2012). Furthermore, MSCA1/Abph2 (Aberrantphyllotaxy 2) can interact with FEA4 (FASCIATEDEAR4) a TGA protein, and regulate the shoot apicalmeristem size in maize (Pautler et al., 2015; Yang et al.,2015). Rice MIL1 is the maize ortholog of MSCA1, butthe mil1 mutant microsporangium exhibits normaldifferentiation of the archesporial cells and the devel-opment of two secondary parietal layers, different fromthose of msca1 and roxy1/2 (Hong et al., 2012b). Fur-thermore, mil1 has the defects in the differentiation ofthe inner secondary parietal cells into middle andtapetal layers as well as the meiosis initiation, andMIL1has a interaction with OsTGA1 (Hong et al., 2012b).These findings suggest that there is functional diver-gence among GRXs and TGAs in regulating the dif-ferentiation of both germinal and somatic anthertissues.

In thiswork, we showed that OsGrx_I1 has a physicalinteraction with OsTGA10 (Fig. 7A), which is consistentwith the previous report that GRXs bind to bZIP-typeTGA transcription factors to activate the expression ofdownstream genes (Li, 2014). Moreover, OsTGA10 hasa dramatically reduced expression in ostdl1a andmsp1-4(Fig. 5I). Strikingly, OsTGA10 shares an expressionpattern similar to that of MSP1 in the primary parietal


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cells and then peaking in middle layers and tapetalcells, implying that OsTGA10 might have a role inregulating anther cell specification directly or indirectlydependent on OsTDL1A-MSP1 pathway. Consistently,almost no mRNA accumulation of OsTGA10 wasdetected in ostdl1a and msp1-4 (Fig. 7, B–M). Therefore,we speculate that OsTGA10 may be modified by theredox modulator OsGrx_I1, activating the transcriptionof downstream targets. Collectively, these results pro-vide new evidence that redox signaling plays a criticalrole in anther specification, broadening our under-standing of how redox-regulated TGA factors contrib-ute to plant development.

The Regulatory Pathway of OsTDL1A-MSP1 Signaling

Increasing evidence reveals the key role of tran-scription factors in regulating tapetal fate and normalmicrosporogenesis. For instance, four bHLH membersUDTI (Jung et al., 2005), TIP2 (Fu et al., 2014), TDR (Liet al., 2006), and EAT1 (ETERNAL TAPETUM1; Niuet al., 2013) were shown to be key regulators for riceanther development (Zhang and Liang, 2016). UDT1specifies the differentiation of secondary parietal cellsto functional tapetal cells (Jung et al., 2005). The ex-pression signal of TIP2 is strongly present in the middlelayer and tapetum andweakly in the endothecium, andTIP2 promotes cell differentiation of three inner somaticlayers. The tip2 mutant displays undifferentiated innerthree anther wall layers and aborted tapetal pro-grammed cell death (Fu et al., 2014). Genetic and bio-chemical evidence indicates that TIP2 functionsupstream of TDR and EAT1, and TDR interacts withboth TIP2 and EAT1, respectively, forming a TIP2-TDR-EAT1 regulatory cascade for anther development (Niuet al., 2013; Fu et al., 2014). Our transcriptome analysisshowed that the expression of UDT1, TIP2, and TDR isreduced in ostdl1a and msp1-4, suggesting that UDT1,TIP2, and TDR are downstream of OsTDL1A-MSP1signaling. In support of this, in situ hybridizationshowed the reduced expression pattern of UDT1 inostdl1a/mil2-1 anthers (Hong et al., 2012a). On the otherhand, the expression levels of MSP1 and OsTDL1Awere not significantly changed in tip2, whileUDT1wasup-regulated in tip2 (Fu et al., 2014). Furthermore, weobserved 21 bHLH members with changed expressionin ostdl1a and/ormsp1-4 (Fig. 5). It will be interesting toinvestigate the function of these bHLHs affected byOsTDL1A-MSP1 in early anther development.Genetic studies have uncovered that RLKs regulate

various steps in anther patterning. Although a fewgenes encoding LRR-RLKs have expression alterationin ostdl1a and/or msp1-4, their biological function re-mains unclear (Supplemental Table S5). In addition, inostdl1a and msp1-4, we observed the down-regulationsof mRNA levels of several OsMAPKKKs and OsMAPK3,whose counterparts also exhibited reduced expression inmac1 (Supplemental Table S2), suggesting possible com-mon and conserved regulatory pathways involving

OsTDL1A-MSP1 in anther development. Consistentwith the hypothesis that MAPK proteins functiondownstream of OsTDL1A-MSP1, Arabidopsis MAPkinases MPK3/6 and the LRR-RLKs ER/ERL1/ERL2were shown to act in the same pathway required fornormal anther lobe formation and cell differentiation(Hord et al., 2008). Future work may reveal whichMAPKs receive the developmental signal fromOsTDL1A-MSP1 pathway and coordinate the devel-opment of anther cell wall layers and germ cells in thefuture.

CONCLUSION

In summary, we used genetic and biochemicalapproaches to support that the peptide OsTDL1A bindsto a LRR-RLK MSP1 and then promotes the develop-ment of parietal cells into the middle layer and tapetallayer. These proteins also limit the extra number ofsporogenous cells during rice anther development. The21-amino acid domain of OsTDL1A that binds MSP1 isevolutionarily conserved from lower plants to higherplants. The OsTDL1A-MSP1 signaling pathway mayspecify anther cell fate by changing the expression ofnumerous genes encoding bHLH, bZIP transcriptionfactors, and redox-related proteins directly or indirectly(Fig. 8). In particular, OsTDL1A-MSP1 signaling dra-matically affects the expression of OsTGA10, whichcould interact with a glutaredoxin OsGrx_I1. Intrigu-ingly, the presence of hydrogen peroxide in the middlelayer highlights a new role of reactive oxygen species inspecifying anther cell identity. These findings provideinsights into signaling pathway important for thespecification of cell identity during early anther devel-opment in plants.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

All the rice (Oryza sativa) plants used in this study are in the background of9522 cultivar (O. sativa ssp. japonica) and were grown in the paddy field ofShanghai Jiao Tong University. Male sterile mutants ostdl1a,msp1-4, andmsp1-6are from our ricemutant librarymade by 60Co g-ray radiation. The ostdl1a msp1-4 double mutant was isolated by crossing and verified by genotyping.

Characterization of the Mutant Phenotype

Morphology of flowers and anthers was photographed with a Leica S8AP0stereomicroscope. Pollen viability analysis was conducted using iodine-potassium iodide solution and then photographed with a Leica DM2500 Mi-croscope. The 49,6-diamino-phenylindole staining of microspores and semithinsection analysis were performed according to a previous study (Li et al., 2006).Anthers from different developmental stages, as defined by Zhang et al. (2011),were collected based on spikelet length and lemma/palea morphology, and thedevelopmental stages of wild-type anthers were further confirmed by semithinsections.

Map-Based Cloning

The F2 mapping population was generated from a cross between ostdl1a/msp1-6 (japonica) and 9311 (indica) for gene mapping. To map theOsTDL1A and



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MSP1 locus, bulked segregated analysis was used as described by Liu et al.(2005), and insertion-deletion (InDel) molecular markers were designed basedon the sequence difference between rice ssp. japonica and rice ssp. indica de-scribed in the National Center for Biotechnology Information. Further fine-mapping was performed using the previously published method (Chu et al.,2005). Markers used for cloning are listed in Supplemental Table S6.

Yeast Two-Hybrid Analysis

In this study, all full-length cDNA and cDNA fragments for cloning wereamplified using primers listed in Supplemental Table S6. PCR sequences werecloned into pGBKT7 and pGADT7 (Clontech). Yeast two-hybrid assayswere performed according to instructions of theMatchmaker GAL4 two-hybridsystem (Clontech). Specific bait and prey constructs were transformed intoyeast strain AH109 simultaneously. Protein interactions were tested by thegrowth conditions on selective synthetic defined (SD)media (-Trp/-Leu/-His/-Ade/+X-a-gal).

Antibody Preparation and Purification

The specific fragment of MSP1 cDNA was synthesized for antigen and wascloned into pGEX-6P-1 vector at the BamHI and NotI sites. The construct wastransformed into Escherichia coli BL21 (DE3; Novagen) and protein expressedand purified using Affinity Resin (Clontech) from a soluble fraction undernatural conditions, according to the manufacturer’s instructions. Recombinantproteins were separated by SDS-PAGE, and gel slices containing the proteinwere used directly for the production of rabbit polyclonal antibodies. MSP1antibodies were purified as described previously (Ritter, 1991).

Pull-Down Assay and Coimmunoprecipitation

The full-length cDNA of OsTDL1A was synthesized by Sangon and wascloned into pGEX-6P-1 vector at the BamHI and NotI sites. Recombinantproteins were expressed in the DE3 strain induced by isopropyl-b-D-1-thiogalactopyranoside. 35S::MSP1 was transiently expressed in tobacco leaves.The purified GST-OsTDL1A and GST proteins were immobilized onto gluta-thione Sepharose beads (Aogma). Then total soluble protein extract of MSP1transgenic leaves was incubated with the immobilized GST-OsTDL1A andGSTproteins. Proteins retained on the beads were washed three times using washbuffer (15mMHEPES-NaOH at pH 7.9, 50mM potassiumGlu, 5mMmagnesiumchloride, and 0.1% [v/v] Nonidet P-40) and were analyzed using 15% SDS-PAGE and tested with MSP1 antibody.

Coimmunoprecipitation analysis was performedwith extracts from 4-week-old tobacco (Nicotiana tabacum) leaves, as described by Bai et al. (2012). To createthe FLAG-tagged OsTDL1A and Myc-tagged MSP1 for protein expression, thefull-length coding regions of these two genes were PCR amplified with theprimer pairs (Supplemental Table S6). The fusion proteins OsTDL1A-FLAGand MSP1-Myc were transiently expressed in tobacco leaves. Proteins wereextracted with ice-cold buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1%Triton X-100, 2.5 mM EDTA, 2 mM benzamidine [Sigma-Aldrich], 10 mMb-mercaptoethanol, 20 mM NaF, 1 mM PMSF, 1% Protease Cocktail [Sigma-Aldrich], and 10% glycerol). After centrifugation at 13,000g for 10 min at 4°C,the supernatant was incubated with anti-FLAG or anti-Myc antibodies andIgG-bound to Protein A/G Sepharose beads (Sigma-Aldrich) for 1 h at 4°C, andthe beads were washed three times with wash buffer. Proteins were eluted byboiling the beads in 23 SDS sample buffer and separated on SDS-PAGE beforeimmunoblotting using anti-FLAG or anti-MYC antibodies.

qRT-PCR and in Situ Hybridization

Total RNAs were isolated from rice tissue using the Trizol reagent (Invi-trogen) following the manufacturer’s instructions. cDNAwas synthesized from1mg of total RNAper sample using the Primescript RT reagent kit with genomicDNA eraser (Takara). qRT-PCR was performed on a CFX96 (Bio-Rad) machineusing SYBR Green qRT-PCR Mix (Bio-Rad) according to the manufacturer’sinstructions. ACTIN was used as an internal control, and all the primers forqRT-PCR are listed in Supplemental Table S6. qRT-PCR results shown arerepresentative of experiments performed at three biological repeats with threetechnique repeats.

For RNA in situ hybridizations, Specific fragments of the coding se-quences were amplified by PCR using specific primers carried with T7

promoter (Supplemental Table S6). Antisense and sense probes weretranscribed using the DIG RNA labeling kit (Roche). Freshly collectedsamples were fixed in FAA, dehydrated in a series of graded ethanol,infiltrated with Histo-clear II, embedded in Paraplast Plus (Sigma-Aldrich),and sectioned into 6-mm-thick sections using a Leica RM2245 rotary mi-crotome. RNA in situ hybridizations were performed as described by Liet al. (2006).

ROS Staining Analysis with DAB

Freshly collected anthers were put in the fixative solution O.C.T. Compound(Sakura) and then frozen into liquid nitrogen. Embed fresh tissues were sec-tioned into 8-mm-thick sections using a Leica CM3050 S cryostat. Production ofhydrogen peroxide was performed by incubating sectioned anthers in the DABsolution (Vector Laboratories) according to the manufacturer’s instructions andphotographed with a Leica DM2500 microscope.

RNA-Seq and GO Enrichment Analysis

Total RNAs were prepared from wild-type, ostdl1a, and msp1-4 spikeletswith three biological replicates. RNA-seq library preparation and sequencingwere performed with Illumina sequencing technology (BGI-Shenzhen); theselibraries and the mapping methods were described (Q Fei, L Yang, W Liang,D Zhang, B Meyers, unpublished data) Genes were considered to be sig-nificantly differentially expressed if both the q-value , 0.01 and the foldchange$ 2.0. GO analysis was performed by R package “topGO” (Alexa andRahnenfuhrer, 2010).

Accession Numbers

Sequence data from this article for the mRNA and genomic DNA can befound in the GenBank/EMBL data libraries under the following accessionnumbers: OsTDL1A (Os12g28750),MSP1 (Os01g68870), OsTGA10 (Os09g31390),OsTGA911 (Os11g05480), OsTGA912 (Os12g05680), OsGrx_I1 (Os01g47760)),OsOsbHLH006 (Os04g23550), OsOsbHLH010 (Os01g50940), OsOsbHLH063(Os03g26210), OsOsbHLH148 (Os03g53020), OsMAPKKK55 (Os01g50400),OsMAPKKK62 (Os01g50420), and OsMAPKKK63 (Os01g50370).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Map-based cloning of OsTDL1A and MSP1.

Supplemental Figure S2. Complementation of the ostdl1a mutant.

Supplemental Figure S3. DAPI-stained chromosome spreads of microspo-rocytes in the wild type, ostdl1a, and msp1-4.

Supplemental Figure S4..Expression pattern of OsTDL1A and MSP1.

Supplemental Figure S5. The LRR repeats of the MSP1 protein and se-quence analysis of OsTDL1A.

Supplemental Figure S6. In vivo interaction between OsTDL1A andMSP1.

Supplemental Figure S7. Transcriptome comparison among rice (ostdl1aand msp1-4), maize (mac1), and Arabidopsis (ems1).

Supplemental Figure S8. Distribution of specific classes of transcriptionfactors in the mutants.

Supplemental Figure S9. Expression pattern for individual members ofTGA subfamily in anther development.

Supplemental Figure S10. The expression analysis of OsMAPKKKs in themutants.

Supplemental Table S1. List of genes that were differentially expressed inmutants compared to the wild type in rice.

Supplemental Table S2. Genes differentially coexpressed among rice (ost-dl1a and msp1-4), maize (mac1), and Arabidopsis (ems1).

Supplemental Table S3. Differentially expressed genes encoding transcrip-tion factors.


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Supplemental Table S4. Differentially expressed genes associated withredox process.

Supplemental Table S5. Differentially expressed genes associated withsignal transduction.

Supplemental Table S6. Lists of primers used in this study.

ACKNOWLEDGMENTS

We thank Zhijing Luo for mutant screening and generation of F2 populationfor mapping and Virginia Walbot for critical reading and comments on themanuscript. Katie Murphy and Virginia Walbot of Stanford University pro-vided detailed protocols for performing the DAB assays on anthers.

Received January 13, 2016; accepted May 18, 2016; published May 20, 2016.

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regulatory role of a receptor-like kinase in specifying ...lrr-rlk, msp1 (multiple sporocyte1),...

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