The Study of Farnesyl Diphosphate and Squalene Synthase Gene Expression Levels in Artemisia absinthium Exposed to Environmental Gamma Radiation

Extended Abstract Background: Artemisia absinthium, commonly known as Afsantin, is an important perennial medicinal herb native to Asia, the Middle East, Europe, and North Africa. During the evolutionary process, plants have developed mechanisms that not only allow them to survive under the most severe radiation doses but also respond effectively to small changes in radiation intensity through physiological adjustments. One of the plant defense mechanisms against radiation is evolutionary changes in gene expression levels. Among these genes, farnesyl diphosphate synthase (FDS) is a key enzyme in the terpenoid metabolic pathway that catalyzes the synthesis of sesquiterpenoid compounds from the farnesyl pyrophosphate (FPP) precursor and plays an important role in regulating plant growth and development. The present study investigated the effects of radiation from radioactive waste in an abandoned uranium mine on the expression changes of the FDS and squalene synthase (SQS) genes in A. absinthium, grown for many years in the radioactive sampling site environment. Methods: Plant material was sampled from the altitudes of Kojanaq village located 18 km northwest of Meshginshahr City with the geographical coordinates of 38° 29' 17.7" N and 47° 30' 15.1" E. All calculations related to the geographical position were performed using a Garmin satellite positioning device (Oregon model, 650). For complete access to radiation contamination information in the study area, a point-by-point radon radiation map was prepared at 10 points using a radiation meter over two consecutive years with a Victoreen 451 radiation meter (Fluke Biomedical Company, USA). These measurements included points at the same altitude on two opposite mountains (mountains with radioactive material points A and non-radioactive B). A. absinthium shoots were sampled systematically in three different biological replicates. Total RNA was extracted from the sampled plant leaves, and the first cDNA strand was synthesized afterward. The Primer 3 Plus online software was used to design qPCR primers, which were then analyzed using the Oligo Analyzer tool and the NCBI/Primer-BLAST plugin in the NCBI genetic database. Initially, the gene regions controlling the farnesyl diphosphate and squalene synthase enzymes were amplified using the synthesized cDNA for all control and non-control samples as templates to ensure the accuracy of the designed primers. Quantitative analysis of the target gene expression was performed by the Real-Time PCR method using the Corbett Real-Time PCR (3000) and the CyberGreen kit (Sinaclon Company). The gene expression pattern changes in all synthesized cDNAs from the studied samples were measured using the Amplicon SYBR Green High ROX master mix. Results: Statistical analysis using GraphPad Prism 10 software showed that the expression levels of both studied genes were higher in samples collected from radioactive points than in non-radioactive points. The highest relative increase in gene expression was observed in the squalene synthase-controlling gene, which is one of the key genes in the biosynthetic pathway of the isoprenoid metabolite group. Statistical comparison of the relative expression pattern of the SQS gene in non-radioactive (Bsqs) and radioactive (Asqs) samples showed a significant difference in the SQS gene expression levels between samples collected from radioactive and non-radioactive areas at a 99% probability level. Furthermore, statistical analysis of the relative expression pattern of the FDS gene in non-radioactive (Bfds) and radioactive (Afds) samples revealed no significant difference in FDS gene expression levels between samples collected from radioactive and non-radioactive areas at a 99% probability level. Additionally, the P-value for the relative expression of the SQS gene in radioactive and non-radioactive area samples was calculated as 0.0077, and it was 0.6039 for the FDS gene. Conclusion: Considering that the expression level of the SQS gene was highest at an altitude of 860-900 m with an average radiation intensity of 0.567 mSv, and at an altitude of 910-930 m with the highest radiation level (1.25 mSv), the squalene gene expression level showed a severe decrease. It can be inferred that the optimal radiation intensity for inducing the SQS gene expression is around 0.5 mSv, and an increase in radiation intensity leads to a significant reduction in the expression of this gene. Presumably, with an increase in radiation intensity and ionizing rays, the plant intelligently modulates the biosynthetic pathway of squalene and directs the biosynthetic pathway toward the synthesis of sesquiterpenoids, which compete with the SQS enzyme for the common precursor farnesyl diphosphate. Since the mutagenic effect of radioactive elements has been proven, it can be concluded that long-term exposure of perennial plants to radioactive radiation leads to genetic changes. These mutations alter the structure and behavior of enzymes involved in the biosynthetic pathways of secondary metabolites and likely represent a genetic-biochemical protective response of the plant to combat the damage caused by free radicals generated by radioactive materials.