Identification of Novel Regulators of Fruit Sugar Accumulation Based on Transcriptome and WGCNA in Citrus sinensis

Abstract

Sweet orange (Citrus sinensis) is recognized as one of the most significant citrus fruits globally. The sugar content of fruits is the most critical internal quality associated with taste in sweet oranges, serving as a vital determinant of fruit quality and commercial value. Therefore, a comprehensive exploration of the regulatory mechanisms governing sugar accumulation during fruit ripening holds substantial value for high-quality fruit breeding. In this study, we investigated citrus sugar accumulation using the flesh of the Newhall navel orange and its high-sugar-content mutant cultivar, Ganmi, as experimental materials. RNA sequencing of the flesh from both Ganmi and Newhall oranges at 180 and 200 days after flowering identified 642 and 493 differentially expressed genes (DEGs), respectively. Functional enrichment analysis indicated that DEGs were mainly enriched in the sugar metabolism pathways, sugar transporters, and plant hormone signal transduction. Important DEGs associated with fruit sugar accumulation in Ganmi included Cs_ont_2g004470 (CsNAC73) and Cs_ont_9g005250 (CsSTP13) involved in sugar accumulation. Weighted gene co-expression network analysis showed that 20 co-expression modules were obtained, and the brown1 module had the strongest correlations with sugar content. Based on gene functionality and gene expression analyses of 1189 genes in this module, three genes (Cs_ont_2g004470 (CsNAC73), Cs_ont_5g050360 (CsMYC2) and Cs_ont_3g002820 (CsBBX21)) were identified as key genes potentially related to sugar accumulation during the ripening. These findings may contribute to elucidating the mechanisms underlying sugar accumulation during ripening and provide insights for the molecular breeding of citrus varieties.

Keywords: WGCNA; citrus; key genes; sugar content; transcriptome analyses.

Figure 1

Comparison of fruit morphology and TSS content between Ganmi and Newhall navel orange: (A) Fruit morphology of Ganmi (GM) and Newhall (NHE) navel orange at 180 and 200 days after flower (DAF), scale bars = 2 cm; (B) TSS of fruit pulps of GM and NHE navel orange at 180 and 200 DAF. ** denote significant difference at the 0.01 probability levels.

Figure 2

Identification and pathway enrichment analysis of DEGs related to fruit sugar accumulation in Ganmi: (A) The DEG numbers of the two groups. (B) Venn diagram analysis showing the number of common and unique DEGs identified at two stages. (C,D) KEGG enrichment pathway (level 1 and 2) annotated classification results of all DEGs in Ganmi and Newhall navel orange at 180, and 200 DAF, respectively.

Figure 3

Gene Ontology (GO) classification of the DEGs, found to be common to both 180 and 200 DAF. Red indicates GO terms related to biological processes, green indicates GO terms related to cell components, and blue indicates GO terms related to molecular function.

Figure 4

Identification of key regulators related to fruit sugar accumulation in Ganmi: (A) Weighted gene coexpression network analysis identifies 22 co-expression modules. (B) Module–trait relationships plot. The module sample correlation and corresponding p-values are shown in parentheses. The panel on the left shows 22 modules. The color code on the right shows the module feature correlation −1 (blue) to 1 (red); (C) KEGG enrichment analysis of genes in the brown1 module.

Figure 5

Gene expression profiling analysis of three key genes related to fruit sugar accumulation in Ganmi: (A) Expression characterization of three genes in fruits at different developmental stages. (B–D) Expression analysis of 3 sugar-accumulating related genes, Cs_ont_2g004470 (B), Cs_ont_5g050360 (C) and Cs_ont_3g002820 (D), in fruits at 180 and 200 DAF. * and ** denote significant differences at the 0.05 and 0.01 probability levels, respectively.