Comparative Transcriptomics of Arabidopsis Sperm Cells

In ﬂowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part in fertilization are crucial goals in the study of plant reproduction. Studies of gene expression in male gametes of maize ( Zea mays ) and Plumbago and in lily ( Lilium longiﬂorum ) generative cells already showed that the previously held view of transcriptionally inert male gametes was not true, but genome-wide studies were lacking. Analyses in the model plant Arabidopsis ( Arabidopsis thaliana ) were hindered, because no method to isolate sperm cells was available. Here, we used ﬂuorescence-activated cell sorting to isolate sperm cells from Arabidopsis, allowing GeneChip analysis of their transcriptome at a genome-wide level. Comparative analysis of the sperm cell transcriptome with those of representative sporophytic tissues and of pollen showed that sperm has a distinct and diverse transcriptional proﬁle. Functional classiﬁcations of genes with enriched expression in sperm cells showed that DNA repair, ubiquitin-mediated proteolysis, and cell cycle progression are overrepresented Gene Ontology categories. Moreover, analysis of the small RNA and DNA methylation pathways suggests that distinct mechanisms might be involved in regulating the epigenetic state of the paternal genome. We identiﬁed numerous candidate genes whose involvement in sperm cell development and fertilization can now be directly tested in Arabidopsis. These results provide a roadmap to decipher the role of sperm-expressed proteins. assessed using an Agilent 2100 Bioanalyser with an RNA 6000 Nano Assay (Agilent Technologies) and processed for use on Affymetrix Arabidopsis ATH1 Genome Arrays (Santa Clara) according to the manufacturer’s Two- Cycle Target Labeling Assay. Brieﬂy, 16 ng of total RNA containing spiked-in Poly-A RNA controls (GeneChip Expression GeneChip Eukaryotic Poly-A RNA Control kit; Affymetrix) was used in an RT reaction (Two-Cycle DNA synthesis kit, Affymetrix) to generate ﬁrst-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in an in vitro transcription reaction to generate cRNA (MEGAscript T7 kit; Ambion). The cRNA obtained was used for a second round of cDNA and cRNA synthesis, resulting in biotinylated cRNA (GeneChip Expression 3 # -Ampliﬁcation Reagents for in vitro transcription labeling; Affymetrix). The size distribution of the cRNA and fragmented cRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Assay. Fifteen micrograms of fragmented cRNA was used in a 300- m L hybridi- zation containing added hybridization controls. A total of 200 m L of the mixturewashybridized onarraysfor16hat45 (cid:3) C.Standardposthybridization washes and double-stain protocols (FS450_0004) were used on an Affymetrix GeneChip Fluidics Station 450, in conjunction with the GeneChip Hybridiza- tion Wash and Stain kit (Affymetrix). Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G. All quality parameters for the arrays were conﬁrmed to be in the recommended range. GeneChip datasets for the nine arrays used in this study are available in a MIAME-compliant format through ArrayExpress (accession no. E-ATMX-35). see detailed protocol in Supplemental Protocol S1). For downstream analyses, only genes called present in at least two replicates of each sample were considered. Hierarchical clustering and PCA were computed using Partek Genomics Suite (Partek). We performed comparative analyses of our samples with datasets of leaves, ﬂowers, and siliques (Pina et al., 2005) and with datasets from ovules and unpollinated pistils (L.C. Boavida, F. Borges, J.D. Becker, and J.A. Feijo´, unpublished data). The graphical representation SnailView (Becker et al., 2003) was applied to visualize the mean pattern of expression for the 5,829 genes detected in sperm cells coplotted with correspondent pollen data.