Identification and expression analysis of PIN genes related to seed coat development in pomegranate

【Objective】The auxin efflux proteins PIN- formed (PIN) is a secondary transporter protein that plays an important role in regulating auxin polarity transport during a variety of developmental processes in plants. The size and hardness of pomegranate seeds directly affect the palatability and flavor of the fruit, and PIN proteins are involved in the formation of pomegranate seed size and hardness through the polar transport of auxin, while research on the effect of PIN gene on the growth and development of pomegranate and its regulatory mechanism is still unclear. The study aimed to understand the distribution, evolution and tissue expression properties of PIN proteins in the pomegranate genome, andto screen candidate PIN genes associated with pomegranate inner seed coat development and their response to exogenous auxin and investigate the effect of exogenous auxin on pomegranate fruit and seed coat development, and the expression characteristics of PIN genes during seed coat development. The identification of pomegranate PIN family members and the characterization of their expression during seed coat development would be conducive to the understanding of the functions of the PIN gene family members in pomegranate and their effects on the development of pomegranate seed coat, which would provide a new theoretical basis for the regulatory mechanism of pomegranate seed coat development, and then lay the foundation for the mechanism of pomegranate seed coat size and hardness formation as well as genetic regulation.【Methods】Eight known PIN proteins and conserved domains from Arabidopsis (Arabidopsis thaliana) were used to identify pomegranate PIN gene family members from the whole pomegranate genome, and the physicochemical properties of pomegranate PIN family members, such as amino acid number, isoelectric point, instability coefficient and so on, were analyzed using bioinformatics methods. The PIN protein sequences of pomegranate, Arabidopsis thaliana, Eucalyptus megacephalus, grapevine and Populus were compared and a phylogenetic tree was constructed. The prediction and analysis of promoter cis- acting elements of pomegranate PIN family members were performed using PlantCARE, representative tissues of hard- seeded pomegranate Dabenzi at different times, Dabenzi and Tunisia transcript levels of each PgrPIN gene were analysed in the inner and outer seed coat at different developmental stages. The roots of Dabenzi pomegranate seedlings were treated with exogenous auxin and the candidate PIN genes were quantitatively analysed by fluorescence in the root system 6 h and 12 h after treatment. At the same time, the fruit and seed morphology indexes of pomegranate at different developmental stages were determined after exogenous NAA treatment, and the expression of PgrPIN gene in the inner seed coat was analysed after treatment.【Results】A total of 15 PIN family members were identified from the whole pomegranate genome, including 7 classical and 8 non- classical ones, encoding 267- 667 amino acids, with protein molecular weights ranging from 29.62 kDa to 74.79 kDa, and isoelectric points between 6.29 and 9.80, and most of the members functioned on the plasma membrane. The analysis of cis-acting elements in the promoter region showed that the PgrPINs gene contained a large number of cis-acting elements related to hormone regulation, light response, stress induction and growth and development. The expression of the PgrPINs genes in different tissues of pomegranate at different developmental stages was analysed using transcriptome data, and the genes with high expression levels in the inner seed coat of the two varieties, Dabenzi and Tunisia, were the PgrPIN1, PgrPIN2, PgrPIN3, PgrPIN5 and PgrPIN14. They were screened as five candidate genes involved in pomegranate seed coat development, among them, the PgrPIN1, PgrPIN3, PgrPIN5 and PgrPIN14 genes were expressed in the roots of Dabenzi pomegranate seedlings in response to exogenous IAA. The exogenous NAA treatment of young fruits 15 days after pollination had a promoting effect on pomegranate fruit development, and both single fruit weight and 100- grain weight showed a trend of significant increase after NAA treatment, and the longitudinal and transverse diameters of pomegranate seeds and 100 grain weight of the inner seed coat increased significantly compared with the control, in which the hardness of hormone-treated seeds at 90 d showed a significant trend of increase compared with the control . Under NAA treatment, the expression levels of the PgrPIN1 and PgrPIN3 were significantly increased after 30 d, and the PgrPIN5 was mainly expressed at a high level in the late developmental stage of the inner seed coat, and the PgrPIN14 was expressed at high level in the early developmental stage of the inner seed coat in both treatments and control groups. The correlation analysis between the expression level of the four candidate PgrPINs and fruit and seed growth relat-ed indices as well as seed hardness showed that the expression of the PgrPIN1 was significantly and positively correlated with fruit and seed size and seed hardness.【Conclusion】The PgrPINs promoters were involved in pomegranate growth and development through light response, multiple hormone signaling response, adversity regulation and growth regulation. The five candidate genes were differentially expressed in two pomegranate varieties, namely, Dabenzi and Tunisia, at different developmental periods, which indicated that they might be differentially expressed in the pomegranate varieties of Dabenzi and Tunisia. This suggested that they might have a regulatory role in the formation of pomegranate grain hardness during different developmental periods. The NAA treatment could promote the growth of pomegranate fruits, and effectively promote the morphological indexes of pomegranate fruits, such as single fruit weight, longitudinal and transverse diameters, and 100-grain weight. Under NAA treatment, the PgPINs genes responded differently at different times, and NAA could regulate the growth and development of pomegranate fruits by inducing the expression of the PIN genes. The changes in the expression of the PgrPIN1 were significantly and positively correlated with the inner and outer seed coat sizes, and even the seed and fruit sizes, and significantly and positively correlated with the seed hardness, which indicated that it might be involved in the development of the seed size as well as fruit size and the formation of seed hardness in pomegranate development as well as the formation of seed hardness in pomegranate. The results of this study would lay a theoretical foundation for further research on the functions of pomegranate PIN gene family members, and at the same time provide a certain research direction for the regulation of pomegranate grain hardness development.