Correlation between oil gland development and essential oil accumulation in citrus peel and varietal differences

【Objective】Citrus essential oils are commercially valuable secondary metabolites synthesized and stored in specialized secretory structures called oil glands within the fruit flavedo. The accumulation patterns of these oils show considerable diversity among citrus species, but the underlying regulatory mechanisms connecting gland development with oil deposition remain inadequately characterized. This research aimed to systematically investigate the relationship between oil gland morphogenesis and essential oil accumulation by examining three representative citrus cultivars: Gaoban pummelo (Citrus grandis), Hamlin sweet orange (C. sinensis), and Ponkan mandarin (C. reticulata). Through comprehensive analysis of the developmental dynamics of oil glands and their correlation with essential oil content, this study sought to provide crucial insights into the species-specific regulation of essential oil accumulation and establish a theoretical foundation for targeted genetic improvement of citrus germplasm.【Methods】The study employed a systematic approach to analyze oil gland development and essential oil accumulation patterns. Fruits were sampled at seven developmental stages: 50, 90, 120, 150, 180, 210, and 240 days after flowering (DAF). For morphological analysis, we integrated fruit mor-phometry with advanced microscopic imaging techniques. High-resolution images of peel surfaces were captured using stereomicroscopy and processed with ImageJ software for quantitative assessment of oil gland number, density and volume. Total oil gland number per fruit was determined by direct counting, while density was calculated as glands per unit area. Individual gland volumes were estimated from cross- sectional measurements using geometric models. Essential oil content was quantified through standardized solvent extraction with n- hexane, followed by volumetric measurement of the extracted oil. Statistical analysis included Pearson correlation to evaluate relationships between morphological parameters and oil content.【Results】The investigation revealed distinct patterns in oil gland development across the three cultivars. All of the species showed a rapid increase in total oil gland number during early developmental stages (50-150 DAF), followed by stabilization as the fruits approached maturity. This pattern indicated that oil gland initiation is primarily confined to the first half of fruit development. In contrast, oil gland density demonstrated a consistent decline throughout development in all cultivars, reflecting the diluting effect of fruit surface expansion. Volumetric development displayed significant interspecific variation. Gaoban pummelo exhibited progressive volume increase throughout development, with glands expanding continuously from 50 DAF until maturity. Hamlin sweet orange showed delayed volumetric growth, maintaining minimal size increase until 120 DAF followed by active expansion thereafter. Technical constraints prevented reliable volume measurement in Ponkan mandarin due to its irregular peel morphology. Essential oil accumulation patterns correlated with gland development strategies. Gaoban pummelo displayed rapid oil accumulation after 180 DAF, coinciding with its progressive gland expansion. Hamlin sweet orange entered rapid accumulation phase earlier, from 90 DAF onward, while Ponkan mandarin initiated substantial accumulation after 150 DAF. Correlation analysis identified species- specific determinants of oil content. In Gaoban pummelo, oil gland volume showed the strongest correlation with essential oil content, indicating a volume- dominant strategy. Hamlin sweet orange demonstrated stronger dependence on oil gland number, suggesting a number- dominant approach. Ponkan mandarin showed weaker correlation with gland number, implying involvement of additional regulatory factors beyond those measured.【Conclusion】This study demonstrated that citrus varieties employ distinct oil gland development strategies to regulate essential oil accumulation. The relationship between gland morphology and oil content is conserved across varieties, but the relative importance of different morphological parameters varied significantly. Gaoban pummelo utilizes continuous gland expansion to enhance storage capacity, while Hamlin sweet orange relies more on maintaining numerous glands. Ponkan mandarin appears to employ an alternative strategy that may involve additional physiological or biochemical regulation. These findings highlight the diversity of essential oil accumulation mechanisms in citrus and provide valuable insights for future research on metabolic regulation in oil glands. The identification of varieties-specific strategies offers practical guidance for breeding programs aimed at modifying essential oil characteristics in different citrus types.