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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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Expression profiles of genes encoding proteins producing or detoxifying reactive oxygen species (ROS) in chickpea roots and nodules. A) In the very intricate ROS pathway in legume nodules, superoxide radicals (O2-) are generated by elevated mitochondrial respiration rates. In turn, leghemoglobin (LB2+), the enzyme keeping the nodules free of molecular oxygen (O2), can spontaneously be converted to ferric LB (LB3+), generating new O2-. These radicals can induce further conversions of LB2+ to LB3+. The generated superoxide radical can be directly dismutated by SOD to H2O2, which is immediately decomposed, as depicted in (B). On the other hand, H2O2 can generate hydroxyl radicals (OH-) in the presence of abundant free Fe+ ions, which are sequestered by metallothionein-like proteins. B) Hydrogen peroxide can be scavenged via the glutathione/ascorbate cycles or the action of catalases (CAT) NC: UniTag expression profiles indicating prevalence in nodules (with various intensities of blue):Organ-specific expression NS and RS: UniTag expression profiles of roots and nodules, and up- and down-regulation under salt stress (with various intensities of red and green, respectively): Stress-specific expression
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Figure 10: Expression profiles of genes encoding proteins producing or detoxifying reactive oxygen species (ROS) in chickpea roots and nodules. A) In the very intricate ROS pathway in legume nodules, superoxide radicals (O2-) are generated by elevated mitochondrial respiration rates. In turn, leghemoglobin (LB2+), the enzyme keeping the nodules free of molecular oxygen (O2), can spontaneously be converted to ferric LB (LB3+), generating new O2-. These radicals can induce further conversions of LB2+ to LB3+. The generated superoxide radical can be directly dismutated by SOD to H2O2, which is immediately decomposed, as depicted in (B). On the other hand, H2O2 can generate hydroxyl radicals (OH-) in the presence of abundant free Fe+ ions, which are sequestered by metallothionein-like proteins. B) Hydrogen peroxide can be scavenged via the glutathione/ascorbate cycles or the action of catalases (CAT) NC: UniTag expression profiles indicating prevalence in nodules (with various intensities of blue):Organ-specific expression NS and RS: UniTag expression profiles of roots and nodules, and up- and down-regulation under salt stress (with various intensities of red and green, respectively): Stress-specific expression

Mentions: After dismutation of superoxide to hydrogen peroxide (H2O2), catalases (CATs), ascorbate peroxidases (APXs), dehydroascorbate reductases (DHARs), glutathione peroxidases (GPXs), glutathione reductases (GRs), and glutathione-S-transferases (GSTs) finish the H2O2 scavenging process via the ascorbate and glutathione cycles [8,9,27]. In salt stressed chickpea roots and nodules, diverse expression profiles were revealed by UniTags annotated to these enzymes. Several of these transcripts are very active in nodules even before the onset of the stress, probably due to the high metabolic activity of these chickpea organs [79]. A total of 59 UniTags annotated to proteins belonging to the ascorbate and glutathione cycles were detected in the present dataset. An overview of the plant genes involved in basic ROS-scavenging mechanisms along with the UniTag transcript levels in salt-stressed roots and nodules is depicted in Figure 10. Fold-regulation, annotation to anonymous chickpea ESTs, sequences, and information on copy numbers per SuperSAGE library for the above detailed pathways are accessible through the filtering options on Additional file 1.


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)

Expression profiles of genes encoding proteins producing or detoxifying reactive oxygen species (ROS) in chickpea roots and nodules. A) In the very intricate ROS pathway in legume nodules, superoxide radicals (O2-) are generated by elevated mitochondrial respiration rates. In turn, leghemoglobin (LB2+), the enzyme keeping the nodules free of molecular oxygen (O2), can spontaneously be converted to ferric LB (LB3+), generating new O2-. These radicals can induce further conversions of LB2+ to LB3+. The generated superoxide radical can be directly dismutated by SOD to H2O2, which is immediately decomposed, as depicted in (B). On the other hand, H2O2 can generate hydroxyl radicals (OH-) in the presence of abundant free Fe+ ions, which are sequestered by metallothionein-like proteins. B) Hydrogen peroxide can be scavenged via the glutathione/ascorbate cycles or the action of catalases (CAT) NC: UniTag expression profiles indicating prevalence in nodules (with various intensities of blue):Organ-specific expression NS and RS: UniTag expression profiles of roots and nodules, and up- and down-regulation under salt stress (with various intensities of red and green, respectively): Stress-specific expression
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3045889&req=5

Figure 10: Expression profiles of genes encoding proteins producing or detoxifying reactive oxygen species (ROS) in chickpea roots and nodules. A) In the very intricate ROS pathway in legume nodules, superoxide radicals (O2-) are generated by elevated mitochondrial respiration rates. In turn, leghemoglobin (LB2+), the enzyme keeping the nodules free of molecular oxygen (O2), can spontaneously be converted to ferric LB (LB3+), generating new O2-. These radicals can induce further conversions of LB2+ to LB3+. The generated superoxide radical can be directly dismutated by SOD to H2O2, which is immediately decomposed, as depicted in (B). On the other hand, H2O2 can generate hydroxyl radicals (OH-) in the presence of abundant free Fe+ ions, which are sequestered by metallothionein-like proteins. B) Hydrogen peroxide can be scavenged via the glutathione/ascorbate cycles or the action of catalases (CAT) NC: UniTag expression profiles indicating prevalence in nodules (with various intensities of blue):Organ-specific expression NS and RS: UniTag expression profiles of roots and nodules, and up- and down-regulation under salt stress (with various intensities of red and green, respectively): Stress-specific expression
Mentions: After dismutation of superoxide to hydrogen peroxide (H2O2), catalases (CATs), ascorbate peroxidases (APXs), dehydroascorbate reductases (DHARs), glutathione peroxidases (GPXs), glutathione reductases (GRs), and glutathione-S-transferases (GSTs) finish the H2O2 scavenging process via the ascorbate and glutathione cycles [8,9,27]. In salt stressed chickpea roots and nodules, diverse expression profiles were revealed by UniTags annotated to these enzymes. Several of these transcripts are very active in nodules even before the onset of the stress, probably due to the high metabolic activity of these chickpea organs [79]. A total of 59 UniTags annotated to proteins belonging to the ascorbate and glutathione cycles were detected in the present dataset. An overview of the plant genes involved in basic ROS-scavenging mechanisms along with the UniTag transcript levels in salt-stressed roots and nodules is depicted in Figure 10. Fold-regulation, annotation to anonymous chickpea ESTs, sequences, and information on copy numbers per SuperSAGE library for the above detailed pathways are accessible through the filtering options on Additional file 1.

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