Expression and functional analysis of MiOFP1 gene in mango

Abstract: 【Objective】Ovate Family Proteins (OFPs) play roles in the growth and development, hormone response and stress response of plants. In our previous study, the MiOFP1 gene was obtained from a screen mate and plate library of mango flowering genes. However, the information and functional characteristics of the MiOFP1 gene in mango were limited. To clarify the function of the MiOFP1 gene, its expression pattern and transgenic function were studied. The exploration of the MiOFP1 gene would provide a reference for further exploring the molecular mechanism of the MiOFP1 gene in mango flowering and resistance to adversity.【Methods】To learn more about the expression and regulation characteristics of the MiOFP1 gene, the promoter sequences in the 2000 bp region upstream of the coding region were analysed, and cis-acting elements of the promoter were analysed by NEW PLACE and illustrated via TBtools. The expression of the MiOFP1 in different tissues and different growth stages was verified and analysed by quantitative real- time PCR (qRT- PCR). The transgenic function of theMiOFP1 was investigated by overexpression of it in Arabidopsis thaliana. The phenotypes of homozygous plants of the T3 generation were observed, and the expression of the FLOWERING LOCUS T (FT) and FLOWERING LOUS C (FLC) in Arabidopsis thaliana was determined. The seeds of the T3 generation and seedlings of the T4 generation were treated with ABA, the germination rate and root length were observed and photographed, the physiological indexes, such as proline (Pro) content and peroxidase (POD) activity, and the expression of ABA- related genes, such as ABA- INSENSITIVE1 (ABI1), MOTHER OF FT AND TFL (MFT), DESICCATION-RESPONSIVE PROTEIN 29B (RD29B) and Ninecis-epoxycarotenoid dioxygenase 3 (NCED3), were determined. All data were analysed with IBM SPSS Statistics Version 22 (SPSS, Inc., Chicago, IL, USA), and the statistical validity was analysed by oneway ANOVA. Duncan’s multiple range test was applied.【Results】The promoter sequences of the MiOFP1 contain many hormone response elements, such as abscisic acid (ABA), gibberellin (GA), salicylic acid (SA) and ethylene response elements, and stress response elements, such as Slat, Dehydration, MYB and MYC binding sites. The results of tissue- specific expression analysis showed that the MiOFP1 gene was expressed in all tissues and organs in different development stages of mango, with the highest expression in the juvenile and adult stems and the lowest expression in the ripe fruit. The temporal expression pattern analysis showed that the MiOFP1 was highest in the vegetative growth stage but lower in the flower induction stage and flower development stage and gradually increased in the leaves during the fruit developmental stage. The transgenic Arabidopsis overexpression of the MiOFP1 gene showed that the bolting time and flowering time of three overexpressing lines were 2-4 days later than those of WT. The detection of the expression level of endogenous flowering genes in Arabidopsis showed that the MiOFP1 significantly increased the expression level of the flowering inhibiting gene AtFLC in transgenic Arabidopsis but significantly decreased the expression level of the flowering promoting gene AtFT. The ectopic expression of the MiOFP1 in transgenic plants resulted in higher tolerance to ABA. More specifically, the transgenic plants showed high ABA level tolerance manifested as increased seed germination and seedling growth. In the medium without ABA, the transgenic seeds germinated slightly faster than WT seeds; on the medium supplemented with ABA, not only containing 2 μmol·L^-1 ABA but also containing 5 μmol·L^-1 ABA, the germination of all seeds was inhibited, and the germination of transgenic seeds was significantly faster than that of WT seeds. For root length, no significant difference was observed in growth phenotype or root length between the transgenic Arabidopsis overexpression of the MiOFP1 gene and the WT plants under normal growth; however, the transgenic plants showed significantly greater root elongation than WT plants under 5 μmol · L^{- 1} ABA and 10 μmol·L^-1 ABA conditions. These results suggested that heterologous overexpression of the MiOFP1 could promote root elongation in Arabidopsis. Furthermore, under 20 μmol · L^-1 ABA stress, the Pro content and POD activity were increased, and the transcript levels of the several stress-responsive genes (AtABI1, AtMFT, AtRD29B and NCED3) in the transgenic lines were significantly upregulated compared with those in wild-type plants. In addition, the ectopic expression of the MiOFP1 in Arabidopsis resulted in insensitivity to ABA.【Conclusion】Promoter sequence analysis showed that the MiOFP1 promoters contained multiple hormone and stress cis-regulatory elements. Tissue expression analysis showed that the MiOFP1 was mainly expressed in juvenile and adult stems. The temporal expression analysis showed that the MiOFP1 was highly expressed in the vegetative growth stage. Furthermore, the overexpression of the MiOFP1 had a significant impact on the bolting time and flowering time. On the other hand, compared with those in WT plants, the overexpression of the MiOFP1 would improve root length, survival rate, Pro content, POD activity, and the expression of the ABA- relatedgenes under ABA conditions. We speculate that it is the key gene that affects mango flowering correlation and participates in the ABA response.