The integrative expression and co-expression analysis of the AGO gene family in rice

GENE-38786; No. of pages: 15; 4C: Gene xxx (2013) xxx–xxx

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The integrative expression and co-expression analysis of the AGO gene family in rice Yang Yang, Jun Zhong, Yi-dan Ouyang, Jialing Yao ⁎

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College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

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Article history: Accepted 3 July 2013 Available online xxxx

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Argonautes (AGOs) play crucial roles in RNAi and related pathways in several species and regulate plant growth and development. However the investigation in rice argonautes (OsAGOs) remains elusive. Here we focused on the expression pattern and co-expression profiles of OsAGO genes. Microarray-based and qRT-PCR expression profiling of 19 OsAGO genes indicated that most OsAGOs expressed specifically and preferentially during stages of reproductive development, and exhibited preferential up-regulation in panicle stages. Six OsAGO genes showed specific up/down-regulation in response to Gibberellin A3 (GA3), Kinetin (KT), or 1-Naphthaleneacetic acid (NAA) treatments. And three OsAGOs presented specific up-regulation in response to light and dark treatments. Ten OsAGOs were co-expressed with Dicer-like (DCL), Double-stranded RNA Binding (DRB) and RNAdependent RNA polymerase (RDR) genes, which were related with RNA processing including RNAi pathways. Twelve OsAGOs were correlated with 17 kinds of transcription factors involving diverse functions. Four OsAGOs RNAi plants were constructed, the expression level of co-expression genes, including DCL3, DRB2, RDR4 etc., were changed while OsAGOs were down-regulated in RNAi lines, providing experimental evidence for coexpression networks. The results provide new insights in understanding the biological pathways of OsAGO genes, as well as in selecting the candidate genes involved in RNA silencing mechanisms. © 2013 Elsevier B.V. All rights reserved.

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Keywords: Argonaute Expression analysis Co-expression analysis Rice

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1. Introduction

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Argonaute (AGO) proteins are known as core components of the RNA interference effectors complex RISC (RNA-induced silencing complex) (Song et al., 2004). Small RNA guided AGO proteins in modulating gene expression at various levels, including RNA cleavage (all eukaryotes), internal genomic DNA sequence elimination (in ciliates) and translational repression (animals). In some cases such regulations were followed by DNA methylation and chromatin remodeling (Vaucheret, 2008). Several AGO proteins cleaved target mRNAs in the middle of their small interfering RNA (siRNA) or microRNA (miRNA) complementary sequence, leading to RNA interference (RNAi) (Vaucheret, 2008). AGO proteins contain several functional domains, which are DUF1785, PAZ and PIWI domains (Hutvagner and Simard, 2008). A remarkable feature of the PAZ domain is that it can recognize the 3′-ends of

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Abbreviations: AGO, Argonaute; RISC, RNA-induced silencing complex; RNAi, RNA interference; MH63, MingHui63 (O. sativa ssp. Japonica); ZS97, Zhenshan97 (O. sativa ssp. Japonica); ZH11, Zhonghua11 (O. sativa ssp. Japonica); GA3, Gibberellin A3; KT, Kinetin; NAA, 1-Naphthaleneacetic acid; PCCs, Pearson's correlation coefficients; GO, Gene Ontology; qRT-PCR, Quantitative Reverse Transcriptase Polymerase Chain Reaction; DCL, Dicer-like; DRB, Double-stranded RNA Binding; RDR, RNA-dependent RNA polymerase. ⁎ Corresponding author. Tel.: +862787282866. E-mail address: [email protected] (J. Yao).

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small RNAs (Hutvagner and Simard, 2008; Tolia and Joshua-Tor, 2007), in contrast to the PIWI domain which has an RNase-H-like fold (Hutvagner and Simard, 2008). It is suggested that PAZ binds the miRNA or the siRNA, whereas the PIWI domain of some AGO proteins may function as an RNaseH domain that cleaves the mRNA (Lingel and Sattler, 2005; Tang, 2005). Expression profiles in female gametophyte of Arabidopsis suggested that genes encoding PAZ, PIWI domain or DUF1785 protein were involved in tRNA, rRNA, or mRNA processing (Wuest et al., 2010). The AGO family genes have been identified in several plants, such as Arabidopsis, rice, maize, and tomato (Bai et al., 2012; Kapoor et al., 2008; Qian et al., 2011), but the number of AGO genes varies greatly among different species. AGOs play important roles in regulation of development and stress responses, antiviral immune response, transposons mediated by miRNA and/or siRNA, and regulation of chromatin structure (Vaucheret, 2008). They can affect the growth and development as well as the response to abiotic and biotic stress (Vaucheret, 2008). In addition, the mutants in Arabidopsis are valuable in identifying their biological functions individually (Carmell et al., 2002). For example, AtAGO1 is involved in antiviral defense (Vaucheret, 2008), while AtAGO7 is responsible for the transition from juvenile to adult vegetative stage (Adenot et al., 2006; Fahlgren et al., 2006; Hunter et al., 2006). The Arabidopsis protein AGO9 controls female gamete formation by restricting the specification of gametophyte precursors in a dosagedependent non-cell-autonomous manner (Olmedo-Monfil et al., 2010).

0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.07.002

Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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Protein subcellular locations were analyzed by using WoLF 98 PSORT (http://psort.nibb.ac.jp/), an extension of the PSORT II pro- 99 gram (http://www.psort.org). 100

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2.1. Analysis in physical and chemical properties

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qRT-PCR. These formed the basis for further functional research of OsAGOs during rice growth and reproductive development, and contribute to screen candidate genes in RNAi-related pathways. The construction of the co-expression networks of several OsAGOs and their related genes provided valuable clues in understanding the biological functions of the rice AGOs.

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The studies in maize AGO104 pointed out that AGO104 played a crucial role in epigenetic regulation in sexual development, which suggested an important link to apomictic development (Singh et al., 2011). Rice, a model of monocotyledonous plants, is one of the major food crops all over the world. Though 19 AGO genes are identified in rice (Kapoor et al., 2008), only two OsAGOs genes, OsMEL1 and OsPNH1, have been characterized. OsMEL1 is involved specifically in rice male meiosis (Nonomura et al., 2007). OsPNH1 functions not only in stem apical meristem (SAM) maintenance, but also in leaf formation through vascular development (Nishimura et al., 2002). In this study, we focused on an integrative analysis of expression and co-expression profiling using 25 tissues/developmental stages in 2 different rice varieties, MingHui63 (MH63) and Zhenshan97 (ZS97), respectively. Moreover, the expression pattern in Zhonghua11 (ZH11) was investigated by

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Fig. 1. Expression patterns of OsAGO genes found in segmentally duplicated regions of the rice genome (A) and present as tandem duplicates (B). X-axis represents the developmental stages as given in the following table. Y-axis represents the raw expression values obtained from microarray. The detailed information of the samples is listed in Additional files 3: Table S3.

Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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Fig. 2. Expression patterns of OsAGO genes during the life cycle of the rice plant. Hierarchical cluster display the expression profile for 19 OsAGO genes with probes in the Affymetrix microarray. (Color bar at the base represents log2 expression values: green, representing low expression; black, medium expression; red, high expression.) The detailed information of the samples is listed in Additional files 1: Table S1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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2.3. Genome-wide expression analysis of OsAGO in rice

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The expression profile data and co-expression profile data of OsAGO in different tissue samples of ZS97 (Oryza sativa L. ssp. indica) and MH63 (O. sativa L. ssp. indica) were obtained from the CREP database (http:// crep.ncpgr.cn) (Wang et al., 2010). The developmental stages and organs of the tissues were described in Table S1. After normalization and variance stabilization (Gautier et al., 2004; Gentleman et al., 2004; Wang et al., 2010a), the average signal value of two biological was replicated for each sample. When more than one probe sets were available for one gene, the higher signal value of the probe set was used for analysis. For phytohormone treatments, seedlings at trefoil stage were treated with 0.1 mM NAA, GA3 and KT, respectively. Then samples were harvested at the time points of 5, 15, 30 and 60 min after that. For light treatments, samples were extracted after 48 h light or dark treatments. Expression value of each gene was logarithmized, and cluster analyses were performed using R with Euclidean distances and hierarchical cluster method of “complete linkage”. For data analysis, expression level in each tissue was compared against that in seed by using a student-t test. The genes, which were up- or down-regulated by more

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The permutation test was done to determine the optimal threshold of the Pearson's correlation coefficients (PCCs) (Butte et al., 2000; Carter et al., 2004). We used probes in the Affymetrix microarray of all the correlated genes in two sets of transcriptomes to calculate the PCCs for all pairs of OsAGO genes with (expression profiles for two varieties ZS97 and MH63 in CREP database, http://crep.ncpgr.cn) comprising a total of 190 microarray experiments. The distribution of the PCCs was observed to choose the optimal thresholds. In order to eliminate the interference of the constitutive expression pattern with the correlation of expression level OsAGO genes with standard errors greater than 500 were used for further co-expression analysis. The correlated genes with PCCs higher than the optimal thresholds were extracted from the CREP database (http://crep.ncpgr.cn) (Wang et al., 2010) and considered as the putative co-expression genes. Statistical significance for candidate genes in co-expression network construction was further determined by using a student-t-test. The co-expression network was constructed with a visualization tool of Cytoscape. A web-based tool Singular Enrichment Analysis (SEA) in agriGO (http://bioinfo.cau.edu.cn/agriGO/index.php) was applied to analyze the Gene Ontology (GO) enrichment with default parameters (Du et al., 2010), which used the rice MSU6.1 genome annotation as

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The duplicated genes were elucidated from the segmental genome duplication and tandem duplications of rice in MSU (http://rice. plantbiology.msu.edu/).

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than two-fold and with p values b0.05, were considered to express dif- 125 ferentially. The average expression of more than two biological replica- 126 tions for each sample was used for analysis. 127

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Fig. 3. Comparison of OsAGO gene expression during vegetative and reproductive development. Red, up-regulated in both MH63 and ZS97 during vegetative development; orange, upregulated in MH63 during vegetative development; purple, up-regulated in ZS97 during vegetative development; dark blue, down-regulated in both MH63 and ZS97 during reproductive development; green, down-regulated in MH63 during reproductive development; light blue, down-regulated in ZS97 during reproductive development; black, up-regulated; grey, upregulated and down-regulated; light grey, down-regulated; white, no change. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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Gene-specific primers were designed for all the OsAGO genes with Primer 5 (Table S4). The samples were collected from Zhonghua 11 (O. sativa ssp. Japonica), and total RNA was isolated from root, stem, leaf, panicle at stage 3, panicle at stage 5, panicle at stage 8, endosperm1 (3 days after pollination), and endosperm 2 (7 days after pollination). The tissues were ground in liquid nitrogen using a mortar and pestle. Total RNA (3 mg) was treated with 3 units of DNase (Promega) and then put into use in first-strand synthesis with an oligo (dT) primer (20 mer) and reverse transcriptase according to the manufacturer's instructions (TransGen, China). For real-time PCR, an amount of cDNA corresponding to 25 ng of input RNA was used in each reaction. Reactions were performed in a final volume of 0.5 μM of each primer, 10 μg of cDNA and 20 μl containing 10 μl of 2× SYBR Green Master Mix. PCR conditions were as follows: 3 min at 95 °C for predenaturation, followed by 29 cycles of 20 s at 95 °C, 20 s at 60 °C and 30 s at 72 °C, and a final 5 min extension at 72 °C. Fluorescence threshold cycle (Ct) data were analyzed by using the MJ Opticon Monitor Software version 3.1 (Bio-Rad, USA) and then exported to Microsoft Excel for further analysis. Relative expression levels in

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The construct pDS1301 vector was used as an RNAi vector (Chu et al., 2006). Specific cDNA fragments of OsAGOs (OsAGO1b, OsAGO1c, OsAGO4a and OsAGO17) from the coding region of the 5′-end were applied. The dsF contained KpnI and BamhI restriction enzyme sites (underlined) at the 5′-end, while dsR contained SacI and SpeI restriction enzyme sites (under-lined) at the 3′-end (Table S4). RNAi constructs were transferred into Agrobacterium tumefaciens strain EHA105 by electroporation. Agrobacterium-mediated transformation was performed using calli derived from mature embryos of rice variety ZH11 (O. sativa ssp. japonica) (Lin and Zhang, 2005). The expression of OsAGOs and co-expression gene in transgenic negative plants and RNAi plants was detected by qRT-PCR. The total RNA of young leaves was isolated from 3 independent RNAi T2 generation plants for each OsAGO gene. The primers of OsAGO genes were showed in Table S4, and co-expression gene-specific primers were designed using Primer 5 (Table S4). qRT-PCR was performed according to Section 2.5 protocol.

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2.6. Construction of OsAGOs RNAi interference (RNAi) plants and related 174 genes expression detection 175

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each cDNA sample were normalized to an ubiquitin reference gene 172 (Table S4). 173

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the background. Statistical significance was done through the Fisher's exact test and the Yekutieli multi-test adjustment (Du et al., 2010).

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Fig. 4. Quantitative RT-PCR verification of the expression of OsAGO genes in vegetative and reproductive organs of ZH11. R, roots of 7-day-seeding; S, stems of 7-day-seeding; L, leaves of 7-day-seeding; P3, panicles at stage 3; P5, panicles at stage 5; P8, panicles at stage 8; E5, endosperm of 5 days after pollination; E7, endosperm of 7 days after pollination. The results are consistent with the data from database, OsAGOs expressed especially high in panicle stage.

Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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3.2. Gene duplication

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Eleven out of the nineteen OsAGO genes were found to be duplicated genes. Four pairs were suggested as segmental duplicated genes (AGO1a and AGO1b, AGO1a and AGO1d, AGO1b and AGO1d, MEL1 and AGO14), while three pairs were classified as tandemly duplicated genes (AGO4a and AGO15, AGO11 and AGO12, AGO2 and AGO3). The segmental genes were located in different chromosomes. AGO1a, 1b, 1d, 14 and MEL1 was located in chromosome 2, 4, 6, 7 and 3, respectively. In contrast, the tandemly duplicated gene pairs were located either next to each other, or separated with one gene (Fig. 1, Table S3).

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The expression profiling of OsAGO genes covering 24 developmental stages (Fig. 2, Table S1) in MH63 and ZS97 was analyzed using Affymetrix rice microarray data in CREP database (Wang et al., 2010). OsAGOs could be divided into two groups at the expression level. The average expression level of group one was nearly as twice as another one. In MH63, the high expression level group contained 10 members, which were AGO1a, AGO1b, AGO1c, AGO1d, AGO2, AGO4a, AGO4b, AGO16, AGO18, and MEL1. The low expression level group comprised AGO3, AGO7, AGO11, AGO12, AGO13, AGO14, AGO15, AGO17, and PNH1. In ZS97, 8 OsAGO genes (AGO1a, AGO 1b, AGO1c, AGO1d, AGO2, AGO4a, AGO4b, and MEL1) belonged to the high expression level group and the remaining (AGO3, AGO7, AGO11, AGO12, AGO13, AGO14, AGO15, AGO16, AGO17, AGO18, and PNH1) were in the low group. The majority of OsAGOs had the similar expression pattern in MH63 and ZS97, except OsAGO16 and OsAGO18, which expressed higher in MH63 than that in ZS97. Remarkably, all the OsAGOs were significantly up-regulated in panicles when comparing with other tissues (Fig. 2). We compared the expression levels of OsAGOs in panicle 1, panicle 2, panicle 3 and panicle 5 with those in seeds (control samples) in both MH63 and ZS97 during vegetative development and reproductive development (Fig. 3, Table S2). Eleven genes, OsAGO1a, OsAGO1b, OsAGO2, OsAGO4a, OsAGO12, OsAGO13, OsAGO14, OsAGO16, OsAGO17, OsAGO18 and MEL1 showed up-regulated pattern during reproductive development (Fig. 3, Table S2). It can be seen from the results that AGO1a, AGO1b, AGO1c, AGO1d and AGO4a, AGO4b belonged to high expression level group and had close evolutionary relationship although

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In order to characterize the properties of OsAGOs, the protein length, molecular weight, isoelectric point (pI), and subcelluler localization were predicted. The protein lengths of most OsAGOs were expected to range from 877 to 1119 amino acids and molecular weights were from 100,639 to 123,592 Da. The predicted pI ranged from 8.3197 to 10.0018. According to the results, these 19 OsAGOs might have similar physical properties. Most of OsAGOs (13 of 19) were expected to localize in nuclei and the other four OsAGOs were suggested to be in cytoplasm. The rest two genes were predicted to be in mitochondria and peroxisomes, respectively. Only 2 (OsAGO3 and OsAGO16) of the 19 OsAGO proteins were predicted to be stable. The results implied that these OsAGOs might play different roles.

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3.1. Physical and chemical properties of OsAGOs

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3.3. Expression profiles of OsAGO genes during vegetative and reproductive 219 development 220

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Fig. 5. The response to hormones of OsAGO genes. The hormones included GA3, KT, NAA. Red, up-regulated in both MH63 and ZS97; orange, up-regulated in MH63; purple, up-regulated in ZS97; dark blue, down-regulated in both MH63 and ZS97; green, down-regulated in MH63; light blue, down-regulated in ZS97; black, up-regulated; light grey, down-regulated; white, no change. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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As phytohormones play critical roles in rice growth and development, we analyzed the expression of OsAGO genes under GA3, KT, and NAA treatments by microarray database. We identified that 6 OsAGO genes were differentially expressed with treatments of one or more of the phytohormone NAA, KT, GA3 in seedlings when compared with the control ones (Fig. 5). The fold change values with respect to control are given in Table S2. Under the treatment of GA3, the expression levels of OsAGO1a and OsAGO1d were up-regulated in MH63 but there was no change in ZS97. The expression of OsAGO3 decreased in MH63, while the expression of OsAGO16 increased in ZS97. Under the treatment of KT, the expression of OsAGO1a in MH63 and OsAGO1d and OsAGO13 in ZS97 was up-regulated, while other OsAGOs showed no response to KT treatment. When it comes to NAA treatment, OsAGO1a and OsAGO1d in MH63, OsAGO13 and OsAGO16 in ZS97 were up-regulated, whereas

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3.5. Responses of OsAGO genes under NAA, KT, and GA3 treatments

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To confirm the microarray database and investigate the abundance of OsAGOs in ZH11 (Oryza sativa L. ssp. Japonica), the expression levels of all OsAGOs were analyzed by qRT-PCR in 8 organs and tissues (root, stem, leaf, panicle at stage 3, panicle at stage 5, panicle at stage 8, endosperm: 5 days after pollination, endosperm: 7 days after pollination).

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The results showed that all the OsAGOs expression could be detected in tissues examined. Remarkably, for 17 OsAGOs, the expression levels in at least one stage of the panicles were higher than those in the other organs and tissues (Fig. 4, Table S4). Among which, OsAGO4a, OsAGO4b, OsAGO7, OsAGO11, OsAGO12, OsAGO15, OsAGO16, OsAGO17, OsPNH1 and OsMEL1 showed quantity peaks in P3, while OsAGO1b, OsAGO1c, OsAGO3, OsAGO13 were with highest abundance in P5 and OsAGO1a, OsAGO1d, OsAGO14 were in P8. In contrast, OsAGO2 showed a quantity peak in stems while OsAGO18 peaked in leaves. When compared with microarray data, qRT-PCR revealed the same expression patterns for most genes, despite some quantitative differences in expression levels in MH63 and ZS97.

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their expression patterns showed some differences. In MH63, AGO1a and AGO1b were up-regulated in stem of heading stage and panicle 1, 2, 3 and 5 stages. AGO1c was up-regulated in panicle 1, panicle 3 and panicle 5 while AGO1d was up-regulated in 15 out of 25 analyzed tissues, such as root 1 (seedling with 2 tillers), sheath1 (secondary branch primordium differentiation stage), leaf 2 (4–5 cm young panicle stage), stem 1 (days before heading stage), stem 2 (heading stage), flag leaf 3 (days before heading stage), flag leaf 4 (14 days after heading stage), panicle 1,2,3,5 and panicle 8 (heading stage), glume (one day before flowering), stamen (one day before flowering) and spikelet (3 days after pollination). The AGO4a was up-regulated from panicle development stage 1 to stage 3 and stage 5, endosperm 1 (7 days after pollination), endosperm 2 (14 days after pollination), and endosperm 3 (21 days after pollination), but AGO4b was up-regulated from panicle development stage 1 to stage 3 (Fig. 3, Table S2). Similarly, in ZS97, AGO1a, AGO1b, AGO1c, AGO1d and AGO4a, AGO4b also showed some differences at the expression level. In a word, the expression characters of OsAGOs suggested their important roles involved in the growth and development of rice. It is noticeable that the expression levels of more than half genes in reproductive tissues were higher than those in vegetative ones. Especially, most OsAGOs preferentially expressed in panicles at 5 development stages.

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Fig. 6. OsAGOs expression pattern after light treatment in MH63 and ZS97.

Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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Here the distribution of the PCCs suggested that the p value higher than 0.75 was significant (Table S5). Based on this, we found that 1707 genes were tightly correlated with the expression of 15 OsAGO genes which were with the PCC higher than 0.75 and p value lower than 0.05. Then according to the functional annotation and web-based GO analysis, 7 networks were constructed concerning correlations between OsAGO genes and their co-expressed genes to reflect functional bias and possible molecular pathways that they involved in. Fig. 7 showed the networks containing 10 OsAGO genes and 62 genes involved in RNA processing. Considering the large information in such

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Comparing with the expression levels in seeds, AGO1b and AGO17 were up-regulated both in plumule and radicle of seedling under light and dark treatments in MH63 and ZS97. The expression levels of AGO4a, AGO4b and PNH1 were up-regulated in plumule, which were down-regulated in radicle under both treatments (Fig. 6). No matter under light treatment or dark treatment, expression levels of OsAGOs showed more changes in plumule than those in radical (Fig. 6), which indicated that these OsAGOs might be more active in plumule than those in radicle. It is likely that OsAGOs responded to light treatment in different pathways.

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complex network, we reconstructed the sub-networks among these 10 OsAGOs (Fig. S1). It is noted that these OsAGOs showed co-expression relationship with DCL3, a member of the Dicer family (Fig. 11D). In Fig. 8, the GO analysis showed biological processes and molecular function of these genes that involved in RNA metabolic processing, RNA polymerase activity, ATP-dependent helicase activity and so on. The networks in Fig. 9 contained 12 OsAGO genes and a number of transcription factors, including 12 C2H2 genes, 10 PHD genes, 10 MYB genes, 8 SNF2 genes, 5 FHA genes, 4 HMG genes, 3 AP2 genes, 3 MADS genes (MADS5, MADS15, MADS32), 2 basic helix-loop-helix (bHLH) genes, 2 SET genes, 2 SWI/SNF genes, 2 bZIP genes, 2 GNAT genes, 1 WRKY gene, 1 GRF2 gene, 1 CCAAT gene, and 1 TCP gene. The networks of each OsAGO gene were presented in Fig. S2. It showed that each OsAGO gene was co-expressed with different transcription factors, and vice verse, one transcription factor might regulate a series of OsAGOs. The GO analysis in Fig. 10 suggested that the co-expression genes in Fig. 9 were involved in multiple biological processes, such as regulation of biological process, regulation of cellular process, regulation of metabolic process, and regulation of transcription. The regulation processes and molecular function such as nucleic acid binding, DNA binding, sequence-specific DNA binding, hydrolase activity, pyrophosphatase activity, nucleoside-triphosphatase activity, and helicase activity were included as well. Besides, 12 out of the 19 OsAGOs were co-expressed with each other (Fig. 12A), 11 OsAGOs have co-expression relationships with 10 auxin related genes (Fig. 12B), and 10 OsAGOs were co-expressed with 3 anther development related genes and 5 pollen development related genes (Fig. 12C). 10 out of the 19 OsAGOs, named OsAGO1a, OsAGO1b, OsAGO1c, OsAGO1d, OsAGO4a, OsAGO4b, OsAGO7, OsAGO16, OsAGO17and OsPNH1were co-expressed with more than 1000 other genes including DCL3, RNA processing genes, transcription factor genes, F-box genes,

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OsAGO7 in MH63 was down-regulated. It can be seen from the results that OsAGO1a, OsAGO1d, OsAGO13, and OsAGO16 responded actively to hormones (GA3, KT, NAA), and OsAGO3 and OsAGO7 were sensitive to hormones (Fig. 5), although differences existed among varieties. Therefore, these OsAGOs which responded to hormones might participate in different hormone pathways.

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Fig. 7. Co-expression network comprehensive analysis of OsAGO genes and RNA processing genes.

Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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Fig. 8. Significant GO annotations for genes indicated in the co-expression networks in Fig. 7. The boxes in the graph list the GO identifier, the statistical significance, and the description of the GO term. The color of the box indicates the significance of the term (p b 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Based on the constructed co-expression networks of OsAGOs, we selected randomly four genes from ten OsAGO genes which co-expressed with more than 1000 other genes. And co-expression genes which involved in RNA related and transcription factors were selected, for further verification of putative co-expression networks, RNA interference (RNAi) was used to down-regulate OsAGO1b, OsAGO1c, OsAGO4b and OsAGO17 transcript levels. And expression level of the four OsAGO genes and some genes that were co-expression with these OsAGOs in transgenic plants were analyzed by qRT-PCR (Fig. 13). As the expression levels of AGO1b were dramatically down-regulated in RNA interfered T2 plants, OsRDR4 and OsDCL3 were down-regulated while OsDRB2 and OsZOS1-12 were remarkably up-regulated. The expression levels of OsAGO1c were down-regulated in OsAGO1c RNAi T2 plants, expression increase of OsZOS9-15, OsMADS5 and Os06g16400

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pollen development related genes, and anther development related genes (Table S5). Meanwhile these ten genes were co-expressed with each other (Fig. 11A). The whole rice genome annotation displayed that these OsAGO genes seemed to affect the regulation of multiple biological processes and functions in molecular pathways (Fig. 12). It infers that these ten OsAGOs with co-expression genes play active roles in the vegetative and reproductive development in rice.

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(encoding bHLH protein) was noteworthy but expression of OsDCL3 was decreased. In OsAGO4b RNA interfered T2 plants, OsAGO4b was down-regulated, all the tested co-expression genes including OsDCL3, OsDRB2, OsRDR4, OsMADS5 and OsZOS1-12 were up-regulated. The expression levels of OsAGO17 were down-regulated in RNAi T2 plants, all the tested co-expression genes including OsDCL3, OsDRB2, OsRDR4 and OsZOS9-15 were down-regulated. Interestingly, expression levels of OsDCL3 in three tested OsAGOs RNAi lines were significantly decreased. In a word, the expression levels of all the tested co-expression genes that presented in putative co-expression networks showed changes along with the OsAGOs silencing. These results indicated that the coexpression networks based on the expression data were credible. It implied that the four OsAGO genes may participate in pathways which the co-expressed genes involved, and also provided more clues to study the roles of AGOs in rice.

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4.1. OsAGO evolution and classification

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Gene families were extended by three major mechanisms: segmental duplication, tandem duplication and retroposition (Kong et al., 2007). In this study, the fate of duplicated genes in OsAGOs could be described as nonfunctionalization, subfunctionalization

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Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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Fig. 9. Co-expression network comprehensive analysis of OsAGO genes and transcription factor encoding genes.

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The expression patterns are likely to suggest the possible functions and metabolism pathways of the corresponding genes. Thus the coexpression networks provide valuable clues for identifying genes involved in the same pathway. The results from the co-expression networks indicated that the 10 rice AGO genes mentioned in Fig. 11D might participate in the regulation of various RNA processing including RNA interference. There are several evidences to support our inferences. First of all, OsDCL3 was found to be co-expressed with 10 rice AGO genes (Fig. 11D), and the expression level of OsDCL3 indeed changed in four

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and neofunctionalization. The expression patterns of three pairs of the segmental genes (OsAGO1a and OsAGO1b, OsAGO1a and OsAGO1d, OsAGO1b and OsAGO1d) were overlapped, suggesting that these gene pairs might undergo neofunctionalization. The expression patterns of another pair of the segmental genes (MEL1 and OsAGO14) were partial complementary, which might indicate the process of subfunctionalization. In two cases of tandem duplication genes (OsAGO2 and OsAGO3, OsAGO4a and OsAGO15), one of the duplicated genes (OsAGO2 and OsAGO4a) was not expressed at significant levels in all tissues. We might infer that one of the copies lost its function during the evolution by nonfunctionalization. The last pair (OsAGO11 and OsAGO12) of the tandem genes could be described as neofunctionalization because of their similar expression patterns. During the process of evolution, the functions of the genes might reserve, or lose, or gain new functions.

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OsAGOs RNA interference plants (Fig. 13). OsDCL3 was homologous to AtDCL3 in Arabidopsis, which was involved in RNA metabolism with dsRNA-binding activity (Hiraguri et al., 2005). The processing of 24-nucleotide phased small RNA required the participation of DCL3 (Song et al., 2012). Besides, it is reported that OsDCL4 was the major dicer responsible for the 21-nucleotide siRNAs associated with inverted repeat transgenes and for trans-acting siRNA (ta-siRNA) from the endogenous TRANS-ACTING siRNA3 (TAS3) gene (Liu et al., 2007). Therefore, the OsDCL3 might regulate RNA processing pathway in relation with rice AGO genes. Secondly, the Arabidopsis AGO genes, which have already been functionally characterized, provide useful information in understanding OsAGOs. It is noticeable that four rice AGO genes, OsAGO1a, OsAGO1b, OsAGO1c, and OsAGO1d, had close relationship with AtAGO1 and OsAGO7, while PNH1 was homologous to AtAGO7 and AtAGO10, respectively (Kapoor et al., 2008). Besides, OsAGO4a and OsAGO4b are in close relationship with AtAGO4 and ZmAGO104, respectively (Fig. 14). AtAGO1 was normally limiting, and the level of AtAGO1 is maintained in equilibrium by the dual effect of miR168 on AtAGO1 mRNA, as well as that of AtAGO1 protein on miR168 (Vaucheret et al., 2004; Vaucheret et al., 2006). AtAGO7 preferentially associated with miR390, which guided the cleavage of TAS3 precursor RNA (Montgomery et al., 2008). AtAGO6 specifically acted in the heterochromatin siRNA and TGS pathways, and the activity of AtAGO6 was partially redundant with the activity of AGO4 (Zheng et al., 2007). The molecular function of AtAGO10 remained undetermined, but it was the closest paralogue of AtAGO1 (Kapoor et al., 2008). Since AtAGO1 acted in the miRNA and siRNA pathways, it was likely that AtAGO10 might also participate in these pathways, at least in some tissues (Vaucheret, 2008).

Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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Fig. 10. Significant GO annotations for genes indicated in the co-expression networks in Fig. 10. The boxes in the graph list the GO identifier, the statistical significance, and the description of the GO term. The color of the box indicates the significance of the term (p b 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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In addition, in this study, we found that OsAGOs were co-expressed with 62 RNA processing genes, which would further support the inference that OsAGOs might be involved in RNA pathways (Fig. 7). The GO analysis also confirmed the inference that these OsAGOs are involved in important RNA-related molecular functions. For example, three OsRDR genes and OsDRB2 were contained in the 62 RNA processing genes. OsRDR genes encoded RNA-dependent RNA polymerase and OsDRB were double-stranded RNA binding motif containing protein. Although, the specific functions of these genes are still unknown in rice, the function of the homologous gene AtDRB2 in Arabidopsis is

clear. In the SAM region, AtDRB2 is both antagonistic and synergistic to the role of DRB1 in miRNA biogenesis, adding an additional layer of gene regulatory complexity in this developmentally important tissue (Eamens et al., 2012). In rice, seven genes, DRB2, DRB4, DRB5, DRB1, DCL1, DCL3, and HYL1, exhibited significant dsRNA-binding activity, which indicated that these proteins might be involved in RNA metabolism (Hiraguri et al., 2005). Concerted activities of DCLs, DRBs, and RDRs are required in RNA silencing pathways (Bai et al., 2012; Mallory and Vaucheret, 2010). In this study, the three RNA processes related important genes all showed changed expression levels in OsAGOs RNAi plants,

Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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implied that OsAGOs with OsDCLs, OsRDRs, OsDRBs performed their functions in RNA silencing pathways. However, the trend of expression level changes of OsDCLs, OsRDRs, OsDRB in different OsAGO interference plants was different, suggesting that the relationship of OsAGOs with OsDCLs, OsRDRs, OsDRBs could be complicated. Furthermore, direct molecular evidence suggested that the rice AGO gene MEL1 regulated the cell division of premeiotic germ cells, the proper modification of meiotic chromosomes, and the faithful progression of meiosis, probably via small RNA-mediated gene silencing, but not the initiation and establishment of germ cells themselves (Nonomura et al., 2007). Analysis in AGO1a, AGO1b, and AGO1c also suggested that the three AGO1s had a strong preference for binding small RNAs (sRNAs) with 5′U and had slicer activity (Wu et al., 2009). Thus in all, the ten OsAGOs mentioned above might be involved in RNA pathways with a number of RNA-related processing genes.

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Fig. 11. Co-expression network comprehensive analysis of OsAGO genes and correlated genes. A, OsAGOs co-expressed with each other. B, OsAGOs co-expressed with auxin genes. C, OsAGOs co-expressed with anther and pollen related genes. D, OsAGOs co-expressed with OsDCL3.

4.3. OsAGO genes may initiate their diverse functions by performing interaction with various transcription factors Transcription factors (TFs) are DNA-binding proteins that regulate gene expression at the level of mRNA transcription (Xue et al., 2012). Co-expression analysis in this study showed that 12 OsAGO genes

were correlated with 17 groups of transcription factors (Figs. 9 and S2). Each of the 12 OsAGOs co-expressed with more than one kind of the transcription factors. It was possible that OsAGOs and these transcription factors were related within these various biological processes and molecular functions, such as flower and seed development, and in response to abiotic stress. Among the co-expression TFs, the largest group contains 12 C2H2 transcription factors which belonged to ZOS gene family. Previous reports showed that 6 out of the 12 ZOS genes of rice, ZOS1-12, ZOS1-21, ZOS3-01, ZOS3-03, ZOS5-01 and ZOS9-15, were up-regulated in panicle and seed development, while ZOS12-04 was down-regulated in both panicles and seed development (Agarwal et al., 2007). Our analysis showed that OsAGOs were co-expressed with these ZOS genes and upregulated during panicle development stages. Furthermore, OsZOS9-15 was up-regulated in OsAGO1c RNAi T2 plants but down-regulated in OsAGO17 RNAi T2 plants (Fig. 13). And ZOS1-12 was remarkably upregulated in OsAGO1b and OsAGO4b RNAi T2 plants (Fig. 13). These co-expression and expression characters of OsAGOs and ZOS might imply that they played roles in the same pathway during panicle development. MYB proteins constitute a diverse class of DNA-binding proteins of particular importance in transcriptional regulation in plants.

Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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Fig. 12. Significant GO annotations for genes indicated in the co-expression networks, including OsAGOs and all the co-expressed genes. The boxes in the graph list the GO identifier, the statistical significance, and the description of the GO term. The color of the box indicates the significance of the term (p b 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Functions of MYB proteins in plants include regulation of secondary metabolism, control of cellular morphogenesis and regulation of meristem formation, and the cell cycle (Suzuki et al., 1997). Thus, we inferred that MYB proteins might interact with a number of AGO proteins, and they either activated or repressed the expression of sets of target genes that were increasingly being identified through a diversity of high-throughput genomic approaches. For example, Os02g42870 co-expressed with OsAGOs, and it is a member of R2R3-MYBs, which control phenylpropanoid biosynthesis, glucosinolate biosynthesis and in response to hormone- and pathogen-mediated stress, also involve the control of cell shape and the formation of root hairs and trichomes (Feller et al., 2011). However, what was the interaction of OsAGOs and MYB proteins, and how do they control biosynthesis, response to stress and involve the development of the rice, were needed to answer this question in future.

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MADS-box transcription factors are essential for various aspects of pathways in reproductive transition and flower development in dicotyledon and monocotyledon (Kater et al., 2006). For example, OsMADS5 gene was expressed preferentially in anthers, and OsMADS5 is structurally related to the AGL2 family and may be involved in controlling flowering time (Kang and An, 1997). In rice, FAC induces transcription of OsMADS15, a homologue of AP1 of Arabidopsis, which led to early flowering (Taoka et al., 2011). In this study, from the co-expression networks, AGO1b, AGO1c, AGO1d, AGO4a, AGO4b, AGO7, AGO16, AGO17, MEL1, PNH1 had co-expression relationship with MADS5, and AGO1a, AGO2 co-expressed with MADS15, implying that these 12 OsAGO genes may involve in flowering processing and flower development in rice. In confirmed co-expression networks of this study, the expression levels of OsMADS5 were changed in OsAGO1c and OsAGO4b RNAi T2 plants (Fig. 13), prompting us to suppose that OsAGOs and OsMADS5 may be related with anther development and flowering time controlling.

Please cite this article as: Yang, Y., et al., The integrative expression and co-expression analysis of the AGO gene family in rice, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.002

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Fig. 13. Quantitative RT-PCR verification of the expression of OsAGO genes and co-expression genes.

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In this study, we performed a validated approach in finding out useful clues in OsAGOs function and interaction. The approach relies on a comprehensive phylogenetic analysis, in addition with integrating the expression profiling and co-expression networks. The results revealed that the OsAGO proteins played important roles in siRNA and miRNA pathways, regulating the vegetative and reproductive development of

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Fig. 14. Phylogenetic analysis of OsAGO4a, 4b proteins and AtAGO4, ZmAGO104. The tree was generated using ClustalX by neighbor-joining method with the alignments of OsAGO protein sequences and AtAGO4, ZmAGO104 protein sequences.

rice, and in response to hormones. These results would provide insights into functional analysis of AGO family in rice. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.07.002.

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This research was supported by grants from the National Natural Science Foundation of China (Grant No. 30971551), the Fundamental Research Funds for the Central Universities (Grant No. 2012MBDX012) and Huazhong Agricultural University Scientific & Technological Selfinnovation Foundation (Grant No. 2012SC10).

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