Functional Genomics of Cold Tolerance in Bermudagrass Cynodon Dactylon L.

Bermudagrass ( Cynodon dactylon L pers.) is one of the most widely adapted warm-season grasses with its usage and geographic distribution limited by cold temperatures. The goal of this research was to identify genes that are differentially expressed during cold acclimation in two bermudagrass genotypes that differ in tolerance to low temperature stress. Cold tolerant genotype (MSU) and cold sensitive experimental line (Zebra) were used. Plants were cold acclimated at 8 � C/2 � C day/night temperatures. Crown tissue was sampled at 2 and 28 days after cold acclimation from both acclimated and non-acclimated plants. Total RNA was extracted from crown tissues and suppression subtraction hybridization (SSH) was performed to create eight normalized cDNA libraries enriched for expressed sequence tags (ESTs) representing up or down-regulated genes. A total of 3,853 clones were isolated from the eight cDNA libraries and sequenced. Sequenced ESTs were assigned putative functions and deposited in the GenBank database. ESTs were printed on the microarray slide and hybridization analysis was done for identification of differential gene expression profiles. A total of 566 differentially expressed genes were identified. Among them, genes involved in kinase signal cascade (MAPKKK, MK5 , SERK2 protein precursor) and in metabolism (sucrose synthase2, ACBP, aspartate aminotransferase) were identified as being associated with cold tolerance because as they were up-regulated only in the cold tolerant genotype MSU during cold acclimation. Further characterization of these genes will help us better understand cold tolerance in bermudagrassDepartment of Plant and Soil Science

Transcriptome profiling, physiological, and biochemical analyses provide new insights towards drought stress response in sugar maple (Acer saccharum Marshall) saplings

Sugar maple (Acer saccharum Marshall) is a temperate tree species in the northeastern parts of the United States and is economically important for its hardwood and syrup production. Sugar maple trees are highly vulnerable to changing climatic conditions, especially drought, so understanding the physiological, biochemical, and molecular responses is critical. The sugar maple saplings were subjected to drought stress for 7, 14, and 21 days and physiological data collected at 7, 14, and 21 days after stress (DAS) showed significantly reduced chlorophyll and Normalized Difference Vegetation Index with increasing drought stress time. The drought stress-induced biochemical changes revealed a higher accumulation of malondialdehyde, proline, and peroxidase activity in response to drought stress. Transcriptome analysis identified a total of 14,099 differentially expressed genes (DEGs); 328 were common among all stress periods. Among the DEGs, transcription factors (including NAC, HSF, ZFPs, GRFs, and ERF), chloroplast-related and stress-responsive genes such as peroxidases, membrane transporters, kinases, and protein detoxifiers were predominant. GO enrichment and KEGG pathway analysis revealed significantly enriched processes related to protein phosphorylation, transmembrane transport, nucleic acids, and metabolic, secondary metabolite biosynthesis pathways, circadian rhythm-plant, and carotenoid biosynthesis in response to drought stress. Time-series transcriptomic analysis revealed changes in gene regulation patterns in eight different clusters, and pathway analysis by individual clusters revealed a hub of stress-responsive pathways. In addition, qRT-PCR validation of selected DEGs revealed that the expression patterns were consistent with transcriptome analysis. The results from this study provide insights into the dynamics of physiological, biochemical, and gene responses to progressive drought stress and reveal the important stress-adaptive mechanisms of sugar maple saplings in response to drought stress

Identification and analysis of common bean (Phaseolus vulgaris L.) transcriptomes by massively parallel pyrosequencing

Common bean (Phaseolus vulgaris) is the most important food legume in the world. Although this crop is very important to both the developed and developing world as a means of dietary protein supply, resources available in common bean are limited. Global transcriptome analysis is important to better understand gene expression, genetic variation, and gene structure annotation in addition to other important features. However, the number and description of common bean sequences are very limited, which greatly inhibits genome and transcriptome research. Here we used 454 pyrosequencing to obtain a substantial transcriptome dataset for common bean

Identification and analysis of common bean (<it>Phaseolus vulgaris </it>L.) transcriptomes by massively parallel pyrosequencing

<p>Abstract</p> <p>Background</p> <p>Common bean (<it>Phaseolus vulgaris</it>) is the most important food legume in the world. Although this crop is very important to both the developed and developing world as a means of dietary protein supply, resources available in common bean are limited. Global transcriptome analysis is important to better understand gene expression, genetic variation, and gene structure annotation in addition to other important features. However, the number and description of common bean sequences are very limited, which greatly inhibits genome and transcriptome research. Here we used 454 pyrosequencing to obtain a substantial transcriptome dataset for common bean.</p> <p>Results</p> <p>We obtained 1,692,972 reads with an average read length of 207 nucleotides (nt). These reads were assembled into 59,295 unigenes including 39,572 contigs and 19,723 singletons, in addition to 35,328 singletons less than 100 bp. Comparing the unigenes to common bean ESTs deposited in GenBank, we found that 53.40% or 31,664 of these unigenes had no matches to this dataset and can be considered as new common bean transcripts. Functional annotation of the unigenes carried out by Gene Ontology assignments from hits to <it>Arabidopsis </it>and soybean indicated coverage of a broad range of GO categories. The common bean unigenes were also compared to the bean bacterial artificial chromosome (BAC) end sequences, and a total of 21% of the unigenes (12,724) including 9,199 contigs and 3,256 singletons match to the 8,823 BAC-end sequences. In addition, a large number of simple sequence repeats (SSRs) and transcription factors were also identified in this study.</p> <p>Conclusions</p> <p>This work provides the first large scale identification of the common bean transcriptome derived by 454 pyrosequencing. This research has resulted in a 150% increase in the number of <it>Phaseolus vulgaris </it>ESTs. The dataset obtained through this analysis will provide a platform for functional genomics in common bean and related legumes and will aid in the development of molecular markers that can be used for tagging genes of interest. Additionally, these sequences will provide a means for better annotation of the on-going common bean whole genome sequencing.</p

The highly differentially up and down-regulated genes in resistant MSU and susceptible Zebra crown tissues during cold acclimation treatment of 2 and 28 days.

<p>Bolded values represent maximum levels of expression</p><p>^aNCBI Acc: Accession numbers starting with B were associated with spring dead spot library. Numbers with D were associated with the cold acclimation libraries.</p><p>^bM2D- MSU 2 days cold acclimation treatment</p><p>^cM28D- MSU 28 days cold acclimation treatment</p><p>^dZ2D- Zebra 2 days cold acclimation treatment</p><p>^eZ28D- Zebra 28 days cold acclimation treatment</p><p>^fSAP DIN1- Senescence-associated protein DIN1</p><p>^gNs E value was not significant <0.001</p><p>The highly differentially up and down-regulated genes in resistant MSU and susceptible Zebra crown tissues during cold acclimation treatment of 2 and 28 days.</p

Transcriptional Analysis of Resistance to Low Temperatures in Bermudagrass Crown Tissues

<div><p>Bermudagrass (<i>Cynodon dactylon</i> L pers.) is one of the most geographically adapted and utilized of the warm-season grasses. However, bermudagrass adaptation to the Northern USA is limited by freeze damage and winterkill. Our study provides the first large-scale analyses of gene expression in bermudagrass regenerative crown tissues during cold acclimation. We compared gene expression patterns in crown tissues from highly cold tolerant “MSU” and susceptible “Zebra” genotypes exposed to near-freezing temperatures. Suppressive subtractive hybridization was used to isolate putative cold responsive genes Approximately, 3845 transcript sequences enriched for cold acclimation were deposited in the GenBank. A total of 4589 ESTs (3184 unigenes) including 744 ESTs associated with the bermudagrass disease spring dead spot were printed on microarrays and hybridized with cold acclimated complementary Deoxyribonucleic acid (cDNA). A total of 587 differentially expressed unigenes were identified in this study. Of these only 97 (17%) showed significant NCBI matches. The overall expression pattern revealed 40% more down- than up-regulated genes, which was particularly enhanced in MSU compared to Zebra. Among the up-regulated genes 68% were uniquely expressed in MSU (36%) or Zebra (32%). Among the down-regulated genes 40% were unique to MSU, while only 15% to Zebra. Overall expression intensity was significantly higher in MSU than in Zebra (p value ≤ 0.001) and the overall number of genes expressed at 28 days was 2.7 fold greater than at 2 days. These changes in expression patterns reflect the strong genotypic and temporal response to cold temperatures. Additionally, differentially expressed genes from this study can be utilized for developing molecular markers in bermudagrass and other warm season grasses for enhancing cold hardiness.</p></div

Gene expression comparisons in resistant and susceptible backgrounds.

<p>Bolded values represent maximum level of expression</p><p>^a NCBI accession number: Numbers starting with B were associated with spring dead spot library, numbers starting with D associated with cold acclimation libraries</p><p>^b MSU Ave: Average level of Log₂ ratio for cultivar MSU between treated and control crown tissues averaged across time points</p><p>^c Zebra Ave: Average level of Log₂ ratio for cultivar Zebra between treated and control crown tissues averaged across time points</p><p>^d ABS Value M-Z: The absolute value of the difference between cultivars MSU and Zebra in Log₂ ratio of treated and control tissues</p><p>^e ABS Fold change: The absolute value of the fold change difference between MSU and Zebra treated and control tissues</p><p>^f Highest Expression: The cultivar MSU or Zebra with the highest level of expression</p><p>Values are expressed as Log₂ ratio of expression between cold treated and control tissues.</p

Differential gene expression in resistant and susceptible bermudagrass exposed to cold temperatures for 2 and 28 days.

<p><b>A</b> Venn diagram showing overlapping and unique genes in MSU and Zebra, <b>B</b> Average up and down expression values for each genotype and cold acclimation treatment.</p

Genes with NCBI sequence similarities and the most up and down regulated in MSU and Zebra bermudagrass crown tissues when exposed to cold acclimation treatments.

<p>Bolded values represent maximum level of expression</p><p>^a NCBI accession number: Numbers starting with B were associated with spring dead spot library, numbers starting with D associated with cold acclimation libraries</p><p>^b M2D: MSU 2 days cold acclimation treatment</p><p>^c M28D: MSU 28 days cold acclimation treatment</p><p>^d Z2D: Zebra 2 days cold acclimation treatment</p><p>^e Z28D: Zebra 28 days cold acclimation treatment</p><p>^f SAP DIN1: Senescence-associated protein DIN1</p><p>Genes with NCBI sequence similarities and the most up and down regulated in MSU and Zebra bermudagrass crown tissues when exposed to cold acclimation treatments.</p

Differences in expression between 2 and 28 days (temporal difference) for MSU cultivar only.

<p>^a NCBI accession number: Numbers starting with B were associated with spring dead spot library. Numbers starting with D associated with cold acclimation libraries</p><p>^b MSU2: Log2 ratio of treated vs control for MSU 2 day treatment</p><p>^c MSU28: Log2 ratio of treated vs control for MSU 28 day treatment</p><p>^d ABS value 28d-2d: Absolute value of the Log₂ ratio of treated vs control difference between MSU 28 day and 2 day treatments</p><p>^e Direction: Direction in terms of greatest change between treated and control</p><p>^f DPCS: Delta-1-Pyrroline-5-Carboxylate Synthetase</p><p>Differences in expression between 2 and 28 days (temporal difference) for MSU cultivar only.</p