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Cell type-specific transcriptome of Brassicaceae stigmatic papilla cells from a combination of laser microdissection and RNA sequencing.

Osaka M, Matsuda T, Sakazono S, Masuko-Suzuki H, Maeda S, Sewaki M, Sone M, Takahashi H, Nakazono M, Iwano M, Takayama S, Shimizu KK, Yano K, Lim YP, Suzuki G, Suwabe K, Watanabe M - Plant Cell Physiol. (2013)

Bottom Line: Pollination is an early and critical step in plant reproduction, leading to successful fertilization.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.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life Sciences, Tohoku University, Sendai, 980-8577 Japan.

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.

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Isolation of a papilla cell by LM in Arabidopsis and Brassica. The paraffin-embedded stigmas were cut into 6–8 µm thick sections using a microtome and mounted on PEN membrane frame slides. After removing the paraffin, LM was performed using an Arcturus XT Laser Capture Microdissection System. (A–C) Arabidopsis thaliana, (D–F) B. rapa, (A, D) before LM, (B, E) after LM, and (C, F) isolated papilla cells.
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pct133-F1: Isolation of a papilla cell by LM in Arabidopsis and Brassica. The paraffin-embedded stigmas were cut into 6–8 µm thick sections using a microtome and mounted on PEN membrane frame slides. After removing the paraffin, LM was performed using an Arcturus XT Laser Capture Microdissection System. (A–C) Arabidopsis thaliana, (D–F) B. rapa, (A, D) before LM, (B, E) after LM, and (C, F) isolated papilla cells.

Mentions: Using optimal fixation conditions for pistils, embedded specimens were prepared using 2–6 floral buds from each plant species. Serial paraffin sections of pistil specimens were prepared at 6–8 µm in thickness and papilla cells were isolated by LM, using approximately 60 pistil sections from each plant sample (Fig. 1). Total RNAs were extracted from the LM-collected papilla cells, confirmed to be acceptable quality for RNA-seq analysis (Supplementary Fig. S1), and 1.5 rounds of mRNA linear amplification were performed for each sample. Subsequently, 50–200 ng µl−1 cDNAs were obtained from each sample, and cDNA fragment libraries were subjected to SOLiD5500xl sequencing. As a result of the RNA-seq analysis, 65,690,858, 101,509,276 and 74,175,637 raw reads of 75 bp in length were obtained in A. thaliana, A. halleri and B. rapa, respectively. On the alignment of raw reads to the A. thaliana and B. rapa reference genomes, a total of 21,310, 23,414 and 26,852 unigenes were defined, respectively, after removing low-quality raw reads (MapQ ≤10) and adaptor sequences. Of these, 17,240, 19,260 and 21,026 unigenes were found to be >10 read tags and could be assigned an annotation based on database sequence assembly in A. thaliana, A. halleri and B. rapa, respectively (Table 2; Supplementary Tables S1–S3). In A. thaliana, approximately 50–60% of the genes have previously been predicted to be expressed in stigmas (Suwanson et al. 2005), and the remaining ∼50% of the genes have never been predicted to be expressed in stigmas, and thus they are likely to be novel stigma-expressed genes, including papilla-expressed genes. The fact that approximately half of the annotated genes from the RNA-seq have not been detected previously in microarray and cDNA subtraction analyses highlights the advantages, including greater sensitivity, of the combination of LM and RNA-seq analysis for the tissue of interest.Fig. 1


Cell type-specific transcriptome of Brassicaceae stigmatic papilla cells from a combination of laser microdissection and RNA sequencing.

Osaka M, Matsuda T, Sakazono S, Masuko-Suzuki H, Maeda S, Sewaki M, Sone M, Takahashi H, Nakazono M, Iwano M, Takayama S, Shimizu KK, Yano K, Lim YP, Suzuki G, Suwabe K, Watanabe M - Plant Cell Physiol. (2013)

Isolation of a papilla cell by LM in Arabidopsis and Brassica. The paraffin-embedded stigmas were cut into 6–8 µm thick sections using a microtome and mounted on PEN membrane frame slides. After removing the paraffin, LM was performed using an Arcturus XT Laser Capture Microdissection System. (A–C) Arabidopsis thaliana, (D–F) B. rapa, (A, D) before LM, (B, E) after LM, and (C, F) isolated papilla cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814185&req=5

pct133-F1: Isolation of a papilla cell by LM in Arabidopsis and Brassica. The paraffin-embedded stigmas were cut into 6–8 µm thick sections using a microtome and mounted on PEN membrane frame slides. After removing the paraffin, LM was performed using an Arcturus XT Laser Capture Microdissection System. (A–C) Arabidopsis thaliana, (D–F) B. rapa, (A, D) before LM, (B, E) after LM, and (C, F) isolated papilla cells.
Mentions: Using optimal fixation conditions for pistils, embedded specimens were prepared using 2–6 floral buds from each plant species. Serial paraffin sections of pistil specimens were prepared at 6–8 µm in thickness and papilla cells were isolated by LM, using approximately 60 pistil sections from each plant sample (Fig. 1). Total RNAs were extracted from the LM-collected papilla cells, confirmed to be acceptable quality for RNA-seq analysis (Supplementary Fig. S1), and 1.5 rounds of mRNA linear amplification were performed for each sample. Subsequently, 50–200 ng µl−1 cDNAs were obtained from each sample, and cDNA fragment libraries were subjected to SOLiD5500xl sequencing. As a result of the RNA-seq analysis, 65,690,858, 101,509,276 and 74,175,637 raw reads of 75 bp in length were obtained in A. thaliana, A. halleri and B. rapa, respectively. On the alignment of raw reads to the A. thaliana and B. rapa reference genomes, a total of 21,310, 23,414 and 26,852 unigenes were defined, respectively, after removing low-quality raw reads (MapQ ≤10) and adaptor sequences. Of these, 17,240, 19,260 and 21,026 unigenes were found to be >10 read tags and could be assigned an annotation based on database sequence assembly in A. thaliana, A. halleri and B. rapa, respectively (Table 2; Supplementary Tables S1–S3). In A. thaliana, approximately 50–60% of the genes have previously been predicted to be expressed in stigmas (Suwanson et al. 2005), and the remaining ∼50% of the genes have never been predicted to be expressed in stigmas, and thus they are likely to be novel stigma-expressed genes, including papilla-expressed genes. The fact that approximately half of the annotated genes from the RNA-seq have not been detected previously in microarray and cDNA subtraction analyses highlights the advantages, including greater sensitivity, of the combination of LM and RNA-seq analysis for the tissue of interest.Fig. 1

Bottom Line: Pollination is an early and critical step in plant reproduction, leading to successful fertilization.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.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life Sciences, Tohoku University, Sendai, 980-8577 Japan.

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.

Show MeSH
Related in: MedlinePlus