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

Primer sequences utilized for Real-Time Quantitative Reverse Transcription PCR (qRT-PCR).

<p>Primer sequences utilized for Real-Time Quantitative Reverse Transcription PCR (qRT-PCR).</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

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

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

Overall GO functional analysis of bermudagrass gene expression under cold temperature conditions.

<p><b>A</b> describes biological process domain, <b>B</b> cellular component domain and <b>C</b> molecular function domain, Sub domain descriptions are described on the y axis and numbers of genes with GO functions on the x axis. Data labels to the right of each bars reflect the up/mixed/or down numbers of regulated genes for each description.</p

Average change in gene expression among cultivars for top few genes.

<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>^g SAP DIN1: Senescence-associated protein DIN1</p><p>Values are expressed as Log₂ ratio of expression between cold treated and control.</p

Additional file 2: Figure S2. of Adoptive transfer of immune cells from glaucomatous mice provokes retinal ganglion cell loss in recipients

Representative images of H&E stained retinal cross –sections. Examinations of retinal cross-sections show the typical layered structure without any indications of severe pathological changes. The morphology of the retina in age-matched naïve control animals and in nee splenocyte recipients, four months after transfer, are in accordance with the retinal phenotype examined through OCT. (rgcl, including the nerve fiber layer: inl: inner nuclear layer; onl: outer nuclear layer; is/os: inner and outer photoreceptor cell segments). (TIFF 3627 kb