Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression analysis in microdissected shoot meristems of Brassica napus microspore-derived embryos with altered levels of SHOOTMERISTEMLESS

ABSTRACT: SHOOTMERISTEMLESS (STM) is a well characterized key regulator of the shoot apical meristem (SAM) activity in diverse plant species. We used a transgenic approach to investigate how the altered expression of Brassica napus (Bn) STM affects the morphology and behavior of microspore-derived embryos (MDEs). Down-regulation of BnSTM repressed the formation of the SAM and reduced the number of MDEs able to regenerate viable plants at germination. This was in contrast to embryos over-expressing BnSTM which displayed enlarged SAMs with an improved regeneration frequency. To further investigate the regulatory mechanisms underpinning these different responses, global changes in transcript levels were analysed in laser micro dissected SAMs of the transformed MDEs. The induction of BnSTM up-regulated the expression of many transcription factors (TFs) some of which directly involved in the formation of the SAM, i.e. CUPSHAPED COTYLEDON1 and WUSCHEL, and regulatory components ofthe antioxidant response, hormone signalling, and cell wall synthesis and modification. Opposite expression patterns for some of these genes were observed in the SAMs of MDEs down-regulating BnSTM. These data were interpreted in relation to the ability of BnSTM to modulate the size and functionality of the SAM. Structural and expression studies were further conducted to confirm the role of BnSTM in mediating the regulatory network of cell wall and lignin synthesis. Besides lowering the transcript levels of early components of cell wall and lignin biosynthetic pathways in the SAM, BnSTM had a remarkable repressing effect on the expression of PHENYLALANINE AMMONIA LYASE2, CINNAMATE 4-4HYDROXYLASE, andCINNAMYL ALCOHOL DEHYDROGENASE. Since lignin formation is a feature of irreversible cell differentiation, these results suggest that one way in which BnSTM promotes indeterminate cell fate may be by preventing the expression of components of biochemical pathways involved in the accumulation of lignin in the meristematic cells. Overall these studies provide evidence for a novel function of BnSTM in enhancing the quality of in vitro produced meristems, and propose that this gene can be used as a potential target to improve regeneration of cultured embryos. 2 mutants and 1 wild type

ORGANISM(S): Brassica napus

SUBMITTER: Raju Datla

PROVIDER: E-GEOD-35992 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:SHOOTMERISTEMLESS (STM) is a well characterized key regulator of the shoot apical meristem (SAM) activity in diverse plant species. We used a transgenic approach to investigate how the altered expression of Brassica napus (Bn) STM affects the morphology and behavior of microspore-derived embryos (MDEs). Down-regulation of BnSTM repressed the formation of the SAM and reduced the number of MDEs able to regenerate viable plants at germination. This was in contrast to embryos over-expressing BnSTM which displayed enlarged SAMs with an improved regeneration frequency. To further investigate the regulatory mechanisms underpinning these different responses, global changes in transcript levels were analysed in laser micro dissected SAMs of the transformed MDEs. The induction of BnSTM up-regulated the expression of many transcription factors (TFs) some of which directly involved in the formation of the SAM, i.e. CUPSHAPED COTYLEDON1 and WUSCHEL, and regulatory components ofthe antioxidant response, hormone signalling, and cell wall synthesis and modification. Opposite expression patterns for some of these genes were observed in the SAMs of MDEs down-regulating BnSTM. These data were interpreted in relation to the ability of BnSTM to modulate the size and functionality of the SAM. Structural and expression studies were further conducted to confirm the role of BnSTM in mediating the regulatory network of cell wall and lignin synthesis. Besides lowering the transcript levels of early components of cell wall and lignin biosynthetic pathways in the SAM, BnSTM had a remarkable repressing effect on the expression of PHENYLALANINE AMMONIA LYASE2, CINNAMATE 4-4HYDROXYLASE, andCINNAMYL ALCOHOL DEHYDROGENASE. Since lignin formation is a feature of irreversible cell differentiation, these results suggest that one way in which BnSTM promotes indeterminate cell fate may be by preventing the expression of components of biochemical pathways involved in the accumulation of lignin in the meristematic cells. Overall these studies provide evidence for a novel function of BnSTM in enhancing the quality of in vitro produced meristems, and propose that this gene can be used as a potential target to improve regeneration of cultured embryos.

Project description:Plant growth is the result of cell proliferation in the meristems, which requires a dynamic balance between the formation of new tissue and the maintenance of a set of undifferentiated stem cells. There is much that remains unknown about this vital developmental process. In this study, we have reported the generation of 2735 ESTs from three cDNA libraries derived from dissected garden pea (Pisum sativum cv Torsdag) shoot apical meristems. Clustering analysis of the resulting sequences gave rise to 1686 non-redundant ESTs. The unique sequences generated together with 500 other pea transcripts randomly selected from the GenBank pea protein database have been utilized to construct a 2K oligonucleotide array. Using this pea array, we have obtained the transcript profiles of pea shoot apical meristems in comparison to non-apical-meristem tissues. A total of 181 or 174 transcripts were identified to be significantly up- or down-regulated in the pea shoot apical meristem, respectively. As expected, close to 61% of the transcripts down-regulated in the shoot apical mersitem are those retrieved from the public database, whereas sequences from the same source only made up 12% of the genes that were expressed at higher levels in SAMs. This revelation highlights the under-representation of transcripts from the meristmatic tissues in the current public protein database. Manual inspection of the list of up-regulated transcripts reveals the presence of sequences predicted to encode products associated with cell division and proliferation, epigenetic regulation, auxin-mediated responses and miRNA regulation. Similar genes have been implicated in the regulation of plant stem cell activity. Taken together, our EST collection as well as the microarray data provides useful starting points for more in depth analysis of the meristem function and maintenance. Keywords: shoot apical meristem transcript profiling Total RNA was extracted from dissected SAM (approximately 80 SAMs per extraction) or other plant parts (primary stem, primary roots and mature leaves) using Qiagen RNeasy Mini Kit. Four independent tissue collections and RNA extractions (designated A, B, C and D) were performed for each of the microarray hybridization experiment.The Cy5- or Cy3-labelled cDNA was then hybridized to different sector of the chip according to a balanced block design dual label experiment scheme (Cochran & Cox, 1992): Sector 1: Cy3-SAM A vs Cy5-NM A Sector 2: Cy5-SAM B vs Cy3-NM B Sector 3: Cy3-SAM C vs Cy5-NM C Sector 4: Cy5-SAM D vs Cy3-NM D

Project description:Knowledge about an organismâs cell and tissue-specific transcriptional repertoire is essential for understanding the gene regulatory circuits that control key developmental events. The shoot apical meristem (SAM) is responsible for development of all the above ground parts of plants. Our understanding of SAM at the molecular level is far from complete. The present work investigates the global gene expression repertoire of SAMs in the garden pea (Pisum sativum). To this end, 10,346 EST sequences representing 7611 unique genes were generated from pea SAM cDNA libraries. These sequences, together with previously reported ESTs, were used to construct a 12K oligonucleotide array used to identify genes exhibiting differential SAM expression, as compared to the axillary meristem, root apical meristem, and non-meristematic tissues. We identified a number of genes that are predominantly expressed in specific cell layers or domains of the SAM, and thus are likely components of the gene networks involved in stem cell maintenance and initiation of lateral organ primordial cells. In situ hybridization confirmed the spatial localisation of some of these key genes within the SAM. Our data also indicate the diversification of some gene expression patterns and functions in legume crop plants. RNA samples were hybridized to two-colour Combimatrix arrays manufactured with a custom library of 11958 probes. The limma software package for R (Smyth, 2005) was used in subsequent statistical analysis of the data generated. On examination of the data during quality control assessment, it became evident that the Cy3 channel showed little dynamic range and background correction and normalisation could not correct for this. Therefore only the Cy5 channel was used in the subsequent analysis. This resulted in somewhat unbalanced sample sizes with six replicates of SAM and one each of RAM, AM and NM. Nevertheless, linear modelling and empirical Bayes moderated t-statistics (Smyth, 2004) used in the statistical analysis allows variance information to be borrowed across both samples and genes, resulting in reliable statistical inference even for the groups with only one sample.

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Gene expression analysis in microdissected shoot meristems of Brassica napus microspore-derived embryos with altered levels of SHOOTMERISTEMLESS

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