SuperSAGE: the drought stress-responsive transcriptome of chickpea roots

BackgroundDrought is the major constraint to increase yield in chickpea (Cicer arietinum). Improving drought tolerance is therefore of outmost importance for breeding. However, the complexity of the trait allowed only marginal progress. A solution to the current stagnation is expected from innovative molecular tools such as transcriptome analyses providing insight into stress-related gene activity, which combined with molecular markers and expression (e)QTL mapping, may accelerate knowledge-based breeding. SuperSAGE, an improved version of the serial analysis of gene expression (SAGE) technique, generating genome-wide, high-quality transcription profiles from any eukaryote, has been employed in the present study. The method produces 26 bp long fragments (26 bp tags) from defined positions in cDNAs, providing sufficient sequence information to unambiguously characterize the mRNAs. Further, SuperSAGE tags may be immediately used to produce microarrays and probes for real-time-PCR, thereby overcoming the lack of genomic tools in non-model organisms.ResultsWe applied SuperSAGE to the analysis of gene expression in chickpea roots in response to drought. To this end, we sequenced 80,238 26 bp tags representing 17,493 unique transcripts (UniTags) from drought-stressed and non-stressed control roots. A total of 7,532 (43%) UniTags were more than 2.7-fold differentially expressed, and 880 (5.0%) were regulated more than 8-fold upon stress. Their large size enabled the unambiguous annotation of 3,858 (22%) UniTags to genes or proteins in public data bases and thus to stress-response processes. We designed a microarray carrying 3,000 of these 26 bp tags. The chip data confirmed 79% of the tag-based results, whereas RT-PCR confirmed the SuperSAGE data in all cases.ConclusionThis study represents the most comprehensive analysis of the drought-response transcriptome of chickpea available to date. It demonstrates that – inter alias – signal transduction, transcription regulation, osmolyte accumulation, and ROS scavenging undergo strong transcriptional remodelling in chickpea roots already 6 h after drought stress. Certain transcript isoforms characterizing these processes are potential targets for breeding for drought tolerance. We demonstrate that these can be easily accessed by micro-arrays and RT-PCR assays readily produced downstream of SuperSAGE. Our study proves that SuperSAGE owns potential for molecular breeding also in non-model crops.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[3]  Derekt . A. Lamport,et al.  Extensin: repetitive motifs, functional sites, post-translational codes, and phylogeny. , 1994, The Plant journal : for cell and molecular biology.

[4]  Aaron P. Campbell,et al.  Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[5]  A. Hanson,et al.  Betaine-aldehyde dehydrogenase from amaranth leaves efficiently catalyzes the NAD-dependent oxidation of dimethylsulfoniopropionaldehyde to dimethylsulfoniopropionate. , 1997, Archives of biochemistry and biophysics.

[6]  J. Claverie,et al.  The significance of digital gene expression profiles. , 1997, Genome research.

[7]  J. Massagué.  TGF-beta signal transduction. , 1998, Annual review of biochemistry.

[8]  D. Bastola,et al.  Transgenic overexpression of the transcription factor alfin1 enhances expression of the endogenous MsPRP2 gene in alfalfa and improves salinity tolerance of the plants , 1999, Plant physiology.

[9]  C. Brownlee,et al.  Communicating with Calcium , 1999, Plant Cell.

[10]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[11]  D. Gage,et al.  Feedback Regulation of GA5 Expression and Metabolic Engineering of Gibberellin Levels in Arabidopsis , 1999, Plant Cell.

[12]  D. Hardie.  PLANT PROTEIN SERINE/THREONINE KINASES: Classification and Functions. , 1999, Annual review of plant physiology and plant molecular biology.

[13]  K. Shinozaki,et al.  A stress-inducible gene for 9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis under water stress in drought-tolerant cowpea. , 2000, Plant physiology.

[14]  O. Vitolo,et al.  Improved NlaIII digestion of PAGE-purified 102 bp ditags by addition of a single purification step in both the SAGE and microSAGE protocols. , 2000, Nucleic acids research.

[15]  F. Chen,et al.  Expression of an expansin is associated with endosperm weakening during tomato seed germination. , 2000, Plant physiology.

[16]  R. R. Samaha,et al.  Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. , 2000, Science.

[17]  Hong Wang,et al.  Gene Expression Profiles during the Initial Phase of Salt Stress in Rice , 2001, Plant Cell.

[18]  Signal transduction. Calcium channels--link locally, act globally. , 2001, Science.

[19]  C. Larsson,et al.  Data mining the Arabidopsis genome reveals fifteen 14-3-3 genes. Expression is demonstrated for two out of five novel genes. , 2001, Plant physiology.

[20]  S. Ikeda.  Calcium Channels--Link Locally, Act Globally , 2001, Science.

[21]  Alex Levine,et al.  Oxidative Stress Increased Respiration and Generation of Reactive Oxygen Species, Resulting in ATP Depletion, Opening of Mitochondrial Permeability Transition, and Programmed Cell Death1 , 2002, Plant Physiology.

[22]  J. Bewley,et al.  In vivo characterization of the effects of abscisic acid and drying protocols associated with the acquisition of desiccation tolerance in alfalfa (Medicago sativa L.) somatic embryos. , 2002, Annals of botany.

[23]  K. Shinozaki,et al.  Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. , 2002, The Plant journal : for cell and molecular biology.

[24]  Zheng-Hua Ye,et al.  Mutation of a Chitinase-Like Gene Causes Ectopic Deposition of Lignin, Aberrant Cell Shapes, and Overproduction of Ethylene Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010278. , 2002, The Plant Cell Online.

[25]  T. G. Owens,et al.  Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Ji Huang,et al.  [Serial analysis of gene expression]. , 2002, Yi chuan = Hereditas.

[27]  Jian-Kang Zhu,et al.  Cell Signaling during Cold, Drought, and Salt Stress Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.000596. , 2002, The Plant Cell Online.

[28]  Jian-Kang Zhu,et al.  Salt and drought stress signal transduction in plants. , 2002, Annual review of plant biology.

[29]  A. Lapthorn,et al.  Plant glutathione transferases , 2002, Genome Biology.

[30]  Peter Winter,et al.  Gene expression analysis of plant host–pathogen interactions by SuperSAGE , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[31]  P. Christou,et al.  Reduction in the endogenous arginine decarboxylase transcript levels in rice leads to depletion of the putrescine and spermidine pools with no concomitant changes in the expression of downstream genes in the polyamine biosynthetic pathway , 2003, Planta.

[32]  Patrick S Schnable,et al.  Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize. , 2003, The Plant cell.

[33]  Kazuo Shinozaki,et al.  Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) Function as Transcriptional Activators in Abscisic Acid Signaling Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.006130. , 2003, The Plant Cell Online.

[34]  T. Munnik,et al.  Phospholipid-based signaling in plants. , 2003, Annual review of plant biology.

[35]  Christopher J. Lee.  Generating Consensus Sequences from Partial Order Multiple Sequence Alignment Graphs , 2003, Bioinform..

[36]  D. Shasha,et al.  A Gene Expression Map of the Arabidopsis Root , 2003, Science.

[37]  H. Hirt,et al.  Reactive oxygen species: metabolism, oxidative stress, and signal transduction. , 2004, Annual review of plant biology.

[38]  J. Thevelein,et al.  The Arabidopsis Trehalose-6-P Synthase AtTPS1 Gene Is a Regulator of Glucose, Abscisic Acid, and Stress Signaling1 , 2004, Plant Physiology.

[39]  J. Salinas,et al.  14-3-3 proteins and the response to abiotic and biotic stress , 2002, Plant Molecular Biology.

[40]  J. Thevelein,et al.  The Arabidopsis Trehalose-6P Synthase AtTPS 1 Gene Is a Regulator of Glucose , Abscisic Acid , and Stress Signaling 1 , 2004 .

[41]  Mark Stitt,et al.  Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. , 2004, The Plant journal : for cell and molecular biology.

[42]  Y. Kamiya,et al.  The Arabidopsis cytochrome P450 CYP707A encodes ABA 8′‐hydroxylases: key enzymes in ABA catabolism , 2004, The EMBO journal.

[43]  K. Shinozaki,et al.  Arabidopsis stress-inducible gene for arginine decarboxylase AtADC2 is required for accumulation of putrescine in salt tolerance. , 2004, Biochemical and biophysical research communications.

[44]  A. Hanson,et al.  Salt-inducible betaine aldehyde dehydrogenase from sugar beet: cDNA cloning and expression , 2004, Plant Molecular Biology.

[45]  N. Soranzo,et al.  Organisation and structural evolution of the rice glutathione S-transferase gene family , 2004, Molecular Genetics and Genomics.

[46]  K. Shinozaki,et al.  Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression , 2000, Plant Molecular Biology.

[47]  Klaus F. X. Mayer,et al.  Crosstalk and differential response to abiotic and biotic stressors reflected at the transcriptional level of effector genes from secondary metabolism , 2004, Plant Molecular Biology.

[48]  P. Verma,et al.  Long Term Transcript Accumulation during the Development of Dehydration Adaptation in Cicer arietinum1 , 2004, Plant Physiology.

[49]  K. Hirschi.  The Calcium Conundrum. Both Versatile Nutrient and Specific Signal1 , 2004, Plant Physiology.

[50]  H. Matsumura,et al.  SuperSAGE: Interaction transcriptomics with SuperSAGE , 2004 .

[51]  D. Inzé,et al.  Extensin gene expression is induced by mechanical stimuli leading to local cell wall strengthening in Nicotiana plumbaginifolia , 2004, Planta.

[52]  M. Mewies,et al.  Defining substrate specificity and catalytic mechanism in ascorbate peroxidase. , 2004, Biochemical Society symposium.

[53]  P. Christou,et al.  Promoter strength influences polyamine metabolism and morphogenic capacity in transgenic rice tissues expressing the oat adc cDNA constitutively , 2000, Transgenic Research.

[54]  Patrik Edén,et al.  Comparing Functional Annotation Analyses with Catmap Comparing Functional Annotation Analyses with Catmap , 2004 .

[55]  Ji-yeon Lee,et al.  Use of SAGE technology to reveal changes in gene expression in Arabidopsis leaves undergoing cold stress , 2003, Plant Molecular Biology.

[56]  P. Christou,et al.  Modulation of the polyamine biosynthetic pathway in transgenic rice confers tolerance to drought stress. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[57]  P. Christou,et al.  A transgenic rice cell lineage expressing the oat arginine decarboxylase (adc) cDNA constitutively accumulates putrescine in callus and seeds but not in vegetative tissues , 2000, Plant Molecular Biology.

[58]  S. Kim,et al.  ABF2, an ABRE-binding bZIP factor, is an essential component of glucose signaling and its overexpression affects multiple stress tolerance. , 2004, The Plant journal : for cell and molecular biology.

[59]  K. Hirschi,et al.  The Protein Kinase SOS2 Activates the Arabidopsis H+/Ca2+ Antiporter CAX1 to Integrate Calcium Transport and Salt Tolerance* , 2004, Journal of Biological Chemistry.

[60]  K. C. Oliveira,et al.  Transcription profiling of signal transduction-related genes in sugarcane tissues. , 2005, DNA research : an international journal for rapid publication of reports on genes and genomes.

[61]  O. Borsani,et al.  Endogenous siRNAs Derived from a Pair of Natural cis-Antisense Transcripts Regulate Salt Tolerance in Arabidopsis , 2005, Cell.

[62]  S. Tabata,et al.  Comparison of the transcript profiles from the root and the nodulating root of the model legume Lotus japonicus by serial analysis of gene expression. , 2005, Molecular plant-microbe interactions : MPMI.

[63]  K. Weising.  DNA fingerprinting in plants: principles, methods, and applications , 2005 .

[64]  H. Matsumura,et al.  SuperSAGE combined with PCR walking allows global gene expression profiling of banana (Musa acuminata), a non-model organism , 2005, Theoretical and Applied Genetics.

[65]  Peter Winter,et al.  SuperSAGE , 2005, Cellular microbiology.

[66]  Kazuo Shinozaki,et al.  Effects of free proline accumulation in petunias under drought stress. , 2005, Journal of experimental botany.

[67]  M. Winfield.  DNA Fingerprinting in Plants: Principles, Methods and Applications. 2nd Edition. By K. Weising, H. Nybom, K. Wolff and G. Kahl. London: CRC Press (2005), pp. 444, £56.99 (paperback). ISBN 0-8493-1488-7 , 2006, Experimental Agriculture.

[68]  P. Winter,et al.  Chickpea molecular breeding: New tools and concepts , 2006, Euphytica.

[69]  M. Bilgin,et al.  Arabidopsis cytochrome P450s through the looking glass: a window on plant biochemistry , 2006, Phytochemistry Reviews.

[70]  W. Shi,et al.  Expression profiling of the 14-3-3 gene family in response to salt stress and potassium and iron deficiencies in young tomato (Solanum lycopersicum) roots: analysis by real-time RT-PCR. , 2006, Annals of botany.

[71]  A. Camargo,et al.  Sense-antisense pairs in mammals: functional and evolutionary considerations , 2007, Genome Biology.

[72]  V. Brendel,et al.  Genomewide comparative analysis of alternative splicing in plants. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[73]  N. Le Meur,et al.  Transcriptome profiling uncovers metabolic and regulatory processes occurring during the transition from desiccation-sensitive to desiccation-tolerant stages in Medicago truncatula seeds. , 2006, The Plant journal : for cell and molecular biology.

[74]  Francisco Marco,et al.  Involvement of polyamines in plant response to abiotic stress , 2006, Biotechnology Letters.

[75]  Fang Chen,et al.  The rice 14-3-3 gene family and its involvement in responses to biotic and abiotic stress. , 2006, DNA research : an international journal for rapid publication of reports on genes and genomes.

[76]  L. Gentzbittel,et al.  Genetic variability for physiological traits under drought conditions and differential expression of water stress-associated genes in sunflower (Helianthus annuus L.) , 2006, Theoretical and Applied Genetics.

[77]  P. Nilsson,et al.  The genetics and genomics of the drought response in Populus. , 2006, The Plant journal : for cell and molecular biology.

[78]  Günter Kahl,et al.  SuperSAGE array: the direct use of 26-base-pair transcript tags in oligonucleotide arrays , 2006, Nature Methods.

[79]  M. Klimecka,et al.  Structure and functions of plant calcium-dependent protein kinases. , 2007, Acta biochimica Polonica.

[80]  Sophie Alvarez,et al.  Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes , 2007, Proceedings of the National Academy of Sciences.

[81]  K. Shinozaki,et al.  Gene networks involved in drought stress response and tolerance. , 2006, Journal of experimental botany.

[82]  R. E. Sharp,et al.  Comparing regional transcript profiles from maize primary roots under well-watered and low water potential conditions , 2007 .

[83]  Sung-ju Ahn,et al.  Transgenic Arabidopsis and tobacco plants overexpressing an aquaporin respond differently to various abiotic stresses , 2007, Plant Molecular Biology.

[84]  Kazuo Shinozaki,et al.  Regulatory metabolic networks in drought stress responses. , 2007, Current opinion in plant biology.

[85]  Xinxiang Peng,et al.  Identification of aluminum‐responsive proteins in rice roots by a proteomic approach: Cysteine synthase as a key player in Al response , 2007, Proteomics.

[86]  Sunia A Trauger,et al.  Correlating the Transcriptome, Proteome, and Metabolome in the Environmental Adaptation of a Hyperthermophile , 2022 .