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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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Related in: MedlinePlus

Confirmation of gene expression patterns by in situ hybridization. The pistils for in situ hybridization were collected 1 d before anthesis. Both antisense and sense probes were synthesized using a T7/SP6 digoxigenin RNA labeling kit. Insets show the signal area in papilla cells at high magnification. (A) AT1G09690, (B) AT1G53130, (C) AT2G41430, (D) Bra004382, (E) Bra019017 and (F) Bra028171.
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pct133-F3: Confirmation of gene expression patterns by in situ hybridization. The pistils for in situ hybridization were collected 1 d before anthesis. Both antisense and sense probes were synthesized using a T7/SP6 digoxigenin RNA labeling kit. Insets show the signal area in papilla cells at high magnification. (A) AT1G09690, (B) AT1G53130, (C) AT2G41430, (D) Bra004382, (E) Bra019017 and (F) Bra028171.

Mentions: To assess the validity and reliability of our LM–RNA-seq data from papilla cells, quantitative real-time PCR (qRT-PCR) analysis was performed on 12 representative genes belonging to different classification groups (Supplementary Table S4). Expression levels of the 12 genes were compared between papilla cells and the leaf to determine whether these genes were expressed in papilla specifically or preferentially (Fig. 2). The results from qRT-PCR showed that these 12 genes were expressed exclusively in papilla cells, and this result correlated with those obtained by LM–RNA-seq. To confirm further the expression and localization of the genes in papilla cells, in situ hybridizations were performed with six representative genes, of stress-related, metal tolerance and unknown functions (Supplementary Table S5). Probes from all six genes exhibited hybridization signals in papilla cells and the transmitting tract of the stigma, confirming that the corresponding gene transcripts were detected in papilla cells by in situ hybridization (Fig. 3).Fig. 2


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)

Confirmation of gene expression patterns by in situ hybridization. The pistils for in situ hybridization were collected 1 d before anthesis. Both antisense and sense probes were synthesized using a T7/SP6 digoxigenin RNA labeling kit. Insets show the signal area in papilla cells at high magnification. (A) AT1G09690, (B) AT1G53130, (C) AT2G41430, (D) Bra004382, (E) Bra019017 and (F) Bra028171.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

pct133-F3: Confirmation of gene expression patterns by in situ hybridization. The pistils for in situ hybridization were collected 1 d before anthesis. Both antisense and sense probes were synthesized using a T7/SP6 digoxigenin RNA labeling kit. Insets show the signal area in papilla cells at high magnification. (A) AT1G09690, (B) AT1G53130, (C) AT2G41430, (D) Bra004382, (E) Bra019017 and (F) Bra028171.
Mentions: To assess the validity and reliability of our LM–RNA-seq data from papilla cells, quantitative real-time PCR (qRT-PCR) analysis was performed on 12 representative genes belonging to different classification groups (Supplementary Table S4). Expression levels of the 12 genes were compared between papilla cells and the leaf to determine whether these genes were expressed in papilla specifically or preferentially (Fig. 2). The results from qRT-PCR showed that these 12 genes were expressed exclusively in papilla cells, and this result correlated with those obtained by LM–RNA-seq. To confirm further the expression and localization of the genes in papilla cells, in situ hybridizations were performed with six representative genes, of stress-related, metal tolerance and unknown functions (Supplementary Table S5). Probes from all six genes exhibited hybridization signals in papilla cells and the transmitting tract of the stigma, confirming that the corresponding gene transcripts were detected in papilla cells by in situ hybridization (Fig. 3).Fig. 2

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