The publication of several high-quality genomes has contributed greatly to clarifying the evolution of citrus. However, due to their complex genetic backgrounds, the origins and evolution of many citrus species remain unclear. We assembled de novo the 294-Mbp chromosome-level genome of a more than 200-year-old primitive papeda (DYC002). Comparison between the two sets of homologous chromosomes of the haplotype-resolved genome revealed 1.2% intragenomic variations, including 1.75 million SNPs, 149,471 insertions and 154,215 deletions. Using this genome as a reference, we resequenced and performed population and phylogenetic analyses of 378 representative citrus accessions. Our study confirmed that the primary origin center of core Citrus species is in South China, particularly in the Himalaya–Hengduan Mountains. Papeda species are an ancient Citrus type compared with C. ichangensis. We found that the evolution of the Citrus genus followed two radiations through two routes (to East China and Southeast Asia) along river systems. Evidence for the origin and evolution of some individual citrus species was provided. Papeda probably played an important role in the origins of Australian finger lime, citrons, Honghe papeda and pummelos; Ichang papeda originated from Yuanjiang city of Yunnan Province, China, and C. mangshanensis has a close relationship with kumquat and Ichang papeda. Moreover, the Hunan and Guangdong Provinces of China are predicted to be the origin center of mandarin, sweet orange and sour orange. Additionally, our study revealed that fruit bitterness was significantly selected against during citrus domestication. Taken together, this study provides new insight into the origin and evolution of citrus species and may serve as a valuable genomic resource for citrus breeding and improvement.

Ethylene treatment promotes orange coloration in the flavedo of Satsuma mandarin (Citrus unshiu Marc.) fruit, but the corresponding regulatory mechanism is still largely unknown. In this study, we identified a C2H2-type zinc-finger transcription factor, CitZAT4, the expression of which was markedly induced by ethylene. CitZAT4 directly binds to the CitPSY promoter and activates its expression, thereby promoting carotenoid biosynthesis. Transient expression in Satsuma mandarin fruit and stable transformation of citrus calli showed that overexpressing of CitZAT4 inhibited CitLCYE expression, thus inhibiting α-branch yellow carotenoid (lutein) biosynthesis. CitZAT4 overexpression also enhanced the transcript levels of CitLCYB, CitHYD, and CitNCED2, promoting β-branch orange carotenoid accumulation. Molecular biochemical assays, including yeast one-hybrid (Y1H), electrophoretic mobility shift (EMSA), chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR), and luciferase (LUC) assays, demonstrated that CitZAT4 directly binds to the promoters of its target genes and regulates their expression. An ethylene response factor, CitERF061, which is induced by ethylene signaling, was found to directly bound to the CitZAT4 promoter and induced its expression, thus positively regulating CitZAT4-mediated orange coloration in citrus fruit. Together, our findings reveal that a CitZAT4-mediated transcriptional cascade is driven by ethylene via CitERF061, linking ethylene signaling to carotenoid metabolism in promoting orange coloration in the flavedo of Satsuma mandarin fruit. The molecular regulatory mechanism revealed here represents a significant step toward developing strategies for improving the quality and economic efficiency of citrus crops.

Fruit taste quality is greatly influenced by the content of soluble sugars, which are predominantly stored in the vacuolar lumen. However, the accumulation and regulation mechanisms of sugars in most fruits remain unclear. Recently, we established the citrus fruit vacuole proteome and discovered the major transporters localized in the vacuole membrane. Here, we demonstrated that the expression of tonoplast sugar transporter 2 (CsTST2) is closely associated with sugar accumulation during sweet orange (Citrus sinensis) ripening. It was further demonstrated that CsTST2 had the function of transporting hexose and sucrose into the vacuole. Overexpression of CsTST2 resulted in an elevation of sugar content in citrus juice sac, calli, and tomato fruit, whereas the downregulation of its expression led to the reduction in sugar levels. CsTST2 was identified as interacting with CsCIPK23, which binds to the upstream calcium signal sensor protein CsCBL1. The phosphorylation of the three serine residues (Ser277, Ser337, and Ser354) in the loop region of CsTST2 by CsCIPK23 is crucial for maintaining the sugar transport activity of CsTST2. Additionally, the expression of CsCIPK23 is positively correlated with sugar content. Genetic evidence further confirmed that calcium and CsCIPK23-mediated increase in sugar accumulation depends on CsTST2 and its phosphorylation level. These findings not only unveil the functional mechanism of CsTST2 in sugar accumulation, but also explore a vital calcium signal regulation module of CsCBL1/CIPK23 for citrus sweetness quality.

Starch biosynthesis is a critical factor in wheat (Triticum aestivum L.) quality and yield. However, the full scope of its regulation is not fully understood. Here we report that TaDL interacts with TaB3 and TaNF-YB1 to synergistically regulate starch biosynthesis and quality in wheat. Genome-edited tadl mutant lines had smaller and lighter grains with lower total starch and amylose contents compared to wild type (WT). Correspondingly, the transcript levels of starch biosynthesis-related genes, including TaSUS1, TaSUS2, TaAGPL2, TaSBEIIa, TaGBSSII, and TaSWEET2a, were markedly lower at 15 d after flowering (DAF) in tadl mutants. TaDL physically interacted with TaB3 and TaNF-YB1 and activated the transcription of TaSUS2 and TaAGPL2 through direct binding to their promoter regions. A null mutant of TaB3 also affected grain filling, with phenotypes similar to those of tadl mutants, whereas overexpression of TaNF-YB1 promoted grain filling. Our study demonstrated that TaDL plays an essential role in starch biosynthesis and identified an elite allele (TaDL-BI) associated with starch content, providing insights into the underlying molecular mechanism of wheat grain filling, which may be useful in breeding of high-yielding wheat and quality improvement.