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The salt-responsive transcriptome of chickpea roots and nodules via deepSuperSAGE.

Molina C, Zaman-Allah M, Khan F, Fatnassi N, Horres R, Rotter B, Steinhauer D, Amenc L, Drevon JJ, Winter P, Kahl G - BMC Plant Biol. (2011)

Bottom Line: From several filtered pathways, here we focus exemplarily on transcripts associated with the generation and scavenging of reactive oxygen species (ROS), as well as on transcripts involved in Na+ homeostasis.Newly identified transcript isoforms are potential targets for breeding novel cultivars with high salinity tolerance.We demonstrate that these targets can be integrated into breeding schemes by micro-arrays and RT-PCR assays downstream of the generation of 26 bp tags by SuperSAGE.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular BioSciences, Biocenter, Johann Wolfgang Goethe University, Max-von-Laue-Str, 9, D-60439 Frankfurt am Main, Germany. carlos.molina@dijon.inra.fr

ABSTRACT

Background: The combination of high-throughput transcript profiling and next-generation sequencing technologies is a prerequisite for genome-wide comprehensive transcriptome analysis. Our recent innovation of deepSuperSAGE is based on an advanced SuperSAGE protocol and its combination with massively parallel pyrosequencing on Roche's 454 sequencing platform. As a demonstration of the power of this combination, we have chosen the salt stress transcriptomes of roots and nodules of the third most important legume crop chickpea (Cicer arietinum L.). While our report is more technology-oriented, it nevertheless addresses a major world-wide problem for crops generally: high salinity. Together with low temperatures and water stress, high salinity is responsible for crop losses of millions of tons of various legume (and other) crops. Continuously deteriorating environmental conditions will combine with salinity stress to further compromise crop yields. As a good example for such stress-exposed crop plants, we started to characterize salt stress responses of chickpeas on the transcriptome level.

Results: We used deepSuperSAGE to detect early global transcriptome changes in salt-stressed chickpea. The salt stress responses of 86,919 transcripts representing 17,918 unique 26 bp deepSuperSAGE tags (UniTags) from roots of the salt-tolerant variety INRAT-93 two hours after treatment with 25 mM NaCl were characterized. Additionally, the expression of 57,281 transcripts representing 13,115 UniTags was monitored in nodules of the same plants. From a total of 144,200 analyzed 26 bp tags in roots and nodules together, 21,401 unique transcripts were identified. Of these, only 363 and 106 specific transcripts, respectively, were commonly up- or down-regulated (>3.0-fold) under salt stress in both organs, witnessing a differential organ-specific response to stress.Profiting from recent pioneer works on massive cDNA sequencing in chickpea, more than 9,400 UniTags were able to be linked to UniProt entries. Additionally, gene ontology (GO) categories over-representation analysis enabled to filter out enriched biological processes among the differentially expressed UniTags. Subsequently, the gathered information was further cross-checked with stress-related pathways. From several filtered pathways, here we focus exemplarily on transcripts associated with the generation and scavenging of reactive oxygen species (ROS), as well as on transcripts involved in Na+ homeostasis. Although both processes are already very well characterized in other plants, the information generated in the present work is of high value. Information on expression profiles and sequence similarity for several hundreds of transcripts of potential interest is now available.

Conclusions: This report demonstrates, that the combination of the high-throughput transcriptome profiling technology SuperSAGE with one of the next-generation sequencing platforms allows deep insights into the first molecular reactions of a plant exposed to salinity. Cross validation with recent reports enriched the information about the salt stress dynamics of more than 9,000 chickpea ESTs, and enlarged their pool of alternative transcripts isoforms. As an example for the high resolution of the employed technology that we coin deepSuperSAGE, we demonstrate that ROS-scavenging and -generating pathways undergo strong global transcriptome changes in chickpea roots and nodules already 2 hours after onset of moderate salt stress (25 mM NaCl). Additionally, a set of more than 15 candidate transcripts are proposed to be potential components of the salt overly sensitive (SOS) pathway in chickpea. Newly identified transcript isoforms are potential targets for breeding novel cultivars with high salinity tolerance. We demonstrate that these targets can be integrated into breeding schemes by micro-arrays and RT-PCR assays downstream of the generation of 26 bp tags by SuperSAGE.

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Venn Mapper output detailing shared responses (number of UniTags) between salt-stressed roots and nodules, respectively, and non-stressed nodules relative to roots.
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Figure 4: Venn Mapper output detailing shared responses (number of UniTags) between salt-stressed roots and nodules, respectively, and non-stressed nodules relative to roots.

Mentions: Setting a minimum threshold of 3-fold differential expression, from the 2,098 26 bp tags prevalent in non-stressed nodules, 515 (24.5%) were also at least 3-fold up-regulated in roots under salt stress. These 515 UniTags represented 23.3% of the root transcripts >3-fold up-regulated by salt. On the other hand, only 10 out of the 2,098 UniTags were more than 3-fold up-regulated in salt-stressed nodules. In both salt-stressed roots and nodules, 363 common 26 bp tags were more than 3-fold up-regulated (16.7% from nodules, and 16.4% from roots; Figure 4, upper panel). As far as down-regulation is concerned, 1,729 out of 1,936 UniTags prevalent in non-stressed roots were more than 3-fold down-regulated in roots after 2 h of salt treatment. A total of 275 tags were commonly more than 3-fold down-regulated in both roots and nodules under salt-stress.


The salt-responsive transcriptome of chickpea roots and nodules via deepSuperSAGE.

Molina C, Zaman-Allah M, Khan F, Fatnassi N, Horres R, Rotter B, Steinhauer D, Amenc L, Drevon JJ, Winter P, Kahl G - BMC Plant Biol. (2011)

Venn Mapper output detailing shared responses (number of UniTags) between salt-stressed roots and nodules, respectively, and non-stressed nodules relative to roots.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045889&req=5

Figure 4: Venn Mapper output detailing shared responses (number of UniTags) between salt-stressed roots and nodules, respectively, and non-stressed nodules relative to roots.
Mentions: Setting a minimum threshold of 3-fold differential expression, from the 2,098 26 bp tags prevalent in non-stressed nodules, 515 (24.5%) were also at least 3-fold up-regulated in roots under salt stress. These 515 UniTags represented 23.3% of the root transcripts >3-fold up-regulated by salt. On the other hand, only 10 out of the 2,098 UniTags were more than 3-fold up-regulated in salt-stressed nodules. In both salt-stressed roots and nodules, 363 common 26 bp tags were more than 3-fold up-regulated (16.7% from nodules, and 16.4% from roots; Figure 4, upper panel). As far as down-regulation is concerned, 1,729 out of 1,936 UniTags prevalent in non-stressed roots were more than 3-fold down-regulated in roots after 2 h of salt treatment. A total of 275 tags were commonly more than 3-fold down-regulated in both roots and nodules under salt-stress.

Bottom Line: From several filtered pathways, here we focus exemplarily on transcripts associated with the generation and scavenging of reactive oxygen species (ROS), as well as on transcripts involved in Na+ homeostasis.Newly identified transcript isoforms are potential targets for breeding novel cultivars with high salinity tolerance.We demonstrate that these targets can be integrated into breeding schemes by micro-arrays and RT-PCR assays downstream of the generation of 26 bp tags by SuperSAGE.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular BioSciences, Biocenter, Johann Wolfgang Goethe University, Max-von-Laue-Str, 9, D-60439 Frankfurt am Main, Germany. carlos.molina@dijon.inra.fr

ABSTRACT

Background: The combination of high-throughput transcript profiling and next-generation sequencing technologies is a prerequisite for genome-wide comprehensive transcriptome analysis. Our recent innovation of deepSuperSAGE is based on an advanced SuperSAGE protocol and its combination with massively parallel pyrosequencing on Roche's 454 sequencing platform. As a demonstration of the power of this combination, we have chosen the salt stress transcriptomes of roots and nodules of the third most important legume crop chickpea (Cicer arietinum L.). While our report is more technology-oriented, it nevertheless addresses a major world-wide problem for crops generally: high salinity. Together with low temperatures and water stress, high salinity is responsible for crop losses of millions of tons of various legume (and other) crops. Continuously deteriorating environmental conditions will combine with salinity stress to further compromise crop yields. As a good example for such stress-exposed crop plants, we started to characterize salt stress responses of chickpeas on the transcriptome level.

Results: We used deepSuperSAGE to detect early global transcriptome changes in salt-stressed chickpea. The salt stress responses of 86,919 transcripts representing 17,918 unique 26 bp deepSuperSAGE tags (UniTags) from roots of the salt-tolerant variety INRAT-93 two hours after treatment with 25 mM NaCl were characterized. Additionally, the expression of 57,281 transcripts representing 13,115 UniTags was monitored in nodules of the same plants. From a total of 144,200 analyzed 26 bp tags in roots and nodules together, 21,401 unique transcripts were identified. Of these, only 363 and 106 specific transcripts, respectively, were commonly up- or down-regulated (>3.0-fold) under salt stress in both organs, witnessing a differential organ-specific response to stress.Profiting from recent pioneer works on massive cDNA sequencing in chickpea, more than 9,400 UniTags were able to be linked to UniProt entries. Additionally, gene ontology (GO) categories over-representation analysis enabled to filter out enriched biological processes among the differentially expressed UniTags. Subsequently, the gathered information was further cross-checked with stress-related pathways. From several filtered pathways, here we focus exemplarily on transcripts associated with the generation and scavenging of reactive oxygen species (ROS), as well as on transcripts involved in Na+ homeostasis. Although both processes are already very well characterized in other plants, the information generated in the present work is of high value. Information on expression profiles and sequence similarity for several hundreds of transcripts of potential interest is now available.

Conclusions: This report demonstrates, that the combination of the high-throughput transcriptome profiling technology SuperSAGE with one of the next-generation sequencing platforms allows deep insights into the first molecular reactions of a plant exposed to salinity. Cross validation with recent reports enriched the information about the salt stress dynamics of more than 9,000 chickpea ESTs, and enlarged their pool of alternative transcripts isoforms. As an example for the high resolution of the employed technology that we coin deepSuperSAGE, we demonstrate that ROS-scavenging and -generating pathways undergo strong global transcriptome changes in chickpea roots and nodules already 2 hours after onset of moderate salt stress (25 mM NaCl). Additionally, a set of more than 15 candidate transcripts are proposed to be potential components of the salt overly sensitive (SOS) pathway in chickpea. Newly identified transcript isoforms are potential targets for breeding novel cultivars with high salinity tolerance. We demonstrate that these targets can be integrated into breeding schemes by micro-arrays and RT-PCR assays downstream of the generation of 26 bp tags by SuperSAGE.

Show MeSH
Related in: MedlinePlus