The Rice Receptor-Like Kinases DWARF AND RUNTISHSPIKELET1 and 2 Repress Cell Death and Affect SugarUtilization during Reproductive Development

Cui-Xia Pu,a,b,c,1 Yong-Feng Han,a,b,1 Shu Zhu,a,b,c,1 Feng-Yan Song,a,b Ying Zhao,a,b Chun-Yan Wang,a,b,c

Yong-Cun Zhang,a,b Qian Yang,a,b Jiao Wang,a,b,c Shuo-Lei Bu,a,b,c Li-Jing Sun,a,b Sheng-Wei Zhang,a,b,c

Su-Qiao Zhang,a,b,c Da-Ye Sun,a,b,c and Ying Suna,b,c,2

a Hebei Key Laboratory of Molecular and Cellular Biology, Hebei 050024, P.R. Chinab Key Laboratory of Molecular and Cellular Biology of Ministry of Education, College of Life Science, Hebei Normal University, Hebei050024, P.R. ChinacHebei Collaboration Innovation Center for Cell Signaling, Shijiazhuang, Hebei 050024, P.R. China

ORCID IDs: 0000-0001-5460-8925 (C.-X.P.); 0000-0002-5626-668X (Y.-F.H.); 0000-0001-6308-8511 (S.Z.); 0000-0002-1444-5057 (Y.S.)

Cell-to-cell communication precisely controls the creation of new organs during reproductive growth. However, the sensormolecules that mediate developmental signals in monocot plants are poorly understood. Here, we report that DWARF ANDRUNTISH SPIKELET1 (DRUS1) and DRUS2, two closely related receptor-like kinases (RLKs), redundantly controlreproductive growth and development in rice (Oryza sativa). A drus1-1 drus2 double knockout mutant, but not either singlemutant, showed extreme dwarfism and barren inflorescences that harbored sterile spikelets. The gibberellin pathway was notimpaired in this mutant. A phenotypic comparison of mutants expressing different amounts of DRUS1 and 2 revealed thatreproductive growth requires a threshold level of DRUS1/2 proteins. DRUS1 and 2 maintain cell viability by repressingprotease-mediated cell degradation and likely by affecting sugar utilization or conversion. In the later stages of antherdevelopment, survival of the endothecium requires DRUS1/2, which may stimulate expression of the UDP-glucosepyrophosphorylase gene UGP2 and starch biosynthesis in pollen. Unlike their Arabidopsis thaliana ortholog FERONIA,DRUS1 and 2 mediate a fundamental signaling process that is essential for cell survival and represents a novel biologicalfunction for the CrRLK1L RLK subfamily.

INTRODUCTION

In flowering plants, the sequential generation of new organs reliesoncell fatedeterminationand the formationof specificcell types inresponse to developmental and physiological cues from neigh-boring cells. Receptor-like kinases (RLKs) possess diverse ex-tracellular domains that are linked to a conserved kinase domainvia a transmembrane region and function as the major sensormolecules at thecell surface (Osakabeet al., 2013; Engelsdorf andHamann, 2014). RLK-mediated cell signaling induces cellulardifferentiation by activating distinct pathways and regulatesa wide range of biological processes to shape the plant (De Smetet al., 2009).

Inflorescence development involves the sequential initiation ofmeristems and primordia, the specification of cell lineages, thedifferentiation of floral organs, and the production of game-tophytes for eventual sexual reproduction (Ikeda et al., 2004;Hake, 2008). These processes are tightly regulated by a tran-scriptionnetwork (Zhangetal., 2013;ZhangandYuan,2014;Dreniand Zhang, 2016) and require the frequent exchange of signals

between cells. Several RLK-peptide pairs play roles in male re-productive development (Zhang and Yang, 2014), such asBARELYANYMERISTEM1/2-CLE9andEXTRASPOROGENOUSCELLS1 (EXS1)/EXCESS MICROSPOROCYTES1-TAPETUMDETERMINANT1 (TPD1) (Hord et al., 2006; Jia et al., 2008;Shinohara et al., 2012; Uchida et al., 2012). In the monocot rice(Oryza sativa), MULTIPLE SPOROCYTE1 (MSP1), a homolog ofArabidopsis thaliana EXS1 (Nonomura et al., 2003), interacts withthe peptide MICROSPORELESS2 (MIL2)/TPD-Like 1A to specifyearlyanthercell fatebymaintaining redoxstatus (Hongetal., 2012;Yang et al., 2016). FLORALORGANNUMBER1 (FON1), a putativeortholog of CLAVATA1 (CLV1; Suzaki et al., 2004; Moon et al.,2006), maintains the inflorescence meristem by interacting withthe putative ligand FON2/FON4, a CLV3-related protein (Chuet al., 2006; Suzaki et al., 2006, 2008). In maize (Zea mays), Thicktassel Dwarf1 (TD1), a counterpart of CLV1, controls reproduc-tive meristem development (Bommert et al., 2005), but its ligandis unknown. The fasciclin glycoprotein MICROSPORE ANDTAPETUM REGULATOR1, which is specifically secreted fromgametophyte cells during early development, controls sporo-phytic and reproductive cell development (Tan et al., 2012);however, its receptor is unknown. In summary, accumulatingevidence from Arabidopsis and other plants strongly suggeststhat members of the RLK superfamily are extensively involved inevery aspect of sexual reproduction in plants. Rice has twice asmany RLK genes as Arabidopsis (Shiu and Bleecker, 2003; Shiuet al., 2004), but the roles of these genes in reproductive growth are

1 These authors contributed equally to this work.2 Address correspondence to yingsun@mail.hebtu.edu.cn.The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Ying Sun(yingsun@mail.hebtu.edu.cn) and Cui-Xia Pu (pucx@hebtu.edu.cn).www.plantcell.org/cgi/doi/10.1105/tpc.16.00218

The Plant Cell, Vol. 29: 70–89, January 2017, www.plantcell.org ã 2017 American Society of Plant Biologists. All rights reserved.

largely unknown.Functional annotationof theseRLKgenes,alongwith temporal and spatial expression profiling in reproductiveorgans, will be important for analyzing these genes and manip-ulating their expression to optimize grain formation.

Members of the Catharanthus roseus RLK1-like (CrRLK1L)subfamily have a putative carbohydrate binding malectin-likedomain and function in diverse biological processes (Nissen et al.,2016), including male and female interactions mediated by thesynergid-expressed gene FERONIA (FER) and the pollen-specific genes ANXUR1 (ANX1) and ANX2 (Escobar-Restrepo

et al., 2007; Boisson-Dernier et al., 2009; Miyazaki et al., 2009);cell wall sensingmediated by THESEUS1 (THE1) (Hématy et al.,2007); cell elongationmediated by FER, THE1, andHERCULES(HERK1 and HERK2) (Guo et al., 2009b; Guo et al., 2009a);cytoskeleton dynamics mediated by CURVY1 (Gachomo et al.,2014); polarized growth in root hairs mediated by FER and [Ca2+]cyt-associated protein kinase 1/ERULUS (Duan et al., 2010;Bai et al., 2014); and seed size control, powdery mildew infection,and mechanical signal transduction mediated by FER (Kessleret al., 2010; Shih et al., 2014; Yu et al., 2014) in Arabidopsis.

Figure 1. The Expression of DRUS1 and DRUS2 in Rice.

(A) Immunoblot analysis of DRUS1 and 2 proteins in the indicated tissues using anti-DRUS1 C-terminal (CT) antibodies. The asterisk marks a nonspecific band.Sh, shoots of 20-d-old plants; Sm, the elongating stembefore bolting; In 7 and In 8, young inflorescences at stages 7 and 8; St 8 to St 12, anthers at stages 8 to12;Ci, calli.(B) to (Q) In situhybridizationofDRUS1andDRUS2 in the inflorescence ([B] to [I]) andanther ([J] to [Q]) at the indicatedstage. Thearrows in (C)and (G)markthe secondary branch meristem. The arrows in (D) and (H) mark the spikelet meristem. (E), (I), (M), and (Q) are the sense probes.An, anther; IM, inflorescencemeristem; In 1, 5, and6, inflorescence stages 1, 5, and6; St 4, 6, and9, anther stages 4, 6, and 9; T, tapetum; V, vascular tissue.Bars = 50 mm in (B) to (I) and 20 mm in (J) to (Q).

DRUS1 and 2 Redundantly Control Reproductive Growth 71

RUPTURED POLLEN TUBE was recently shown to control pollentube growth and integrity in rice (Liu et al., 2016). However, thebiological functions of other CrRLK1L RLKs in rice are unknown.

In this study, we characterized two CrRLK1L RLKs, DWARF ANDRUNTISH SPIKELET1 (DRUS1) and DRUS2, the orthologs of FER.These two proteins, acting as key regulators, redundantly controlreproductive growth by inhibiting cell death and affecting sugarutilization, and play distinct biological roles in rice.

RESULTS

Os03g21540 and Os01g56330 Encode RLKs and HaveWidespread, Overlapping Expression Patterns

To explore the roles of cell surface-localized RLKs in sexual re-production in rice, we used genomic annotations, protein domain

predictions, andmicroarray data (5.5K) to identify candidate RLK-encoding genes that are strongly expressed during the headingstage. Among the 33 genes identified, Os03g21540 andOs01g56330 (designated DRUS1 and 2, respectively, based ontheir stature and spikelet phenotypes) showed 83% amino acidsequence identity. RT-PCR showed that DRUS1 and 2 werebroadly expressed in nearly all tissues examined, includingseedlings, spikelets, the uppermost internode, and the flag leafblade and sheath (Supplemental Figure 1A). GUS staining ofProDRUS1:GUS and ProDRUS2:GUS plants also showedwidespread DRUS1 and 2 promoter activity (SupplementalFigures 1B to1I). DRUS1 and 2 proteins accumulated in shoots,stems, axillary buds, young inflorescences, anthers, and calli(Figure 1A; Supplemental Figure 1O). In situ hybridization showedthat DRUS1 and 2 are expressed in the inflorescence meristem,branchmeristem,spikeletmeristem,anther,microsporemothercell,microspore, anther wall, and vascular bundle (Figures 1B to 1Q),

Figure 2. In Vitro Kinase Assay and Subcellular Localization of DRUS1 and DRUS2.

(A) Diagram of the Pro35S:DRUS1DK-GFP and Pro35S:DRUS2DK-GFP constructs in which the kinase domain was replaced by GFP (green fluorescentprotein). SP, signal peptide; TM, transmembrane domain; amino acid positions are marked below the constructs.(B)TransientexpressionofDRUS1DK-GFPandDRUS2DK-GFPintobacco (N.benthamiana) leafepidermalcells.Pro35S:GFPwasusedasacontrol.Bars=50mm.(C) Immunoblot analysis of the DRUS1-7Myc-6His fusion protein in ProDRUS1:DRUS1-7Myc-6His overexpression plants (DRUS1-OE). M, microsomalproteins; S, soluble proteins; T, total proteins. Arrow marks target protein. Asterisk marks the nonspecific protein used as a loading control.(D) Alignment of the conserved motif from the SIT1(Os02g42780), BRI1(At4g39400), SERK1(At1g71830), DRUS1, and DRUS2 kinase domains. Arrowsindicate the point mutations K569E in m1DRUS1 and K667E in m2DRUS1.(E)Phosphoimage showing autophosphorylation and substrate (myelin basic protein [MyBP]) phosphorylation byGST-kinase fusion proteins, but not GSTalone. Top panel, autoradiograph; bottom panel, Coomassie blue staining of the protein gel.

72 The Plant Cell

Figure 3. Phenotypic Comparison of Six Genotypes at the Reproductive Stage.

(A) Schematic representation of the DRUS1 and DRUS2 cDNAs and T-DNA insertion sites (triangle) in drus1-1, drus1-2, and drus2. Black and red arrowsdenote the primers used for genotyping by PCR andmRNA detection by RT-PCR, respectively. Filled box, coding region; empty box, 59- and 39-UTRs;SP, signal peptide; TM, transmembrane domain; KD, kinase domain; Mal-L, malectin-like domain; ATG and TGA indicate the start and stop codon,respectively.(B)Stature of plants of six genotypes segregated fromBC2F2 progeny of a drus1-1 and drus2 cross at the flowering stage. The numbers in (B) to (I) indicategenotype, as defined in the key above the images.(C) Close-up view of the inflorescences of the six genotypes shown in (B). Red asterisks mark white sterile spikelets.(D) Comparison of the spikelets in the six genotypes. GS, green spikelet; WS, white spikelet.(E) and (F) Comparison of the primary branch meristem number between DJ (E) and the drus1-1 drus2 mutant (F). Red asterisks mark the primarybranch meristem.(G) and (H) The panicle length, the number of primary branches (G), and the green and white spikelet number per panicle (H) in (C). Error bars show themeans 6 SD; n = 30 panicles from 10 plants. Asterisks indicate a significant difference by Student’s t test; ***P < 0.001.

DRUS1 and 2 Redundantly Control Reproductive Growth 73

as well as in mature pistil, including the integument, nucellus, andembryo sac (Supplemental Figures 1J to 1N). These expressionpatterns suggest that both genes function during reproductivegrowth.

DRUS1 and 2 Are Plasma Membrane-Localized,Kinase-Active RLKs That Can Complement Arabidopsisfer-4 Plants

We next investigated the subcellular localization of DRUS1 and 2.Due to the toxicity of Pro35S:DRUS1/2-GFP in Agrobacteriumtumefaciens, we deleted regions of these proteins encoding thekinase domain and the C-terminal end to produce the constructsPro35S:DRUS1/2DK-GFP (Figure 2A), which we introducedinto tobacco (Nicotiana benthamiana) leaves via Agrobacterium-mediated transformation. The fusion proteins mainly localized tothe plasma membranes of epidermal cells (Figure 2B); this wasconfirmed by visualizing DRUS1DK-GFP in onion epidermal cellsafter plasmolysis (Supplemental Figure 2). In addition, we de-tected DRUS1-7Myc-6His fusion protein in total and microsomalfractions from rice plants, but not in the cytosolic fraction (Figure2C). These data indicate that DRUS1 is localized to the plasmamembrane in rice. We then performed an in vitro kinase assayusing the intracellular fragments of DRUS1, DRUS2, and twomutated DRUS1s, m1DRUS1K569E and m2DRUS1K667E, in whichthe conserved lysine residue in the ATP binding domain and incatalytic domain ismutated to glutamine, respectively (Figure 2D),expressed as GST-tagged fusion proteins. DRUS1 and 2, butnot GST alone, catalyzed protein autophosphorylation and thephosphorylation of myelin basic protein (a common substrate).The kinase activity of m1DRUS1K569E, but not m2DRUS1K667E,was dramatically reduced (Figure 2E), indicating that the con-served lysine (Lys-569) in the ATP binding region is important forthe catalytic function of these kinases.

DRUS1 and 2 have extracellular malectin-like domains, in-dicating that they belong to the CrRLK1L subfamily. Phylogeneticanalysis revealed that among the 20 members in rice and 17 inArabidopsis, DRUS1 and 2 are putative orthologs of ArabidopsisFER (Supplemental Figures3Aand3BandSupplementalDataSet1), sharing more than 50% amino acid sequence identity with theFER extracellular domain and nearly 80% identity with the FERintracellular domain (Supplemental Figure 3C). The T-DNA in-sertionmutant fer-4 (GK-106A06) is dwarf and sterile; even fer-4+/2

heterozygotes are semisterile (Duan et al., 2010) (SupplementalFigures 4A, 4C, 4F, and 5A). To investigate themolecular functionof DRUS1, we introduced wild-type DRUS1 and kinase activity-impaired m1DRUS1 into fer-4+/2 under the control of the FERpromoter and theFER39-untranslated region (UTR; SupplementalFigures 4B to 4E). ProFER:DRUS1-FER39 transgenic lines inthe fer-4 background expressed DRUS1 mRNA and protein(Supplemental Figures 5C and 5E) and displayed recovered

vegetative growth and fertility (Supplemental Figures 4F, 5A, and5B and Supplemental Table 1). Furthermore, DRUS1 was de-tected in the microsomal fraction, rather than in the cytosolicfraction, of the R4 plant (a recovery T1 line) (Supplemental Figure5F), indicating its function at the membrane.In contrast to the plants expressing the wild-type DRUS1

transgene, the ProFER:m1DRUS1K569E-FER39 fer-4 plants dis-played recovered vegetative growth but only partially recoveredfertility (Supplemental Figures 4F, 5A, and 5B and SupplementalTable 2). Notably, m1DRUS1 was indeed expressed in linesmR3 and mR7a, the T1 and T2 recovery lines, respectively, atthe mRNA and protein levels (Supplemental Figures 5D and5G). The failure of m1DRUS1 to completely rescue fer-4fertility is likely due to incorrect localization of m1DRUS1within the synergid cells in some embryo sacs or reducedkinase activity. Taken together, our data show that plasmamembrane-localized DRUS1 has the same function as FERand that the kinase activity is important for the recovery offertility in fer.

drus1 drus2 Double Mutants and RNAi Lines Show Defectsin Stem Elongation, Inflorescence, Spikelet Development,and Seed Setting

To investigate the functions of DRUS1 and 2 in rice, we obtainedthe T-DNA insertion mutants drus1-1 (Dongjin [DJ], PFG_2C-80049.R), drus1-2 (Hwayoung, PFG_2A-40140.R), and drus2 (DJ,PFG_3A-60178.L). The insertions in drus1-1 and drus1-2 are lo-cated in the kinase domain, and the insertion in drus2 is in theextracellular domain (Figure 3A). After backcrossing each mutanttwice to eliminate unrelated mutations, we examined the phe-notypes of the BC2F2 progeny (Supplemental Figures 6A and 6B).The drus1-1, drus1-2, and drus2 single mutants, which lack full-length transcripts but possess partial transcripts of the re-spective genes (Supplemental Figures 6C and 7C), did notappear significantly different from thewild typeduring vegetativeand reproductive growth (Figures 3B to 3D; SupplementalFigures 6D to 6J). Because drus1-1 and drus2 were producedin the same ecotype (DJ), we crossed drus1-1 with drus2 toproduce a BC2F2 population (Supplemental Figures 7A and 7B).The double mutant drus1-1 drus2 (dk) displayed extremedwarfism (Figure 3B) due to reduced internode elongation(Supplemental Figures 7D and 7E) and very small inflorescenceswith reduced branching (Figures 3C and 3G), due to the reducednumber of primary branches initiated (Figures 3E and 3F). Thesparsepaniclescontainednomore than20spikelets,whichweregreen or white and much slimmer than those of the wild type(Figures 3D and 3H). We therefore concluded that drus1-1 anddrus2 are recessive loss-of-function mutants and that DRUS1and 2 redundantly control rice stature and inflorescence andspikelet development.

Figure 3. (continued).

(I) Immunoblot analysis showed theDRUS1and2protein levels in youngpanicles (at the secondbranchprimordia stage) of the six genotypes. Thenumbersbeneath the blot show the total amount of protein (DRUS1 plus 2) normalized to a nonspecific protein (marked with an asterisk) in each lane.Bars = 10 cm in (B) and (C), 0.5 cm in (D), and 100 mm in (E) and (F).

74 The Plant Cell

Notably, the drus1-1+/2 drus2-/- plants displayed a weakphenotype, including a significant reduction in stature(Supplemental Figures 7D and 7E), panicle length, and number ofprimary branches (Figures 3C and 3G), and a small number ofwhite, slim spikelets (Figures 3D and 3H), suggesting the oc-currence of haploinsufficiency of DRUS1 in the absence of

DRUS2. We detected lower levels of DRUS1 protein in drus1-1+/2

drus22/2 than in drus2 (Figure 3I; Supplemental Figure 7H),indicating that DRUS1 has a dosage effect on the observedphenotype. Similarly, DRUS2 protein levels were lower indrus1-12/2 drus2+/2 than in drus1-1 (Figure 3I), but DRUS2haploinsufficiency in drus1-12/2 drus2+/2 only affected seedsetting.Theseedsetting indrus1-12/2drus2+/2anddrus1-1+/2drus22/2

plants was reduced (Supplemental Figures 7F and 7G). All whitespikelets were sterile, and some green spikelets also harboredabortedseedsandunfertilizedpistils (SupplementalFigure7G).Thedkmutant did not set seeds (Supplemental Figures 7F and 7G), butthe mutant segregated from well-developed drus1-12/2 drus2+/2

and drus1-1+/2 drus22/2 spikelets at a lower ratio than expected(Supplemental Table 3). The fertility of dk male and female game-tophytes was nearly normal (Supplemental Table 4). These resultssuggest that the sterility in dkmutants is caused by a defect in thesporophyte rather than thegametophyte and that thepartial sterilityofdrus1-12/2drus2+/2anddrus1-1+/2drus22/2plants isdue to theabortion of dk seeds during early embryogenesis.To confirm the double mutant analysis, we used RNA in-

terference (RNAi) to knock down DRUS1 and 2, using the extra-cellular fragment edRi as the target sequence (SupplementalFigure 8A). This fragment does not affect the related CrRLK1Lfamily memberOs04g49690 (Supplemental Figure 8K). The RNAiplants displayed a semidwarf stature (Supplemental Figures 8Band 8E) and sparse inflorescences that barely extended past theflag leaf (Supplemental Figures8Cand8F to8I). Thespikeletswereruntish and varied in width and color (Supplemental Figure 8C, inbox) andset nomore than10seeds (Supplemental Figures8Dand8J). These phenotypes are consistent with the phenotypes of dkplants; thus, the RNAi lines provided a weak allele.To demonstrate the function of DRUS1, we introduced a Pro

DRUS1:DRUS1-7Myc-6His construct into dk (SupplementalFigures 9A to 9C). The homozygous expression of exogenousDRUS1 (DRUS1+/+/dk) (Supplemental Table 5) largely rescued theobserved defects in plant stature, spikelet number (Figures 4A to4D), and seed setting (Supplemental Figure 9E). However, theheterozygous expression ofDRUS1 (DRUS1+/2/dk) (SupplementalTable 5) only partially recovered the dk phenotype, especially intermsof the inflorescences,which carrieda certainnumber ofwhitespikelets (Figures 4C and 4D) and fewer seeds (SupplementalFigure 9E). Immunoblot analysis showed that the DRUS1 fusionprotein levelwashigher inDRUS1+/+/dkplants than inDRUS1+/2/dkplants, and no endogenous DRUS1 or 2 was present (Figure 4E;Supplemental Figure 9D). These results support the functionalredundancyofDRUS1and2and thedosageeffect ofDRUS1 in theabsence of DRUS2.

drus1 drus2 Double Mutants and RNAi Lines Also ShowDefects in Floral Organs and Pollen Viability

We next examined the floral organs and pollen of themutants andfound that, consistent with the phenotypes described above, thedifferent genotypes showed a phenotypic series. The dk plantshad the most severe phenotype: The white spikelets containedfragile anthers and tiny pistils and the green spikelets containedshort, pale anthers but normal pistils (Figures 5A and 5B).

Figure 4. Complementation of the drus1-1 drus2 Double Mutant byProDRUS1:DRUS1-7Myc-6His.

(A) to (D)Phenotypiccomparisonofdoubleknockoutmutant (dk) anddkplantswith homozygous and hemizygous ProDRUS1:DRUS1-7Myc-6His trans-genes at the flowering stage. Plant stature ([A] and [B]), inflorescence archi-tecture, and spikelet number ([C] and [D]) showed nearly total phenotypicrecovery inDRUS1+/+/dkandpartialphenotypic recovery inDRUS1+/2/dk.Redasterisksmarkwhite sterile spikelets. Inset shows themagnification of a greenspikelet (GS) and a white spikelet (WS) in (C). The error bars indicate themeans6 SD; n = 10 to 14 plants in (B) and 30 panicles from 10 plants in (D).(E) Immunoblotanalysisshowed theexogenousDRUS1-7Myc-6His fusionprotein in the complemented plants and endogenous DRUS1 and DRUS2in DJ plants. The nonspecific band marked by an asterisk was used asa loading reference.Bar = 10 cm in (A), 5 cm in (C), and 0.5 cm in the inset of (C).

DRUS1 and 2 Redundantly Control Reproductive Growth 75

Figure 5. Flower Organs and Pollen Viability Test.

(A) and (B) Morphology of the anthers (A) and pistils (B) in four genotypes.(C) to (G) Scanning electron microscopy of the anther epidermis in DJ (C) and dk ([D] to [G]) at stage 12.(H) I2-KI (upper panel) and TTC (lower panel) staining of pollen grains from the anthers in (A). Arrows indicate sterile pollen grains that lackstaining.(I) and (J) Statistical analysis of pollen viability based on the I2-KI and TTC staining intensities in (H). The pollen staining grades areshown above the graphs. The total number of pollen grains counted for each genotype is indicated in parentheses below the graphs. Error bars:means 6 SD.(K) DAPI staining of the representative pollen grains in (H).Numbers 1, 4, 5, and 6 indicate the genotypes shown in Figure 3B.GS, green spikelet;WS,white spikelet. Bars = 1mm in (A) and (B), 20mm in (C) to (G) and(K), and 100 mm in (H).

76 The Plant Cell

Figure 6. Transverse Sections of Mutant and edRi Anthers at Various Developmental Stages.

DRUS1 and 2 Redundantly Control Reproductive Growth 77

Scanning electron microscopy revealed that the anther epithelialcells collapsed to varying degrees (Figures 5C to 5G). Moreover,the dk anther produced sterile pollen that did not stain with I2-KI,which stains starch, or triphenyl tetrazolium chloride (TTC), whichindicates the activity of mitochondrial succinodehydrogenase(Figures 5H to 5J). Furthermore, 49,6-diamidino-2-phenylindole(DAPI; which stains DNA) staining revealed no obvious nuclei inmost dk pollen grains (Figure 5K), indicating that pollen de-velopment had terminated prematurely.

The edRi RNAi line showed an intermediate phenotype, withsmall, irregular anthers and pistils (Supplemental Figures 10A and10B). The edRi anthers also produced many nonviable pollengrains that stained poorly with I2-KI or TTC (Supplemental Figures10C and 10D). Transmission electron microscopy (TEM) revealedfewer and smaller starch granules with little starch deposition anda thin intine in edRi pollen compared with many large starchgranules filled with starch and a thick intine in wild-type pollen(Supplemental Figures 10E to 10H), suggesting a defect in starch,cellulose, pectin, and callose biosynthesis.

White drus1-1+/2 drus22/2 spikelets possessed short and paleanthers, and shrunken pistils with a twisted ovary and poorly de-velopedstigmas (Figures 5Aand5B). Thepaledrus1-1+/2drus22/2

anthers produced sterile pollen that did not stain with I2-KI, TTC, orDAPI (Figures 5H to 5K). The white DRUS1+/2/dk spikelet showeda similar phenotype (Supplemental Figures 9F and 9G). Finally, themorphology of anthers and pistils in green drus1-1+/2 drus22/2

spikelets and in drus1-12/2drus2+/2, drus1-1, drus1-2, and drus2spikelets were all indistinguishable from those of the wild type(Figures 5A and 5B; Supplemental Figures 11A to 11D). Pollengrains produced by the single mutants were well stained with I2-KI,TTC, or DAPI, aswere those of thewild type (Supplemental Figures11E to 11H), while pollen grains from drus1-1+/2 drus22/2 anddrus1-12/2drus2+/2 plants werewell stained with I2-KI or DAPI, butpartially stained with TTC (Figures 5H to 5K).

Cell Death Occurs in dk and edRi Anthersthroughout Development

We examined transverse sections of anthers from dk, edRi, andwhite drus1-1+/2drus22/2 spikelets from stages 1 to 12 (Zhangand Wilson, 2009). In the wild type from stages 1 to 5, the antherprimordium divided and differentiated, forming four-loculeanthers containing sporogenous cells surrounded by the four celllayers of the anther wall (Figures 6A1 to 6A5). From stages 6 to 10,

the microspore mother cells underwent meiosis, forming haploidmicrospores. Subsequently, callose degradation led to micro-spore release and vacuolation (Figures 6A6 to 6A10). At stages11 and 12, the microspores underwent two rounds of mitosis togenerate three-nucleus pollen in the locules of the two-layered(epidermis and endothecium) wall (Figures 6A11 and 6A12).By contrast, indkplants, only a fewanthers developed normally

(Figures6B1 to6B5), andmostof theanther primordiumdegraded(Figures 6C1 to 6C5). Cell degradation continued throughoutall stages; consequently, few anthers survived to stage 12. InedRi, the anther primordia developed normally from stages 1 to5 (Supplemental Figures 12A to 12E); thereafter, approximatelytwo-thirds of the locules developed from stages 6 to 10 to gen-erate vacuolated microspores (Figures 6D6 to 6D10), whereasone-third of the locules degraded (Figures 6E6 to 6E10). Of thenormal anthers, nearly half terminated at stage 10, with degradedendothecium (Supplemental Figure 12F). Ultimately, only one-third of the anthers reached stage 12 and produced a smallamount of mature pollen (Figures 6D11 and 6D12).In white drus1-1+/2 drus22/2 spikelets, the anthers developed

normally until stage 10 (Supplemental Figure 12G). From stage11 to 12, the endothecium degraded, leaving premature pollengrains or debris enclosed by a thin, fragile epidermis (Figures 6F11and 6F12). In green drus1-1+/2 drus22/2, drus1-12/2 drus2+/2,drus1-1, and drus2 spikelets, anthers developed normally(Supplemental Figures 12H to 12K). Consistent with the pheno-typic series, cell degradation was severe in dk anthers throughoutdevelopment, moderate in edRi from stages 6 to 12, and weakin drus1-1+/2 drus22/2 from stage 11 to 12 (as summarized inFigure 6G).We further compared theultrastructuresof anther cellsbetween

dk and DJ by TEM. At stage 3, the epithelium and primary parietallayers of the locule underwent active cell division.DJanther cellshad large nuclei, dense nucleoli, and many organelles. Bycontrast, in dk, many cells in the epithelial and primary parietallayers displayed typical features of cell death: The nuclei werecondensed or digested; the nucleoli disappeared; the nuclearenvelope swelled or disintegrated; the cytoplasmwas shrunkenwith many digesting vacuoles, and no organelles were ob-served; plasmolysis occurred, resulting in a largecavitybetweenthe cell wall and plasma membrane, which was disintegrated,and a loss of cell-to-cell connection (Figures 7A to 7F). Terminaldeoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) assays further demonstrated DNA fragmentation in the

Figure 6. (continued).

(A1) to (A12) DJ anthers at stages (St) 1 to 12.(B1) to (B5) dk anthers at stages 1 to 5 showing normal morphogenesis.(C1) to (C5) dk anthers at stages 1 to 5 showing abnormal morphogenesis.(D6) to (D12) edRi anthers at stages 6 to 12 showing a weak defect.(E6) to (E10) edRi anthers at stages 6 to 10 showing a severe defect.(F11) and (F12) drus1-1+/2 drus22/2 anthers in white spikelets at stages 11 and 12 showing a severe defect.(G) Schematic representation of the proportion of impaired locules for each genotype during development.Ar, archesporial cell; BP, bicellular pollen; DBP, degraded bicellular pollen; DMC, degraded meiotic cell; DMMC, degraded microspore mother cell;DMP, degradedmature pollen; DMsp, degeneratedmicrospores; DTds, degraded tetrads; E, epidermis; En, endothecium; L1, the first cell layer in thestamenprimordia;MC,meiotic cell;ML,middle layer;MMC,microsporemother cell;MP,mature pollen;Msp,microspores; 1°P, primary parietal layer;2°P, secondary parietal layer; pMP, premature pollen; Sp, sporogenous cell; T, tapetum; Tds, tetrads. Bars = 20 mm.

78 The Plant Cell

dk nucleus. TUNEL signals were observed in dk anthers atstages 2 to 5 but were not found in DJ anthers until stage10 (Figures 7G to 7R), which is consistent with our TEM andsemi-thin-section microscopy observations. These resultsclearly indicate that DRUS1 and 2 are essential for repressingcell death.

The Cysteine Protease Inhibitor E-64 Blocks Cell Death butDoes Not Rescue Callus Growth in dk Mutants

Toelucidate thecauseof thesevere cell degradation indkanthers,weanalyzed themRNA levelsofOsCP1,whichencodesacysteineprotease that functions in tapetumdegradation (Lee et al., 2004; Liet al., 2006), in edRi anthers, which exhibit moderate phenotypes.We also measured the expression of tapetal degradation-relatedgenes: TAPETUM DEGENERATION RETARDATION (TDR),ETERNAL TAPETUM1 (EAT1), TDR INTERACTING PROTEIN2(TIP2)/bHLH142, Defective in Exine Formation 1 in rice (OsDEX1),PERSISTENTTAPETALCELL1 (PTC1),APOPTOSIS INHIBITOR5(API5), and API5-INTERACTING PROTEIN (AIPs), and GAMYB(Kaneko et al., 2004; Li et al., 2006; H. Li et al., 2011; X. Li et al.,2011; Niu et al., 2013; Fu et al., 2014; Ko et al., 2014; Yu et al.,2016). RT-qPCR analysis revealed that the total levels of DRUS1plus 2 mRNA were reduced to one-third those in the wild typefrom stages 8 to 12 (Supplemental Figure 13A), and the mRNAlevels of tapetal PCD regulatory genes were either not affectedor slightly changed at some stages, but not dramatically so(Supplemental Figures 14). By contrast, OsCP1 mRNA levelsincreased from stages 9 to 12 (Supplemental Figure 13B). In situhybridization revealed that OsCP1 mRNA levels were muchhigher in the anther walls and microspores at stages 6, 8, and10 in edRi compared with the wild type (Supplemental Figures13C to 13J). These results suggest that high levels ofOsCP1maycause anther degradation in plants lacking DRUS1 and 2, whichmay be independent of the known tapetal PCD regulatorypathway.

To test this hypothesis and topinpoint thedefects indkcells,weinduced embryonic calli from individual drus1-1+/2drus22/2 andDJ seeds on Murashige and Skoog (MS)-sucrose medium andmeasured their growth. At 5 d after induction (DAI), DJ calli werelarge, drus1-1+/2 drus22/2 calli were small, and dk calli were tiny.Their calli are 5 3 7 mm2, 3 3 6 mm2, and 1.5 3 3 mm2, re-spectively, in size (Figure 8A). DJ calli grew rapidly, but dkcalli grew slowly and remained small at 25 DAI. The growth ofdrus1-1+/2 drus22/2 calli was in between that of DJ and dk calli(Figure 8B).

We examined cell viability by double staining with fluoresceindiacetate (FDA), which indicates the activity of cytoplasmic es-terase, and propidium iodide (PI), which stains DNA when theplasma membrane is disintegrated, or the cell wall when theplasma membrane is intact. The DJ calli had light-green FDAstaining in thecytosoland redPIstaining in thecellwall (Figure8C),indicating vigorous cell division and cell membrane integrity. Bycontrast, thedk calli had large, swollen cells or a tiny cellmass andexhibited little or no FDA staining but strong nuclear PI staining(Figure 8C), suggesting that the cytoplasm degraded and cellmembrane disintegrated during cell growth or cell division. The

resulting empty cells were similar to those observed in dk anthers(Figures 6C1 to 6C5 and 7D to 7F).We next attempted to block cell death with the cysteine pro-

tease inhibitor E-64. Indk calli grownonMS-sucrosemediumwith0.5 mM E-64, cell death was largely blocked, as indicated by theincreased cytosolic FDA staining and reduced nuclear PI staining(Figures 8C and 8D), but cell growth did not recover to wild-typelevels, only slightly improving (Figure 8E). These results imply thatin addition to protease-induced cell degradation, dk cells havedefects in cell growth signaling.

Lack of DRUS1 and 2 Affects Sugar Utilizationor Conversion

Increases in callus tissue biomass depend on sugar utilizationand conversion. As expected, the DJ calli (with seeds at-tached) grew slowly on MS medium without sugar but rapidlyon MS-glucose or -sucrose medium. However, the dk calligrew slowly on the media regardless of sugar content (Figure8F). To separate callus growth from callus induction, whichoccurred more slowly in dk than in DJ, we subcultured thesame amount of dk or DJ callus (no seeds attached) onmediumwith or without sugars and calculated the time course of in-crease in biomass under each treatment. Again, we observeda sharp increase in growth in DJ calli but little increase ingrowth indk calli at 25 d after subculturing on sugar-containingmediumcomparedwith non-sugar-containingmedium (Figure8G). These data suggest that dk cells cannot use sucrose orglucose as a building block, even after blocking cell death(Figure 8E).To determine which molecular pathway was affected, we

examined the expression of several anther development-related genes, i.e., CARBON STARVED ANTHER (CSA),UNDEVELOPED TAPETUM1 (UDT1), OsMADS3, MSP1,DEFECTIVE POLLEN WALL1 (DPW1), DPW2, and CYP704B2(Nonomura et al., 2003; Jung et al., 2005; Li et al., 2010; Zhanget al., 2010; Hu et al., 2011; Shi et al., 2011; Xu et al., 2016), in edRianthers. The expression of CSA, UDT1, DPW1, DPW2, andCYP704B2 was dramatically altered in edRi relative to the wildtype at some developmental stages (Supplemental Figure 15).CSA regulates sugar partitioning during male reproduction(Zhang et al., 2010) and seed development (Zhu et al., 2015),and its expression pattern in anthers is similar to that of DRUS1and 2. We therefore examined the expression of CSA andsugar metabolism-associated genes in anthers of greendrus1-1+/2 drus22/2 spikelets, which appeared similar to thoseof DJ (Figure 5A) but possessed lower DRUS1 protein levels(Figure 3I; Supplemental Figure 7H). The expression of CSA andits target gene MONOSACCHARIDE TRANSPORTER8, su-crose cleavage gene INV4, starch synthase gene GBSS1, andUDP-glucose synthesis gene UGP2, but not UGP1, was sig-nificantly altered in these anthers at later stages comparedwithDJ (Supplemental Figure 16). Upon further examinationofwhitedrus1-1+/2 drus22/2 spikelets, which were poorly developedlike those of dk (Figures 3D), we found that UGP2, which ismainly expressed in anthers but also somewhat expressedin other floral organs at stages 11 and 12 (Mu et al., 2009),was totally suppressed, while GBSS1 was less affected

DRUS1 and 2 Redundantly Control Reproductive Growth 79

(Figures 9A and 9B). MST8 expression was maintained at highlevels until stage 12, whereas it was dramatically reduced in DJ(Figure 9C).

Since the expression ofCSA and its target genes was affected,we performed a 14C-sucrose feeding assay as described (Zhanget al., 2010) to detect sugar partitioning in stems and floral organsin the weak allele drus1-1+/2 drus22/2, which shows the mutantphenotype after stage 10. The levels of 14C signals in palea andlemma tissue, anthers, and segments from internodes II (S2) and I(S3), except for segment from internode III (S1), of drus1-1+/2

drus22/2 at stages 11 and 12, was not significantly different fromthatofDJ (Supplemental Figure17). These results suggest that thedefective development of dk spikelets is not caused by failedsugar transport, but is instead caused by disrupted sugar utili-zation or conversion. We detected dramatically reduced ex-pression of UGP2, but not UGP1, even in young inflorescences(anther stage 1) (Figures 9D and 9E), indicating that DRUS1 and2 are key factors that modulate glucose metabolism-associatedgenes in reproductive tissues.

DISCUSSION

DRUS1 and 2 Are Typical RLKs and Orthologs ofArabidopsis FER but Have Distinct Biological Functions inRice Reproduction

In rice, the number of RLK genes is nearly double that inArabidopsis due to gene duplication; these genes are es-sential for regulating growth and development and for de-fense responses to various pathogens (Shiu and Bleecker,2003; Shiu et al., 2004). DRUS1 and 2 appear to be duplicatedgenes based on their similar gene structures and proteinsequences, and they are closely related to FER (Hématyand Höfte, 2008) (Supplemental Figure 3). During sexual repro-duction, FER controls pollen tube perception in synergid cells(Escobar-Restrepo et al., 2007). Here, we demonstrated thatDRUS1 and 2 are cell surface-localized RLKs with kinase activity(Figures 2B and 2E; Supplemental Figure 2) and that DRUS1can fully rescue the fer-4 phenotype (Supplemental Figures

Figure 7. Cell Death in dk Anthers.

(A) to (F)Transmission electronmicrographsofDJ ([A] to [C]) anddk ([D] to [F]) anthers at stage 3. (B)and (C)are highermagnificationsof the redandyellowsquares in (A), respectively. (E)and (F)arehighermagnificationsof the redandyellowsquares in (D), respectively.CW,cellwall; E, epidermis;N,nucleus;NE,nuclear envelope; No, nucleolus; PM, plasma membrane; V, vacuole; 1°P, primary parietal layer.(G) to (R)TUNELassay.Wild-type ([G] to [K]) anddk ([L] to [P]) antherswerecomparedat the indicatedstages. In (Q)and (R), aDJanther treatedwithDNaseIwas used as the positive control. The arrows indicate the TUNEL signal in (L) and (M).Bars = 10 mm in (A) and (D), 2 mm in (B), (C), (E), and (F), and 20 mm in (G) to (R).

80 The Plant Cell

Figure 8. Callus Growth on Different Conditional Media.

(A) The embryonic callus (yellow arrows) produced from seeds of the indicated genotypes at 5 and 25 DAI in MS-sucrose medium. Bars = 1 mm.(B)Thegrowth curvesof the calli in (A). An individual callus togetherwith thedegrading seedwasweighedandweightswereplotted to show the timecourseof the increase in biomass using a colored line.(C) FDA andPI double staining of DJ anddk calli subcultured inMS-sucrose liquidmediumwith orwithout a cysteine protease inhibitor E-64. Bars = 20mm.(D) The percentage of viable cells in DJ and dk calli in (C) based on the cell wall PI staining and cytoplasm FDA staining. The total number of cells counted isindicated in parentheses below the graph. Error bars indicate the means 6 SD.(E)ThegrowthofDJanddk calli onMS-sucrosemediumwithorwithoutE-64. Thepercentageoffivecallus gradesat 10DAI (on the top)wascalculated. Thetotal number of seeds used for the analysis is shown in parentheses below the graph. Error bars indicate the means 6 SD. Bar = 1 mm.(F) and (G)Comparison of DJ and dk callus growth onMSmediumwith or without sugar. In (F), callus was weighed with seeds attached; in (G), callus wassubcultured and weighed without seeds attached. Error bars indicate the means 6 SD; n = 10 to 33 in (F) and 3 to 6 in (G).

4F and 5A and Supplemental Table 1), indicating that the mo-lecular function of DRUS1 is the same as that of FER.

Our data indicate that the biological functions of DRUS1 andFER likely diverged when they developed different tissue ex-pression patterns in rice and Arabidopsis. In the ovule, FER ismainly expressed in synergid cells, and the female gametophytesof fer-1 are sterile, with only 14.5% transmission efficiency(Huck et al., 2003). DRUS1 and 2 are highly and ubiquitouslyexpressed in the ovule (Supplemental Figures 1J to 1N). The fe-male gametophytes of dk are nearly normal in well-developedpistils, with 85 to 100% transmission efficiency (SupplementalTable 4). DRUS1 and 2 may also function in synergid cells.However, unlike that in fer-1, female fertility in dk plants is de-termined by the sporophyte, not the gametophyte. On the otherhand, FER is expressed in early anthers, but not in mature pollen(Escobar-Restrepo et al., 2007). The reported transmission effi-ciency of fer-1 pollen is 78.5% (Huck et al., 2003), but partialsterility has not been described. Mutant dk pollen shows 100%transmission efficiency (Supplemental Table 4), and its fertility isalso sporophyte dependent. In addition, DRUS1 and 2 play

essential roles in inflorescence, spikelet, and anther developmentand pollen maturation.

DRUS1 and 2 Are Crucial Factors in Reproductive Growthand Development and Mediate a Gibberellin-IndependentSignaling Pathway in Rice

Meristem transition, floral organ initiation and subsequent out-growth, and elongation at each internode are characterized byrapid cell proliferation, differentiation, and expansion (Itoh et al.,2005). These processes rely on a sharp increase in sugar andenergy supply and are highly sensitive to environmental con-ditions, including light, temperature, andwater (Muller et al., 2011;Kakumanu et al., 2012; Yuan et al., 2012; Lu et al., 2014). Theevolution of two RLK genes, DRUS1 and 2, with overlappingexpression patterns in the meristems of inflorescences and floralorgans (Figures 1B to1Q;Supplemental Figures1J to1N) and littlefunctional divergence,may have helped ensure inflorescence andspikelet development while avoiding signaling paralysis. Indeed,we found thata threshold level of totalDRUS1/2protein is requiredto sustain the initiation and differentiation of a sufficient number ofprimary branches and spikelets. The dosage effect of DRUS1 and(probably) 2 on spikelet development suggests that their down-streamsignaling eventsmaybeassociatedwith nutrient or energy

Figure 9. Altered Expression of Sugar Metabolism-Associated Genes inDRUS1- and DRUS2-Deficient Mutants.

(A) to (C) Starch synthesis-related gene expression in spikelets of DJ anddrus1-1+/2 drus22/2 plants at stages 10 to 12 as determined by qRT-PCR.GS, green spikelet; WS, white spikelet.(D) and (E) UGP1 (D) and UGP2 (E) expression in the inflorescence ofDJ and dk at the early inflorescence stage, when the anther primordiumappeared.OsUBE2wasusedasan internal control. For (A) to (C), themRNA level inDJspikelets at stage 10 was set to 1; for (D) and (E), the mRNA level in DJinflorescences was set to 1. NA, not available. Error bars indicate themeans 6 SD of three biological replicates. Asterisks indicate a significantdifference by Student’s t test; ***P < 0.001.

Figure 10. Model for the Role of DRUS1 and 2 in Viability in the Endo-thecium and Starch Accumulation in Pollen at Anther Stages 11 and 12.

(A) In green spikelet anthers, the endothecium, which expresses DRUS1or 2, may receive an unknown signal after complete degradation of thetapetum to trigger cell growth and cell wall thickening. The viable endo-thecium cell in turn sends an effector to stimulate UGP2 expression inpollen through an unknown pathway, leading to a sharp increase in UGP2,which is crucial for starch synthesis.(B) In white spikelet anthers, which do not express DRUS1 and 2, theendothecium is unable to receive the survival signal; thus, it degrades tooearly to stimulate UGP2 expression in pollen, resulting in failed starchsynthesis. This starch deficiency in turn causes upregulation of themonosaccharide transporterMST8 to compensate for the sugar shortage.

82 The Plant Cell

supply because mutant plants showed reductions in spikeletnumber and floral organ size, but no alterations in floral organidentity (Figures 3C to 3H, 5A, and 5B).

The plant hormone gibberellin (GA) promotes stem elongationand inflorescence and spikelet development in rice (Peng et al.,1999; Ueguchi-Tanaka et al., 2007; Yamaguchi, 2008). Never-theless, DRUS1- andDRUS2-mediated growth signaling appearsto occur via a different pathway from that of GA. First, their proteinlevels were not affected by GA treatment or in GA-deficient andinsensitive mutants d18, sd1, and gid1, which display a dwarfstature (Sakamoto et al., 2004; Ueguchi-Tanaka et al., 2005)(Supplemental Figures 18Aand18B), indicating thatDRUS1and2are not regulated at the protein level by GA. Furthermore, GApromoted leaf sheath elongation, and GA-responsive gene ex-pression is not affected in dk (Supplemental Figures 18C to 18E),suggesting thatGAsignaling is not damaged in thedkmutant. Thefinding that GA mutants and the dk mutant investigated in thisstudy showed different organ phenotypes when their leaves,spikelets, and floral organs were closely compared also supportsthis point.

DRUS1 and 2 Mediate Survival Signaling and RedundantlyControl Anther Development and Pollen Maturation

Anther and pollen development are complicated and controlledby a conserved pathway (Gómez et al., 2015). Correct cellfate determination is a crucial part of this process, in which LRR-RLK-mediated cell-to-cell communication plays key roles. InArabidopsis, more than 1100 small peptide coding genes forcysteine-rich peptides and non-cysteine-rich peptides have beenidentified (Huanget al., 2015), providing potential ligands forRLKsthat regulate plant reproduction. In rice, the LRR-RLK MSP1 in-teracts with the peptide signal MIL2, playing an important role inmicrospore and tapetum differentiation during early development(Nonomura et al., 2003;Hong et al., 2012; Yang et al., 2016). In thisstudy, we found that DRUS1 and 2 mediate survival signaling tomaintain cell viability and repress cell death in all cell typesthroughout anther development (Figures 6 and 7) by controllingthe expression or activation of protease and regulating sugarutilization. Indeed, despite the normal sugar partitioning in anthersand spikelets (Supplemental Figures 17A and 17B), starch de-position and intine formation were dramatically reduced in edRipollen grains (Supplemental Figures 10E to 10H), indicating im-paired polysaccharide biosynthesis.

Two amylogenic processes occur in the microspore after itsrelease from the tetrad. Toward the end of stage 10, transientlysynthesized starch is used to generate osmotic potential, leadingto an increase in vacuole size in the uninucleate microspore,whereas starch synthesized in binucleate pollen during stage11 ismainly used for storage (Pacini andViegi, 1995;Pacini et al., 2006).A temporary increase in the expression of sucrose-to-starchconversion-associated genes inmaize (Datta et al., 2002) and riceanthers (Supplemental Figure 16) during pollen maturation allowsthe plant to meet its demands for starch synthesis. Among thesegenes, the expression of UGP2 sharply increases in binucleateand mature pollen, which is essential for starch accumulation andpollen fertility (Mu et al., 2009). However, UGP2mRNA levels werenotelevated indk-likeanthers (anthersofwhitedrus1-1+/2drus22/2

spikelets) (Figure 9A). These findings, combined with the poor I2-KIstaining in white edRi and drus1-1+/2 drus22/2 spikelets and in dkpollen (Supplemental Figures 10C and 10D; Figures 5H and 5I),suggest thatUGP2,butnotUGP1orGBSS1, is thekeydownstreamgene regulated by DRUS1 and 2.DRUS1 is mainly expressed in the endothecium of the anther

wall, with little expression observed in pollen during anther stages11 and 12 (Supplemental Figures 19E1, 19E2, 19F1, and 19F2)(Tang et al., 2010), when the tapetum is completely degraded.Given this, how does DRUS1 affect the expression of UGP2 inpollen? Gametophyte development depends on its coordinationwith the sporophyte. During late anther development, cell-to-cellcommunicationbetween theendotheciumanddevelopingpollen isimportant for establishing male fertility. Based on our results, wepropose that upon receiving an as yet unknown signal in the locule,cell surface-localized DRUS1 or 2 maintain the viability of endo-theciumcells,which in turn releaseanunknowneffector toenhanceUGP2 expression and stimulate starch synthesis in pollen (Figure10A). When there is little or no DRUS1 and 2 expression (as inwhite drus1-1+/2 drus22/2 spikelets, edRi severe spikelets, or dkanthers), the endothecium fails to sense the necessary signal andis thus degraded too early (Figures 6F11 and 6F12; SupplementalFigure 12F) to enhance UGP2 expression (Figure 9A), resulting infailedstarch-filling inpollenandsubsequentupregulationofMST8to compensate for the sugar shortage (Figure 10B). By contrast,FERwas recently found to limit starch accumulation by interactingwith cytoplasmic glyceraldehyde-3-phosphate dehydrogenase,a key enzyme in glycolysis (Yang et al., 2015).Considering these findings, we propose that DRUS1 and

2 mediate a fundamental pathway that ensures cell survival,growth, and division, similar to tyrosine receptor kinases, whichsensesurvival factors andgrowth factors in animal cells (Ritter andArteaga, 2003; Kumar et al., 2006; Camarena et al., 2010), butprobably different from other CrRLK1Ls, which regulate cell ex-pansion (Liu et al., 2016; Nissen et al., 2016). The ligands forDRUS1 and 2 that function in cell-to-cell communication duringreproductive growth remain to be elucidated.

METHODS

Prediction of RLK Domain Organization

The full-length amino acid sequences of RLK were obtained, and thepresence of signal peptides at their N termini were predicted using SignalP3.0 (http://www.cbs.dtu.dk/services/SignalP-3.0/). The transmembraneregions were predicted using TMpred (http://www.ch.embnet.org/software/TMPRED_form.html) and HMTMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/), and the kinase domains at the cytoplasmic regions werepredicted using PlantsP (http://plantsp.genomics.purdue.edu/) and ProteinBLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Plant Materials and Growth Conditions

Transgenic and wild-type rice (Oryza sativa japonica variety Nipponbare)plants were grown in a paddy field under natural conditions. Rice T-DNAinsertion mutants were obtained from POSTECH RISD (http://signal.salk.edu/cgi-bin/RiceGE). Arabidopsis thaliana plants were grown in a growthroom at 22°C under a 16-h light (80 mmol m22 s21 white light)/8-h darkcycle. The T-DNA insertion mutant fer-4 (GK-106A06; Col-0 background)

DRUS1 and 2 Redundantly Control Reproductive Growth 83

was obtained from the Nottingham Arabidopsis Stock Centre. Transgenicfer-4 plants carrying ProFER:DRUS1-FER39 or ProFER:m1DRUS1-FER39were screened on plates containing 7.5 mg/L sulfadiazine and 25 mg/Lhygromycin and genotyped.

Vector Construction and Plant Transformation

To produce the DRUS1-edRi constructs, 638-bp edRi (nucleotides 131–768)(Supplemental Figure 8A) fragments were amplified from genomic DNAusing the primer pair edRi-f/edRi-r and cloned into pTCK303 between theSpeI andSacI sites in the forward direction and theKpnI andBamHI sites inthe reverse direction (Wang et al., 2004). To produce the ProDRUS1:GUS andProDRUS2:GUS reporter constructs, 2338- and 2280-bp fragments up-streamof theDRUS1 andDRUS2 start codon, respectively, were amplifiedfrom genomic DNA using the primer pairs pDRUS1-f/pDRUS1-r andpDRUS2-f/pDRUS2-r and cloned into pCAMBIA1391Z using BamHI andEcoRI. To create the Pro35S:DRUS1DK-GFP and Pro35S:DRUS2DK-GFPexpression vectors, the nucleotide sequences encoding amino acids1 to 501 of DRUS1 and 1 to 504 of DRUS2 were amplified using primerpairs DRUS1-f/DRUS1-r2 and DRUS2-f/DRUS2-r2, followed bycloning into pCAMBIA1300-YFP using XbaI and BamHI, respectively.To create the ProFER:DRUS1-FER39 and ProFER:m1DRUS1-FER39constructs, a 1832-bp FER promoter fragment and a 758-bp FER39-UTR fragment with the stop codon were amplified using theprimer pair pFER-f/pFER-r and FER-39f/FER-39r, respectively, andcloned into pCAMBIA1300 to generate pCAMBIA1300-ProFER-FER39. Finally, the 2679-bp full-length coding sequence of DRUS1 orm1DRUS1 without the stop codon was amplified using the primer pairDRUS1-f/DRUS1-r and cloned into pCAMBIA1300-ProFER-FER39between the XbaI and BamHI sites. To produce ProDRUS1:DRUS1-7Myc-6His, a 1700-bp DRUS1 promoter fragment plus the DRUS1coding sequence was amplified with pDRUS1-f2/DRUS1-r and in-serted into pCAMBIA1300-7Myc-6His. All of the constructs were se-quencedbefore being introduced into rice or Arabidopsis viaAgrobacteriumtumefaciens EHA105- or GV3101-mediated transformation (Clough andBent, 1998; Yang et al., 2004). The sequences of the primers are listed inSupplemental Table 6.

Genotyping of the T-DNA Insertion Mutants

The T-DNA insertions in rice drus1-1 and drus1-2 were identified byPCRusing the right border primer Ngus-r and gene-specific primer R2.The T-DNA insertion in drus2was identified using the left border primer2715-LB and the gene-specific primer R5. Partial transcripts ofDRUS1 and DRUS2 were detected using the primers shown in Figure3A and Supplemental Figure 7C. The T-DNA insertion in Arabidopsisfer-4 was identified using the left border primer T-DNAr and gene-specific primer 550-f. The sequences of the primers are given inSupplemental Table 6.

RNA Isolation and RT-PCR and RT-qPCR Analyses

Total RNA was isolated from different rice tissues using Trizol reagent(Invitrogen) according to the manufacturer’s instructions. The expressionpattern of DRUS1 and 2, the mRNA levels of DRUS1, DRUS2, andOs04g49690 in the edRi plants, and full-length and partial transcripts ofDRUS1and2 indrus1-1anddrus2weredetectedbyRT-PCRusinganRNAPCR (AMV) kit version 3.0 (Takara Bio). The mRNA levels of DRUS1,DRUS2, and anther-related genes in the anthers or spikelets of wild-type,edRi, anddrus1-1+/2drus22/2plantsand in the inflorescencesofDJanddkwere detected by RT-qPCR using a PrimeScript RT reagent kit with gDNAEraser (Takara Bio). OsActin1 (Os03g50890), OsUBQ (Os03g13170), andOsUBE2 (Os01g28480) were used as internal controls in rice; AtActin7(At5g09810) was used as an internal control in Arabidopsis.

Antibody Preparation and Immunoblot Analysis

To examine the protein levels of DRUS1 and DRUS2 in wild-type andmutant plants, polyclonal anti-DRUS1 antibodies were raised in rabbit,respectively, against the DRUS1-specific extracellular domain (aminoacids 171–327) and DRUS1 C-terminal region (amino acids 690–893),which shares 96% identity with that of DRUS2. The coding sequences ofthe extracellular domain and C-terminal region ofDRUS1were cloned intothepET28bbacterial expressionvector usingEcoRIandSacI, respectively.The resulting fusion proteins, 6His-DRUS1ED and 6His-DRUS1CT, werepurified in His-select nickel affinity gels (Sigma-Aldrich) according to themanufacturer’s instructions and used to immunize rabbits. The anti-DRUS1ED and anti-DRUS1CT antibodies were purified from serumusing GST-DRUS1ED and GST-DRUS1CT antigen-affinity purification,respectively.

For immunoblot analysis, total proteins extracted from Arabidopsis orrice tissues with 23 SDS protein sample buffer were separated by 6% or7.5%SDS-PAGE. Blots carrying the transferred proteinswere probedwithanti-DRUS1ED or anti-DRUS1CT antibodies for endogenous protein orwith monoclonal anti-cMyc antibodies (M4439; Sigma-Aldrich) for exog-enous protein, then with IRDye 800CW goat anti-rabbit IgG (925-32211;LI-CORBiosciences) or anti-mouse IgG (925-32210;LI-CORBiosciences),and developed using an Odyssey infrared imaging system (LI-CORBiosciences). Microsomal proteins were collected from the centrifugedpellet at 100,000g.

In Situ Hybridization

Spikelets with the upper half cut off were fixed in FAA for 4 h, followed byfresh solution overnight at 4°C. After dehydration in a graded ethanolseries, the tissues were embedded in Paraplast (Sigma-Aldrich). Anthersections (8-mm thick) prepared using a microtome (Leica) were usedfor in situ hybridization. A DRUS1 probe (from 2205 to +10), DRUS2probe (from 2224 to +32), and OsCP1 probe (encompassing 103 bpbefore and 134 bp after the stop codon) were amplified using primerpairs DRUS1insitu-f/DRUS1insitu-r, DRUS2insitu-f/DRUS2insitu-r, andOsCP1insitu-f/OsCP1insitu-r, respectively and cloned into pGEM-T Easy(Promega). The resulting constructs were identified by PCR and se-quencing to determine forward or reverse insertion. The T7 promoter plusinsert was amplified and the PCR fragment was used to transcribe senseand antisense probes using a DIG-labeled RNA labeling kit (Roche). RNAhybridization and immunological detection were performed as describedpreviously (Kouchi and Hata, 1993). The hybridization signals were ob-served by microscopy (Zeiss Imager M2).

Determining the Subcellular Localizations of DRUS1 and DRUS2

ThePro35S:DRUS1DK-GFPandPro35S:DRUS2DK-GFPconstructswereintroduced into tobacco (Nicotiana benthamiana) leaves through Agro-bacteriumGV3101-mediated infiltration as described previously (Gampalaet al., 2007). For bombardment, 15mgplasmids ofPro35S:DRUS1DK-GFPwas coatedwith gold bymixingwith 90mL solution containing60mgmL21

AuCl2, 2.5 M CaCl2, and 0.1 M spermidine. The gold particles werebombarded into onion epidermal cells with a helium burst at 1800 p.s.i. inaPDS-1000/He instrument (Bio-Rad).Transient expressionof theDRUS1DK-GFP and DRUS2DK-GFP fusion proteins in tobacco leaf or onionepidermal cells wasmonitored by confocal laser scanningmicroscopy(Zeiss META 510).

In Vitro Kinase Assays

The full-length intracellular regions of the DRUS1 (encoding amino acids476–893) and DRUS2 (amino acids 479–896) coding sequences were am-plifiedusingprimerpairsK-DRUS1-f/K-DRUS1-randK-DRUS2-f/K-DRUS2-r

84 The Plant Cell

andclonedintopGEX-4T-1usingEcoRIandSalI, respectively.Pointmutations(m1DRUS1K [K569E] and m2DRUS1K [K667E]) in the construct DRUS1K-pGEX-4T-1 were created using the Fast Mutagenesis System (TransgenBiotech). GST fusions of the wild-type kinases (DRUS1K and DRUS2K) andtwo mutant kinases were expressed in Escherichia coli and purified withglutathione Sepharose 4B beads (GE Healthcare). An in vitro kinaseassay was performed as described previously with some modifications(Escobar-Restrepo et al., 2007). GST or a GST-fusion protein (1 mg) wasincubated with 5 mg of MyBP (myelin basic protein) in kinase buffer(50mMHEPES-KOH,pH7.5,150mMNaCl, 0.1mMEGTA,10mMMgCl2,10% glycerol, 1 mM DTT, 0.1 mM ATP, and 37 MBq of [g-32P]ATP) for30 min at 25°C and heated at 100°C for 5 min in 43 SDS sample buffer.The denatured samples were subjected to SDS-PAGE, stained withCoomassie Brilliant Blue, and autoradiographed with a phosphor imager(Typhoon 9400; GE Healthcare).

Pollen Viability Assays

Six anthers before dehiscence were crushed in 150 mL of 1% (w/v) I2-KI or0.6% (w/v) TTC in 50 mM potassium phosphate buffer (pH 7.4) andincubated for 15 to 20 min at room temperature for I2-KI staining or at37°C in the dark for TTC staining. After washing with the same buffer,the pollen grains were observed by microscopy (Zeiss Imager M2), andthe viability grades were determined based on the extent of staining.DAPI staining for trinucleate pollen was performed as described (Hanet al., 2006).

Histological Analysis

GUS staining was performed as described (Pu et al., 2012). Samplesfor semithin sections were embedded in Spurr’s resin (Structure Probe),and ;1.5-mm-thick microsections were stained with 0.25% toluidineblue-O (Sigma-Aldrich) before microscopy imaging (Zeiss Imager M2).

Scanning Electron Microscopy and TransmissionElectron Microscopy

Scanning electron microscopy was performed as previously described(Pu et al., 2012).

For transmission electronmicroscopy, anthers at stages 3 and 12 werefixed in 2% paraformaldehyde and 2% glutaraldehyde in PBS (pH 7.4) at4°C overnight, followed by postfixing in 1% osmium tetroxide for 4 h. Afterdehydration in an ethanol series, the samples were embedded in Spurr’sresin (StructureProbe), andultrathin sections (;70nm)wereproducedandstainedwith2%(w/v) saturateduranyl acetate in70%ethanol and2%(w/v)lead citrate before observation under a transmission electron microscope(Hitachi H-7650).

TUNEL and Immunofluorescence Assays

Anthers with half of a lemma attached at different stages of developmentwere fixed in 4% paraformaldehyde in PBS, dehydrated in an ethanolseries, and embedded in Paraplast (Sigma-Aldrich). The anthers weresectioned every 4mm (stages 8–11) or 10mm (stage 12) using amicrotome(Leica). After stripping the paraffin with xylene, the sections for the TUNELassay were treated and apoptosis signals were detected with a TUNEL kit(DeadEnd Fluorometric TUNEL system; Promega) according to themanufacturer’s instructions. The sections for the immunofluorescenceassay were blocked with blocking buffer (3% BSA in PBS) and incubatedwith anti-DRUS1EDantibodies, followedbyFITC-labeled goat IgG (F6258;Sigma-Aldrich). Antibody-depleted serumwas used as a negative control.The samples were stained with 1% toluidine blue-O (Sigma-Aldrich) toblock spontaneous fluorescence before observation. All sections werecovered with 50% glycerol for confocal imaging (Zeiss META 510).

Sugar and E-64 Treatment, and FDA and PI Staining of Calli

Sterilized DJ and drus1-1+/2 drus22/2 seeds were placed on MS,MS-sucrose, orMS-glucosemediumcontaining 2mg/Lof 2,4-D, 0.1 g/L ofinositol, and0.3g/L of casein hydrolysate, 2.8g/L L-proline, pH5.8,withoutor with 30 g/L of sucrose or glucose, and incubated in darkness at 30°C.Embryonic drus1-1 drus2 dk and drus1-1+/2 drus22/2 calli were identifiedby genotyping 5-d-old coleoptiles, and their biomass was weighed on theindicated days. For subculture, calli at 10 DAI were divided into three equalparts, each part was placed onto the above-mentioned media, and thegrowing calli were weighed on the indicated days.

For the protease inhibitor experiment, 0.5 mM E-64 (Sigma-Aldrich) ormocksolution (water)wasadded toMS-sucrosemedium.Thegrowingcalliwere graded and the number of calli in each grade was counted.

For FDA and PI staining, a suspension cell culture was produced bytransferring 12-d-old calli to MS-sucrose liquid medium and shaking theculture at 140 rpm for 7 d in darkness at 30°C. The cells were double-stained with FDA (0.1 mg/mL in acetone) and PI (0.02 mg/mL in PBS) for30 min in darkness and visualized by confocal laser scanning microscopy(Zeiss META 510). The viable callus cells exhibiting FDA staining in thecytoplasm and PI staining in the cell wall under different growth conditionswere counted.

Radiolabeling

The [14C]sucrose feeding assay was performed according to a previouslypublished protocol (Zhang et al., 2010) with some modifications. The DJand drus1-1+/2 drus22/2 stems were each incubated in 2 mL watercontaining2mCi [14C]sucrose (9.14–25.9GBqmmol-1 in9:1ethanol:water;Perkin-Elmer), and their panicles were incubated in 2 mL water containing6 mCi [14C]sucrose for 12 h at room temperature. The radioactivity (cpm)was measured with a liquid scintillation counter (Wallac Trilux,1450 Microbeta).

GA Treatment

DJ and drus1-1+/2 drus22/2 seeds were germinated in water with orwithout 1026 M uniconazol for 3 d at 28°C in darkness and transferred toHoagland medium with or without uniconazol for two additional weeks.Uniconazol-treatedDJanddkseedlingswere treatedwith2mL1023MGA3

or mock solution, which was dropped onto the adaxial surface of thesecond sheath. The length of the last leaf sheath was measured 1 weeklater. The expression of GA-responsive genes was also analyzed byRT-qPCR after 2 h of GA3 treatment.

Accession Numbers

Sequencedata in this article canbe found in theGenBank/EMBL/Gramenedatabase and at http://cdna01.dna.affrc.go.jp/cDNA under the followingaccession numbers: DRUS1, LOC_Os03g21540 and AK099791; DRUS2,LOC_Os01g56330 and AK101775; Os04g49690 (AK106447); FER(At3g51550); OsCP1 (Os04g0670500); TDR (LOC_Os02g02820); EAT1(Os04g0599300); TIP2 (Os01g0293100);OsDEX1 (Os03g0825700); PTC1(Os09g0449000); OsAPI5 (Os02g0313400); OsAIP1 (Os01g0549700);OsAIP2 (Os01g0550000); GAMYB (Os01g0812000); CSA (Os01g0274800);UDT1 (Os07g0549600);OsMADS3 (Os01g0201700);MSP1 (Os01g0917500);DPW1 (Os03g0167600);DPW2 (LOC_Os01g70025);CYP704B2 (Os03g0168600);MST8 (Os01g0567500); INV4 (Os04g0413200); UGP2 (Os02g0117700); UGP1(Os09g0553200); and GBSS1 (Os06g0133000).

Supplemental Data

Supplemental Figure 1. Expression patterns of DRUS1 and DRUS2in rice.

DRUS1 and 2 Redundantly Control Reproductive Growth 85

Supplemental Figure 2. Transient expression of DRUS1DK-GFP inonion epidermal cells.

Supplemental Figure 3. Protein sequence analysis of the CrRLK1Lsubfamily.

Supplemental Figure 4. Molecular characterization of Arabidopsisfer-4 complementation plants with DRUS1 and m1DRUS1.

Supplemental Figure 5. DRUS1 rescues fer-4 fertility better than didm1DRUS1.

Supplemental Figure 6. Phenotypic analysis of drus1-2 mutants froma BC2F2 population.

Supplemental Figure 7. Molecular characterization and phenotypeanalysis of six genotypes.

Supplemental Figure 8. Phenotype of edRi plants at the reproductivestage.

Supplemental Figure 9. Molecular characterization and floral organphenotypes of DRUS1 complementation plants.

Supplemental Figure 10. Defects in floral organs and pollen grains inedRi plants.

Supplemental Figure 11. Floral organs and pollen viability in thedrus1-1 and drus2 single mutants.

Supplemental Figure 12. Transverse sections of edRi, drus1-1, drus2,drus1-1+/2drus22/2, and drus1-12/2drus2+/2anthers at the indicated stages.

Supplemental Figure 13. OsCP1 was upregulated in edRi anthers.

Supplemental Figure 14. The expression of tapetal PCD-relatedgenes in edRi anthers, as determined by RT-qPCR.

Supplemental Figure 15. The expression of anther developmentalgenes in edRi anthers, as determined by RT-qPCR.

Supplemental Figure 16. The altered expression of sugar metabo-lism-associated genes in drus1-1+/2 drus22/2 anthers, as determinedby RT-qPCR.

Supplemental Figure 17. 14C-signal accumulation in the flowers and stem.

Supplemental Figure 18. The relationship between the DRUS1/2 andGA signaling pathway.

Supplemental Figure 19. Immunofluorescence assay of DRUS1protein expression in wild-type anthers at the indicated stages.

Supplemental Table 1. fer-4 fertility is rescued by ProFER:DRUS1-FER39.

Supplemental Table 2. fer-4 fertility is rescued by ProFER:m1DRUS1-FER39.

Supplemental Table 3. Segregation of drus1-12/2 drus2+/2 anddrus1-1+/2drus22/2 selfed progeny.

Supplemental Table 4. Transmission efficiency of the drus1-1 drus2male and female gametophyte.

Supplemental Table 5. Complete and partial complementation ofdrus1-1 drus2 with ProDRUS1:DRUS1-7Myc-6His.

Supplemental Table 6. Primers used in this study.

Supplemental Data Set 1. Alignment used to generate the phylogenyshown in Supplemental Figure 3A.

ACKNOWLEDGMENTS

We thank Dabing Zhang and the members of the Zhang lab at Shang HaiJiao Tong University for providing technical assistance with the in situhybridization assay; Yuhong Hu and Jibiao Li at the Instrumental Analysis

Center inHebei Normal University for helpwith TEMand scanning electronmicroscopy, respectively; and Dejiu Zhang at the Institute of Biophysics ofChinese Academic Science (CAS) for help with the sucrose feeding assay.We also thank Gynheung An at the POSTECH Biotech Center, Republic ofKorea, for providing the rice mutants drus1-1, drus1-2, and drus2; theNottingham Arabidopsis Stock Centre for providing the Arabidopsis mu-tant fer-4; and Xiangdong Fu at the Institute of Genetics and Developmentat CAS for the gibberellin mutants. This work was supported financially bythe National Key Program on the Development of Basic Research inChina (2013CB126900 and 2014CB943404), the National Science Foun-dation of China (31500238), China Postdoctoral Science Foundation(2012M520594), the National Program of High Technology DevelopmentofChina (2002AA2Z1001-10), and theOutstandingResearcherProgramofHebei in China (SPRC046), Hebei Province Department of Education(ZD2010130).

AUTHOR CONTRIBUTIONS

Y.S. and D.-Y.S. conceived the project. Y.S. and C.-X.P. designed theexperiments. C.-X.P., Y.-F.H., and S.Z. carried out most of the experi-ments. F.-Y.S. and C.-X.P. performed the anther sections and antherscanning electron microscopy and contributed to Figures 5 and 6 andSupplemental Figures 4C, 4F, and 5A. Y.Z. contributed to Figures 4A to 4Eand Supplemental Figure 9 with Y.-F.H. and performed in situ hybridizationtogether with C.-X.P. C.-Y.W. performed GA treatment assay. Y.-C.Z. did thecrossbetweendrus1-1anddrus2 togetherwithF.-Y.S.andY.Z.Q.Y.andL.-J.S.generated the edRi transgenic plants. S.-L.B. constructed the vectorProFER:DRUS1-FER39. S.-W.Z. assistedwith theexperiment designanddataanalysis. J.W.assistedwith thecollectionof anther.S.-Q.Z. selectedthe RLK genes frommicroarray data. Y.S. andC.-X.P. organized the dataand wrote the paper. D.-Y.S. supervised and completed writing thearticle.

Received March 21, 2016; revised December 19, 2016; accepted January7, 2017; published January 12, 2017.

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DRUS1 and 2 Redundantly Control Reproductive Growth 89

DOI 10.1105/tpc.16.00218; originally published online January 12, 2017; 2017;29;70-89Plant Cell

Ying SunQian Yang, Jiao Wang, Shuo-Lei Bu, Li-Jing Sun, Sheng-Wei Zhang, Su-Qiao Zhang, Da-Ye Sun and

Cui-Xia Pu, Yong-Feng Han, Shu Zhu, Feng-Yan Song, Ying Zhao, Chun-Yan Wang, Yong-Cun Zhang,and Affect Sugar Utilization during Reproductive Development

The Rice Receptor-Like Kinases DWARF AND RUNTISH SPIKELET1 and 2 Repress Cell Death

 This information is current as of November 23, 2020

 

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References /content/29/1/70.full.html#ref-list-1

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