RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing      

Abstract

(S.M., P.Q.)Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 proteincoding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 59 and 39 untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser.

Pollen-Specific Activation of Arabidopsis Retrogenes Is Associated with Global Transcriptional Reprogramming

“…Gene transcription in mature pollen grains was compared with that in seedling tissues (Loraine et al, 2013). Plotting the mean reads per kilobase per million reads values for the entire set, quantile 1 (lowest transcribed), and quantile 4 (highest transcribed) of all genes, retrogenes, and parents confirmed the microarray data ( Figures 3A, 3E, and 3F; Supplemental Figure 3B).…”

“…Therefore, somatic retroposition events in the shoot apical meristems may also be transmitted to the next generations. Thus, we tested for tissuespecific transcription of retrogenes in Arabidopsis using a developmental transcription data series (Schmid et al, 2005) and validated these findings using RNA sequencing data sets (Loraine et al, 2013). Surprisingly, this revealed that retrogenes are overtranscribed in pollen while overall transcription was not increased at this stage.…”

“…Significant differences (P < 0.05) in the x 2 test relative to background are indicated by asterisks. stages assembled by the AtGenExpress consortium (Schmid et al, 2005) and validated for selected tissues by RNA sequencing (Loraine et al, 2013). In total, 209 retrogenes and 245 parents are present on the ATH1 cDNA microarray (Supplemental Data Sets 4 and 5).…”

“…The transcription borderline for transcribed genes (gcRMA $ 5) was based on the minimal density of genes between peaks indicating absent or background signals versus high transcription signals (Supplemental Figure 2). The Arabidopsis mRNA half-life data and rosette-and pollen-specific RNA sequencing data were extracted from previously published data sets (Narsai et al, 2007;Loraine et al, 2013). Randomized sets of genes or gene pairs were generated, plots drawn, and statistical tests calculated in R. The significance of density distributions was tested using the MWW rank-sum test with correction and cotranscription correlation by the Pearson productmoment correlation coefficient (r).…”

Combinatorial interactions between LBD10 and LBD27 are essential for male gametophyte development in Arabidopsis

“…25 Arabidopsis pollen RNA-seq data sets have also shown that in addition to LBD10 and LBD27, the mRNAs from other LBDs including LBD22, LBD25, and LBD36, were significantly detected in pollen. 26 Heterodimerization between 2 different transcription factors facilitates nuclear localization and contributes to transcriptional regulation by modulating DNA-binding specificity and affinity and/ or the recruitment of different binding partners. 27 Thus the protein-protein interactions among these LBD transcription factors may provide redundancy as well as diversity to their biological functions during pollen developmental process.…”