Identification and Comprehensive Analysis of OFP Genes for Fruit Shape Influence in Mango

Abstract

OVATE family proteins (OFPs) are a class of plant-specific proteins with a conserved OVATE domain that play fundamental roles in fruit development and plant growth. Mango (Mangifera indica L.) is an economically important subtropical fruit tree characterized by a diverse array of fruit shapes and sizes. Despite extensive research on OFPs across various species, there remains a scarcity of information regarding OFPs in mango. Here, we have successfully identified 25 OFP genes (MiOFPs) in mango, each of which exhibits the conserved OVATE domains. The MiOFP gene exhibit a range of 2-6 motifs, with all genes containing both motif 1 and motif 2. Phylogenetic analysis on 97 OFPs (including 18 AtOFPs, 24 SlOFPs, 25 MiOFPs, and 30 OsOFPs) indicated that MiOFPs could be divided into three main clades: clade I, II, and III. Comparative morphological analysis identified significant variations in fruit longitudinal diameter, fruit transverse diameter, and fruit shape index between two distinct shaped mango cultivars ('Hongxiangya' and 'Jingpingmang') at DAP5, DAP7, and DAP10 stages. The subsequent examination of paraffin sections revealed distinct patterns of cell elongation. The majority of MiOFP genes exhibited predominantly expressed in developing organs, specifically flowers and immature fruits, while displaying distinct expression patterns. RNA-Seq analysis revealed significant disparities in the expression levels of several OFP genes, including MiOFP5, MiOFP11, MiOFP21, MiOFP22, MiOFP23, and MiOFP25, between the two mango cultivars. These findings suggest that these six genes may play a crucial role for fruit shape in mango, especially the MiOFP22. The findings of this study have established a basis for future investigations into MiOFPs in mango, offering a solid foundation for further research in this field.

Keywords: OVATE family proteins; expression pattern; fruit development; mango (Mangifera indica L.).

Figure 1

Locations, multiple-sequence alignment of MiOFP in mango: (A) Chromosomal locations of mango MiOFP genes. The intensity of the color of the chromosome is directly proportional to the gene density in that particular region; high gene density: red; low gene density: blue; (B) Two conserved motifs in OVATE domain. The overall height of each stack represents the conservation of the sequence at that position, and the height of the letters within each stack indicates the relative frequency of the respective amino acid; (C) The OVATE domain sequence alignment of mango OFPs. Identical or similar amino acids were shaded in same color box.

Figure 2

Phylogenetic relationship, conserved domains, conserved motif and promoter analyses of MiOFPs: (A) Phylogenetic relationship among the mango OFP proteins. Four clusters are labeled as Class 1, Class 2, Class 3, and Class 4; (B) Conserved domains of MiOFPs; (C) Conserved motifs of MiOFPs; (D) Analysis of cis-acting elements in the promoter region of the MiOFP gene family.

Figure 3

Phylogenetic and collinearity analysis of OFPs: (A) Numbers on branches indicate bootstrap values carried out with 1000 bootstrap replicates. Red star, OVATE, SlOFP1, and SlOFP20 of tomato (S. lycopersicum); circle, mango (M. indica). AtOFP, A. thaliana OFPs; SlOFP, tomato OFPs; OsOFP, rice OFPs; and MiOFP, mango OFPs; (B) Genome collinearity analysis was performed in A. thaliana, M. indica, and Solanum lycoperaicum. Black line represent the conserved OFP pairs between genomes.

Figure 4

Heatmap of mango MiOFPs gene expression in four tissues (five samples). The color key represents the relative transcript abundance of the MiOFP genes mango tissues.

Figure 5

Morphological changes in the fruit shape of elongated and round mango throughout fruit development: (A) Fruit shapes at different developmental stages. Fruits of elongated mango ‘HXY’ and round mango ‘JPM’ are present in the top and bottom rows, respectively. DAP1, one day after pollination. Bar = 10 mm; (B) Fruit longitudinal length, transverse width, and lateral thickness of ‘HXY’; (C) Fruit longitudinal length, transverse width, and lateral thickness of ‘JPM’; (D) Fruit shape index of ‘JPM’ and ‘HXY’; (E) Paraffin sections of the fruit pulp were examined at developmental stages DAP5, DAP7, and DAP10. The black arrow represents the direction of the fruit stalk. The scale bar represents 50 μm. Sections were stained with hematoxylin–eosin staining (HE).

Figure 6

Expression pattern analysis of MiOFPs in mango: (A) Heat map of 25 OFP genes from three developmental stages in ‘HXY’ and ‘JPM’; (B) PCA of global gene expression levels of all 18 samples data; (C) Differentially expressed genes between elongated and round-shaped fruits at the DAP5, DAP7, and DAP10 stages; (D) Venn diagram showing number of differentially expressed MiOFP genes (log2FC > 2; padj < 0.05); (E) RT-qPCR validation of MiOFP5, MiOFP11, MiOFP15, MiOFP21, MiOFP22, MiOFP23, and MiOFP25 in the development of different mango fruit. Error bar represents standard deviation from three replicates. Different stars number above the bars represent significant differences (p < 0.05, LSD) among two varieties.* = p < 0.05, ** = p < 0.01, *** = p < 0.001.