IC4R001-Microarray-2011-221106719

Project Title

Distinct Gene Expression Profiles in Egg and Synergid Cells of Rice as Revealed by Cell Type-Specific Microarrays

The Background of This Project

Figure 1. Isolation of rice egg and synergid cells.

• The molecular machineries underlying plant reproduction are becoming an area of active research. How- ever, the small number and inaccessibility of these cells have hampered molecular and genome-wide studies; thus, we know little about the molecular basis of cell specification, differentiation, and function in the female gametophyte. The identification of genes expressed in the female gametophyte is essential to understanding how female gametophyte cells become specified and acquire their unique features and functions. Previous research in this field has identified genes that are expressed in specific cells of the female gametophyte in various plant species. With the exception of Arabidopsis (Arabi- dopsis thaliana), microarray-based, comprehensive screens for genes exhibiting female gametophyte expression have never been done for plants.
• In rice, several methods for isolating egg cells have been reported. Most require a step to degrade the cell wall with catalytic enzymes, such as cellulase (Han et al., 1998; Zhao et al., 2000; Khalequzzaman and Haq, 2005). Egg and synergid cells can also be isolated from unfertilized ovaries by manual manipulation without using enzymes (Zhang et al., 1999; Uchiumi et al., 2006). In this project, the researchers collected a large number of rice egg and synergid cells by using manual manipulation instead of enzymes. The quality and quantity of the RNA obtained from the isolated cells were enough to per- form transcriptome analysis, and high-quality expression data for rice egg and synergid cells were obtained without cross-contamination. Direct comparisons between the data obtained and the transcriptomes of a variety of diploid tissues showed that the egg and synergid cell transcriptome was distinct. We also identified and characterized a large number of the genes expressed in rice egg and synergid cells. This study provides insights into the roles of genes ex- pressed in the female gametes before, during, and possibly after double fertilization.

Plant Materials & Treatment

• Wild-type rice plants (Oryza sativa 'Nipponbare') were grown in pots under natural conditions. Transgenic rice plants were grown and maintained in a biohazard greenhouse at 30°C (day) and 25°C (night) under natural light conditions.

Research Findings

Figure 2.Scatterplot and correlation plot analyses.

• Egg and synergid cells can be isolated from unferilized ovaries by manual manipulation without using enzymes (Zhang et al., 1999; Uchiumi et al., 2006). We chose this nonenzymatic method with some modifications (Takanashi et al., 2010) for the isolation of egg and synergid cells. The sizes of rice egg and synergid cells range between 30 and 50 mm in diameter, which exceeds the size of other cells released from dissected ovaries. Since many vacuoles, ranging in size from 2 to 25 mm, were present in the peripheral region of the egg cells, egg cells can be distinguished from synergid cells by their internal contrast (Fig. 1, D–G). Because an intact egg apparatus composed of a single egg cell and two synergid cells was obtained only rarely, we picked up one or two targeted cell(s) from every ovule. As the synergid cells are more delicate than egg cells, a large number of synergid cells were disrupted during the isolation procedure.
• The researchers obtained 3,000 egg cells and 1,000 synergid cells from the basal portions of dis- sected ovaries several days before flowering (Figure 1) and then extracted the RNA from the isolated cells for microarray analysis. RNA samples derived from egg and synergid cells were of sufficient quality for use in microarray analysis. We conducted a 44K microarray analysis of egg cell, synergid cell, ovary, and whole plant of japonica rice (cv Nipponbare). In the cell isolation procedure, isolated cells were first washed in mannitol solution and then stored transiently in it. Therefore, we needed to account for any changes in gene expression in the isolated cells caused by the transient storage in mannitol solution. To pay attention to fluctuations in gene expression caused by these washing steps, we prepared two types of ovary RNA: one was derived from ovaries just after cutting the middle portion, and the other was derived from ovaries treated with mannitol after cutting.
• To examine for correlations in gene expression be- tween samples, we created scatterplot diagrams and correlation plots for the microarray data sets. When replicates from the same cell type were compared, the points fell on the 45° identity line (Fig. 2, A and B), showing that the data was highly reproducible for microarray replicates of samples with the same bio- logical origin (egg or synergid cell). In contrast, the points were spread widely when we compared the averaged microarray data set from egg cell samples with that from synergid cell samples (Fig. 2C). This means that the gene expression profiles of egg and synergid cells are very different. Therefore, the cell types are already differentiated with genome-wide transcriptional responses within the egg apparatus at the micropylar end of the female gametophyte during the period comprising the three sequential mitotic nuclear divisions after the creation of the megaspore. These conclusions were supported by correlation plot analysis (Fig. 2D). The expression profiles of the synergid cell were less correlated with the profiles of other samples than were the expression profiles of the egg cell.
• To clearly demonstrate that the egg cell and the synergid cell had already differentiated from the diploid cells that conferred ovule identity, we created a heat map based on the expression patterns of MADS box genes that control floral organ identity (Fig. 3). We confirmed that the levels of the transcripts derived from the three classes of genes (C, D, and E) were higher in both types of ovary samples than in whole plant samples (Fig. 3). Although both egg and synergid cells develop within the ovule, our results show that transcripts from the three classes of genes do not accumulate in these haploid cell samples. In particular, the expression of OsMADS13 was clearly suppressed. OsMADS13 is specifically expressed in the ovule, and its expression is first detected in the ovule primordium, where it persists during further development of the ovule. Inside the ovule, OsMADS13 is expressed in integuments and nucellus tissues (Lopez-Dee et al., 1999). This heat map is consistent with the fact that the egg and synergid cells had already differentiated from the diploid cells that form the ovule and that they had acquired unique gametic and nongametic cell identities, respectively.

Labs working on this Project

• Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113–8657, Japan (T. Ohnishi, H. Takanashi, M.M., H. Takahashi, N.T.);
• Department of Life Sciences, Faculty of Agriculture, Meiji University, Kawasaki, Kanagawa 214–8571, Japan (S.K., K.Y.);
• Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo 192–0397, Japan (T. Okamoto);
• Genetic Strain Stock Center, National Institute of Genetics, Mishima, Shizuoka 411–8540, Japan (M.F., N.K.)

Corresponding Author

Nobuhiro Tsutsumi(email: atsutsu@mail.ecc.u-tokyo.ac.jp.)