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Differential SAGE analysis in Arabidopsis uncovers increased transcriptome complexity in response to low temperature.

Robinson SJ, Parkin IA - BMC Genomics (2008)

Bottom Line: Abiotic stress, including low temperature, limits the productivity and geographical distribution of plants, which has led to significant interest in understanding the complex processes that allow plants to adapt to such stresses.Novel genes and cis-acting sequences have been identified as compelling targets to allow manipulation of the plant's ability to protect against low temperature stress.The analyses performed provide a contextual framework for the interpretation of quantitative sequence tag based transcriptome analysis which will prevail with the application of next generation sequencing technology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agriculture and Agri-Food Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, SK, S7N 0X2, Canada. robinsons@agr.gc.ca

ABSTRACT

Background: Abiotic stress, including low temperature, limits the productivity and geographical distribution of plants, which has led to significant interest in understanding the complex processes that allow plants to adapt to such stresses. The wide range of physiological, biochemical and molecular changes that occur in plants exposed to low temperature require a robust global approach to studying the response. We have employed Serial Analysis of Gene Expression (SAGE) to uncover changes in the transcriptome of Arabidopsis thaliana over a time course of low temperature stress.

Results: Five SAGE libraries were generated from A. thaliana leaf tissue collected at time points ranging from 30 minutes to one week of low temperature treatment (4 degrees C). Over 240,000 high quality SAGE tags, corresponding to 16,629 annotated genes, provided a comprehensive survey of changes in the transcriptome in response to low temperature, from perception of the stress to acquisition of freezing tolerance. Interpretation of these data was facilitated by representing the SAGE data by gene identifier, allowing more robust statistical analysis, cross-platform comparisons and the identification of genes sharing common expression profiles. Simultaneous statistical calculations across all five libraries identified 920 low temperature responsive genes, only 24% of which overlapped with previous global expression analysis performed using microarrays, although similar functional categories were affected. Clustering of the differentially regulated genes facilitated the identification of novel loci correlated with the development of freezing tolerance. Analysis of their promoter sequences revealed subsets of genes that were independent of CBF and ABA regulation and could provide a mechanism for elucidating complementary signalling pathways. The SAGE data emphasised the complexity of the plant response, with alternate pre-mRNA processing events increasing at low temperatures and antisense transcription being repressed.

Conclusion: Alternate transcript processing appears to play an important role in enhancing the plasticity of the stress induced transcriptome. Novel genes and cis-acting sequences have been identified as compelling targets to allow manipulation of the plant's ability to protect against low temperature stress. The analyses performed provide a contextual framework for the interpretation of quantitative sequence tag based transcriptome analysis which will prevail with the application of next generation sequencing technology.

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Promoter sequence analysis for COR-like genes. Distribution of 6 mer promoter elements over-represented among the 63 genes clustered according to the COR-gene like profile, described in Figure 3C. Shaded box indicates presence of a cis-element. DRE – CRT/DRE; AUX – auxin responsive; ABRE – ABA responsive; ARE – antioxidant responsive.
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Figure 4: Promoter sequence analysis for COR-like genes. Distribution of 6 mer promoter elements over-represented among the 63 genes clustered according to the COR-gene like profile, described in Figure 3C. Shaded box indicates presence of a cis-element. DRE – CRT/DRE; AUX – auxin responsive; ABRE – ABA responsive; ARE – antioxidant responsive.

Mentions: An analysis of the promoter sequences (within 1500 bp 5' of the ATG) for the 63 genes in the COR gene-like profile (Figure 3C) revealed that 24 possessed the CRT/DRE cis-acting element (CCGAC) recognised by CBF (Figure 4). No correlation was found between the number of CRT/DRE elements in each promoter and the magnitude of gene expression. The promoter sequences of the remaining 39 genes did not possess this element. A comprehensive analysis of 6 mer motifs found to be significantly over-represented within the promoter elements of this cluster identified two additional known cis-acting regulatory elements, 'AUX' and 'ABRE' that are found in the promoters of auxin and absisic acid (ABA) regulated genes respectively [27]. It has been shown that COR gene expression responds to the application of ABA, which is reflected by the presence of the ABRE sequence in a high proportion (83%) of those genes whose promoters also contained the CRT/DRE element. However, there were a number of predicted ABA responsive genes that were independent of the CBF regulon (Figure 4). In addition, two further groups of genes appeared to be independent of either CBF or ABA regulation and were characterised by the preponderance of unknown motifs 'GGCCCA' and 'ATAACC'. A similar promoter analysis of the 126 genes found to be up-regulated at 30 minutes only (Figure 3A) identified 193 6 mers which were significantly more abundant in the selected genes compared to the random set (Additional file 4). The majority of the elements were uninformative due to sequence ambiguity. However, for those which could be assigned to a known motif, they were predominantly annotated as light responsive or under circadian control, which may be expected for this timepoint.


Differential SAGE analysis in Arabidopsis uncovers increased transcriptome complexity in response to low temperature.

Robinson SJ, Parkin IA - BMC Genomics (2008)

Promoter sequence analysis for COR-like genes. Distribution of 6 mer promoter elements over-represented among the 63 genes clustered according to the COR-gene like profile, described in Figure 3C. Shaded box indicates presence of a cis-element. DRE – CRT/DRE; AUX – auxin responsive; ABRE – ABA responsive; ARE – antioxidant responsive.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2568001&req=5

Figure 4: Promoter sequence analysis for COR-like genes. Distribution of 6 mer promoter elements over-represented among the 63 genes clustered according to the COR-gene like profile, described in Figure 3C. Shaded box indicates presence of a cis-element. DRE – CRT/DRE; AUX – auxin responsive; ABRE – ABA responsive; ARE – antioxidant responsive.
Mentions: An analysis of the promoter sequences (within 1500 bp 5' of the ATG) for the 63 genes in the COR gene-like profile (Figure 3C) revealed that 24 possessed the CRT/DRE cis-acting element (CCGAC) recognised by CBF (Figure 4). No correlation was found between the number of CRT/DRE elements in each promoter and the magnitude of gene expression. The promoter sequences of the remaining 39 genes did not possess this element. A comprehensive analysis of 6 mer motifs found to be significantly over-represented within the promoter elements of this cluster identified two additional known cis-acting regulatory elements, 'AUX' and 'ABRE' that are found in the promoters of auxin and absisic acid (ABA) regulated genes respectively [27]. It has been shown that COR gene expression responds to the application of ABA, which is reflected by the presence of the ABRE sequence in a high proportion (83%) of those genes whose promoters also contained the CRT/DRE element. However, there were a number of predicted ABA responsive genes that were independent of the CBF regulon (Figure 4). In addition, two further groups of genes appeared to be independent of either CBF or ABA regulation and were characterised by the preponderance of unknown motifs 'GGCCCA' and 'ATAACC'. A similar promoter analysis of the 126 genes found to be up-regulated at 30 minutes only (Figure 3A) identified 193 6 mers which were significantly more abundant in the selected genes compared to the random set (Additional file 4). The majority of the elements were uninformative due to sequence ambiguity. However, for those which could be assigned to a known motif, they were predominantly annotated as light responsive or under circadian control, which may be expected for this timepoint.

Bottom Line: Abiotic stress, including low temperature, limits the productivity and geographical distribution of plants, which has led to significant interest in understanding the complex processes that allow plants to adapt to such stresses.Novel genes and cis-acting sequences have been identified as compelling targets to allow manipulation of the plant's ability to protect against low temperature stress.The analyses performed provide a contextual framework for the interpretation of quantitative sequence tag based transcriptome analysis which will prevail with the application of next generation sequencing technology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agriculture and Agri-Food Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, SK, S7N 0X2, Canada. robinsons@agr.gc.ca

ABSTRACT

Background: Abiotic stress, including low temperature, limits the productivity and geographical distribution of plants, which has led to significant interest in understanding the complex processes that allow plants to adapt to such stresses. The wide range of physiological, biochemical and molecular changes that occur in plants exposed to low temperature require a robust global approach to studying the response. We have employed Serial Analysis of Gene Expression (SAGE) to uncover changes in the transcriptome of Arabidopsis thaliana over a time course of low temperature stress.

Results: Five SAGE libraries were generated from A. thaliana leaf tissue collected at time points ranging from 30 minutes to one week of low temperature treatment (4 degrees C). Over 240,000 high quality SAGE tags, corresponding to 16,629 annotated genes, provided a comprehensive survey of changes in the transcriptome in response to low temperature, from perception of the stress to acquisition of freezing tolerance. Interpretation of these data was facilitated by representing the SAGE data by gene identifier, allowing more robust statistical analysis, cross-platform comparisons and the identification of genes sharing common expression profiles. Simultaneous statistical calculations across all five libraries identified 920 low temperature responsive genes, only 24% of which overlapped with previous global expression analysis performed using microarrays, although similar functional categories were affected. Clustering of the differentially regulated genes facilitated the identification of novel loci correlated with the development of freezing tolerance. Analysis of their promoter sequences revealed subsets of genes that were independent of CBF and ABA regulation and could provide a mechanism for elucidating complementary signalling pathways. The SAGE data emphasised the complexity of the plant response, with alternate pre-mRNA processing events increasing at low temperatures and antisense transcription being repressed.

Conclusion: Alternate transcript processing appears to play an important role in enhancing the plasticity of the stress induced transcriptome. Novel genes and cis-acting sequences have been identified as compelling targets to allow manipulation of the plant's ability to protect against low temperature stress. The analyses performed provide a contextual framework for the interpretation of quantitative sequence tag based transcriptome analysis which will prevail with the application of next generation sequencing technology.

Show MeSH
Related in: MedlinePlus