Flavor formation in citrus fruit is governed by complex and dynamic changes in primary and secondary metabolism during development and ripening. Here, we integrated metabolomic, hormonal, and transcriptomic analyses to elucidate the regulatory landscape underlying soluble sugar and organic acid metabolism in ‘Kiyomi’ citrus fruit. A total of 1679 metabolites were identified, revealing stage-specific reprogramming of metabolic pathways, including a sharp decline in citric acid after 90 days after flowering (DAF) and peak accumulation of sucrose at 180 DAF. Hormonal profiling showed that abscisic acid (ABA) progressively accumulated from 120 to 210 DAF, while 1-aminocyclopropane-1-carboxylic acid (ACC) peaked at 120 DAF and declined thereafter, suggesting distinct and temporally coordinated roles in ripening regulation. Transcriptomic profiling uncovered widespread temporal shifts in gene expression, with the most pronounced changes occurring between 180 and 210 DAF. Co-expression network analysis identified gene modules associated with sugar and acid accumulation, and highlighted transcription factors from the ERF, MYB, NAC, and HSF families as candidate regulators of ripening-related metabolic transitions. These findings provide a comprehensive framework for understanding the coordinated molecular and metabolic programs underlying flavor development in non-climacteric citrus fruit and offer candidate genes for the genetic improvement of fruit quality.

Plant melatonin is widely recognized as a pleiotropic regulator. As a growth-regulating hormone, it extensively participates in various growth and developmental processes and has significant functions in stress responses and disease resistance. Plant melatonin is synthesized primarily through the catalytic actions of five enzymes: TDC (tryptophan decarboxylase), T5H (tryptamine-5-hydroxylase), SNAT (serotonin N-acetyltransferase), ASMT (N-acetylserotonin methyltransferase), and COMT (caffeic acid-O-methyltransferase). There are multiple genes for each of these five enzymes in citrus genomes, however, with the exception of COMT5—whose function has recently been elucidated—and SNAT, which has only been preliminarily identified, the remaining genes have not been unequivocally characterized or functionally annotated. Hence, we carried out a genome-wide analysis of melatonin biosynthesis enzyme-related gene families in trifoliate orange (Poncirus trifoliata), one of the most common citrus rootstock varieties. Through bioinformatics approaches, we identified 96 gene family members encoding melatonin biosynthetic enzymes and characterized their protein sequence properties, phylogenetic relationships, gene structures, chromosomal distributions, and promoter cis-acting elements. Furthermore, by analyzing expression patterns in different tissues and under various stresses, we identified multiple stress-responsive melatonin synthase genes. These genes likely participate in melatonin synthesis under adverse conditions, thereby enhancing stress adaptation. Specifically, PtCOMT5, PtASMT11, and PtTDC9 were significantly induced by low temperature; PtSNAT1, PtSNAT14, PtSNAT18, and PtTDC10 were markedly responsive to drought; and PtASMT15, PtSNAT15, PtASMT16, and PtSNAT3 were strongly induced by ABA. Among them, PtASMT23 expression was induced up to 120-fold under low temperature, while PtSNAT18 showed over 100-fold upregulation under dehydration treatment. These findings strongly suggest that PtASMT23 and PtSNAT18 play critical roles in regulating melatonin biosynthesis in response to cold and drought stress, respectively. Collectively, these findings pinpoint novel genetic targets for enhancing stress resilience in citrus breeding programs and lay the foundation for the functional characterization of specific melatonin biosynthesis pathway gene family members in citrus and other horticultural crop species.

Introduction: Citrus is one of the most widely cultivated fruit trees worldwide, and protected cultivation has become increasingly prevalent in recent years. Cover cropping improves orchard soil health, yet its mechanisms in protected citrus cultivation remain unclear. This study investigated how white clover ( Trifolium repens L.) and ryegrass ( Lolium perenne L.) affect soil properties, rhizosphere microbiota, and fruit quality in greenhouse-grown ‘Kanpei’ citrus through integrated analyses of soil physicochemical properties, high-throughput amplicon sequencing, and microbial isolation. Results: Both cover crops significantly increased total soluble solids (TSS) and vitamin C levels in mature fruits. Ryegrass enhanced the availability of nitrogen, phosphorus, calcium, magnesium, and manganese, whereas white clover more effectively acidified the soil and increased iron (Fe) availability. Each cover crop distinctively altered the rhizosphere microbial community. Notably, white clover specifically enriched Pseudomonas, which strongly correlated with elevated soil available Fe, TSS, and vitamin C. Screening with Chrome Azurol S (CAS) agar identified Pseudomonas as the dominant siderophore-producing genus. Inoculation with a representative strain, Pseudomonas sp. PA9, significantly enhanced Fe uptake, chlorophyll content, and fruit quality, offering insights into its potential role in promoting fruit quality under protected cultivation. Conclusions: This work provides a comprehensive understanding of how white clover promotes fruit quality via fostering siderophore-producing Pseudomonas that enhance Fe mobilization, suggesting new avenues for developing microbiome-based management strategies in protected citrus cultivation. These findings underscore the potential of cover crop-mediated microbial recruitment in advancing sustainable citrus production and soil health improvement.

Citrus fruits are widely cultivated all over the world. Due to climatic conditions, citrus fruits are frequently exposed to periodic low temperatures, which poses a serious threat to their yield and quality. Cold not only restricts plant growth and deteriorates fruit quality but also leads to fruit abscission and tree mortality, posing severe constraints on large-scale citrus production. The TIFY family gene plays crucial roles in plant development and stress adaptation. However, the genome-wide identification and functional analysis of TIFY genes in cold stress adaptation of citrus plants remain largely unexplored. Here, we performed a systematic genome-wide analysis of the TIFY family in sweet orange (Citrus sinensis (L.) Osbeck) and identified 14 CsTIFY members. We conducted a comprehensive study on the protein characteristics, phylogenetic relationships, gene structure, chromosome distribution, promoter cis-acting elements, and subcellular localization of these genes. Phylogenetic analysis classified the CsTIFYs into ZML (ZML1–ZML4), JAZ (JAZ1–JAZ7), PPD (JAZ8, JAZ9), and TIFY (TIFY1) subfamilies, and they are distributed on seven chromosomes. Collinearity analysis revealed that segmental duplication is the primary driver for CsTIFY family expansion. Expression profiling under cold stress identified JAZ1, JAZ2, and JAZ3 as the most cold-inducible members. All three CsTIFY proteins are targeted to the nucleus, as confirmed by subcellular localization analysis. Overexpression of JAZ1, JAZ2, or JAZ3 in citrus calli significantly enhanced cold sensibility. Collectively, this study elucidates the gene function of CsTIFYs under cold stress and provides new insight for molecular breeding of cold-tolerant citrus varieties.