Biol 526 Exam 2 - Ice

What is cold? Free atmosphere conditions that deviate substantially (negatively) from optimum temperatures for growth (varies by species)
1. Cold Hardiness - the ability of a plant to adapt to and withstand freezing temperatures at a structural or cellular level
  1. Adaptations
    1. Avoidance - physical attributes of a plant that determine if, when, or where ice forms within the plant (ice nucleators, supercooling, ice barriers)
    2. Tolerance - biochemical adaptations brought about or regulated by a specific set of genes (membrane changes, cryoprotective compounds, compatible solute and sugar production)
    3. Acclimation - "nonheritable" changes in gene expression and protein accumulation that enable the plant to tolerate dehyrdative stress during freezing temps
  2. Acute Stress - single freezing event
  3. Chronic Stress - prolonged period of freezing or ice encasement
2. Multiple components: time, latitude/elevation, and temperature (gradual vs. extreme)
3. Evolutionary adaptations for a cold-adapted genotype
  1. Freezing resistance - must resist local low temperature extremes
  2. Fine-tuning of plant phenology - "seasonality" avoids damaging freezing temperatures in fall/spring
  3. Plant stature - short/tall as is environmentally appropriate
Experiments
1. Overexpression of Chinese cabbage ICE1 gene
  1. Plant - Chinese Cabbage (Brassica campestris) & rice host
  2. Conditions - 20 day old rice seedlings exposed to 4°C & -2°C for 48 h, respectively, then 10 days of recovery at normal temps
  3. Treatments - ICE1 homolog (BcICE1) from Chinese cabbage was overexpressed with a ubiquitin promoter to assess freezing tolerance. Wild type rice and mainly two transgenic lines with the BcICE1 gene (L03 & L07) were observed.
  4. What did they look for? To see if the overexpression of an ICE! gene from Chinese cabbage could increase the freezing tolerance of rice in an ICE1-CBF cold responsive signal pathway,
  5. What did they measure? Survival rates, electrolyte leakage, the expression of COR genes, and the relative content of proline, soluble sugars, MDA, and chlorophyll.
  6. Results - The overexpression of the BcICE1 gene in rice increased the survival rate, proline content, and sugar content. Electrolyte leakage and chlorophyll were better with the gene present, and there was a drastic increase in the production of COR genes.
2. Mutational Evidence for the Critical Role of CBF Transcription Factors
  1. Plant - Arabidopsis
  2. Conditions - Cold acclimation (4° C) for 7 days, some trials exposed to -7° C for 1 hr, 23° C for recovery
  3. Treatments - CRISPR/Cas 9 system to generate single, double and triple CBF mutants, electrolyte leakage assay, freezing survival assay, & RNA sequencing assay
  4. What did they look for? The role of CBF proteins in freezing tolerance, plant development, and confirmation that COR genes are regulated by CBF transcription factors
  5. What did they measure? Germination rates, growth, and flowering of mutants, survival rate and electrolyte leakage, COR gene expression, and the regulation of CBF-activated genes
  6. Results - Plant development worsened, electrolyte leakage was much greater, and COR genes were not expressed in the mutants compared to wild type due to the accumulation of DELLA proteins via reduction of GA, indicating that CBF genes play a critical role in freezing tolerance; CBF genes are redundant, however, CBF2 is more important in cold acclimation
3. Rice mutants deficient in omega-3 fatty acid desaturase (FAD8)
  1. Plant - rice (Oryza sativa L..)
  2. Treatments - 3 T-DNA insertional knockout mutants of FAD8 (osfad8-Ex3, osfad8-Ex7, osfad8-int4) and wildtype for control
  3. Conditions - 5 days at 28°C and 4°C
  4. What did they look for? Changes in fatty-acid composition and changes in thylakoid membrane fluidity to determine if the photosynthetic mechanism was intact
  5. Results - At low temps, 18:3/18:2 ratio decreased in mutants (lost their ability to desaturate), photosystem efficiency was much lower in the mutants, more degradation of D1 in the mutants, and less light absorbed in the mutants (failure to acclimate to light due to decreased desaturation)
  6. What did they measure? 18:3/18:2 ratio and DBI (double bond index) in chloroplast membrane lipids (MGDG, DGDG, SQDG, & PG), chlorophyll fluorescence, degradation of D1 proteins, and PS1 activity (amount of P700)
4. Plant traits co-vary with altitude
  1. Plant - 701 species in the Swiss Alps
  2. Conditions - 5 sites in the Swiss Alps, transects in grasslands and forests, 120 vegetative plots total
  3. Treatments - none, mountain altitude was a substitution for temperature change
  4. What did they look for? Variations of 13 plant traits along altitudinal gradients (mainly life form, leaf morphology, & reproduction)
  5. What did they measure? Applied PCA exploratory tool to compare variables with one another at the same time to look for trends
  6. Results - 11/13 traits showed trends, mainly plant size decreases and vegetative reproduction is more frequent as altitude increases
Cold Stress Regulation of Gene Expression (Arabidopsis)
1. Step One: Cold temperatures reduce membrane fluidity, increased rigidity
2. Step Two: Membrane rigidification, ABA, or ROS activate Ca2+ channels, increasing Ca2+ in the cytosol
3. Step Three: Increased levels of Ca2+ activate protein kinases, which activate ICE proteins (Inducer of CBF Expression) transcription factors
4. Step Four: ICE proteins induce the expression of CBF genes (C-repeat binding factor)
5. Step Five: CBF proteins induce the expression of COR genes (Cold Responsive)
6. Step Six: COR proteins alter the physiology of the plant for cold acclimation

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Biol 526 Exam 2 - Ice

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