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First published online June 7, 2007; 10.1104/pp.107.101600 Plant Physiology 144:1797-1812 (2007) © 2007 American Society of Plant Biologists
Genome-Wide Gene Expression Profiling Reveals Conserved and Novel Molecular Functions of the Stigma in Rice1,[W]Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences and National Centre for Plant Gene Research, Beijing 100080, China (M.L., W.X., W.Y., Z.K., Y.X.); and Graduate University of Chinese Academy of Sciences, Beijing 100049, China (M.L., W.Y., Z.K.)
In angiosperms, the stigma provides initial nutrients and guidance cues for pollen grain germination and tube growth. However, little is known about the genes that regulate these processes in rice (Oryza sativa). Here, we generate rice stigma-specific or -preferential gene expression profiles through comparing genome-wide expression patterns of hand-dissected, unpollinated stigma at anthesis with seven tissues, including seedling shoot, seedling root, mature anther, ovary at anthesis, seeds 5 d after pollination, 10-d-old embryo, 10-d-old endosperm, and suspension-cultured cells by using both 57 K Affymetrix rice whole-genome array and 10 K rice cDNA microarray. A high reproducibility of the microarray results was detected between the two different technology platforms. In total, we identified 548 genes to be expressed specifically or predominantly in the stigma papillar cells of rice. Real-time quantitative reverse transcription-polymerase chain reaction analysis of 34 selected genes all confirmed their stigma-specific expression. The expression of five selected genes was further validated by RNA in situ hybridization. Gene Ontology analysis shows that several auxin-signaling components, transcription, and stress-related genes are significantly overrepresented in the rice stigma gene set. Interestingly, most of them also share several cis-regulatory elements with known stress-responsive genes, supporting the notion of an overlap of genetic programs regulating pollination and stress/defense responses. We also found that genes involved in cell wall metabolism and cellular communication appear to be conserved in the stigma between rice and Arabidopsis (Arabidopsis thaliana). Our results indicate that the stigmas appear to have conserved and novel molecular functions between rice and Arabidopsis.
In flowering plants, pollination is the first major reproduction process that results in the production of seeds. The process begins with the adhesion of pollen grains to the stigmatic tissue of style. Then, the highly desiccated pollen grains are rehydrated to reach a certain water content that allows them to regain active metabolisms. Once the pollen grain cell has established its internal polarity relative to an external signal, the pollen germinates and breaches the exine wall to emit a pollen tube. The newly formed pollen tube subsequently penetrates the stigma, grows down through the transmitting tissue of the style, and ultimately reaches an ovule, allowing fertilization to take place (Lord, 2003  ; Sanchez et al., 2004; Swanson et al., 2004; Boavida et al., 2005).
Successful pollination requires continued communication and coordination between the pollen and stigma and is essential to maximize the seed set. The stigma, the uppermost part of the pistil, is generally considered to be a passive structure for pollen grain capture and reception, germination, and initial growth of the pollen tubes. There are two major types of stigma, the dry and the wet types, which differ by the presence or absence of the secreting exudate compounds (Edlund et al., 2004
Nevertheless, the molecular roles of the stigma in pollination are largely unknown. Several stigma-specific expressed proteins have been identified, a few of which are found to be directly involved in the pollen-stigma interaction. SRK (S-locus receptor kinase) encoding a glycoprotein localizes to the stigmatic plasma membrane and interacts with the pollen coat S-locus Cys-rich protein (SCR) in the self-incompatibility response in Brassica (Nasrallah, 2000
Additional factors have been found to be involved in pollination in several species. Auxin has been proposed to operate as a pollination signal and promote the differentiation and development of ovules in flowers of orchid of the genus Phalaenopsis (O'Neill, 1997
However, the molecular mechanisms of these events involved in pollination remain obscure. It is anticipated that, given recent rapid developments in functional genomics, genome-wide identification of stigma-specific genes could provide important initial steps in dissecting molecular control of pollination. Recently, two groups have carried out the stigma-specific gene profiling of Arabidopsis using whole-genome microarray analysis (Swanson et al., 2005
Cultivated rice is by far one of the most important food crops and is also considered the model monocot plant for molecular and genetic studies. Rice is primarily an autogamous, self-pollinating plant and has the typical dry stigma. But very little is known about stigma-specific genes that are required for ensuring successful pollination in rice. In this study, we generate rice stigma-specific or -preferential gene expression profiles by using both the commercially available 57 K Affymetrix rice whole-genome array and 10 K rice cDNA microarray (Lan et al., 2004
Gene Expression Profiles of Stigma versus Ovary by Two Independent Microarray Platforms To generate stigma-specific and ovary-specific gene expression profiles, two independent microarrays were applied in this study. The stigma samples were hand collected by cutting the pistil just below the base of the plumose stigma, and the remainder of the pistil containing the style was used as the ovary samples. The important criterion for the dissection was that the stigma samples had the papillar cells but not the ovary samples.
To identify the genes preferentially expressed in the stigma, 57 K Affymetrix rice whole-genome array was used to generate the genome expression patterns across eight representative organs or tissues and suspension-cultured cells. They included seedling shoots (Sh), seedling roots (Rt), mature anthers (An), unpollinated stigmas at anthesis (St), ovaries at anthesis (Ov), seeds 5 d after pollination (5DAP), 10-d-old embryos (10EM), and 10-d-old endosperms (10EN). We performed three biological replicates for the stigma and ovary samples, respectively. The correlation coefficient value of each experiment was larger than 0.99. By using P value
We also used the 10 K rice cDNA microarray constructed previously (Lan et al., 2004 The microarray results showed a high reproducibility using the two different platforms (cDNA microarray versus Affymetrix oligo array). For the 453 cDNAs preferentially expressed in the stigma identified using the 10 K rice cDNA microarray, 201 cDNAs' (using E value = 1e-20 and identity >80% as cutoff) expression patterns were confirmed by the Affymetrix GeneChip results (Supplemental Table S4), with about a 44.37% match. For the 257 cDNA clones highly expressed in the ovary, 109 cDNAs' expression patterns (using E value = 1e-20 and identity >80% as cutoff) were confirmed by the Affymetrix GeneChip results, with about a 42.41% match (Supplemental Table S5).
To identify rice stigma preferentially expressed genes, two statistical methods were conducted for data processing. First, Significance Analysis of Microarrays software package analysis was conducted for three biological samples replicates between the stigma and ovary. Using q value
Among the identified 548 genes preferentially expressed in the stigma, 410 genes had putative functions, 103 genes were assigned as expressed proteins, 23 genes were assigned as hypothetical proteins, and 12 genes had no hits when blasted against The Institute for Genomic Research rice genome annotation database (http://www.tigr.org). The 410 genes were classified into 11 groups according to their annotations and one unclassified group: transcription, cell wall related, stress/defense, signal transduction, lipid metabolism, transport, hormone related, protein metabolism, carbohydrate and energy metabolism, nucleic acid metabolism, amino acid metabolism, and unclassified. The categories are listed in the order of gene numbers in each group (Supplemental Table S7). We list 149 genes derived from 173 probe sets with putative annotated functions and their ratios of the stigma/ovary of more than 10 (P < 0.05) in Table I
. The largest category was transcription-related proteins, including two MADS-box proteins, four zinc-finger proteins, and six Myb- or Myb-like transcription factors. These proteins accounted for nearly 15% of the 410 putative functional genes. In the second category, including putative cell wall-localized enzymes (e.g.
GO analysis of the rice stigma-preferential genes was performed, and the biological process term enrichment status and hierarchy are shown in Supplemental Figure S1. The results showed that the DNA-dependant transcription may play an important role in the stigma, and the biological processes that respond to hormone stimuli, especially to auxin, were overrepresented and seemed to be more significant in the rice stigma-specific or -preferential gene data set.
We compared the stigma preferentially expressed genes in rice with that identified in Arabidopsis (Tung et al., 2005
Real-time quantitative reverse transcription (qRT)-PCR analysis was employed to validate the candidate genes. In total, 34 of the identified genes preferentially expressed in stigma were selected, including 30 putative function genes belonging to different classification groups, two hypothetical proteins, one expressed protein, and one unknown function gene. The signal intensity range of the selected genes was from 107.8 to 2,8284.3 and the ratios (stigma versus ovary) from 2.6 to 2,188.5 (see Supplemental Table S1). To confirm whether these genes were stigma-specific by qRT-PCR analysis, the expression of the 34 candidate genes was compared between the stigma and the ovary, anther, seeding-shoot, or flag leaf (heading stage) samples (Table II ). The results showed that their expression patterns detected by 57 K Affymetrix rice whole-genome array were in good correlation with those obtained by qRT-PCR.
Confirmation of the Candidate Stigma-Specific Genes by RNA in Situ Hybridization To further examine the expression of the candidate stigma-specific genes, we selected five genes to perform RNA in situ hybridization, including a Ser carboxypeptidase 1 precursor gene (LOC_Os02g46260), an extracellular ribonuclease LE precursor gene (LOC_Os09g36700), a putative CER1 gene (LOC_Os10g33250), a metal tolerance gene C3 (LOC_Os01g62070), and an unknown function gene (AK071040). They exhibited relatively high hybridization signals in the stigma microarray data sets, ensuring that their transcripts could be detected by the RNA in situ hybridization technology. A CCCH-type zinc finger protein gene (LOC_Os01g09620) expressed in the entire mature pistil according to the microarray results was used as a reference. Longitudinal sections through the center of pistil just before pollination were used for all the hybridizations. The results showed that the five selected genes exhibited unique expression patterns in the stigma papilla cells (Fig. 1 ) and that the hybridization signals of the examined genes were in good correlation with their intensities in 57 K Affymetrix rice GeneChip. Taken together, our results showed that the 548 genes preferentially expressed in the stigma thus identified represented good candidates for the stigma-specific genes in rice.
cis-Acting Regulatory Element Analyses of the Candidate Stigma-Specific Genes
To identify possible cis-acting regulatory elements responsible for the transcription regulation of the candidate stigma-specific genes, we first did the hierarchical clustering analysis and found that nearly 35% of the identified genes (193) were mainly located in three clades (Supplemental Fig. S2). The common features were that all the genes in the three clades were specific or highly expressed in the stigma, and the correlation coefficients of the three clusters were 0.9277, 0.9389, and 0.8263, respectively. We named the three clades as Cluster I, II, and III, respectively. Then, 1,000-bp regions located in the upstream of the start codons of the genes from the three clusters were used for analysis. Several conserved motifs were subsequently identified using the MEME (Multiple Em for Motif Elicitation)/MAST (Motif Alignment and Search Tool) system (Bailey and Elkan, 1994
In this study, we have identified 548 genes expressed specifically or predominantly in the stigma papillar cells of rice by using 57 K Affymetrix rice whole-genome array and 10 K rice cDNA microarray. It is highly likely that they represent good candidates for the stigma-specific genes in rice. First, for the two different technological platforms we used, there existed good correlations, and the variables observed were reasonable according to several recent reports (Maruyama et al., 2004 ; Yauk et al., 2004; Hannah et al., 2005). Second, the qRT-PCR and RNA in situ hybridization confirmed the microarray results (Table II; Fig. 1). Third, the rice stigma gene data set is similar to that identified in Arabidopsis (Tung et al., 2005). Thus, the careful selection of the starting tissues is an effective approach to identify the stigma-specific genes using the whole-genome and cDNA microarrays.
The functional annotation of both rice and Arabidopsis genes specifically or preferentially expressed in stigma suggests that several groups of the genes appear to play conserved roles in the stigma. Forty-two Arabidopsis stigma-specific genes representing 36% of the gene set are highly similar to 83 genes representing about 15% of the rice stigma gene dataset (Supplemental Table S8). The majority of them belong to the cell wall-related and signal transduction groups, indicating that these two classes of genes have conserved functions in the stigma. Swanson et al. (2005)
Auxin is the central growth regulator of a myriad of aspects of plant growth and developmental processes and appears to be actively transported throughout the plant to control cell division, extension, and differentiation (Benjamins et al., 2005
However, it is unclear whether auxin is involved directly in the interaction of the pollen and stigma. In the hormone-related genes group from our data set, the auxin-related genes appear to be overrepresented (Table I; Supplemental Table S7), and the results of the GO analysis of the rice stigma also suggested that the auxin signaling plays a significant role in the rice stigma function (Supplemental Fig. S1). The five of the identified auxin-related genes are SAURs (Jain et al., 2006
The transport-related proteins are mostly abundant in the rice stigma gene dataset and appear to be actively involved in the stigma function. The rice pollen is not fully dehydrated and metabolically active when shed from the anthers. They likely require the stigma to be fully developed to supply all the nutrition and metabolites for pollen germination and tube growth. The different kinds of transporters appear to be involved in the exchange of materials and information between pollen and stigma. The ABC transporter is one of the active transport systems of the cell, which is widespread in prokaryotes and eukaryotes. In tobacco, the gene NtWBC1 is developmentally regulated in the stigma/style, with mRNA accumulation increasing toward anthesis (Otsu et al., 2004
Previously, we detected an extensive overlap of the genes involved in pollination and abiotic stress responses (Lan et al., 2005
Our analysis also revealed that the stigma-specific genes share some common cis-regulatory elements with the stress-responsive genes. A large percentage of genes in cluster I possess the same motif, named GCC box (Fig. 2). The GCC box has been found in many pathogen-responsive genes (Brown et al., 2003 In conclusion, we have identified a large set of genes that are specifically or highly enriched in the rice stigma. In addition to the conserved roles of the cell wall metabolism and cellular communication in the stigma, the identification of genes involved in the auxin signaling, transcription functions, and possible cross talk between pollination and stress/defense responses provides new insights into the molecular functions of the stigma in rice.
Plant Materials Rice (Oryza sativa) ‘Nipponbare’ seeds were germinated and grown in a growth container for 2 weeks (28°C, 16 h light, 8 h dark, and photo intensity of 240 µm photos m–2 s–1). Then, shoot and root were harvested for Sh and Rt, respectively. Other plant materials used in this study described below were harvested from rice plants grown under natural conditions in the field of the Institute of Genetics and Developmental Biology, Beijing, during May to October and Hainan, China, during December to April. An were collected at a stage 1 to 2 d before floret flowering; unpollinated St and Ov were dissected at a stage 0 to 1 d before floret flowering; pistils of 5DAP were dissected 5 d after anthesis, 10EM and 10EN were dissected from grains 10 d after flowering, respectively.
The method used for suspension cell culture was described by Wang et al. (2005)
Total RNA was isolated using RNeasy kit (Qiagen). Double-strand cDNA was synthesized from 5 µg total RNA using cDNA Synthesis kit (TaKaRa). In vitro transcription from cDNA to cRNA was performed using T7 RiboMAX Express Large Scale RNA Production system (Promega). Then the cRNA was converted to DNA by using Superscript II RT kit (Invitrogen) and random primers. One microgram DNA product and random primers (9-mers) were annealed to denatured DNA template and extended by Klenow fragment (TaKaRa) in the presence of Cy5-dCTP/Cy3-dCTP (Amersham Pharmacia) for target preparation in microarray analysis. Hybridization and washing were performed as described in CyScribe Post-Labeling kit (Amersham Pharmacia) and CMTTM hybridization chamber (Corning) user manuals. Scanning and data acquisition were performed on a GenePix 4000B scanner using GENEPIX 4.0 software (Axon Instruments). GenePix Pro 4.0 output files were converted to The Institute for Genomic Research Multi Experiment Viewer file (.mev) by ExpressConverter V1.4 (http://www.tigr.org/software/tm4/utilities.html) and normalized (local lowess) by MicroArray Informatics Discovery System (http://www.tigr.org/software/tm4/). Then, spots flagged bad or not found by Genepix software were removed from further data analysis, and only those spots that showed fluorescent intensity levels in at least one channel above the background (local) + 2SD were used for further analysis. Those spots that exhibited a large difference between the duplicate experiments (dye swap) were regarded as outliers and removed manually. Hierarchical clustering was performed as described by Eisen et al. (1998)
Total RNA was isolated using TRIzol reagent (Invitrogen) and purified by using Qiagen RNeasy columns (Qiagen). For Affymetrix GeneChip (Affymetrix) analysis, 8 µg total RNA was used for making biotin-labeled cRNA targets. All the processes for cDNA and cRNA synthesis, cRNA fragmentation, hybridization, washing and staining, and scanning were conducted according to the GeneChip standard protocol (Eukaryotic Target Preparation, Affymetrix). Poly-A RNA Control kit and the One-Cycle cDNA Synthesis kit were used in this experiment as described in the Web site: http://www.affymetrix.com/products/arrays/specific/rice.affx. The information about GeneChip Rice Genome Array (MAS 5.0) could be accessed from the Affymetrix Web site: http://www.affymetrix.com/products/arrays/specific/rice.affx. Affymetrix GeneChip Operating software (GCOS) was used for data collection and normalization. The overall intensity of all probe sets of each array was scaled to 500 to guarantee that hybridization intensity of all arrays was equivalent, each probe set was assigned with P, A, and M and a P value from the algorithm in GCOS. To identify differentially expressed genes, the log2 transformed signal ratio of each gene was calculated using the GCOS baseline tool, and log2 (ratio)
Two statistical methods were used for data processing. First, analysis using the Significance Analysis of Microarrays software package was conducted for rice triplicate samples between stigma and ovary using q value
In total, we identified 665 probe sets preferentially expressed in stigma using Z score
We searched GO information for the 665 probe sets using EasyGO software (http://bioinformatics.cau.edu.cn/easygo/category_treeBrowse.html). Using the rice ‘Japonica’ gene database, we applied
Total RNA preparation and real-time PCR were performed as previously described (Lan et al., 2004
RNA in situ hybridization was performed as previously described (Lai et al., 2002
The 1,000-bp regions located upstream of the start codons of genes of interest were used for analysis with the MEME/MAST system (http://meme.sdsc.edu/meme/meme.html) and PLACE (http://www.dna.affrc.go.jp/PLACE). Sequence logos of cis-acting regulatory elements were created by weblogo (http://weblogo.berkeley.edu/logo.cgi).
The following materials are available in the online version of this article.
We thank Kang Chong and Lingfeng Chen (Institute of Botany, Chinese Academy of Sciences) for technical assistance in microarray scanning and Zhen Su and Xin Zhou (China Agricultural University) for assisting on microarray analysis. Received April 26, 2007; accepted May 24, 2007; published June 7, 2007.
1 This work was supported by the Ministry of Science and Technology of China (grant no. 2005CB120800) and by the Chinese Academy of Sciences.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Yongbiao Xue (ybxue{at}genetics.ac.cn).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.101600 * Corresponding author; e-mail ybxue{at}genetics.ac.cn; fax 86–10–62537814.
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ABSTRACT
RESULTS
0.05 and fold change 
2 as cutoff to identify differentially expressed genes, 3,300 probe sets were up-regulated in the stigma compared to the ovary, and 4,125 probe sets were up-regulated in the ovary compared to the stigma (Supplemental Table S1). All microarray data are available online (
-expansin, pectinesterase, and peroxidase), cellulose synthase, glycerol-3-P acyltransferase, and UDP-glucosyltransferase, the third category consisted of proteins that were potentially involved in stress/defense responses, such as glutathione S-transferase, phospholipase D, and wound-induced protein WI12. Many signal transduction- and transport-related proteins were enriched in the stigma. There was a large percentage of genes that may be related to lipid metabolism, including lipid binding protein, triacylglycerol lipase, and fatty acid elongase. The auxin signaling related proteins were identified to be highly expressed in the stigma, including auxin-responsive factor, five members of the auxin-responsive small auxin-up RNA (SAUR) gene family, and the highly expressed IAA-amido synthetase GH3.1 gene (see 
is the average of expression level in stigma tissue sample; 
is the average of expression level in the ovary tissue sample; and T1, T2, ...,Tn are the expression levels in other tissue samples. The ratios of all 665 probe sets were more than a 2-fold change. 
2 tests for the biological process search, and the cutoff for false discovery rate was adjusted and the P value was 0.0001.