Transcriptome Analysis of the Arabidopsis Megaspore Mother Cell Uncovers the Importance of RNA Helicases for Plant Germline Development

Abstract

Germ line specification is a crucial step in the life cycle of all organisms. For sexual plant reproduction, the megaspore mother cell (MMC) is of crucial importance: it marks the first cell of the plant “germline” lineage that gets committed to undergo meiosis. One of the meiotic products, the functional megaspore, subsequently gives rise to the haploid, multicellular female gametophyte that harbours the female gametes. The MMC is formed by selection and differentiation of a single somatic, sub-epidermal cell in the ovule. The transcriptional network underlying MMC specification and differentiation is largely unknown. We provide the first transcriptome analysis of an MMC using the model plant Arabidopsis thaliana with a combination of laser-assisted microdissection and microarray hybridizations. Statistical analyses identified an over-representation of translational regulation control pathways and a significant enrichment of DEAD/DEAH-box helicases in the MMC transcriptome, paralleling important features of the animal germline. Analysis of two independent T-DNA insertion lines suggests an important role of an enriched helicase, MNEME (MEM), in MMC differentiation and the restriction of the germline fate to only one cell per ovule primordium. In heterozygous mem mutants, additional enlarged MMC-like cells, which sometimes initiate female gametophyte development, were observed at higher frequencies than in the wild type. This closely resembles the phenotype of mutants affected in the small RNA and DNA-methylation pathways important for epigenetic regulation. Importantly, the mem phenotype shows features of apospory, as female gametophytes initiate from two non-sister cells in these mutants. Moreover, in mem gametophytic nuclei, both higher order chromatin structure and the distribution of LIKE HETEROCHROMATIN PROTEIN1 were affected, indicating epigenetic perturbations. In summary, the MMC transcriptome sets the stage for future functional characterization as illustrated by the identification of MEM, a novel gene involved in the restriction of germline fate.

Plant germline formation: common concepts and developmental flexibility in sexual and asexual reproduction

“…Nevertheless, recent methodological advances have allowed the transcriptional profiling of such rare cell types by combining laserassisted microdissection or micromanipulation with microarray and/ or RNA-Seq analyses. These studies have provided novel insights into the transcriptional basis of germline specification and development (Wüest et al, 2010;Schmidt et al, 2011Schmidt et al, , 2012Schmidt et al, , 2014Okada et al, 2013;Wüest et al, 2013;Abiko et al, 2013;Anderson et al, 2013;Chettoor et al, 2014).…”

“…AGO/PIWI proteins, and potentially other proteins involved in small RNA pathways, interact with VASA or VASA-like RNA helicases that are preferentially expressed in the germline (Yajima and Wessel, 2011). Although neither the PIWI clade of AGOs nor VASA RNA helicases have been identified in plants, recent evidence suggests that similar regulatory mechanisms evolved in the plant reproductive lineage, likely by convergence (Wüest et al, 2010;Schmidt et al, 2011).…”

Somatic small RNA pathways promote the mitotic events of megagametogenesis during female reproductive development in Arabidopsis

“…NUC2 and OV2 samples were captured from the same slides in three independent replicate collections. RNA was extracted using a Picopure kit (Acturus) and amplified twice using MessageAmp III (Applied Biosystems), similar to previous methods (Schmidt et al, 2011). RNA was labelled using a ULS aRNA labelling kit (Kreatech) and hybridised to Agilent 4x44K arrays (Institute for Molecular Bioscience, Brisbane, Australia).…”

“…Transcriptome profiling in Arabidopsis has identified pathways acting in the megaspore mother cell (Schmidt et al, 2011) and in dividing or mature female gametophytes (Yu et al, 2005;Johnston et al, 2007;Jones-Rhoades et al, 2007). Little is known, however, about pathways involved in the transition from megasporogenesis to megagametogenesis.…”

Reproductive Versatility and the Epigenetic Control of Female Gametogenesis

“…During the past few years, methods that allow the isolation of developing ovules or specific cells through laser-capture microdissection have opened up the possibility to conduct largescale transcriptional analysis of female reproductive development and harvesting of developing ovules of Arabidopsis thaliana (L.) Heynh (Arabidopsis) at specific developmental stages that encompass events that precede meiosis to fully differentiated organs (Schmidt et al 2011;Sanchez-Leon et al 2012;Schmid et al 2012). Some of these efforts have resulted in the possibility of globally analyzing gene expression in whole developing ovule primordia, allowing the generation of full-length cDNA libraries, RNA-seq profiles, and large-scale analysis of DNA methylation states.…”