Dynamic differential evolution schemes of WRKY transcription factors in domesticated and wild rice.

WRKY transcription factors play key roles in stress responses, growth, and development. We previously reported on the evolution of WRKYs from unicellular green algae to land plants. To address recent evolution events, we studied three domesticated and eight wild species in the genus Oryza, an ideal model due to its long history of domestication, economic importance, and central role as a model system. We have identified prevalence of Group III WRKYs despite differences in breeding of cultivated and wild species. Same groups of WRKY genes tend to cluster together, suggesting recent, multiple duplication events. Duplications followed by divergence may result in neofunctionalizations of co-expressed WRKY genes that finely tune the regulation of target genes in a same metabolic or response pathway. WRKY genes have undergone recent rearrangements to form novel genes. Group Ib WRKYs, unique to AA genome type Oryza species, are derived from Group III genes dated back to 6.76 million years ago. Gene tree reconciliation analysis with the species tree revealed details of duplication and loss events in the 11 genomes. Selection analysis on single copy orthologs reveals the highly conserved nature of the WRKY domain and clusters of fast evolving sites under strong positive selection pressure. Also, the numbers of single copy orthologs under positive or negative selection almost evenly split. Our results provide valuable insights into the preservation and diversification of an important gene family under strong selective pressure for biotechnological improvements of the world's most valued food crop.

Metabolomic Profiling of Soybeans (Glycine max L.) Reveals the Importance of Sugar and Nitrogen Metabolism under Drought and Heat Stress.

Soybean is an important crop that is continually threatened by abiotic stresses, especially drought and heat stress. At molecular levels, reduced yields due to drought and heat stress can be seen as a result of alterations in metabolic homeostasis of vegetative tissues. At present an incomplete understanding of abiotic stress-associated metabolism and identification of associated metabolites remains a major gap in soybean stress research. A study with a goal to profile leaf metabolites under control conditions (28/24 °C), drought [28/24 °C, 10% volumetric water content (VWC)], and heat stress (43/35 °C) was conducted in a controlled environment. Analyses of non-targeted metabolomic data showed that in response to drought and heat stress, key metabolites (carbohydrates, amino acids, lipids, cofactors, nucleotides, peptides and secondary metabolites) were differentially accumulated in soybean leaves. The metabolites for various cellular processes, such as glycolysis, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, and starch biosynthesis, that regulate carbohydrate metabolism, amino acid metabolism, peptide metabolism, and purine and pyrimidine biosynthesis, were found to be affected by drought as well as heat stress. Computationally based regulatory networks predicted additional compounds that address the possibility of other metabolites and metabolic pathways that could also be important for soybean under drought and heat stress conditions. Metabolomic profiling demonstrated that in soybeans, keeping up with sugar and nitrogen metabolism is of prime significance, along with phytochemical metabolism under drought and heat stress conditions.

Effect of Spectral Quality of Monochromatic LED Lights on the Growth of Artichoke Seedlings.

Indoor farming is becoming a popular alternative approach in food production to meet the demand of a growing world population. Under this production system, artificial light provides the main source of illumination in sustaining plant growth and development. The use of light-emitting diodes (LEDs) is a popular source of artificial light for indoor farms due to its narrow light spectra, modular design and energy efficiency. This study purposely assessed the effect of monochromatic LED light quality on the growth of three varieties of artichoke seedlings compared to greenhouse condition. Spectral quality assessment showed that photosynthetic photon flux density (PPFD) was highest under red LED light, but only a third of the total PPFD under natural light. Seedlings grown under red light showed 60-100% more shoot dry weight and were 67-115% taller than seedlings grown in the greenhouse. However, seedlings under blue or white light conditions showed 67-76% less in biomass compared to greenhouse-grown seedlings. Overall, plant response of seedlings under red light condition was much better compared to greenhouse-grown seedlings emphasizing the importance of red light spectral quality in plant growth and development.

Comparative Metabolome Profile between Tobacco and Soybean Grown under Water-Stressed Conditions.

Understanding how plants respond to water deficit is important in order to develop crops tolerant to drought. In this study, we compare two large metabolomics datasets where we employed a nontargeted metabolomics approach to elucidate metabolic pathways perturbed by progressive dehydration in tobacco and soybean plants. The two datasets were created using the same strategy to create water deficit conditions and an identical metabolomics pipeline. Comparisons between the two datasets therefore reveal common responses between the two species, responses specific to one of the species, responses that occur in both root and leaf tissues, and responses that are specific to one tissue. Stomatal closure is the immediate response of the plant and this did not coincide with accumulation of abscisic acid. A total of 116 and 140 metabolites were observed in tobacco leaves and roots, respectively, while 241 and 207 were observed in soybean leaves and roots, respectively. Accumulation of metabolites is significantly correlated with the extent of dehydration in both species. Among the metabolites that show increases that are restricted to just one plant, 4-hydroxy-2-oxoglutaric acid (KHG) in tobacco roots and coumestrol in soybean roots show the highest tissue-specific accumulation. The comparisons of these two large nontargeted metabolomics datasets provide novel information and suggest that KHG will be a useful marker for drought stress for some members of Solanaceae and coumestrol for some legume species.

What Have We Learned About Synthetic Promoter Construction?

The molecular components of transcriptional regulation are modular. Transcription factors have domains for specific functions such as DNA binding, dimerization, and protein-protein interactions associated with transcriptional activation and repression. Similarly, promoters are modular. They consist of combinations of cis-acting elements that are the binding sites for transcription factors. It is this promoter architecture that largely determines the expression pattern of a gene. The modular nature of promoters is supported by the observation that many cis-acting elements retain their activities when they are taken out of their native promoter context and used as building blocks in synthetic promoters. We therefore have a large collection of cis-acting elements to use in building synthetic promoters and many minimal promoters upon which to build them. This review discusses what we have learned concerning how to use these building blocks to make synthetic promoters. It has become clear that we can increase the strength of a promoter by adding increasing numbers of cis-acting elements. However, it appears that there may be a sweet spot with regard to inducibility as promoters with increasing numbers of copies of an element often show increased background expression. Spacing between elements appears important because if elements are placed too close together activity is lost, presumably due to reduced transcription factor binding due to steric hindrance. In many cases, promoters that contain combinations of cis-acting elements show better expression characteristics than promoters that contain a single type of element. This may be because multiple transcription factor binding sites in the promoter places it at the end of multiple signal transduction pathways. Finally, some cis-acting elements form functional units with other elements and are inactive on their own. In such cases, the complete unit is required for function in a synthetic promoter. Taken together, we have learned much about how to construct synthetic promoters and this knowledge will be crucial in both designing promoters to drive transgenes and also as components of defined regulatory networks in synthetic biology.

Transcript structure and domain display: a customizable transcript visualization tool.

UNLABELLED: Transcript Structure and Domain Display (TSDD) is a publicly available, web-based program that provides publication quality images of transcript structures and domains. TSDD is capable of producing transcript structures from GFF/GFF3 and BED files. Alternatively, the GFF files of several model organisms have been pre-loaded so that users only needs to enter the locus IDs of the transcripts to be displayed. Visualization of transcripts provides many benefits to researchers, ranging from evolutionary analysis of DNA-binding domains to predictive function modeling. AVAILABILITY AND IMPLEMENTATION: TSDD is freely available for non-commercial users at http://shenlab.sols.unlv.edu/shenlab/software/TSD/transcript_display.html CONTACT: : jeffery.shen@unlv.nevada.edu.

Proteomic Responses of Switchgrass and Prairie Cordgrass to Senescence.

Senescence in biofuel grasses is a critical issue because early senescence decreases potential biomass production by limiting aerial growth and development. 2-Dimensional, differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometry of selected protein spots was used to evaluate differences between leaf proteomes of early (ES)- and late- senescing (LS) genotypes of Prairie cordgrass (ES/LS PCG) and switchgrass (ES/LS SG), just before and after senescence was initiated. Analysis of the manually filtered and statistically evaluated data indicated that 69 proteins were significantly differentially abundant across all comparisons, and a majority (41%) were associated with photosynthetic processes as determined by gene ontology analysis. Ten proteins were found in common between PCG and SG, and nine and 18 proteins were unique to PCG and SG respectively. Five of the 10 differentially abundant spots common to both species were increased in abundance, and five were decreased in abundance. Leaf proteomes of the LS genotypes of both grasses analyzed before senescence contained significantly higher abundances of a 14-3-3 like protein and a glutathione-S-transferase protein when compared to the ES genotypes, suggesting differential cellular metabolism in the LS vs. the ES genotypes. The higher abundance of 14-3-3 like proteins may be one factor that impacts the senescence process in both LS PCG and LS SG. Aconitase dehydratase was found in greater abundance in all four genotypes after the onset of senescence, consistent with literature reports from genetic and transcriptomic studies. A Rab protein of the Ras family of G proteins and an s-adenosylmethionine synthase were more abundant in ES PCG when compared with the LS PCG. In contrast, several proteins associated with photosynthesis and carbon assimilation were detected in greater abundance in LS PCG when compared to ES PCG, suggesting that a loss of these proteins potentially contributed to the ES phenotype in PCG. Overall, this study provides important data that can be utilized toward delaying senescence in both PCG and SG, and sets a foundational base for future improvement of perennial grass germplasm for greater aerial biomass productivity.

A toolbox of genes, proteins, metabolites and promoters for improving drought tolerance in soybean includes the metabolite coumestrol and stomatal development genes.

BACKGROUND: The purpose of this project was to identify metabolites, proteins, genes, and promoters associated with water stress responses in soybean. A number of these may serve as new targets for the biotechnological improvement of drought responses in soybean (Glycine max). RESULTS: We identified metabolites, proteins, and genes that are strongly up or down regulated during rapid water stress following removal from a hydroponics system. 163 metabolites showed significant changes during water stress in roots and 93 in leaves. The largest change was a root-specific 160-fold increase in the coumestan coumestrol making it a potential biomarker for drought and a promising target for improving drought responses. Previous reports suggest that coumestrol stimulates mycorrhizal colonization and under certain conditions mycorrhizal plants have improved drought tolerance. This suggests that coumestrol may be part of a call for help to the rhizobiome during stress. About 3,000 genes were strongly up-regulated by drought and we identified regulators such as ERF, MYB, NAC, bHLH, and WRKY transcription factors, receptor-like kinases, and calcium signaling components as potential targets for soybean improvement as well as the jasmonate and abscisic acid biosynthetic genes JMT, LOX1, and ABA1. Drought stressed soybean leaves show reduced mRNA levels of stomatal development genes including FAMA-like, MUTE-like and SPEECHLESS-like bHLH transcription factors and leaves formed after drought stress had a reduction in stomatal density of 22.34 % and stomatal index of 17.56 %. This suggests that reducing stomatal density may improve drought tolerance. MEME analyses suggest that ABRE (CACGT/CG), CRT/DRE (CCGAC) and a novel GTGCnTGC/G element play roles in transcriptional activation and these could form components of synthetic promoters to drive expression of transgenes. Using transformed hairy roots, we validated the increase in promoter activity of GmWRKY17 and GmWRKY67 during dehydration and after 20 µM ABA treatment. CONCLUSIONS: Our toolbox provides new targets and strategies for improving soybean drought tolerance and includes the coumestan coumestrol, transcription factors that regulate stomatal density, water stress-responsive WRKY gene promoters and a novel DNA element that appears to be enriched in water stress responsive promoters.

The WRKY transcription factor family and senescence in switchgrass.

BACKGROUND: Early aerial senescence in switchgrass (Panicum virgatum) can significantly limit biomass yields. WRKY transcription factors that can regulate senescence could be used to reprogram senescence and enhance biomass yields. METHODS: All potential WRKY genes present in the version 1.0 of the switchgrass genome were identified and curated using manual and bioinformatic methods. Expression profiles of WRKY genes in switchgrass flag leaf RNA-Seq datasets were analyzed using clustering and network analyses tools to identify both WRKY and WRKY-associated gene co-expression networks during leaf development and senescence onset. RESULTS: We identified 240 switchgrass WRKY genes including members of the RW5 and RW6 families of resistance proteins. Weighted gene co-expression network analysis of the flag leaf transcriptomes across development readily separated clusters of co-expressed genes into thirteen modules. A visualization highlighted separation of modules associated with the early and senescence-onset phases of flag leaf growth. The senescence-associated module contained 3000 genes including 23 WRKYs. Putative promoter regions of senescence-associated WRKY genes contained several cis-element-like sequences suggestive of responsiveness to both senescence and stress signaling pathways. A phylogenetic comparison of senescence-associated WRKY genes from switchgrass flag leaf with senescence-associated WRKY genes from other plants revealed notable hotspots in Group I, IIb, and IIe of the phylogenetic tree. CONCLUSIONS: We have identified and named 240 WRKY genes in the switchgrass genome. Twenty three of these genes show elevated mRNA levels during the onset of flag leaf senescence. Eleven of the WRKY genes were found in hotspots of related senescence-associated genes from multiple species and thus represent promising targets for future switchgrass genetic improvement. Overall, individual WRKY gene expression profiles could be readily linked to developmental stages of flag leaves.

Transcriptome profiling of tobacco under water deficit conditions.

Drought is one of the limiting environmental factors that affect crop production. Understanding the molecular basis of how plants respond to this water deficit stress is key to developing drought tolerant crops. In this study we generated time course-based transcriptome profiles of tobacco plants under water deficit conditions using microarray technology. In this paper, we describe in detail the experimental procedures and analyses performed in our study. The data set we generated (available in the NCBI/GEO database under GSE67434) has been analysed to identify genes that are involved in the regulation of tobacco's responses to drought.

Transcriptomics analyses of soybean leaf and root samples during water-deficit.

Drought being a major challenge for crop productivity and yield affects multigenic and quantitative traits. It is also well documented that water stress shows a cross talk with other abiotic stresses such as high temperature and high light intensities (Tripathi et al., 2013) [1]. In this report, we documented the details of the methods and quality controls used and considered in our time course-based transcriptome profile of soybean plants under water deficit conditions using microarray technology. The findings of this study are recently published by the Rushton lab in BMC Genomics for a comparative study of tobacco and Soybean (Rabara et al., 2015) [2]. The raw microarray data set is deposited in GEO database with accession number GSE49537.

Tobacco drought stress responses reveal new targets for Solanaceae crop improvement.

BACKGROUND: The Solanaceae are an economically important family of plants that include tobacco (Nicotiana tabacum L.), tomato, and potato. Drought is a major cause of crop losses. RESULTS: We have identified major changes in physiology, metabolites, mRNA levels, and promoter activities during the tobacco response to drought. We have classified these as potential components of core responses that may be common to many plant species or responses that may be family/species-specific features of the drought stress response in tobacco or the Solanaceae. In tobacco the largest increase in any metabolite was a striking 70-fold increase in 4-hydroxy-2-oxoglutaric acid (KHG) in roots that appears to be tobacco/Solanaceae specific. KHG is poorly characterized in plants but is broken down to pyruvate and glyoxylate after the E. coli SOS response to facilitate the resumption of respiration. A similar process in tobacco would represent a mechanism to restart respiration upon water availability after drought. At the mRNA level, transcription factor gene induction by drought also showed both core and species/family specific responses. Many Group IX Subgroup 3 AP2/ERF transcription factors in tobacco appear to play roles in nicotine biosynthesis as a response to herbivory, whereas their counterparts in legume species appear to play roles in drought responses. We observed apparent Solanaceae-specific drought induction of several Group IId WRKY genes. One of these, NtWRKY69, showed ABA-independent drought stress-inducible promoter activity that moved into the leaf through the vascular tissue and then eventually into the surrounding leaf cells. CONCLUSIONS: We propose components of a core metabolic response to drought stress in plants and also show that some major responses to drought stress at the metabolome and transcriptome levels are family specific. We therefore propose that the observed family-specific changes in metabolism are regulated, at least in part, by family-specific changes in transcription factor activity. We also present a list of potential targets for the improvement of Solanaceae drought responses.

The interactome of soybean GmWRKY53 using yeast 2-hybrid library screening to saturation.

Soybean GmWRKY53 functions in both biotic and abiotic stress signaling. Using GmWRKY53 as a bait yeast 2-hybrid library screening to saturation isolated multiple independent fragments for many interacting proteins, enabling delineation of minimal interacting domains and computation of a confidence score. Multiple independent clones coding for the LATE ELONGATED HYPOCOTYL clock protein GmLCL2 (MYB114) were isolated and the binding site for GmWRKY53 was mapped to 90 amino acids separate from the MYB domain. This suggests a direct input from the clock on GmWRKY53 activity. The GmWRKY53-interacting proteins also included 3 water stress-inducible AP2/ERF transcription factors. One of these (Glyma03g26310) is one of the most strongly water stress induced genes in soybean roots, suggesting that GmWRKY53/ERF complexes regulate water stress responses.

Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells.

Members of the WRKY transcription factor superfamily are essential for the regulation of many plant pathways. Functional redundancy due to duplications of WRKY transcription factors, however, complicates genetic analysis by allowing single-mutant plants to maintain wild-type phenotypes. Our analyses indicate that three group I WRKY genes, OsWRKY24, -53, and -70, act in a partially redundant manner. All three showed characteristics of typical WRKY transcription factors: each localized to nuclei and yeast one-hybrid assays indicated that they all bind to W-boxes, including those present in their own promoters. Quantitative real time-PCR (qRT-PCR) analyses indicated that the expression levels of the three WRKY genes varied in the different tissues tested. Particle bombardment-mediated transient expression analyses indicated that all three genes repress the GA and ABA signaling in a dosage-dependent manner. Combination of all three WRKY genes showed additive antagonism of ABA and GA signaling. These results suggest that these WRKY proteins function as negative transcriptional regulators of GA and ABA signaling. However, different combinations of these WRKY genes can lead to varied strengths in suppression of their targets.

The evolution of WRKY transcription factors.

BACKGROUND: The availability of increasing numbers of sequenced genomes has necessitated a re-evaluation of the evolution of the WRKY transcription factor family. Modern day plants descended from a charophyte green alga that colonized the land between 430 and 470 million years ago. The first charophyte genome sequence from Klebsormidium flaccidum filled a gap in the available genome sequences in the plant kingdom between unicellular green algae that typically have 1-3 WRKY genes and mosses that contain 30-40. WRKY genes have been previously found in non-plant species but their occurrence has been difficult to explain. RESULTS: Only two WRKY genes are present in the Klebsormidium flaccidum genome and the presence of a Group IIb gene was unexpected because it had previously been thought that Group IIb WRKY genes first appeared in mosses. We found WRKY transcription factor genes outside of the plant lineage in some diplomonads, social amoebae, fungi incertae sedis, and amoebozoa. This patchy distribution suggests that lateral gene transfer is responsible. These lateral gene transfer events appear to pre-date the formation of the WRKY groups in flowering plants. Flowering plants contain proteins with domains typical for both resistance (R) proteins and WRKY transcription factors. R protein-WRKY genes have evolved numerous times in flowering plants, each type being restricted to specific flowering plant lineages. These chimeric proteins contain not only novel combinations of protein domains but also novel combinations and numbers of WRKY domains. Once formed, R protein WRKY genes may combine different components of signalling pathways that may either create new diversity in signalling or accelerate signalling by short circuiting signalling pathways. CONCLUSIONS: We propose that the evolution of WRKY transcription factors includes early lateral gene transfers to non-plant organisms and the occurrence of algal WRKY genes that have no counterparts in flowering plants. We propose two alternative hypotheses of WRKY gene evolution: The "Group I Hypothesis" sees all WRKY genes evolving from Group I C-terminal WRKY domains. The alternative "IIa + b Separate Hypothesis" sees Groups IIa and IIb evolving directly from a single domain algal gene separate from the Group I-derived lineage.

Understanding Water-Stress Responses in Soybean Using Hydroponics System-A Systems Biology Perspective.

The deleterious changes in environmental conditions such as water stress bring physiological and biochemical changes in plants, which results in crop loss. Thus, combating water stress is important for crop improvement to manage the needs of growing population. Utilization of hydroponics system in growing plants is questionable to some researchers, as it does not represent an actual field condition. However, trying to address a complex problem like water stress we have to utilize a simpler growing condition like the hydroponics system wherein every input given to the plants can be controlled. With the advent of high-throughput technologies, it is still challenging to address all levels of the genetic machinery whether a gene, protein, metabolite, and promoter. Thus, using a system of reduced complexity like hydroponics can certainly direct us toward the right candidates, if not completely help us to resolve the issue.

The potential of transcription factor-based genetic engineering in improving crop tolerance to drought.

Drought is one of the major constraints in crop production and has an effect on a global scale. In order to improve crop production, it is necessary to understand how plants respond to stress. A good understanding of regulatory mechanisms involved in plant responses during drought will enable researchers to explore and manipulate key regulatory points in order to enhance stress tolerance in crops. Transcription factors (TFs) have played an important role in crop improvement from the dawn of agriculture. TFs are therefore good candidates for genetic engineering to improve crop tolerance to drought because of their role as master regulators of clusters of genes. Many families of TFs, such as CCAAT, homeodomain, bHLH, NAC, AP2/ERF, bZIP, and WRKY have members that may have the potential to be tools for improving crop tolerance to drought. In this review, the roles of TFs as tools to improve drought tolerance in crops are discussed. The review also focuses on current strategies in the use of TFs, with emphasis on several major TF families in improving drought tolerance of major crops. Finally, many promising transgenic lines that may have improved drought responses have been poorly characterized and consequently their usefulness in the field is uncertain. New advances in high-throughput phenotyping, both greenhouse and field based, should facilitate improved phenomics of transgenic lines. Systems biology approaches should then define the underlying changes that result in higher yields under water stress conditions. These new technologies should help show whether manipulating TFs can have effects on yield under field conditions.

NtERF32: a non-NIC2 locus AP2/ERF transcription factor required in jasmonate-inducible nicotine biosynthesis in tobacco.

Nicotine biosynthesis in tobacco (Nicotiana tabacum L.) is highly regulated by jasmonic acid (JA). Two nuclear loci, A and B (renamed NIC1 and NIC2) have been identified that mediate JA-inducible nicotine formation and total alkaloid accumulation. NIC2 was recently shown to be a cluster of seven genes encoding Apetala2/Ethylene-Response Factor (AP2/ERF)-domain transcription factors (TFs) in Group IX of the tobacco AP2/ERF family. Here we report the characterization of several NtERF TF genes that are not within the NIC2 locus, but required for methyl JA (MeJA)-induced nicotine biosynthesis. Expression of NtERF1, NtERF32, and NtERF121 is rapidly induced (<30 min) by MeJA treatment. All three of these TFs specifically bind the GCC box-like element of the GAG motif required for MeJA-induced transcription of NtPMT1a, a gene encoding putrescine N-methyltransferase, the first committed step in the synthesis of the nicotine pyrrolidine ring. Ectopic overexpression of NtERF32 increases expression of NtPMT1a in vivo and elevates total alkaloid contents, whereas RNAi-mediated knockdown of NtERF32 reduces the mRNA levels of multiple genes in the nicotine biosynthetic pathway including NtPMT1a and quinolinate phosphoribosyltransferase (NtQPT2), and lowers nicotine and total alkaloid levels. We conclude that NtERF32 and related ERF genes are important non-NIC2 locus associated transcriptional regulators of nicotine and total alkaloid formation.

A systems biology perspective on the role of WRKY transcription factors in drought responses in plants.

Drought is one of the major challenges affecting crop productivity and yield. However, water stress responses are notoriously multigenic and quantitative with strong environmental effects on phenotypes. It is also clear that water stress often does not occur alone under field conditions but rather in conjunction with other abiotic stresses such as high temperature and high light intensities. A multidisciplinary approach with successful integration of a whole range of -omics technologies will not only define the system, but also provide new gene targets for both transgenic approaches and marker-assisted selection. Transcription factors are major players in water stress signaling and some constitute major hubs in the signaling webs. The main transcription factors in this network include MYB, bHLH, bZIP, ERF, NAC, and WRKY transcription factors. The role of WRKY transcription factors in abiotic stress signaling networks is just becoming apparent and systems biology approaches are starting to define their places in the signaling network. Using systems biology approaches, there are now many transcriptomic analyses and promoter analyses that concern WRKY transcription factors. In addition, reports on nuclear proteomics have identified WRKY proteins that are up-regulated at the protein level by water stress. Interactomics has started to identify different classes of WRKY-interacting proteins. What are often lacking are connections between metabolomics, WRKY transcription factors, promoters, biosynthetic pathways, fluxes and downstream responses. As more levels of the system are characterized, a more detailed understanding of the roles of WRKY transcription factors in drought responses in crops will be obtained.

Extending MapMan Ontology to Tobacco for Visualization of Gene Expression.

Microarrays are a large-scale expression profiling method which has been used to study the transcriptome of plants under various environmental conditions. However, manual inspection of microarray data is difficult at the genome level because of the large number of genes (normally at least 30,000) and the many different processes that occur within any given plant. MapMan software, which was initially developed to visualize microarray data for Arabidopsis, has been adapted to other plant species by mapping other species onto MapMan ontology. This paper provides a detailed procedure and the relevant computing codes to generate a MapMan ontology mapping file for tobacco (Nicotiana tabacum L.) using potato and Arabidopsis as intermediates. The mapping file can be used directly with our custom made NimbleGen oligoarray, that contains gene sequences from both the tobacco gene space sequence and the tobacco gene index 4 (NTGI4) collection of ESTs. The generated data set will be informative for scientists working on tobacco as their model plant by providing a MapMan ontology mapping file to tobacco, homology between tobacco coding sequences and that of potato and Arabidopsis, as well as adapting our procedure and codes for other plant species where the complete genome is not yet available.