Cell Type-Specific Transcriptome of Brassicaceae Stigmatic Papilla Cells From a Combination of Laser Microdissection and RNA Sequencing

Abstract

Pollination is an early and critical step in plant reproduction, leading to successful fertilization. It consists of many sequential processes, including adhesion of pollen grains onto the surface of stigmatic papilla cells, foot formation to strengthen pollen–stigma interaction, pollen hydration and germination, and pollen tube elongation and penetration. We have focused on an examination of the expressed genes in papilla cells, to increase understanding of the molecular systems of pollination. From three representative species of Brassicaceae (Arabidopsis thaliana, A. halleri and Brassica rapa), stigmatic papilla cells were isolated precisely by laser microdissection, and cell type-specific gene expression in papilla cells was determined by RNA sequencing. As a result, 17,240, 19,260 and 21,026 unigenes were defined in papilla cells of A. thaliana, A. halleri and B. rapa, respectively, and, among these, 12,311 genes were common to all three species. Among the17,240 genes predicted in A. thaliana, one-third were papilla specific while approximately half of the genes were detected in all tissues examined. Bioinformatics analysis revealed that genes related to a wide range of reproduction and development functions are expressed in papilla cells, particularly metabolism, transcription and membrane-mediated information exchange. These results reflect the conserved features of general cellular function and also the specific reproductive role of papilla cells, highlighting a complex cellular system regulated by a diverse range of molecules in these cells. This study provides fundamental biological knowledge to dissect the molecular mechanisms of pollination in papilla cells and will shed light on our understanding of plant reproduction mechanisms.

Molecular characterization of genes encoding isoamylase-type debranching enzyme in tuberous root of sweet potato, <i>Ipomoea batatas</i> (L.) Lam.

“…Generally, genes encoding β-actin (ACT) and/or α-tublin (TUB) are used as reference genes (Osaka et al 2013), but there are no ideal references genes able to fulfill all experimental requirements, and so the selection of optimum reference genes is essential (Bustin et al 2009;Park et al 2012). In the case of sweet potato, according to variety and/ or environmental conditions, expression of reference genes differed (Park et al 2012).…”

Single cell subtractive transcriptomics for identification of cell-specifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis

“…But a concern limiting the widespread use of these methods is that RNA integrity may be compromised (Goldsworthy et al, 1999). Protocols in the literature show that paraffin-embedding has to be optimized for each species and tissue type (Osaka et al, 2013;Schmid et al, 2012). Cryo-sectioning is described as being less prone to lead to RNA degradation (Goldsworthy et al, 1999;Ludwig and Hochholdinger, 2014).…”

Laser Microdissection of Plant Tissues

“…To date, there are two commonly used histological methods for processing and sectioning plant material for LMD: tissue freezing for cryostat sectioning [5][6][7] and paraffin embedding [8][9][10]. Cryosectioning may be performed on fresh-frozen tissue, or tissue fixed and infused with a cryoprotectant, such as 10-15 % sucrose.…”

“…Although excellent for preserving the molecular profile of cells, perturbations to cell morphology may make target cell and tissue identification difficult [10,11]. Conversely, paraffin embedding of plant tissues allows for excellent retention of cellular morphology and high-quality nucleic acid integrity for directed quantitative PCR [12], microarray [7,8], and RNA-sequencing strategies [9,13].…”