Whole-genome duplication (WGD) plays important roles in plant evolution and function, yet little is known about how WGD underlies metabolic diversification of natural products that bear significant medicinal properties, especially in nonmodel trees. Here, we reveal how WGD laid the foundation for co-option and differentiation of medicinally important ursane triterpene pathway duplicates, generating distinct chemotypes between species and between developmental stages in the apple tribe. After generating chromosome-level assemblies of a widely cultivated loquat variety and Gillenia trifoliata, we define differentially evolved, duplicated gene pathways and date the WGD in the apple tribe at 13.5 to 27.1 Mya, much more recent than previously thought. We then functionally characterize contrasting metabolic pathways responsible for major triterpene biosynthesis in G. trifoliata and loquat, which pre- and postdate the Maleae WGD, respectively. Our work mechanistically details the metabolic diversity that arose post-WGD and provides insights into the genomic basis of medicinal properties of loquat, which has been used in both traditional and modern medicines. Significance Plants are a primary source of both traditional and modern drugs due to their astounding capability to synthesize diverse molecules. The fruit tree loquat in the apple tribe has been long used in medicine to treat cough, chronic bronchitis, and asthma, yet why loquat—but not its relatives—evolved these medicinal properties is unknown. Here, we generate high-quality genomes of loquat and a relative, which are separated by a recent whole-genome duplication (WGD) around 13.5 to 27.1 Mya. We revealed the post-WGD diversification of triterpene biosynthesis and the exceptionally high levels of bioactive ursane-type triterpenes specifically in loquat. Our work underscores the importance of WGD-associated metabolic diversification underlying the bioactivity of some medicinal plants.

The age pathway is important for regulating flower bud initiation in flowering plants. The major regulators in this pathway are miR156 and SPL transcription factors. To date, SPL genes have been identified in many species of plants. Loquat, as a woody fruit tree of Rosaceae, is unique in flowering time as it blooms in winter. However, the study of its SPL homologous genes on the regulation mechanism of flowering time is still limited. In this study, four SPL homologs—EjSPL3, EjSPL4, EjSPL5, and EjSPL9—are cloned from loquat, and phylogenetic analysis showed that they share a high sequence similarity with the homologues from other plants, including a highly conserved SQUAMOSA promoter binding protein (SBP)-box domain. EjSPL3, EjSPL4, EjSPL5 are localized in the cytoplasm and nucleus, and EjSPL9 is localized only in the nucleus. EjSPL4, EjSPL5, and EjSPL9 can significantly activate the promoters of EjSOC1-1, EjLFY-1, and EjAP1-1; overexpression of EjSPL3, EjSPL4, EjSPL5, and EjSPL9 in wild-type Arabidopsis thaliana can promote flowering obviously, and downstream flowering genes expression were upregulated. Our work indicated that the EjSPL3, EjSPL4, EjSPL5, and EjSPL9 transcription factors are speculated to likely participate in flower bud differentiation and other developmental processes in loquat. These findings are helpful to analyze the flowering regulation mechanism of loquat and provide reference for the study of the flowering mechanism of other woody fruit trees.

Loquat (Eriobotrya japonica) is a subtropical tree that bears fruit that ripens during late spring. Fruit size is one of the dominant factors inhibiting the large-scale production of this fruit crop. To date, little is known about fruit size regulation. In this study, we first discovered that cell size is more important to fruit size than cell number in loquat and that the expression of the EjBZR1 gene is negatively correlated with cell and fruit size. Virus-induced gene silencing (VIGS) of EjBZR1 led to larger cells and fruits in loquat, while its overexpression reduced cell and plant size in Arabidopsis. Moreover, both the suppression and overexpression of EjBZR1 inhibited the expression of brassinosteroid (BR) biosynthesis genes, especially that of EjCYP90A. Further experiments indicated that EjCYP90A, a cytochrome P450 gene, is a fruit growth activator, while EjBZR1 binds to the BRRE (CGTGTG) motif of the EjCYP90A promoter to repress its expression and fruit cell enlargement. Overall, our results demonstrate a possible pathway by which EjBZR1 directly targets EjCYP90A and thereby affects BR biosynthesis, which influences cell expansion and, consequently, fruit size. These findings help to elucidate the molecular functions of BZR1 in fruit growth and thus highlight a useful genetic improvement that can lead to increased crop yields by repressing gene expression.

Fruit weight is an integral part of fruit quality and directly influences the commodity value and economic return of fruit crops. Despite the importance of fruit weight, its underlying molecular mechanisms remain understudied, especially for perennial fruit tree crops such as cultivated loquat (Eriobotrya japonica Lindl.). Auxin is known to regulate fruit development, but its role and metabolism during the development of loquat fruit remain obscure. In this study, we used a multi-omics approach, integrating whole-genome resequencing-based quantitative trait locus (QTL) mapping with an F₁ population, population genomics analysis using germplasm accessions, transcriptome analysis, and metabolic profiling to identify genomic regions potentially associated with fruit weight in loquat. We identified three major loci associated with fruit weight, supported by both QTL mapping and comparative genomic analysis between small- and large-fruited loquat cultivars. Comparison between two genotypes with contrasting fruit weight performance by transcriptomic and metabolic profiling revealed an important role for auxin in the regulation of fruit development, especially at the fruit enlargement stage. The multi-omics approach identified homologs of ETHYLENE INSENSITIVE 4 (EjEIN4) and TORNADO 1 (EjTRN1) as promising candidates for the control of fruit weight. Three single nucleotide polymorphism (SNP) markers were also closely associated with fruit weight. Results from this study provide insights into the genetic and metabolic controls of fruit weight in loquat from multiple perspectives. The candidate genomic regions, genes, and sequence variants will facilitate our understanding of the molecular basis of fruit weight and lay a foundation for future breeding and manipulation of fruit weight in loquat.