Designed by: Anika Kapur, Thanakorn (Gunn) Vajirakachorn, Ananya Dharna, Vin Pungprasert, Dhirath (Kaka) Thanglerdsumpan   Group: iGEM23_Thailand-RIS   (2023-10-10)

Durio zibethinus glutamate-cysteine ligase, mitochondrial

    Glutathione (GSH) is an important antioxidant which combats reactive oxygen species (ROS)[1]. ROS accumulation is one of the main causes of plant stress. When undergoing oxidative stress, organisms experience increased levels of ROS, which can cause damage to the basic building blocks of the plant cell. This accumulation of ROS occurs in the mitochondria where GSH is one of the molecules that helps mitigate this. Glutamate-cysteine ligase (GCL) catalyzes the production of gamma-glutamyl cysteine (γ-EC), a precursor for GSH and acts as a rate-limiting enzyme in this pathway. GSH then gets transported to the mitochondria to neutralize ROS. In this experiment, the team aimed to relocate the GCL protein to the mitochondria, so the GSH synthesis was done in the mitochondria instead. This improved the efficiency of GSH action, thus increasing the stress response in plants. This was done to align with the goal of this project: to enhance plants' response to stress.

    For the GCL gene, Durio zibethinus was chosen. This was because D. zibethinus displays high levels of γ-EC and GSH in its ripe fruit pulp compared to other plants. [2,3] indicating that D. zibethinus might have a GCL with higher activity than other plants. D. zibethinus GCL (DzGCL) is originally located in chloroplasts to address plant stress tolerance. In this project, DzGCL was modified to localize to the mitochondria allowing for the production of gamma-glutamyl cysteine (γ-EC)/GSH within the mitochondria for a more efficient plant stress response.

    Transit peptides are responsible for translocating proteins to target organelles. Naturally, GCL is localized to the chloroplast. However, changing the localization of GCL by changing the transit peptide sequence to target GCL to mitochondria would enable the production of GSH directly in the mitochondria. This allows for more efficient maintenance of ROS levels.[4] In order to remove the chloroplast transit peptide, identification of the amino acid sequence coding for the transit peptide was required to locate the sequence that had to be replaced. After that, a mitochondrial transit peptide was taken from Arabidopsis thaliana succinate dehydrogenase (SDH). This is due to the fact that its mitochondrial transit peptide has been previously shown to localize to the mitochondria successfully.[5,6] The transit peptide was then substituted into the position of the chloroplast transit peptide. Inserting the mitochondrial transit peptide into DzGCL transforms it into mDzGCL. Subsequently, mDzGCL was integrated into the plant expression vector, pCAMBIA1301 plasmid, which was then introduced into Agrobacterium tumefaciens for delivery into Nicotiana benthamiana plants. This strategic relocation of GCL production in the mitochondria serves to mitigate the high levels of ROS.

    Due to D. zibethinus having a high innate level of GSH,[2,3] (mDzGCL) gene is a solid candidate for inspiration for other plant-based science projects that aim to increase efficiency of plant stress response, as high levels of GSH allow for better maintenance of ROS levels. Since the team showed that localizing GCL in the mitochondria can increase plant resistance to drought, other teams can test more thoroughly and build on this research by using part of or the whole mDzGCL gene in plants. As the team only constructed the mDzGCL transient line, others teams could make a transgenic line of plants containing mDzGCL and collect new data on how mDzGCL impacts plant resistance.

Annotated Bibliography

1. Hasanuzzaman M, Nahar K, Anee TI, Fujita M. Glutathione in plants: biosynthesis and physiological role in environmental stress tolerance. Physiology and Molecular Biology of Plants. 2017;23:249-68. doi:10.1007/s12298-017-0422-2

2. Singcha P, Khaksar G, Sirijan M, Sirikantaramas S. Durian (Durio Zibethinus L.) Fruit: A Superior Dietary Source of Natural Glutathione and Γ-Glutamylcysteine. SSRN 2023; 4566631. doi:10.2139/ssrn.4566631

3. Pinsorn P, Oikawa A, Watanabe M, Sasaki R, Ngamchuachit P, Hoefgen R, Saito K, Sirikantaramas S. Metabolic variation in the pulps of two durian cultivars: Unraveling the metabolites that contribute to the flavor. Food chemistry. 2018; 268:118-125. doi:10.1016/j.foodchem.2018.06.066

4. Zhang R, Lei J, Chen L, Wang Y, Yang G, Yin Z, Luo L. γ-Glutamylcysteine exerts neuroprotection effects against cerebral ischemia/reperfusion injury through inhibiting lipid peroxidation and ferroptosis. Antioxidants. 2022; 11(9):1653. doi:10.3390/antiox11091653

5. Meinke DW, Cherry JM, Dean C, Rounsley SD, Koornneef M. Arabidopsis thaliana: A model plant for genome analysis. Science. 1998 Oct 23;282(5389):662-682. doi:10.1126/science.282.5389.662

6. Figueroa P, Leon G, Elorsa E, Holuigue L, Jordana X. Three different genes encode the iron-sulfur subunit of succinate dehydrogenase in Arabidopsis thaliana. Plant molecular biology. May 2001. Accessed July 15, 2023.

Sequence and Features

Assembly Compatibility:
  • 10
  • 12
  • 21
  • 23
  • 25
  • 1000
    Illegal BsaI site found at 1125
    Illegal SapI.rc site found at 1420