Team:Purdue/Oxygen Sequestration Brainstorming & Thoughts

From 2010.igem.org

OUR PROJECT

Design of Promoter for Analyzing Hypoxic Response in Arabidopis


OLD IDEAS

Contents

Oxygen Sequestration to Inhibit Photorespiration in C3 Plants

The problem

Photorespiration is an unwanted chemical process that occurs in C3 plants that wastes ATP and organic nitrogen. While many species have evolved to avoid photorespiration (C4 and CAM plants), attempts to transfer these methods to C3 plants have been unsuccessful due to the drastic physiological differences between species. Photorespiration occurs when O2 concentrations increase relative to CO2. In relatively dry conditions, the stomata of plants close to preserve moisture. Unfortunately, these stomata are used for the exchange of atmospheric CO2 with photosynthetically-derived O2. When the stomata close, the light-independent reactions of photosynthesis, notably the Calvin cycle, continue. The Calvin cycle's purpose is to fix inorganic carbon (CO2) to generate a three-carbon sugar (G3P). The key enzyme in the carbon-fixation process is RuBisCO. Normally, RuBisCO directly fixes the CO2 to RuBP (an intermediate of the Calvin cycle) to produce G3P. However, RuBisCO also has a significant affinity for O2, and can oxygenate RuBP instead. This generates less G3P and produces a toxic intermediate. Eliminating this toxic intermediate requires 1 ATP and causes the formation of NH3, which diffuses out of the plant.

The solution

Hemoproteins (such as hemoglobin and leghemoglobin) are capable of binding O2 with various affinities. By combining a hemoprotein with a sequence that codes for protein import into the stroma of the chloroplast (a sequence found in the RuBisCO protein), excess oxygen produced during photosynthesis can be sequestered. In theory, this would reduce oxygen concentrations and inhibit photorespiration. A promoter for high dissolved oxygen concentrations could increase production of the hemoprotein during times that photorespiration is likely to occur in excess.

Arabidopsis Metabolism

The Calvin Cycle

Photorespiration

Oxygen-Sensitive Promoters

In Arabidopsis

  • Whole-genome study to determine Arabidopsis response to hypoxic stress [1] --Jmason
  • Expression and evolution of functionally distinct haemoglobin genes in plants [2]
    • Also discusses growing and modifying Arabidopsis to test promoter regions for functionality --Jmason
  • Arabidopsis mutants reveal multiple singlet oxygen signaling pathways involved in stress response and development [3]
    • Specifically, mentions a promoter region for an AAA-ATPase that is activated in the presence of reactive singlet O2 species. --Jmason
  • The low-oxygen-induced NAC domain transcription factor ANAC102 affects viability of Arabidopsis seeds following low-oxygen treatment. [4] --Jmason

In Saccharomyces

  • Ixr1p regulates oxygen-dependent HEM13 transcription [5]
    • Great article that analyzes the effects of different promoters during hypoxia, also explains how they generated hypoxic conditions in lab --Jmason

In Bacteria

  • Sinorhizobium meliloti, a nitrogen-fixing bacterium, forms a symbiotic relationship with legumes in the root nodules of the plant. Because these bacteria must produce enzymes to modify environmental nitrogen, they are also able to detect levels of oxygen using the FixL-FixJ two-component system. [6] --Skearney
  • The DosT/DevS system found in Mycobacterium tuberculosis has an analogous oxygen-sensing function.[7] --Skearney

Resources

Photorespiration and Calvin cycle review

Khan Academy video lecture

Potential Partners and Collaborators

Arabidopsis Links

Potentially useful articles

Processes Relevant to Photorespiration - Addresses metabolic pathways associated to photorespriation

Great article on plant response to abiotic stress - Discusses applications of genetic engineering to improving stress tolerance

Arabidopsis study on gene expression under a variety of conditions - Of particular interest is the gene expression during oxidative and heat stress.

Changes in gene expression in Arabidopsis due to available oxygen

External Links

Search engine for genes and their associated promoters in animals

A similar resource for Arabidopsis


Discussion

I've found a book on abiotic stress response that had a chapter based on the Arabidopsis study mentioned above. The responses to oxidative stress had more to do with Paraquat, an herbicide that interferes with electron transfer. The response to heat stress may be more important, and given what we know about when photorespiration occurs, that's probably a more reasonable pursuit. I'd be somewhat surprised if genes specific to high oxygen concentrations exist. Arabidopsis thaliana heat-shock factor 1 seems like a good candidate. --Jmason

Also, the activity of heat-shock transcription factors seems to be conserved among all eukaryotes. --Jmason

Searching 'oxygen' in the parts registry gives a number of parts related to oxygen sensitive detection, binding, and regulation. It is a good idea to look at these to determine if there is something here that would accomodate our ideas. --Skearney
BBa_K258005

Here are some files on Algal photorespiration, algal biofuels, and another on biofuels. --Lgyoung

This video seems like a good review/introduction to the Calvin cycle.

Note from Professor Rickus

Okay. So I am pretty out of my element with plants. That is OK!! But it means you are going to need to add some plant experts to your advisors. Don't forget about Dr. Evans and friends at Dow Agro.

Some of you are also looking to how you could link this with cancer. Sean and Aaron are a Cancer DURIs for example. Sung has a basic interest. etc etc.

So I am actually thinking that there is A LOT you could do using this O2 sensitive transcription circuit in cancer! One thing cancer cells are really good at is surviving and adapting to hypoxic environments. In addition, the human cancer stem cells we have in the lab have been shown to be sensitive to their oxygen environments.

I think there is potential in oxygen sensitive transcription.


Add your thoughts below

I will look into profs in Hort. that have related experience.

Landon, 5/30/10

There are lots of oxygen binding proteins that exist in nature. I even found a synthetic one, coboglobin, that utilizes cobalt instead of iron. So I don't think we should limit ourselves to just using hemoglobin. John mentioned leghemoglobin which I think is a good idea to look into because its natures way of solving a similar problem. Leghemoglobin binds oxygen so that the enzyme nitrogenase, which doesn't work well when oxygenated, can function properly. We could also use myoglobin which is similar to hemoglobin but its a less complex molecule. Also, the purpose of myoglobin is to store oxygen rather than transport it so it might work better than hemoglobin because we are only trying to sequester oxygen rather than transport it.---MJG, 06/01/2010.

insert brainstorming ideas within these, initial and date (?)


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