Eggplant fruits develop from the ovary and their final shape depends on cell division and expansion at the early stages of fruit development. In the present study, we found that the fruit shape index of the long-fruited FS eggplant at maturity is about 10.3, which is about 10 times higher than that of rounded HM eggplant, and this shape difference is obvious as early as 6 days before anthesis (Fig. 1). Morphological changes are usually associated with significant differences in gene expression levels (Jiang et al. 2015). To investigate the mechanisms of gene regulation in early fruit development that determine the shape of eggplant fruit we chose the early fruits (6 days before anthesis, anthesis day, and 6 days after anthesis) from both eggplant varieties for transcriptome analysis, and identified a total of 28992 expressed genes. The number of differentially expressed genes was found to be significantly greater in FS than in HM (Fig. 2), suggesting that FS fruits undergo more pronounced changes during development. In addition, the number of DEGs within FS and HM at different developmental stages were both greater than those between the two varieties, indicating that the degree of variation within the three development stages was greater than that between varieties.
To identify the biological functions of these DEGs, all of the annotated genes in eggplant were utilized as a background and GO enrichment analysis was performed (Supplementary Table S3). Before anthesis, the DEGs between FS and HM eggplants were mainly enriched in the GO terms of gibberellin metabolic and biosynthetic process, on the day of anthesis, they were mainly enriched in pectin catabolic and metabolic process, and after anthesis, they were mainly enriched in microtubule-based movement and auxin-activated signaling pathway. These results demonstrate that differences in polysaccharide metabolism and cellular microtubule movement between FS and HM begin to manifest themselves early in fruit development. It has been shown that polysaccharides are the main components of plant cell walls and that hemicelluloses and pectin metabolism are involved in the biosynthesis and degradation of cell walls during early apple fruit development, thus contributing to a large extent to the formation of apple fruit texture (Dheilly et al. 2016). In previous studies, microtubule-related genes were found to play special functions in rapid cell division and expansion during early fruit development in cucumbers (Yang et al. 2013). Phytohormones coordinate multiple aspects of plant growth and development, including fruit initiation. Fruit initiation has traditionally been attributed to auxin, GA, and cytokinin (Gillaspy et al. 1993). Coincidentally, the cellulose catabolic process and pectin catabolic process were significantly enriched in FS and HM eggplant between -6 DAA and 0 DAA, which may indicate that plant polysaccharide metabolic pathways play a prominent role in mediating the initial stages of fruit development (M et al. 1977). From 0 DAA to 6 DAA, microtubule-based movement and meiosis II cell cycle process were detected as significantly enriched in FS and HM, respectively (Supplementary Figure S2), which indicated that the cells around the ovary divide and expand rapidly after pollination (Dreesen et al. 2012).
During early fruit development, we detected a total of 4467 and 2919 genes differentially expressed in FS and HM varieties, respectively, which were categorized into 16 profiles based on STEM analysis. Three profiles (0, 6, and 7) were obtained for both FS and HM, with profiles 0 being downregulated and profiles 6 and 7 upregulated. In FS eggplants, we identified several terms that were significantly enriched in upregulated DEGs, namely, ‘cell wall polysaccharide metabolic process’ and ‘auxin-activated signaling pathway’ (Supplementary Figure S3), while ‘polysaccharide metabolic process’ and ‘cell wall organization’ were most enriched in upregulated DEGs of HM eggplants (Supplementary Figure S4). These biological processes that are enriched in upregulated expression of genes have been reported previously and are thought to be particularly beneficial for fruit growth and development. The cell wall is composed of pectin, hemicellulose, and cellulose as well as some structural proteins (Dheilly et al. 2016). In addition to producing the strength required by the plant, the cell wall defines cell shape, cell size, and cell function (Marowa et al. 2016). Furthermore, cell wall structures that contribute to differences in softening rates in apple fruit are formed early in fruit development (Ng et al. 2013). The sucrose transporter Pu SUT and the β-glucanases Pu bglu1, Pu bglu2 and Pu bglu4 are highly expressed during the initial stages of fruit development (Xinyue et al. 2016), which is consistent with the results of the present study that suggest that several genes related to cell wall polysaccharide metabolism are indeed critical during the early stages of fruit development.
The regulation of fruit development by plant hormones has been previously reported. The use of these hormones, either alone or in combination, can stimulate fruit growth in a range of plant species (Ozga and Reinecke 2003; Ozga et al. 2002; Vivian-Smith and Koltunow 1999). Several lines of evidence have demonstrated that an auxin signal is generated after fertilization, which is considered to upregulate GA biosynthesis, which in turn activates GA signaling in the ovules, thereby promoting fruit growth (Mezzetti et al. 2004; Dorcey et al. 2009). In our study, the DEGs with upregulated expression were enriched for phytohormone-related pathways, for example, ‘auxin-activated signaling pathway’ (GO:0009734) and ‘hormone-mediated signaling pathway’ (GO:0009755) in biological processes, indicating important roles of plant hormones in fruit development. Among these hormones, it has been reported that auxin was first triggered after flower opening and promotes floral organ enlargement (Gillaspy et al. 1993). The auxin signal then promotes the biosynthesis of other plant hormones such as GA, and the interplay of auxin with other hormones thereby regulates fruit growth and development (Dorcey et al. 2009). We screened 17 hormone-related genes that were significantly enriched in both FS and HM among the upregulated genes. Expression clustering analysis (Fig. 5), revealed, that these were significantly activated on the day of flowering in both eggplant varieties. For example, the auxin response factor SmARF1 was upregulated in both FS and HM. As key players in the auxin signaling pathway, ARFs regulate cell enlargement and plant growth by activating or repressing the expression of auxin-responsive genes (Supplementary Figure S6). In plants, ARFs have been proven to mediate auxin signal transduction and to regulate growth. In melons, CmARF1 expression was linked to fruit growth during early development (Wu et al. 2020). Furthermore, our results show that the expression of SmPIF4 and SmAUX22 was consistently upregulated in FS eggplants and, similarly, SmIAA26-2, SmEBF2-1, SmEIL3, and SmERF1B-1 were also up-regulated in HM eggplants (Fig. 5), suggesting that these hormone-related genes play a role in promoting early fruit development since their expression is positively correlated with fruit growth. Conversely, differences in gene expression patterns between cultivars may result in alterations in cell division and expansion rates, resulting in varied fruit morphologies.
Several genes have been reported to be involved in the regulation of fruit shape during fruit development. Among these genes, FASCIATED (FAS), which belongs to the YABBY gene family, regulates fruit shape by affecting ovary numbers, while SUN and OVATE are key regulators controlling fruit elongation, all of which have been reported to affect blueberry fruit morphology during pre-flowering and post-pollination stages (Yang et al. 2018). The SUN protein harbors an IQD domain, which plays an important role in plant development processes (Cai et al. 2016). OVATE belongs to a plant-specific transcription factor family that plays a significant role in the growth and development of Arabidopsis and tomato (Zhang et al. 2020). In the present study, we screened for genes that are differentially expressed in FS and HM eggplants and found that most genes were expressed at higher levels before flowering (Fig. 6). Similarly, it has been reported that SlOVATE mRNA was detected only around the flowering time (Liu et al. 2002) and that OVATE represents a class of negative regulatory proteins important in plant development. To verify the molecular functions of the differential expressed genes that we identified in our study, we selected the SmOVATE5 gene, which has the highest homology with the tomato SlOVATE gene, for functional validation in Arabidopsis (Supplementary figure S6). Arabidopsis lines that were overexpressing SmOVATE5 had smaller and rounder leaves and shorter stalks and siliques than wild-type plants (Fig. 8). This result demonstrated that the SmOVATE5 gene inhibits plant growth, in agreement with our transcriptome results. Differential expression of genes may lead to differences in traits. For example, slight changes in CaOVATE expression were sufficient to induce changes in pepper shape (Tsaballa et al. 2011). Our study identified SmYAB2 and SmOVATE10 as more highly expressed in HM than in FS. Therefore, these genes are potentially involved in the regulation of eggplant fruit shape, and in the future, they can serve as candidate genes for genetic transformation and phenotypic characterization to further investigate their role in fruit development.