Polyembryony has posed a significant impediment to the advancement of citrus hybrid breeding. FhRWP is widely regarded as a pivotal factor governing asexual reproduction in citrus, and prior research has demonstrated that FhARID1, acting as an upstream regulator, modulates FhRWP expression. In this study, we performed a genome-wide characterization of the ARID-HMG-related genes using the short juvenile mini-citrus Fortunella hindsii. A total of 20 ARID-HMG-related genes were identified. Protein interaction network and enrichment analysis suggested that ARID-HMG-related proteins might might be involved in chromatin remodeling complexes. Knockout of FhARID1 in F. hindsii did not induce the conversion from polyembryony to monoembryony. However, fharid1 plants in T₁ generation exhibited abnormal proliferation at axillary buds, which is similar to phenotype of fhrwp plants. Expression analysis of fharid1 ovary tissues revealed the downregulation of FhRWP. The results indicated that FhARID1, as an upstream regulator of FhRWP, has an effect on the development of citrus axillary buds. Expression analysis of overexpressed leaves of FhARID1 lines showed that no significant up-regulation of FhRWP, indicating that FhARID1 is not the sole upstream regulatory factor of FhRWP. Only FhARID2 showed a correlation in expression with FhARID1 among the ARID-related genes, further supporting the notion that this gene may be involved in complex formation rather than acting alone. Yeast two-hybrid and MS/MS spectra further indicated that FhARID1 function requires casein kinase II-mediated post-transcriptional phosphorylation. This study elucidated the function of FhARID1 in citrus apomixis and axillary bud development, providing a fundamental basis for understanding the role of ARID-HMG-related genes.

Carotenoids are indispensable for human health, and citrus fruit are a crucial source of dietary carotenoids. Bagging, an important orchard practice to enhance fruit economic value, is widely used in many horticultural crops, including citrus fruit. The bagged 'Majiayou' pomelo (Citrus grandis) produces vivid deeper red pulp, a fantastic agronomic trait, but the underlying molecular regulatory mechanism remains largely unexplored. Here, the enhancement of carotenoids, especially lycopene, was confirmed by HPLC analysis of carotenoids in the pulp of bagged fruit and controls. qRT-PCR analysis of the 14 carotenoid pathway genes further revealed that upregulated PSY and downregulated CCD1 expression in bagged fruit could directly enhance the accumulation of carotenoids. In addition, RNA-seq analysis identified 311 differentially expressed genes (DEGs) in the bagged fruit and controls in five developmental stages. Weighted gene co-expression network analysis (WGCNA) identified 13 critical candidate genes among the DEGs, which are closely associated with lycopene accumulation. The underlying regulatory mechanism of these candidate genes on the transcription of carotenoid pathway genes in the bagged fruit was discussed. Considering that the candidate genes were involved in the corresponding metabolic pathways, the increase in sucrose content and decrease in ABA in bagged fruit were also identified, implying that these candidate genes may be indirectly related to carotenoid enhancement in pulp by regulating phytohormones, primary metabolism, and stress responses. The results provide new insights into the potential regulatory mechanism of lycopene enhancement in the pulp of bagged 'Majiayou' pomelo, facilitating breeding and orchard management efforts to improve the nutritional quality and esthetic value of citrus, and perhaps other fruit crops.

Carotenoids are indispensable for both human health and plant survival. Citrus, is one of the fruit crops richest in carotenoid compounds, with approximately 115 kinds of carotenoids; tremendous diversity in carotenoids composition and concentration exists among various species, showing different colors from nearly white to crimson. The carotenoid biosynthetic pathway and the key carotenogenic genes have been identified in citrus; however, the underlying regulatory mechanisms remain unclear. In this study, among the main species of genus Citrus (primitive, wild, and cultivated), we detected carotenoids in flavedo using High-Performance Liquid Chromatography, and analyzed variations in cis-acting elements in the promoters of key carotenoid pathway genes. Intriguingly, both carotenoid composition and content were generally increased during the evolution of citrus, and the corresponding variations in the promoters were identified, including the gain or loss of critical environmental stress-responsive elements and hormone-responsive elements, which are closely associated with carotenoid enhancement. In addition, pummelo has the most heat-responsive elements, but the Mangshan mandarin does not have this element in the promoters of PSY, which is highly related to their geographical origin and indicate that temperature is a critical environmental signal influencing carotenoid accumulation. Moreover, the abscisic acid-responsive motif was rich in almost all the seven species, but the ethylene-responsive motif was deficient, which demystified the unique phytohormone regulation mechanism of carotenoid accumulation in citrus. Overall, our study provides new insights into the molecular regulatory mechanism of carotenoid enhancement in the evolution of citrus, which can facilitate breeding and cultivation efforts to improve the nutritional quality and esthetic value in citrus and hopefully other fruit crops.