Bioscience Evidence 2024, Vol.14, No.5, 206-217 http://bioscipublisher.com/index.php/be 210 pathogen-induced changes in ALA content in Eucommia ulmoides are not available, it is plausible that such stress could either enhance or inhibit ALA biosynthesis depending on the nature and severity of the pathogen attack. Symbiotic relationships with microorganisms, such as mycorrhizal fungi and nitrogen-fixing bacteria, can influence the nutrient uptake and metabolic activities of plants, potentially affecting ALA biosynthesis. While there is no direct evidence linking symbiotic organisms to ALA production in Eucommia ulmoides, it is known that such relationships can enhance overall plant health and metabolic efficiency, which could indirectly support higher ALA synthesis. In summary, environmental factors such as temperature, soil nutrients, water availability, and biotic stress play significant roles in the biosynthesis of ALA in Eucommia ulmoides. Understanding these factors can help optimize conditions for enhanced ALA production, which is valuable for both nutritional and medicinal purposes. Further research is needed to elucidate the specific mechanisms by which these factors influence ALA biosynthesis in this species (Yuan et al., 2021). 5 Analytical Techniques for α-Linolenic Acid Detection 5.1 Chromatographic methods for ALA analysis Gas chromatography (GC) is a widely used technique for the separation and analysis of fatty acids, including α-linolenic acid (ALA). One study demonstrated the use of a highly polar ionic phase column (SLB-IL111) to resolve minor geometrical isomers of ALA in linseed oil. The identification of these isomers was further confirmed using gas chromatography-electron ionisation mass spectrometry (GC-EIMS) and covalent adduct chemical ionisation tandem mass spectrometry (CACI-MS/MS) (Gómez-Cortés et al., 2016). This method highlights the capability of GC to distinguish between structurally similar polyunsaturated fatty acid isomers, making it a valuable tool for ALA analysis. Liquid chromatography (LC) is another essential technique for the separation of ALA and its isomers. A study utilized a cellulose tris (3,5-dichlorophenylcarbamate)-based chiral stationary phase to successfully separate ALA from its positional isomer, γ-linolenic acid (GLA). The optimized chromatographic conditions included a reversed-phase eluent system and UV detection, which allowed for the effective discrimination of these isomers. This method was further adapted for LC-MS/MS implementation, demonstrating its potential for quantifying ALA in various matrices (Ianni et al., 2020). 5.2 Spectroscopic techniques for ALA quantification Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique used for the structural elucidation and quantification of fatty acids, including ALA. NMR provides detailed information about the molecular structure, including the number and position of double bonds in fatty acids. This technique is particularly useful for confirming the identity of ALA and distinguishing it from other similar fatty acids. Fourier-transform infrared (FTIR) spectroscopy is another valuable tool for the analysis of fatty acids. FTIR can be used to identify functional groups and characterize the molecular structure of ALA. This technique is advantageous due to its rapid analysis time and minimal sample preparation requirements. FTIR spectroscopy can complement other analytical methods, providing additional confirmation of ALA's presence and structure (Yuan et al., 2021). 5.3 Advances in molecular techniques for ALA detection Polymerase chain reaction (PCR)-based methods have been developed to detect and quantify genes involved in the biosynthesis of ALA. These techniques can amplify specific DNA sequences associated with ALA biosynthetic pathways, allowing for the identification of key genes and their expression levels. PCR-based methods are highly sensitive and specific, making them suitable for studying the genetic regulation of ALA production. Next-generation sequencing (NGS) has revolutionized the field of genomics, providing comprehensive insights into the genetic basis of ALA biosynthesis. A study on the chromosome-level genome of Eucommia ulmoides utilized PacBio and Hi-C technologies to assemble high-quality genomes, revealing key genes involved in ALA biosynthesis. The high expression of the ω-3 fatty acid desaturase coding gene (EU0103017) was identified as a significant factor contributing to the high ALA content in E. ulmoides (Figure 2) (Du et al., 2023). NGS enables the detailed analysis of entire genomes, facilitating the discovery of novel genes and regulatory mechanisms associated with ALA production.Through the whole-genome sequencing of
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