The meaning of model for us was to find in the literature promoters that could be induced by high CO2 concentrations and study the metabolic pathways in which they are involved to understand and predict their behavior under our experimental conditions in the wet lab.
In biochemistry, a metabolic pathway is a series of chemical reactions that take place inside a living organism. Most of these reactions are regulated by enzymes, a biomolecule that acts as a catalyst to speed up specific chemical reactions.
In our project, we use different promoters that regulate the expression of different enzymes which are found in different metabolic routes and perform different tasks.
Our project's objective is to use plants as CO2 sensors. This requires the identification of promoter sequences induced by high CO2 concentrations. To develop our project first we selected putative routes in which CO2 could act as an inductor and based on the literature, we chose several promoters to make fusion construction with the gene marker GFP.
As a proof of concept, we have used the GFP reporter gene to measure the transcription level of each promoter under different CO2 concentrations using Nicotiana benthamiana plants transformed with our plasmids.
Glycolysis:
Glycolysis is the metabolic pathway that converts glucose (C6H12O6) into pyruvate (CH3COCO2H). The loose strength launched on this system is used to shape the high-strength molecules adenosine triphosphate (ATP) and decreased nicotinamide adenine dinucleotide (NADH).[1] Glycolysis is a series of ten reactions catalyzed via way of means of enzymes. Storage of ATP.
In this pathway we select the following promoters: Glyceraldehyde-3-phosphate dehydrogenase (G3PDH), Mitochondrial aldehyde dehydrogenase (ALDH2), Phosphoenolpyruvate carboxylase 1 (PEPC1).
TCA cycle:
The citric acid cycle (CAC)—additionally called the Krebs cycle or the TCA cycle (tricarboxylic acid cycle)—is a chain of chemical reactions to launch saved energy via the oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins. The Krebs cycle is utilized by organisms that breathe (in preference to organisms that ferment) to generate energy, both through anaerobic breathing or aerobic breathing.
Citrate synthase 4 (CSY4) and Aconitase 3 (ACO3) are found in that route, both affecting the citrate and energy organelles such as mitochondria and chloroplasts.
Electron transportation chain:
The electron transport chain is a series of four protein complexes that couple redox reactions, creating an electrochemical gradient that leads to the creation of ATP in a complete system named oxidative phosphorylation. It occurs in mitochondria in both cellular processes, respiration, and photosynthesis.
The promoters ATPS, 76 kDa subunit of complex I (CI76) and, 14 kDa subunit of complex III (CIII14) are found in this route.
Other promoters:
We also selected Hemoglobin 1 (Hb1) and Hemoglobin 2 (Hb2) promoters whose genes are carriers of oxygen and participate actively as cellular responders to hypoxia (affection in which a certain part of the organism is deprived from oxygen).
| Pathways | Promoter | Complete name | Function |
|---|---|---|---|
| Glycolysis | G3PDH | Glyceraldehyde-3-phosphate dehydrogenase | Encodes the chloroplast that is important in the synthesis of the serine in the roots of the plant. |
| PEPCI | Phosphoenolpyruvate carboxylase 1 | Plays an important role in carbon and nitrogen metabolism. | |
| ALDH2 | Mitochondrial aldheyde dehydrogenase | Encoded putative aldehyde dehydrogenase. | |
| ADH1 | Alcohol dehydrogenase 1 | Participates in aerobic and anaerobic fermentation processes that convert the pyruvate produced by glycolysis to ethanol, less toxic than lactic acid or acetaldehyde. | |
| TAC cycle | CSY4 | Citrate synthase 4 | Encodes a mitochondrion targeted citrate synthase |
| ACO3 | Aconitase 3 | Converse the citrate into isocitrate. Creating a protein that is believed that accumulates in the mitochondria and the cytosol. | |
| Respiratory chain | CI76 | 76 kDa subunit of complex I | Part of the process of the respiratory chain when the succinate passes to fumarate. |
| CIII14 | 14 kDa subunit of complex III | ||
| ATPS | |||
| HB1 | Hemoglobin 1 | Both have multiple functions, such as oxygen sensors, in stress responses and in the signal transduction pathways of ethylene and other hormones. | |
| HB2 | Hemoglobin 2 |