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http://dx.doi.org/10.1007/s10142-012-0274-3

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1 Article Title Expression dynamics of metabolic and regulatory components

across stages of panicle and seed dev elopment in indica rice

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Springer-Verlag 2012(This will be the copyright line in the final PDF)

4 Journal Name Functional & Integrativ e Genomics

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7 Given Name Sanjay

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9 Organization University of Delhi South Campus

10 Division Interdisciplinary Centre for Plant Genomics andDepartment of Plant Molecular Biology

11 Address Benito Juarez Road, New Delhi 110021, India

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79 Division Plant Molecular Biology and BiotechnologyGroup, Melbourne School of Land andEnvironment

80 Address Parkvil le 3010, Victoria, Australia

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82 Division Plant Molecular Biology and BiotechnologyGroup, Melbourne School of Land andEnvironment

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Received 22 November 2011

146 Revised 2 March 2012

147 Accepted 6 March 2012

148 Abstract Caref ully analy zed expression prof iles can serv e as a v aluable ref erencef or deciphering gene f unctions. We exploited the potential of whole genomemicroarray s to measure the spatial and temporal expression prof iles of ricegenes in 19 stages of v egetativ e and reproductiv e dev elopment. We couldv erif y expression of 22,980 genes in at least one of the tissues.Dif f erential expression analy sis with respect to f iv e v egetativ e tissues andpreceding stages of dev elopment rev ealed reproductiv e stage-pref erential/-specif ic genes. By using subtractiv e logic, we identif ied 354and 456 genes expressing specif ically during panicle and seeddev elopment, respectiv ely . The metabolic/hormonal pathway s andtranscription f actor f amilies play ing key role in reproductiv e dev elopmentwere elucidated af ter ov erlay ing the expression data on the publicdatabases and manually curated list of transcription f actors, respectiv ely .During f loral meristem dif f erentiation (P1) and male meiosis (P3), thegenes inv olv ed in jasmonic acid and pheny lpropanoid biosy nthesis weresignif icantly upregulated. P6 stage of panicle, containing maturegametophy tes, exhibited enrichment of transcripts inv olv ed inhomogalacturonon degradation. Genes regulating auxin biosy nthesis wereinduced during early seed dev elopment. We v alidated the stage-specif icityof regulatory regions of three panicle-specif ic genes, OsAGO3, OsSub42,and RTS, and an early seed-specif ic gene, XYH, in transgenic rice. Thedata generated here prov ides a snapshot of the underly ing complexity ofthe gene networks regulating rice reproductiv e dev elopment.

149 Keywordsseparated by ' - '

Dev elopment - Expression - Meta-analy sis - Metabolic pathway s - Panicle -Promoter - Seed - Transcription f actors

150 Foot noteinformation

The authors Rita Sharma and Pinky Agarwal contributed equally to thiswork.

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The online v ersion of this article (doi:10.1007/s10142-012-0274-3) containssupplementary material, which is av ailable to authorized users.

Electronic supplementary material

Table S1(XLS 12 kb)

Table S2(XLS 24 kb)

Table S3(XLS 1198 kb)

Table S4(XLS 57 kb)

Table S5(XLS 33 kb)

Table S6(XLS 557 kb)

Table S7(XLS 19 kb)

Table S8(XLS 20 kb)

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1

23 ORIGINAL PAPER

4 Expression dynamicsQ1Q5= of metabolic and regulatory components5 across stages of panicle and seed development in indica rice

6 Rita Sharma & Pinky Agarwal & Swatismita Ray &

7 Priyanka Deveshwar & Pooja Sharma &

8 Niharika Sharma & Aashima Nijhawan & Mukesh Jain &

9 Ashok Kumar Singh & Vijay Pal Singh &

10 Jitendra Paul Khurana & Akhilesh Kumar Tyagi &11 Sanjay Kapoor

12 Received: 22 November 2011 /Revised: 2 March 2012 /Accepted: 6 March 201213 # Springer-Verlag 2012

14

15 Abstract Carefully analyzed expression profiles can serve as a16 valuable reference for deciphering gene functions. We exploited17 the potential of whole genome microarrays to measure the18 spatial and temporal expression profiles of rice genes in 1919 stages of vegetative and reproductive development. We could20 verify expression of 22,980 genes in at least one of the tissues.21 Differential expression analysis with respect to five vegetative22 tissues and preceding stages of development revealed reproduc-23 tive stage-preferential/-specific genes. By using subtractive log-24 ic, we identified 354 and 456 genes expressing specifically25 during panicle and seed development, respectively. The meta-26 bolic/hormonal pathways and transcription factor families

27playing key role in reproductive development were elucidated28after overlaying the expression data on the public databases and29manually curated list of transcription factors, respectively.30During floral meristem differentiation (P1) and male meiosis31(P3), the genes involved in jasmonic acid and phenylpropanoid32biosynthesis were significantly upregulated. P6 stage of panicle,33containing mature gametophytes, exhibited enrichment of tran-34scripts involved in homogalacturonon degradation. Genes reg-35ulating auxin biosynthesis were induced during early seed36development. We validated the stage-specificity of regulatory37regions of three panicle-specific genes,OsAGO3,OsSub42, and38RTS, and an early seed-specific gene, XYH, in transgenic rice.

The authorsQ3 Rita Sharma and Pinky Agarwal contributed equally to thiswork.

Electronic supplementary material The online version of this article(doi:10.1007/s10142-012-0274-3) contains supplementary material,which is available to authorized users.

R. Sharma : P. Agarwal : S. Ray : P. Deveshwar : P. Sharma :N. Sharma :A. Nijhawan :M. Jain : J. P. Khurana :A. K. Tyagi :S. Kapoor (*)Interdisciplinary Centre for Plant Genomics and Department ofPlant Molecular Biology, University of Delhi South Campus,Benito Juarez Road,New Delhi 110021, IndiaQ2e-mail: [email protected]

A. K. Singh :V. P. SinghDivision of Genetics, Indian Agriculture Research Institute,New Delhi 110012, India

Present Address:R. SharmaDepartment of Plant Pathology,University of California,Davis, CA 95616, USA

Present Address:P. Agarwal :M. Jain :A. K. TyagiNational Institute for Plant Genome Research,Aruna Asaf Ali Marg,New Delhi 110067, India

Present Address:S. RayBiotechnology and Bioresources Management Division, TataEnergy Research Institute,Lodhi Road,New Delhi 110003, India

Present Address:N. SharmaPlant Molecular Biology and Biotechnology Group, MelbourneSchool of Land and Environment, University of Melbourne,Parkville 3010 Victoria, Australia

Funct Integr GenomicsDOI 10.1007/s10142-012-0274-3

JrnlID 10142_ArtID 274_Proof# 1 - 13/03/2012

http://dx.doi.org/10.1007/s10142-012-0274-3

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39 The data generated here provides a snapshot of the underlying40 complexity of the gene networks regulating rice reproductive41 development.

42 Keywords Development .Expression .Meta-analysis .Metabolic43 pathways . Panicle . Promoter. Seed .Transcription factors

44 Introduction

45 Reproductive development is a dynamic process involving46 complex interplay of various regulatory networks. Spatial47 and temporal transcriptome profiles represent snapshot of gene48 activity and thus have been extensively used for deciphering49 the role of individual genes/pathways or regulatory networks50 and plausible interactions among them (Adams 2008). In fact,51 in the past decade, multitude of microarray-based studies have52 been performed towards elucidating reproductive organ devel-53 opment in Arabidopsis (Alves-Ferreira et al. 2007; Becerra et54 al. 2006; Fait et al. 2006; Hennig et al. 2004; Wellmer et al.55 2006; Wellmer et al. 2004; Zhang et al. 2005; Wilson et al.56 2005b; Day et al. 2008), rice (Endo et al. 2004; Furutani et al.57 2006; Hobo et al. 2008; Kondou et al. 2006; Lan et al. 2004;58 Hirano et al. 2008; Suwabe et al. 2008; Jiao et al. 2009; Wang59 et al. 2010; Fujita et al. 2010; Li et al. 2007a, b; Wang et al.60 2005; Deveshwar et al. 2011), maize (Grimanelli et al. 2005;61 Liu et al. 2008; Lee et al. 2002), wheat (Wilson et al. 2005a),62 and other non-model plant systems (Hansen et al. 2009;63 Laitinen et al. 2005; Endo et al. 2002; Tebbji et al. 2010).64 One of the limitations of these studies is that most of these65 scored the number of probe sets rather than unique transcripts66 as an estimate of gene expression. Moreover, these studies67 mainly focused on analyzing spatial expression profiles in68 various cell/tissue types or restricted time points during devel-69 opment. Therefore, it is very difficult to make cross compar-70 isons due to collection of tissues at different stages of71 development and lack of an internationally accepted staging72 system in rice similar to that of Arabidopsis (Smyth et al.73 1990). Here, we report the time-course analysis of expression74 dynamics in indica rice encompassing the complete series of75 reproductive development, from panicle initiation to seed mat-76 uration. All the analysis was performed on the list of probe IDs77 representing non-TE-related unique transcripts. We believe78 that our experimental design provides more realistic and com-79 plete view of transient as well as long-term developmental80 response, which is not attainable by organ/cell-type specific81 or single time point/stage-based studies.82 We categorized panicle and seed development into nine83 (P1–P6 and P1-I–P1-III) and five (S1–S5) categories, respec-84 tively, and used Affymetrix arrays to generate spatial and85 temporal expression profiles during rice reproductive organ86 development. Itemized comparisons with five vegetative tis-87 sues including 7-day-old seedlings (SD), roots (R), Y leaf

88(YL), mature leaf (ML), and shoot apical meristem (SAM)89revealed reproductive stage preferential/specific genes.90Enrichment of transcription factor coding genes and hormonal91pathways in vegetative, panicle, and seed stages implied their92significance during plant growth and development. The data93have previously been extensively validated by qPCR analysis94of candidates from various gene families encoding transcrip-95tion factors (Agarwal et al. 2007; Arora et al. 2007; Nijhawan96et al. 2008; Jain et al. 2008; Sharma et al. 2010), signal97transduction components (Jain et al. 2006; Jain and Khurana982009; Singh et al. 2010; Jain et al. 2007), RNA interference99machinery (Sharma et al. 2010; Sharma et al. 2009), and stress100responsive factors (Ray et al. 2011). Due to prior submission101in Gene Expression Omnibus database, various researchers102(Cao et al. 2008; Howell et al. 2009; Ma and Zhao 2010; Jiang103et al. 2009; Li et al. 2009) have also used this dataset for104analyzing expression profiles of rice genes depicting the con-105fidence of rice community in the quality of the data. We have106also earlier used part of the data generated here to identify107anther-specific transcripts by comparing the expression pro-108files of rice genes during anther development with those of109vegetative and seed stages analyzed here (Deveshwar et al.1102011). However, the goal of this study is to (1) identify key111genes/pathways regulating various stages of panicle and seed112development, (2) provide an insight into magnitude and re-113dundancy in rice transcriptome during different stages of114vegetative and reproductive development, and (3) provide an115in planta validation of stage specificity of selected genes using116promoter-reporter analysis. Expression profiles of four candi-117date genes, exhibiting varied expression patterns, have been118verified by promoter-GUS analysis in transgenic rice.

119Materials and methods

120Collection of plant material and categorization of panicle121and seed stages

122The vegetative tissues and rice panicles spanning all the stages123of panicle and seed development were collected from field-124grown Oryza sativa indica var. IR64 plants at IARI (Indian125Agricultural Research Institute, New Delhi, India). For evalu-126ating the precise stages of development, we performed the127histochemical analysis with the anthers collected at different128stages of development, correlated with the length of panicles129(for details, see Supplementary Figure S1). Based on the stages130of anther development and information available in literature131(Ikeda et al. 2004; Itoh et al. 2005; Raghavan 1988), panicles132were divided into six groups (P1–P6; Supplementary Table S1,133Figure S1 and S2). To document morphological details,134panicles collected from all six groups were photographed135using a digital camera (Canon PowerShot S1 IS, Singapore).136The P1 stage was further categorized into three sub-groups

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137 (P1-I–P1-III) and photographed under a dissecting stereo-138 zoom microscope (MZ12.5 with DFC320 camera; Leica139 Gmbh, Germany). The seed development was categorized140 into five groups (S1–S5) based on the days after pollination141 (Supplementary Table S1, Figure S2). To document seed142 stages, spikelets at different stages of seed development were143 dehusked and dissected to extract embryos. The morpholog-144 ical details were observed and photographed using the dis-145 secting stereo-zoom microscope. Three biological replicates146 of five vegetative tissues, including ML, YL, R, SD, and147 SAM, were also collected from field-grown plants. For uni-148 formity and simplicity, all the categories of panicle and seed149 development and vegetative tissues are referred as stages150 of development in the manuscript. The details of stages151 used are provided in Supplementary Table S1.

152 Microarray experiments and data analysis

153 Affymetrix GeneChip®Rice Genome arrays containing 51,279154 rice transcripts (approximately 48,564 japonica and 1,260 ind-155 ica transcripts) were used to study the global changes in gene156 expression during rice reproductive development. RNA was157 isolated as described (Arora et al. 2007; Nijhawan et al.158 2008). cDNA synthesis, cRNA synthesis, labeling, and hybrid-159 izations followed by scanning were carried out as per manu-160 facturer’s instructions and described (Affymetrix, Santa Clara,161 CA; (Arora et al. 2007). In total, 57 .CEL files representing162 three biological replicates each of ML, YL, R, SD, nine panicle163 stages (P1-I−P1-III, P1−P6), and five seed stages (S1−S5) were164 imported in ArrayAssist 5.0.0 microarray data analysis soft-165 ware (Stratagene) followed by quantile normalization by using166 GCRMA algorithm and log2 transformated (Wu et al. 2003).167 The correlation coefficient between biological replicates was168 analyzed. The data were separately normalized using MAS 5.0169 algorithm to generate Present/Absent calls. The unique number170 of transcripts represented on the GeneChip® was identified as171 described previously (Deveshwar et al. 2011) and filtered data-172 set was used for downstream analysis.173 Principal component analysis, using 19 principle compo-174 nents, was carried out withmean centering and scaling of all the175 variables to unit variance and presented as three-dimensional176 view using eigenvalues, E1–E2–E3. Differential expression177 analysis was carried out with respect to all four vegetative178 stages (SAM, YL, ML, SD), taken separately, as well as with179 respect to preceding stage by applying an FDR correction based180 on Benjamini and Hochberg method and p value cut off of181 ≤0.05 (Benjamini andHochberg 1995). For early panicle stages182 (P1-I, P1-II, and P1-III), SAMwas taken as reference and the p183 values were given as uncorrected at a cut off of ≤0.005. To184 identify stage-specific genes, log2 expression value cut off of185 ≥5.64 (normalized average expression value (NAEV) ≥50) in186 the stage of interest and ≤3.90 (NAEV ≤15) in rest of the stages187 was employed. However, to identify panicle-specific genes, the

188filter was not applied to S1 stage of seed development due to189high similarity in transcript pool of P6 and S1 stages. GO190annotations for selected datasets were downloaded from the191rice genome annotation project (RGAP) database (http://rice.192plantbiology.msu.edu/). The metabolic pathways induced/sup-193pressed during reproductive development were analyzed using194data available in RiceCyc database of Gramene (http://path195way.gramene.org/expression.html; Jaiswal et al. 2006). We196analyzed meta-profiles of short-listed datasets using Rice197Oligonucleotide Array Database (ROAD; http://www.rice198array.org/expression/meta_analysis.shtml).199A comprehensive list of all the transcription factor family200genes was generated using HMM analysis (Madera and201Gough 2002) as well as keyword search as described previ-202ously (Arora et al. 2007). The differential expression pro-203files were analyzed as described above. Hypergeometric204distribution analysis was also performed to identify tran-205scription factor families enriched in panicle/seed-specific206data sets. Cluster analysis on rows was performed for se-207lected datasets, using Euclidean distance metric and Ward’s208Linkage rule of Hierarchical clustering.

209qPCR analysis

210The expression profiles of four selected genes were validat-211ed using qPCR analysis as described earlier (Jain et al. 2006;212Arora et al. 2007). Three biological and three technical213replicates were performed for each stage and standard error214was calculated between them. ACTIN was used as an en-215dogenous control. The data were normalized to match the216profiles with that of microarray data.

217Promoter-reporter constructs

218About 1.5−2 kb putative promoter regions of selected genes219(−1,560 to +16 of RTS, −1,742 to +20 of OsAGO3, −1,518 to220+10 of OsSub42, −1,266 to +71 of XYH) including partial or221complete 5′ UTR were PCR amplified. The list of primers is222given in Supplementary Table S2. Amplified DNA fragments223were cloned in pENTRTM/D-TOPO vector (Invitrogen Inc.,224USA) and validated by sequencing. LR reaction was per-225formed using Gateway® LR ClonaseTM II enzyme mix226(Invitrogen, USA) as per manufacturer’s instructions to trans-227fer the promoter DNA from pENTRTM/D-TOPO vector to228pMDC164, expression vector carrying the gene encoding229GUS as reporter (Arabidopsis Biological Resource Center;230(Curtis and Grossniklaus 2003).

231Rice transformations and GUS histochemical assay

232Final vectors were mobilized into Agrobacterium tumefaciens233strain AGL1 by electroporation. The PB1 variety of indica rice234was transformed using the protocols described previously



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235 (Mohanty et al. 1999; Toki et al. 2006). The list of primers used236 to confirm the presence of transgene is given in Supplementary237 Table S2. GUS histochemical assay was performed to check the238 activity of GUS (Jefferson et al. 1987). Different tissues of the239 transgenic as well as wild-type plants were incubated in GUS240 histochemical buffer containing 10 % methanol at 37°C for 16241 −20 h followed by incubation in acetone/ethanol (1:3). The242 observations were recorded by using DFC320 mounted on243 MZ12.5 Stereo microscope (Leica Gmbh, Germany).

244 Genomic localization of differentially expressed genes

245 To localize the genes on respective chromosomes, only those246 with NAEVof ≥50 (log2 expression value ≥5.64) in at least one247 of the stages were analyzed. The genes exhibiting ≥2 folds248 differential expression in any of the 19 stages were extracted.249 In-house generated programs (written in “C” and Perl) were250 used to identify Gene Clusters with Similar Expression251 (GCSEs) profiles. All the genes were sorted based on their252 chromosomal positions as given in RGAP database of MSU253 (http://rice.plantbiology.msu.edu/). Based upon the expression254 profiles obtained after differential expression analysis, all the255 genes were assigned the stage preferential/specific profiles and256 then a sequential scan was performed by comparing the ex-257 pression profile of each gene with the next gene in order. If258 expression profiles of two or more contiguous genes match up,259 the regionwas scored as a GCSE. To display the GCSEs on rice260 chromosomes, Differential Gene Locus Mapping (DIGMAP)261 version 2 (http://geneexplorer.mc.vanderbilt.edu/DIGMAP/; Yi262 et al. 2005) was used. The per cent identity between the genes263 falling in the same GCSE was calculated using MegAlign264 software 4.03 (DNASTAR Inc.). Genes sharing ≥70 % identity265 at protein level were considered to be tandemly duplicated. To266 check if the genes comprised in a GCSE were involved in the267 same or different pathways, GOSlim assignments and putative268 functions for all of the genes were obtained from RGAP269 database (http://rice.plantbiology.msu.edu/index.shtml) and270 RiceCyc database of Gramene (http://pathway.gramene.org/271 expression.html; Jaiswal et al. 2006).

272 Results

273 Magnitude of rice transcriptome

274 Since the number of probesets (57,381) on GeneChip® Rice275 Genome Array do not correspond to the number of annotat-276 ed genes, we carried out an extensive curation exercise to277 filter probesets that (1) do not map to any annotated tran-278 scription unit, or (2) represent internal controls or TE-related279 genes, or (3) are not the 3′ most probeset (in case of multiple280 probesets per transcription unit). This resulted in a dataset of281 37,927 probe sets representing unique genes on the chip,

282which was used for all the subsequent analyses (for details,283see Supplementary Fig. S3).284Genome-wide expression profiles of all 14 stages of repro-285ductive development, along with five stages of vegetative286development including ML, YL, SAM, R, and SD, were287analyzed using GeneChip® Rice Genome Arrays288(Affymetrix). A correlation of >0.96 was obtained among289three biological replicates of all the stages analyzed. A prin-290ciple component analysis performed on the dataset clearly291demonstrated, on one hand, the expanse and distinctness of292vegetative transcriptomes and, on the other, underlined the293molecular continuum as well as progression of panicle and294seed development paths emanating from SAM to P1-I and P6-295S1 transitions, respectively (Fig. 1a). The distinct positioning296of both reproductive and vegetative tissues/time points also297provided validation of our staging system.298To comprehend the magnitude of rice transcriptome and299contribution of individual tissues, the number of expressed300genes in each stage/tissue was determined. The analysis ver-301ified expression of 22,980 unique genes in at least one of the302stages/tissues analyzed which is comparable to the number of303genes detected using cDNA arrays (Jiao et al. 2009). The304maximum number of genes (20,421) was called “present” in305panicles implying the need for diverse transcript population306during development of specialized floral organs (Fig. 1b).307Among vegetative tissues, seedlings and roots exhibited308higher number of transcripts in comparison to leaf tissues,309probably because of diverse and higher metabolic activity.310A comparison of expressed genes revealed that 9,021 genes311expressed in all the stages analyzed and thusmight be involved312in basal metabolic pathways. About 35-45 % genes from each313stage exhibited overlapping expression in multiple develop-314mental windows, whereas, only 0.5 % to 3.2 % genes were315specific to each developmental stage (Fig. 1b). Among repro-316ductive stages, P6 stage of panicle, that harbors mature pollen,317had maximum number of specific transcripts as many of them318could be stored for pollen germination and tube growth319(Becker and Feijo 2007). Roots exhibited maximum percent-320age of specific genes (2.3 %, Fig. 1b) among vegetative tissues321suggesting that root being an underground part might have322developed some unique biochemical pathways (Zhang et al.3232005). More than 650 genes are expressed in both panicle and324seed stages, whereas, 321 transcripts were shared between325SAM and panicles. Panicles were found to share the maximum326number of transcriptional entities with roots (146) compared to327other vegetative stages analyzed in this study.

328Differential expression analysis during rice reproductive329development

330Since no single method can identify all the relevant genes331with potential involvement in rice reproductive develop-332ment, two strategies were adopted to identify differentially




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Fig. 1 a PrincipleQ4 component analysis of various developmentalstages. The graph shows all the data points projected in the three-dimensional space formed by three coordinates after rotation. Eachdata point represents an independent tissue with green color represent-ing leaf stages (ML and YL); gray represents root (R) and seedlings(SD); purple represents SAM and early panicle stages (P1-I to P1-III);red represents panicle (P1 to P6) and blue represents seed (S1 to S5)stages. The eigenvalues E1–E2–E3 were used for plotting the data. Theclosely related developmental stages are encircled. b Overview of geneexpression during different stages of development. The number ofgenes called “Present” in each stage as well as the number of genesspecific to one, two, or more than two stages has been shown as the

stacked bar graph. The color legend is given on the topmost left of thegraph. c Differential expression analysis during stages of panicle andseed development with respect to vegetative stages. The number ofgenes showing up- and downregulation in each panicle stage withrespect to each of the four vegetative stages is plotted. Pink columnsrepresent the commonly up- and downregulated genes with respect toall four vegetative stages; whereas, genes specifically upregulated ineach stage are represented by yellow columns. The dotted line repre-sents the pattern exhibited by differentially expressed genes withrespect to all four vegetative stages during temporal stages of repro-ductive development. SD seedling, R root, ML mature leaf, YLY leaf,SAM shoot apical meristem, P panicle, S seed stages



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333 expressed genes during panicle and seed development. In334 the first approach, the transcript levels of all the genes were335 compared with the vegetative stages individually as well as336 collectively. In the second approach, the transcriptome of337 each stage was compared with that of preceding stage to338 identify genes whose expression might have triggered tran-339 siently in response to specific developmental cues.

340 Reproductive vs. vegetative development

341 The datasets were analyzed to identify up/downregulated342 genes in each stage with respect to all four vegetative tissues,343 collectively. The resulting dataset would, therefore, include344 genes whose transcript levels change as a function of repro-345 ductive development. Based on differential expression analysis346 with respect to vegetative stages, early panicle stages (P1 and347 P2) showed higher number of differentially expressed genes348 with respect to R and SD; whereas, later panicle stages (P5 and349 P6) had larger number of differentially expressed genes with350 respect to leaf stages (Fig. 1c). Conversely, the maximum351 number of genes involved in seed development was differen-352 tially regulated with respect to young root; whereas, the seed353 tissues had the least number of differentially expressed genes354 when compared with mature leaf. Among the seed stages, the355 number of downregulated genes with respect to vegetative356 stages gradually increased during development suggesting357 general suppression of transcriptional activity commensurate358 with the onset of the dormant phase in seeds.359 Among panicle stages, the maximum number of differ-360 entially expressed genes was found in P2 stage (that corre-361 sponds to initiation of male meiosis) with 1,101 and 1,053362 genes up- and downregulated, respectively, suggesting363 higher order genic activity at this particular stage; whereas,364 the minimum differential transcript accumulation was ob-365 served at the post-meiotic P4 stage (Fig. 1c). The S1 stage,366 corresponding to 0–2 days post-fertilization development,367 represents the least differentially expressed gene pool,368 which increased in S2 and S3 stages involving enlargement369 of organs. Using subtractive logic, the upregulated genes in370 each stage were compared with those in every other stage to371 identify genes that were exclusively activated in a particular372 stage of reproductive development. This analysis revealed373 13, 32, 67, 87, 81, and 248 genes to be specifically upregu-374 lated in P1–P6 stages of panicle development, and 303, 241,375 213, 72, and 295 genes uniquely induced in S1–S5 stages of376 seed development, respectively (Fig. 1c, yellow bars).377 Notable of these were anther-specific proline rich protein378 coding genes uniquely induced in P3 and P4 stages of379 panicle development. Transcription factors were particularly380 enriched in upregulated datasets during early seed stages;381 whereas, those involved in carbohydrate metabolism and382 ubiquitin-mediated proteolysis were exclusively upregulated383 in later seed stages. As the development of reproductive

384organs progressed, the number of uniquely induced transcripts385also increased.386Similarly, differential expression analysis of early panicle387stages (P1-I to P1-III) with respect to shoot apical meristem388(SAM) identified 425, 710, and 2,032 genes exhibiting ≥2389fold change in P1-I, P1-II, and P1-III stages, respectively. Of390these, 202, 385, and 902 genes were upregulated in P1-I, P1-391II, and P1-III, respectively. In total, 49 genes were induced in392all three stages. Among the differentially upregulated genes, a393large proportion (106, 159, and 691 genes, in P1-I, P1-II, and394P1-III stage, respectively) was specific to individual stages395suggesting the rapid development-dependent molecular396switching in these stages.

397Analysis of cascadial expression during reproductive398development

399As each stage analyzed in this study represents a character-400istic phase in flower and seed development, one would401expect significant upregulation of specific transcriptional402units with respect to preceding stage, many of which will403be important for regulating the precise developmental events404and may have induced for a very short period of time. To405identify these components, we performed differential ex-406pression analysis by comparing each stage with its preced-407ing stage of development. For P1 stage, SAM was taken as408reference. This analysis revealed 2,009, 81, 905, 41, 1,854,409and 3,590 genes getting more that 2 folds upregulated (p410value≤0.05) in P1−P6 stages, respectively (Fig. 2a). Among411seed stages, as the development progressed, the number of412upregulated genes declined. In total, 1,973, 1,064, 289, 137,413and 178 genes were upregulated in S1, S2, S3, S4, and S5414stages, respectively (Fig. 2a). To shortlist the most signifi-415cant genes, the ones with NAEVof ≥50 (or log2 expression416value ≥5.64) in any of the vegetative stages were filtered out417from respective upregulated sets resulting in a set of 65, 2,41866, 3, 79, and 415 genes uniquely upregulated in P1−P6419stages, respectively and 112, 204, 33, 19, and 11 genes in S1420−S5 seed stages, respectively (Fig. 2a). The expression

�Fig. 2 a Differential expression analysis during reproductive develop-mental stages taking preceding stage of development as reference. Blackbars represent total number of genes upregulated by ≥2 folds at p value≤0.05 with respect to the preceding stage. Gray bars represent theupregulated genes with NAEV ≤15 in vegetative stages (SupplementaryTable S3). b Major pathways induced with respect to preceding stageduring rice reproductive development. The genes exhibiting ≥2 foldsupregulation in reproductive stages with respect to preceding stage andinvolved in (a) jasmonic acid, (b) phenylpropanoid, and (c) IAA biosyn-thesis have been presented with their expression profiles plotted in theform of a heat map. Color bar at the base represents log

2expression

values, with green color representing low-level expression, black repre-senting medium-level, and red signifying high-level expression. Devel-opmental stages used for expression profiling are given on the top of eachcolumn (for details, see Supplementary Table S1)



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421 profiles of these genes have been compiled as a heat map422 (Supplementary Fig. S4).423 Integration of upregulated dataset with the publicly avail-424 able RiceCyc database (http://www.gramene.org/pathway/425 ricecyc.html) revealed metabolic and hormonal pathways426 involved in reproductive development (Fig. 2b). The genes427 involved in jasmonic acid biosynthesis were upregulated428 during floral organ differentiation (P1), meiotic stage of429 anther development (P3), and pollen development (P6) and430 those implicated in phenylpropanoid biosynthesis exhibited431 significant upregulation during floral organ differentiation432 (P1), male meiosis (P3), and microspore development (P5).433 The analysis also showed that five genes involved in Indole-434 3-Acetic Acid (IAA) biosynthesis were also upregulated in435 S1 stage (Fig. 2b). The genes involved in salvage pathway436 of purines were upregulated during late panicle (P5 and P6)437 and early seed (S1) stages.438 The ROAD (http://www.ricearray.org) provides gene ex-439 pression data across 1,867 publicly available rice microarray440 hybridizations assembled together to perform meta-analysis.441 Meta-profiles of all three datasets shown in Fig. 2b, ana-442 lyzed using ROAD database, conformed with their signifi-443 cant induction during stages of reproductive development444 (Supplementary Figure S5).

445 Identification of reproduction-specific genes and their446 affiliation to metabolic pathways

447 The dataset of differentially expressed genes was further448 filtered to identify genes that expressed specifically in one449 or more stages of panicle and seed development. Three450 hundred and fifty four genes exhibited panicle-specific ex-451 pression (expression value ≥50 in panicle stages and ≤15 in452 other stages) with 3 % of them coding for transcription453 factors. Whereas, 456 genes were expressed only in seed454 developmental stages (expression value ≥50 in seed stages455 and ≤15 in other stages) with 9 % of them encoding tran-456 scription factors. Interestingly, 40 % genes in both panicle457 and seed-specific datasets have not even been annotated yet,458 probably because of narrow windows of their expression.459 The expression of about 48 % of the panicle-specific genes460 (171) initiates at P1, P2, P3, and P4 stages, which in most461 cases is detected till P5 or P6 stage. However, 183 genes462 expressed only in the P6 stage harboring mature male and463 female gametophytes. Since male gametophytic cells out-464 number the female gametophytic cells in every floret, the465 bulk of these genes represent mature pollen-specific tran-466 scripts. In fact, most of these genes code for pollen aller-467 gens, transporters, and components of cytoskeleton and468 those involved in cell wall metabolism (see Supplementary469 Table S3 for details). These categories of genes might play470 role in pollen–pistil interactions and pollen tube germina-471 tion. Putative homolog of pollen-specific gene, SF3, of

472sunflower exhibited P6 stage-specific expression (Baltz et473al. 1992). Many of panicle stage-specific genes seem to be474involved in flavonoid and lipid biosynthesis.475During seed development, the most prominent domain,476S2–S5, comprised of 93 genes with high representation of477genes coding for seed storage proteins (proline and glute-478lins), seed allergens and those involved in starch biosynthe-479sis and ubiquitin-mediated proteolysis. In total, 19, 46, 28,4807, and 55 genes were found to be specific to S1, S2, S3, S4,481and S5 stages, respectively. The expression profiles of pan-482icle and seed-specific genes have been presented in the form483of hierarchical cluster maps in Supplementary Figure S6.484We compared the dataset of panicle/seed-specific genes485with two available expression data sets generated in differ-486ent rice subspecies using different microarray platforms487(Sato et al. 2011; Wang et al. 2010). A higher number of488panicle and seed-specific genes were common between our489dataset (IR64) and the data generated by Wang et al. (2010)490for other indica varieties (Zhenshan 97 and Minghui 63) in491comparison to those identified in japonica (Nipponbare)492rice by Sato et al. (2011; Supplementary Figure S7). Since493the data generated in earlier studies was limited to fewer494spatial stages of panicle and seed development, many of the495genes detected specifically in our dataset could be specific496to the stages not analyzed previously.497On collating the information from Gramene database, a498significant number of genes involved in biosynthesis of499gibberellins, strictosidine, and fatty acids and their elonga-500tion were found to exhibit panicle-specific expression501(Fig. 3). However, transcripts of those involved in starch502degradation were specially detected in seed stages (Fig. 3).503The genes involved in brassinosteroid biosynthesis were504upregulated in P5 stage, where these may be stored in starch505granules and later released during pollination and fertiliza-506tion (Clouse and Sasse 1998). Various genes involved in507gibberellin biosynthesis were upregulated during male mei-508osis (P3), heading stage (P6) as well as early stages of seed509development (S1 and S2). In addition, few genes involved in510ent-kaurene biosynthesis, which is a common gibberellin511precursor, were also upregulated at S1 stage of seed devel-512opment suggestive of their involvement of GA during early513seed development (Davidson et al. 2003). Analysis of meta-514profiles of the genes highlighted in Fig. 3 supports their515specific expression in limited stages of development516(Supplementary Fig. S8).

517Expression dynamics of transcription factors518during reproductive development

519The number of transcription factor (TF) coding genes in rice520is estimated to be 2,527 categorized into 65 genes families521(Riano-Pachon et al. 2007). However, 216 of these genes are522orphans as their role in transcriptional regulation is



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Fig. 3 Schematic representation of major pathways involving panicle/seed-specific genes. Ten major pathways represented by panicle/seed-specific genes are highlighted. The expression profiles of the genes areshown in the form of heat maps. Color bar at the base represents log2

expression values, where green color represents low-level expression,black shows medium-level expression, and red signifies high-level ex-pression. Developmental stages used for expression profiling are given onthe top of each column (for details, see Supplementary Table S1)



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523 ambiguous. Here, we reanalyzed the rice genome using524 name search and HMM analysis and identified a total of525 2,314 transcription factor genes belonging to 68 gene fam-526 ilies. Except for a few gene families, additional members527 were identified in each gene family (Supplementary Table528 S5). Of these, 2,100 TF genes are represented on rice529 GeneChip® array. Differential expression analysis of these530 genes with respect to vegetative stages (ML, YL, R, and SD)531 revealed 204 and 246 genes exhibiting ≥2 folds upregulation532 (p value≤0.05) in at least one of the panicle and seed stages,533 respectively. Only 80 genes were upregulated in both panicle534 and seed stages, whereas, the rest were exclusive to either535 panicle or seed development. Conversely, 55 genes were536 downregulated in both panicle and seed stages. Differential537 expression analysis revealed 18 TF families including SBP,538 GRF, and DOF exhibited higher number of upregulated genes539 in panicles. Further, hypergeometric distribution analysis540 (Supplementary Table S5) revealed that 14 families, namely541 BBR_GAGA, BTB/POZ, bZIP, GRF, HORMA, MADS,542 NAM, PWWP, SBP, TRIHELIX, WRKY, YABBY, C2H2,543 and DOF, were particularly enriched during panicle develop-544 ment. Out of these, bZIP, MADS, and C2H2 families had more545 than 10 upregulated members. Twenty-seven families includ-546 ing MYB, NAM, HSF, MADS, POZ, and bZIP had relatively547 higher number of genes upregulated in seed stages (Fig. 4).548 Nine TF families, namely, bHLH, BTB/POZ, bZIP, HSF,549 MADS, SRS, TRIHELIX, YABBY, and C2H2, were signifi-550 cantly enriched, with five of them having ten or more upregu-551 lated members. Of these, we have earlier shown the detailed552 expression patterns of bZIP, MADS and C2H2 families,553 wherein certain members are panicle/seed-specific (Agarwal554 et al. 2007; Arora et al. 2007; Nijhawan et al. 2008). Similar555 analysis of downregulated genes revealed 14 gene families556 including WRKY, C2H2, and AP2 with higher number of557 members downregulated in panicles; whereas, 27 gene fami-558 lies including bHLH, TUBBYand C3H had higher number of559 downregulated genes in seeds (Fig. 4). An enrichment analy-560 sis of downregulated members lays emphasis on genes whose561 expression is probably not an essential feature for the devel-562 opment process (Supplementary Table S5).563 In total, 52 TF genes including members of bHLH, bZIP,564 B3, C2H2, HOMEOBOX, MADS, MYB, NAM, SBP, and565 WRKY gene families had vegetative stages-specific expres-566 sion with fifteen of them exhibiting SAM-specific and ten567 genes exhibiting root-specific expression in contrast to only568 one gene specific to each leaf and seedlings. Panicle stages569 were found to have specific expression of only eleven TF570 genes having representation of two genes each from bHLH571 and DOF gene families. Forty-seven TF genes exhibited572 seed-specific expression with a majority of them being573 specific to the S2 stage (2–5 DAP), suggesting that S2 might574 represent an important developmental hot spot that should575 be investigated in detail. A hierarchical cluster map showing

576expression profiles of the transcription factor coding genes577expressing in a developmental stage specific manner is578presented in Supplementary Figure S6.

579Gene Clusters with Similar Expression profiles

580Previous studies have shown correlation between expression581profiles and physical location of genes in eukaryotic582genomes including Drosophila, nematode, mouse, and583humans (Michalak 2008). Such a phenomenon has also been584identified in rice and Arabidopsis (Ren et al. 2005; Ren et al.5852007; Zhan et al. 2006). To get a genome-wide perspective586of the influence of physical proximity of genes on their587expression profiles, we selected 18,180 differentially588expressed genes (≥2 folds with NAEV ≥50) and analyzed589their physical location on rice chromosomes as described in590the “Materials and methods” section. This analysis revealed5911,278 GCSE profiles comprising of 2,792 genes592(Supplementary Tables S6 and S7). In total, 25 varied ex-593pression patterns were observed (Supplementary Table S8),594of which 8 major patterns comprising of more than 80 % of595the data are shown in Fig. 5. The maximum number of596clusters was found to exhibit SAM + panicle-preferential597expression (209) and vegetative stages-preferential expres-598sion (200). One hundred and eighty-five clusters exhibited599panicle-preferential expression; whereas, 147 GCSEs were600found to be seed-preferential. Most of the GCSEs (1,099)601comprised of two genes. The largest GCSE included 12602genes on chromosome 10 (10_43) exhibiting root-603preferential expression followed by a cluster of 7 genes on604chromosome 1 (Supplementary Table S6). We did not ob-605serve any chromosomal bias in the distribution of GCSEs.606To check if the genes comprising a GCSE were involved in607similar function, information related to their affiliation to608various biochemical pathways was retrieved from Gramene609database (http://pathway.gramene.org/expression.html).610Apparently, of the 35 clusters (74 genes), for which pathway611data was available, genes in 18 clusters belonged to same612biochemical pathways. In ten of these GCSEs, however,613some of the genes were found to have >70 % identity at614amino acid level, which could suggest their origin from615tandem duplication events. The annotated genes in other616eight GCSEs were involved in different pathways and617exhibited <16 % identity in their protein sequences. In nine618GCSEs, all the members of a cluster had not yet been619annotated, but, based on the available information; they620seem to be involved in similar pathways.

621Molecular characterization of candidate promoters

622Based on their expression profiles, we selected four genes623with stage-specific/preferential expression in panicle and624seed stages namely, OsAGO3 (LOC_Os04g52550),




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Fig. 4 Graphical representation of a up- and b downregulated tran-scription factor family genes in panicle and seed stages. The totalnumber of genes of each transcription factor family represented onthe GeneChip® is shown in the form of area graph (gray). The graph adepicts the number of upregulated and graph b depicts the number of

downregulated genes, respectively, in panicle (red) and seed (green)stages. The axis on the left represents the number of differentiallyexpressed genes (shown in bar chart); whereas, broken axis withdifferent data ranges on the right represents total number of genesrepresented on the GeneChip® (shown by the area graph)



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625 OsSub42 (LOC_Os04g45960), RTS (LOC_Os01g70440),626 and XYH (AK108917) exhibiting P3–P4, P4–P5, P5–P6,627 and S1 specific expression, respectively, for in planta vali-628 dation of promoter activities. Analysis of meta-profiles of629 these genes in Affymetrix data during developmental stages630 showed consistency in expression profiles of these genes631 with the publicly available data (Supplementary Figure632 S9a). The respective expression profiles were validated us-633 ing qPCR and promoter-reporter constructs were prepared634 using GUS as reporter (Fig. 6). OsAGO3 is an argonaute635 family gene containing PIWI domain (Sharma et al. 2010)636 with high transcript accumulation in P3 and P4 stages. The637 promoter activity was observed specifically in anthers at638 meiotic and tetrad stage suggesting its involvement in639 microsprorogenesis (Fig. 6). Analysis of its meta-profile640 during consolidated data from microarray experiments cov-641 ering anatomical stages showed that its transcripts are par-642 ticularly enriched in 0.7 to 1.0 mm anthers (Supplementary643 Figure S9b). The second gene, OsSub42 encodes a644 subtilisin-like serine protease detected in P4 and P5 stages.645 The GUS activity was observedmainly in anthers, specifically

Fig. 5 Chromosomal localization of co-expressed genes in rice.Microarray-based expression profiles of co-expressed genes at 19stages of vegetative and reproductive development were extractedand plotted in sequential order based on their location on rice

chromosomes. Each profile is shown by a different color. The colorlegend has been given at the base. The chromosome numbers areindicated on the left and Locus ID of first and last gene comprising acluster on each chromosome has been given

�Fig. 6 Expression profiles of candidate genes and respective promoters.The panel on the left presents the expression profiles exhibited by candi-date genes.Black bars represent the values obtained using microarray andwhite bars represent qPCR data. The qPCR values have been normalizedto those obtained from the microarray dataset. The error bars representthe standard error between three biological replicates of each stage. Thegene names are given in the top left corner of each graph. Right panelrepresents the expression of GUS reporter derived by promoters ofcandidate genes in different stages of development in rice. Stages ana-lyzed are as below: a amature leaf, b root, c T.S. stem, d seed, e, f floret atP3, g floret at P4, h floret at P5, i, j floret at P6, k anther of floret at P3,l anther of floret at P4,m anther of floret at P6, nmature gynoecium. Scalebar (a–j) 1 mm, (k–n) 0.1 mm. b a mature leaf, b root, c stem, d matureseed, e, i florets at P2, f, j florets at P3, g, k florets at P4 stage, h, l matureflorets. Scale bar, 0.5 mm. c a mature leaf, b T.S. stem, c root, d matureseed, e floret at P3, f floret at P4, g, h floret at P5, i floret at P6, j maturefloret at P6 after anthesis, k dissected P6 floret showing GUS stainedanthers, l anther at P6, m stigma at P6, n isolated pollen at P6. Scale bar(a–k) 1 mm, (l–n) 0.1 mm. d a callus, b mature leaf, c root at pre-pollination stage, d pollen at P6, e anther at P6, f floret at P6, g gynoeciumat P6, h seed at S1 stage, i 0 DAP seed (S1), j, k 1 DAP seed (S1), l 2 DAPseed (S1),m seed at S2 stage, n seed at S3 stage, o seed at S5 stage. Scalebar, 0.5 mm. “1” represents wild type and “2” denotes the transgenicplant. The scale bar is equal to 0.5 mm



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646 at single-celled microspore and bicellular pollen stage with647 very low-level expression in nodal portion of the stem, stigma648 and embryo (Fig. 6). Upon dissection of anthers at uninucleate649 stage, GUS activity was observed in the anther wall as well as650 in developing pollen. Meta-profile analysis during anatomical651 stages showed accumulation of its transcripts in 1.2 to 1.5 mm652 anthers (Supplementary Figure S9b). The third gene, RTS is653 an anther-specific gene that expresses exclusively in tapetum654 and has been shown to be essential for pollen development in655 rice (Luo et al. 2006). The RTS promoter derived specific656 expression in post-meiotic anthers with low level GUS activ-657 ity in stigma. Meta-profile analysis revealed its specific ex-658 pression in 1.6 to 2.0 mm anthers (Supplementary Figure659 S9b). XYH exhibiting S1 stage-specific expression encodes660 1, 4-beta-D xylan xylanohydrolase. The GUS activity was661 observed specifically in ovary and style in pollinated panicles662 suggesting that it might have role during penetration of pollen663 tube. Meta-profile analysis during anatomical stages conforms664 its specific expression in ovaries at 1 day after fertilization665 (Supplementary Figure S9b). The consistency in the expres-666 sion profiles of these genes in specific developmental events/667 stages and organs strengthens the reliability of our microarray668 data as well as staging system used.

669 Discussion

670 The reproductive phase in rice commences with the transition671 of shoot apical meristem to floral meristem and culminates at672 mature seed about a month after fertilization (Itoh et al. 2005).673 Since genes regulating panicle and seed development are674 major determinants of yield, understanding gene regulation675 is crucial for improving yield potential of rice. Here, we676 analyzed the genome-wide spatial and temporal expression677 profiles of rice genes during nine stages of panicle and five678 stages of seed development, demarcated based on the land-679 mark events involved. The transcriptome of each develop-680 mental stage was compared with the vegetative tissues as681 well as the preceding stage of development to investigate682 stage-specific regulation of gene expression. Analysis of683 stage-specific/preferential gene expression along with litera-684 ture mining revealed that the upregulated genes in our study,685 during panicle initiation stages (P1-I, P1-II, and P1-III) with686 respect to vegetative controls have previously been implicated687 in flowering time control (OsFTL1, OsMADS5, OsMADS14,688 and 15 of rice; Komiya et al. 2008; Jia et al. 2000; Lee et al.689 2003; Moon et al. 1999; Kang and An 1997), meristem690 organization (CLAVATA1 of Arabidopsis; Clark et al. 1997),691 and regulating symmetry (DIV of Antirrhinum; Galego and692 Almeida 2002), thus adding credibility to our data. Homologs693 of previously characterized genes for involvement in estab-694 lishment of polarity (FE of Ipomea; Iwasaki and Nitasaka695 2006), sexual fate of meristems (TS2 of maize; DeLong et

696al. 1993), and brassinosteroid response (BIM2 of Arabidopsis;697Yin et al. 2005) were specifically upregulated in P1-I stage;698whereas, those showing similarity with genes associated with699circadian clock (LHY and ELF3 of Arabidopsis; Ding et al.7002007; Liu et al. 2001), flowering time control (FPF1 and CO701of Arabidopsis; Kania et al. 1997; An et al. 2004), panicle702branch initiation (RFL of rice; Kyozuka et al. 1998), and organ703primordia formation (ZmOCL1; Ingram et al. 2000) were704specifically upregulated in P1-II stage. The list of genes show-705ing P1-II specific upregulation also included those involved706in meristem organization (TSO1 of Arabidopsis; Song et al.7072000) and cell specialization in anthers (TPD1 ofArabidopsis;708Yang et al. 2003). Induction of 80 genes was detected in both709P1-I and P1-II stages including genes involved in initiation710and maintenance of rachis and branch meristem (LAX and,711OSH1 of rice and JUBEL2 of barley; Komatsu et al. 2001;712Sentoku et al. 1999; Muller et al. 2001), panicle branching713(OsTB1 of rice; Takeda et al. 2003), whereas those showing714upregulation in P1-II–PI-III window (146 in number) included715genes involved in flowering time control and floral organ716identity (OsMADS1, 2, 6, 7, 8, and 58 of rice; (Agrawal et717al. 2005; Chung et al. 1994; Chung et al. 1995; Greco et al.7181997; Jeon et al. 2008; Kang et al. 1997; Lee et al. 2003;719Prasad et al. 2005; Prasad et al. 2001; Prasad and720Vijayraghavan 2003; Yamaguchi and Hirano 2006;721Yamaguchi et al. 2006; Jeon et al. 2000), auxiliary meristem722initiation (SPT of Arabidopsis; Komatsu et al. 2003), and723determination of floral organ number and shape (PNH of724Arabidopsis; Lynn et al. 1999).725Similarly, overlaying the data of differentially expressed726genes on RiceCyc database provided important insights into727genes and pathways that are specifically altered during rice728reproductive development. For instance, jasmonic acid and729phenylpropanoid biosynthesis pathways are mainly upregu-730lated during panicle development and IAA biosynthesis731during early seed development. Since several genes in-732volved in jasmonic acid and phenylpropanoid biosynthesis733have already been implicated in anther dehiscence and pol-734len development (Ma 2005; Yang et al. 2007; Millar et al.7351999; Wilson and Zhang 2009), detailed investigation of the736novel candidate genes, related to these pathways, identified737in this study would be useful.738Seed development in rice involves embryo and endo-739sperm development, encompassing cell division, organ ini-740tiation, and maturation (Agarwal et al. 2011; Itoh et al.7412005). The genes homologous to cell differentiation protein742RCD1 of wheat and FIE2 of Arabidopsis exhibit S2–S3–S4743stage-specific expression (Danilevskaya et al. 2003; Drea et744al. 2005; Okazaki et al. 1998). The homolog of Arabidopsis745late embryogenesis protein D-34 expresses specifically in746S2–S3–S4–S5 stages (Hundertmark and Hincha 2008). Rice747genes homologous to Arabidopsis MFT and DC-8 of carrot,748implicated in grain maturation and OlE-5 of coffee involved



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749 in stabilization of oil bodies in embryos were detected750 specifically in S3–S4–S5 stages (Chardon and Damerval751 2005; Cheng et al. 1996; Simkin et al. 2006). It would be752 interesting to examine if these genes in rice have similar753 functions to the ones already deduced in other plants. Also,754 the genes involved in auxin distribution and transport have755 been shown to play a role in establishing embryonic pattern756 in wheat (Fischer-Iglesias et al. 2001). Functional character-757 ization of IAA biosynthesis genes identified in this study758 might, therefore, prove useful in understanding the role of759 IAA in embryo establishment.760 The role of gibberellins in floral induction, bolting and761 development of anther, pistil, and seed has been well docu-762 mented (Hu et al. 2008). Upregulation of genes involved in763 gibberellins biosynthesis during panicle and seed develop-764 mental stages reiterates its role in rice reproductive develop-765 ment. Some of the genes controlling same step of betanidin766 degradation, brassinosteroid biosynthesis, flavonoid biosyn-767 thesis, homogalacturonon degradation, cytokinin glucoside768 biosynthesis, and sucrose degradation exhibited either769 panicle- or seed-specific expression suggesting that probably770 different genes may have taken up similar functions in differ-771 ent tissue types.772 The regulation of these metabolic and hormonal pathways773 at transcriptional level is partly executed by specific transcrip-774 tion factors. Therefore, spatial and temporal expression pat-775 terns of genes encoding transcription factors can reveal the776 target genes serving as nucleation points for building gene777 regulatory networks. The list of seed-specific transcription778 factor genes included those belonging to MYB and NAM779 families followed by noteworthy representation from AP2,780 AUX_IAA, bZIP, C3H, HSF, and MADS-box families.781 Many genes belonging to these families are critical for the782 process of seed development (Agarwal et al. 2011). Similarly,783 significant enrichment of bZIP, MADS, and C2H2 families784 during panicle development highlights potential sites of their785 function. Therefore, this analysis has revealed a large dataset786 of candidate genes, which apparently play important roles in787 different stages of panicle and seed development.788 The gene expression dataset presented here would also be789 a useful resource to mine candidates for promoter function790 validation. The genes exhibiting P3–P6 specific high ex-791 pression are likely to have anther-specific expression. The792 promoters of these genes once verified in transgenic system793 can be used to generate male-sterile lines for hybrid seed794 production (Gupta et al. 2007; Perez-Prat and van Lookeren795 Campagne 2002; Roque et al. 2007; Twell et al. 1990; Xu et796 al. 2006). Since meiosis is the main source of variation797 because of recombination, halting meiosis by targeting P2798 and P3-specific genes could be another approach to multiply799 the elite varieties without variations. Many of the genes800 exhibiting P6 stage-specific expression encode allergens.801 Switching off these genes by knock out mutation/RNAi

802approaches can have significant impact on society by reduc-803ing the allergy cases (Bhalla et al. 2001). Similarly, seed-804specific promoters could have important implications in805improving grain quality and yields in cereal crops and have806been exploited for the production of biologically/commer-807cially relevant products (Aluru et al. 2008; Furtado and808Henry 2005; Furtado et al. 2008; Lamacchia et al. 2001;809Qu le and Takaiwa 2004; Russell and Fromm 1997;810Sunilkumar et al. 2002).811The identification of reproductive stage-specific genes is812not trivial. In past few years, many groups have attempted to813understand transcriptional dynamics during rice panicle and814seed development (Endo et al. 2002; Fujita et al. 2010;815Furutani et al. 2006; Jiao et al. 2009; Kondou et al. 2006;816Ma et al. 2005; Sato et al. 2011; Wang et al. 2010).817However, very less overlap is observed among the datasets818generated across different studies. This is largely because of819differences in tissue sampling and subspecies/varieties used820in different experiments. Moreover, tissue/stage-specific ex-821pression is strongly influenced by choice of microarray822platform and parameters used for data analysis. For instance,823when we compared our panicle and seed-specific datasets824with those extracted by Wang et al. (2010) and Sato et al.825(2011), fewer genes were common between datasets gener-826ated from different rice subspecies. Since we used prepro-827cessed datasets by both the groups for comparison, different828methodologies used would be another cause of less redun-829dancy in these datasets. For extracting specific datasets, both830Wang et al. (2010) and Sato et al. (2011) have used Shannon831entropy values [<3 and <4.5, respectively] as a measure to832assess tissue-specificity. Sato et al. (2011) further filtered the833data by applying a stringent minimum expression value (8 in834log2) cut-off in at least one of stages interrogated. Since835Shannon entropy values do not discriminate between bio-836logically relevant stages, a large number of genes with837relatively high expression in non-reproductive stages have838also been shortlisted in both these reports. We, however,839worked on log2 normalized expression value cut-offs of <15840(3.9 in log2, for a gene to be called not-expressed) and >50841(5.6 in log2, for a gene to be called expressed). Selection of842these cut-offs was based on normalized signal intensity843values of non-rice probes (Magnaporthe grisea) on the rice844Affymetrix chip (Deveshwar et al. 2011), which was used to845discriminate between expressed and non-expressed genes.846In spite of all these differences, significant number of genes847was common between the panicle/seed-specific datasets848extracted in all three studies (Supplementary Figure S7).849These genes would likely have higher reproducibility and850thus would serve as candidates for detailed investigation851(Supplementary Table S4). By generating meta-profiles of852shortlisted genes/datasets, we have shown that large-scale853meta-analysis by integrating data generated in independent854studies can provide cross validation of gene expression.



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855 The next challenge would be to decipher genetic regula-856 tory networks by integrating existing information resources.857 Coupling multiple datasets with co-expression analysis will858 help in elucidating gene clusters underlying biological pro-859 cesses. As a first step towards this, we have made an attempt860 to analyze the correlation between expression and physical861 location of genes on the genome. The close placement of862 2,792 genes on rice chromosomes comprising a total of863 1,278 GCSEs suggests a link between genome organization864 and regulation of expression. Though many of these regions865 could also have resulted due to tandem duplications, pres-866 ence of different set of genes in a cluster suggested that867 these might have co-evolved for regulation of similar bio-868 logical processes. Several groups have tried to explain the869 phenomenon based on tandem duplications, overlapping870 genes, chromatin domains, shared regulatory elements, clus-871 tering of genes involved in similar metabolic pathways, etc.872 but so far no single factor could explain it (Michalak 2008).873 Further analysis of these genes will reveal if they are in-874 volved in a regulatory cascade or their expression could be875 attributed to the ripple effect of transcriptional activation,876 where intensive transcription activity at one locus may cause877 upregulation of its physically neighboring loci (Carninci878 2008; Ebisuya et al. 2008).879

880 Acknowledgements This work was supported by Department of881 Biotechnology, Ministry of Science & Technology, Government of882 India (Project No. BT/AB/FG-I(PH-II)(4)/2009). We acknowledge883 Dr. Ramesh Hariharan and his team at Strand LS Bengaluru, India884 for their help in microarray data analysis and Ms. Manupriya for885 providing the list of transcription factor family genes in rice. Senior886 Research fellowship by the Council for Scientific and Industrial Re-887 search (CSIR) to R.S., S.R., P.D, M.J., A.N., and P.S. and University888 Grants Commissions (UGC) fellowship to P.A. are acknowledged.889 Microarray data used in this study have been deposited in the Gene890 Expression Omnibus database at the National Center for Biotechnolo-891 gy Information under the accession nos. GSE6893 and GSE6901. All892 the datasets shortlisted in this manuscript including list of panicle or893 seed-specific genes, differentially expressed genes during panicle or894 seed development with respect to all four vegetative stages and unique895 genes upregulated with respect to preceding stage of development are896 given in Supplementary Table S3.

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Q1. Kindly check if the article title is correctly captured and presented.Q2. Kindly check if the affiliations are correctly captured and presented.Q3. Kindly check if the article note is correctly captured and presented.Q4. Kindly check if the figure captions (Figs. 1 to 6) are correctly captured and presented.Q5. Kindly check if this section is correctly captured and presented.Q6. Reference Jeon et al. (2008) and Wilson et al. (2009) has been updated, please check if correct.

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