Identification of the FAD gene family in kiwifruit and its expression analysis during postharvest ripening

Abstract: 【Objective】Unsaturated fatty acids play many physiological roles in plants, including the formation of triglycerides to provide energy for plant life activities; maintaining cell membrane homeostasis as a key component of cell membranes; participating in hormone regulation and signaling during biotic stress; playing an important role in responding to adversity such as low temperature and drought; and participating in synthesizing aroma substances as precursors during fruit ripening via the LOX pathway, α-oxidation pathway, or β-oxidation pathway. During fruit ripening, the unsaturated fatty acids are used as precursors to synthesize aroma substances through the LOX pathway, α-oxidation pathway, or β- oxidation pathway. At the same time, the unsaturated fatty acids also help the human body in numerous ways, including lowering lipids and blood pressure, enhancing fat metabolism, decreasing thrombosis, enhancing autoimmunity, and having anti-tumor properties. However, the human body is unable to synthesize unsaturated fatty acids, such as linoleic acid and linolenic acid, which can only be ingested fromthe diet. The animal body contains a high concentration of saturated fatty acids, whereas the plant body contains primarily unsaturated fatty acids. The fatty acid desaturases (FADs) are a group of enzymes that carry out the desaturation process, transforming saturated fatty acids into unsaturated fatty acids. To date, the FAD gene family has been identified in several species; however, it has not been reported in kiwifruit. In addition, most of the current studies on FAD genes have focused on the antistress function of unsaturated fatty acids, with little focus on how these fatty acids affect aroma volatiles. Therefore, the aim of this study was to isolate and characterize the kiwifruit FAD gene family and to clarify its expression pattern in kiwifruit during postharvest ripening and its relationship with the changes in unsaturated fatty acids, so as to lay a theoretical foundation for analyzing the formation of characteristic aroma in kiwifruit during postharvest ripening.【Methods】Based on the Hong Yang v3 genomic data of Actinidia chinensis, we downloaded the Hidden Markov Model (HMM) file corresponding to the structural domains of the fatty acid desaturase from the Pfam protein family database and used Simple HMM Search in TBtools (v2.102) to preliminarily search the AcFAD genes with the kiwifruit protein data, and then used the SMART database to verify the structural domain information of the candidate protein sequences to finalize the kiwifruit FAD gene family members. Then we used the SMART database to validate the structural domain information of the initial screened candidate protein sequences to identify the kiwifruit FAD gene family members; we used the protein molecular weight calculation (SMS2) Nanjing DETA Bio-mirror website to analyze the physicochemical properties of the kiwifruit FAD gene family members; and we used the Cell-PLoc 2.0 website to predict the subcellular localization of the proteins; A phylogenetic evolutionary tree of FAD protein sequences of kiwifruit, Arabidopsis thaliana and cucumber was constructed using MEGA (version 11.0) software; the chromosomal localization was mapped using Advanced Circos in TBtools, and the replication events of kiwifruit and Arabidopsis thaliana FAD genes were analyzed by One Step MCScanX- Super Fast. The covariance analysis was performed; the exon-intron gene structures were analyzed by GSDS, a gene structure display server; the conserved motifs of AcFAD proteins were analyzed using the MEME online website; the promoter cisacting elements of each gene were predicted using PlantCARE; and the gene expression heatmaps were generated using TBtools by normalizing the FPKM values of the transcriptomic data obtained. The expression pattern of the FAD genes was explored; the hardness and fatty acid content were determined using mass spectrometry and gas chromatography; and the expression characteristics of the FAD genes were verified with the help of the real-time fluorescence quantitative PCR during post-harvest ripening. 【Results】In this study, a total of 26 FAD gene family members were identified from the whole Hong Yang v3 genome of Actinidia chinensis. These 26 kiwifruit FAD genes were named based on how closely they resembled the counterparts identified in Arabidopsis thaliana and cucumber. These genes were divided into six subfamilies: FAD3/FAD7/FAD8 (ω- 3/Δ- 15), FAD2/FAD6 (ω- 6/Δ- 12), FAB2 (Δ- 9), FAD4 (Δ-3), DES/SLD, and FAD5/ADS (Δ-7), each with a different number of subfamily members. All subfamilies had kiwifruit FAD family members, suggesting that the AcFAD proteins might be functionally diversified. A handful of them were acidic, but the majority were basic. The subcellular localization prediction revealed that kiwifruit FAD proteins were relatively dispersed in their localization and are distributed in all plant cell structures, with the highest number in the endoplasmic reticulum, followed by chloroplasts and cell membranes; the chromosome localization showed that the kiwifruit Hong Yang v3 genome had a total of 29 chromosomes, and 25 kiwifruit FAD genes were distributed on 19 different chromosomes (LG). The majority of the FAD genes were localized in the anterior- middle end of the chromosomes. The segmental duplication events contributed to the diversity and evolution of the Ac-FADs, as evidenced by the 22 pairs of segmental duplicates and 9 pairs of tandem duplicates found in kiwifruit according to the intraspecific covariance mapping; the covariance mapping between kiwifruit and Arabidopsis thaliana interspecies covariance mapping showed the 32 pairs of covariance between the 23 kiwifruit FAD genes and the 17 Arabidopsis thaliana FAD genes, suggesting that kiwifruit and Arabidopsis thaliana would have more FAD homologous genes. In the same branch, most of the members had similar length and the same number of gene structures. The conserved motif analysis revealed that most kiwifruit FAD proteins contained motif 2, motif 4, and motif 14. Moreover, motif 2 and motif 4 were basically located at the C-terminal end, indicating that these motifs were strongly conserved and were typical FAD structural domains, which might perform similar functions. A large number of the light-responsive, phytohormone-responsive (methyl jasmonate, abscisic acid, growth hormone, salicylic acid, gibberellin), stress- responsive (anaerobic, low temperature, drought), and development- related components were found in the FAD promoter region of kiwifruit, and the heatmap expression pattern analysis and qPCR validation experiments showed that the expression of the AcFAD7.2 and the AcFAD2.2 were significantly up- regulated with maturation. During the postharvest ripening process, the content of monounsaturated fatty acids (oleic acid) decreased significantly, while the content of diunsaturated fatty acids (linoleic acid) increased significantly, and the content of polyunsaturated fatty acids (linolenic acid) did not significantly vary with the early and middle stages of ripening, while the content of polyunsaturated fatty acids (linolenic acid) decreased significantly during the late stage; the hardness of kiwifruit rapidly decreased after harvest, and a distinct post-ripening process took place that resulted in the formation of the distinctive aromatic ester substances. The characteristic fragrance esters were produced as a result of this process, and the primary precursor for the synthesis of the ester aroma compounds was linoleic acid.【Conclusion】A total of 26 kiwifruit FAD gene members were identified. A significant decrease in the oleic acid content was accompanied by a significant increase in the linoleic acid content during postharvest ripening of kiwifruit. The variations in the oleic and linoleic acid were highly and positively linked with the expression of the ω-6 fatty acid desaturase gene AcFAD2.2. This expression was consistently and considerably up- regulated throughout ripening. Although the expression of the ω-3 fatty acid desaturase gene AcFAD7.2 was up- regulated, the linolenic acid content remained essentially unchanged or even decreased. Therefore, the AcFAD2.2 would be a key enzyme gene involved in the synthesis and accumulation of unsaturated fatty acids in kiwifruit during postharvest ripening, and the increase in the linoleic acid content was accompanied by the characteristic aroma emitted by kiwifruit during postharvest ripening. This paper would provide a reference basis for further investigation of the biological functions of the FAD genes involved in the transformation of unsaturated fatty acids and aroma synthesis during the course of the postharvest ripening in kiwifruit.