LauraTerada Individual Journal Assignment Week 11

• Laura Terada
• BIOL398-03/S13:Week 3

Outline

• Transcriptional responses to low temperatures were analyzed in this study
  • Adaptation is the stress-response to a rapid phenomena, while acclimation is the physiological response from a long-term phenomena
  • This study focuses on acclimation
  • There are known genes that have been seen in cold-shock studies (e.g. trehalose-synthesis genes, cell-wall mannoproteins, fatty-acid desaturase, ribosome biogenesis, etc.)
  • Chemostat cultures are better than batch cultures because it allows a controlled environment independent of other culture conditions
  • This study is significant because it shows a difference between chemostat-based and literature data in regards to low-temperature acclimation over long-term and short-term scales
  • Goal is to study stead-state, acclimatized growth of the yeast at cold temperatures through transcriptional regulation data
• Steady-state chemostat conditions
  • Yeast strain used: haploid, S. cerevisiae strain CEN.PK113-7D (MATa)
  • Dilution rate fixed at 0.03 1/h
  • Chemostat at both 12C and 30C with a 1.0 L volume and pH of 5.0
  • Growth medium was limited by either carbon or nitrogen with all other factors controlled in excess
  • Trehalose (triplicate) and glycogen (duplicate) measurements were performed; trehalose n=3, glycogen n=2 under 4 growth conditions
  • Three independently cultured replicates for the 4 growth conditions
  • 4 growth conditions: glucose limiting 12C, glucose limiting 30C, ammonium limiting 12C, ammonium limiting 30C
• Used microarray analysis, promoter analysis, and statistical assessment
  • Microarrays were performed for RNA quality and used microsoft Excel for significance analysis
  • Promoter analysis was used by a web-based software
  • Statistical assessment was performed for overrepresentation of transcription-factor binding sites
  • Used Fisher's exact text, with a Bonferroni correction and a p value threshold of 0.01
  • The datasets generated were compared with other datasets from previous studies
• Growth efficiency was not affected by varying growth temperatures (Table 1)
  • Proven by similar biomass and fermentation yields at 12C and 30C in carbon and nitrogen limiting chemostats
• In glucose limitation, 494 genes changed transcription levels between the two temperatures, while 806 genes did the same but in nitrogen limitation (Fig 1)
  • 16% of the yeast genes was temperature-responsive
• The temperature-responsive genes were screened for functional categories (Fig 2)
• Low temperatures result in changed kinetics for the limiting nutrient and enhanced catabolite repression
  • 12C, residual concentrations of growth-limiting nutrients were higher than at 30C
  • Changes in transport kinetics from changes in transcript levels of genes involved in growth-limiting nutrient uptake (Fig 2)
  • Ex: HXT2 and HXT3 hexose-transport genes increased transcription at 13C compared to 30C
  • Residual concentrations of the limiting nutrient is low to prevent catabolite repression
• The induction of trelahose and glycogen formation genes are not needed for yeast acclimation to low temperatures
  • At 12C, trelahose and glycogen metabolism genes were not upregulated
  • In glucose limitation, gene transcription did not change, and im ammonia limitation it slightly decreased
• When adapted to low temperatures, the yeast cells recede the up-regulation of storage carbohydrate synthesis (Table 3)
• 16 genes in ribosome biogenesis in both conditions and 80 genes in protein synthesis in ammonia limited conditions showed higher transcription levels at 13C
  • Increased protein content compensates for impaired enzyme kinetics at 13C
• In comparison to other studies, gene regulation differs
  • When comparing to 3 other studies, 259 (low-adaptation) genes also responded to decreasing temperature, but the responses were not consistent (Fig 3)
  • 29 of these genes were transcriptionally regulated at adaptation and acclimation to low temperature (Fig 4)
  • Only 4 of these showed consistent regulation patterns
• Environmental stress response is not a response to low temperatures, but it is a response due to adaptation from rapid exposure to low temperatures
• Conclusion
  • Growth-limiting nutrients were higher at 12C shown by gene expression for nutrient transporters
  • Transcription of environmental stress response genes decreased at 12C
  • Trehalose is not involved in steady-state low temperature adaptation
  • Acclimation does not only rely on transcriptional reprogramming, but on changing intracellular metabolite levels as well