Pummelo (Citrus maxima) is one of important fruit trees, which belongs to Citrus species. The fruits of different pummelo cultivars have different colors and differ in the contents of carotenoid. Our results clearly showed that ‘Huangjinmiyou’ (HJMY) has the highest content of β-carotene, followed by ‘Hongroumiyou’ (HRMY) and ‘Guanximiyou’ (GXMY). Lycopene is dominantly accumulated in HRMY. However, the molecular mechanism underlying the carotenoid accumulation in pummelo flesh is not fully understood. In this study, we used the RNA-Seq technique to investigate the candidate genes of carotenoid metabolism in the flesh of pummelo cv. GXMY and its mutants HRMY and HJMY in three development periods of fruit. After data assembly and bioinformatic analysis, a total of 357 genes involved in biosynthesis of secondary metabolites were isolated, of which 12 differentially expressed genes (DEGs) are involved in carotenoid biosynthesis. Among these 12 DEGs, phytoene synthase (PSY2), lycopene β-cyclase (LYCB2), lycopene Ɛ-cyclase (LYCE), carotenoid cleavage dioxygenases (CCD4), 9-cis-epoxycarotenoid dioxygenase (NCED2), aldehyde oxidase 3 (AAO3), and ABA 8′-hydroxylases (CYP707A1) are the most distinct DEGs in three pummelo cultivars. The co-expression analysis revealed that the expression patterns of several transcription factors such as bHLH, MYB, ERF, NAC and WRKY are highly correlated with DEGs, which are involved in carotenoid biosynthesis. In addition, the expression patterns of 22 DEGs were validated by real-time quantitative PCR (RT-qPCR) and the results are highly concordant with the RNA-Seq results. Our results provide a global vision of transcriptomic profile among three pummelo cultivars with different pulp colors. These results would be beneficial to further study the molecular mechanism of carotenoid accumulation in pummelo flesh and help the breeding of citrus with high carotenoid content.

Boron (B) toxicity severely restricts Citrus growth and productivity in high-B soil and irrigation water environments. Although exogenous Zinc (Zn) has been reported to mitigate B toxicity in plants, the underlying physiological regulation mechanisms—particularly in Citrus—remain incompletely understood. In this study, seedlings of two Citrus species differing markedly in B tolerance—Citrus sinensis (sweet orange, B-tolerant) and C. grandis (sour pomelo, B-sensitive)—were subjected to three boric acid treatments (10, 200, and 400 μM) and five ZnSO₄ treatments (0.002, 0.05, 0.1, 0.2, and 0.25 mM) in a fully factorial design over 15 weeks. Comprehensive physiological assessments—including growth parameters, photosynthetic performance, mineral-nutrient profiling (in leaves and roots), and antioxidant enzyme activity—were conducted to decipher the mechanistic basis of Zn-mediated B detoxification. Results showed that 400 μM BA alone triggered severe B toxicity symptoms in the older leaves of C. grandis, whereas 0.25 mM Zn independently led to chlorosis symptoms similar to Fe deficiency in the apical leaves of both Citrus species. Either B or Zn toxicity affected photosynthesis in matured leaves. Exogenous Zn at 0.05–0.20 mM alleviated B toxicity symptoms in B-sensitive C. grandis, yet consistent alleviation of Zn toxicity by excess B was not observed across all measured variables. Severe B toxicity caused a significant reduction in Fe, N, P and Ca contents and a significant increase in Cu and Mn contents in C. grandis roots, yet it only led to a significant reduction in Mn, N, P and Ca contents in C. grandis leaves. Unlike this, B toxic treatments in C. sinensis resulted in a significant reduction in merely Fe content and a significant increase in Cu, Mn, N and K contents in roots, and a significant reduction in Mn and Ca contents in the leaves. Under basal B supply, 0.05–0.20 mM Zn supplementation dose-dependently increased root Zn, Cu and Mn contents as well as leaf Zn and Cu contents in both Citrus species, but concurrently decreased root Fe content and leaf Fe and Ca contents—additionally, in C. grandis, leaf N, P, K and Mg contents were further reduced. Regarding antioxidant responses in C. grandis, B toxicity significantly suppressed leaf CAT activity while enhancing APX and GPX activities. Under B toxicity conditions, 0.2 mM Zn supplement significantly restored CAT activity and suppressed APX and GPX activities. These antioxidant modulations were highly dependent on species identity and treatment combination, and were markedly more pronounced in B-sensitive C. grandis. Collectively, this work clarifies the physiological interplay between Zn and B in Citrus, uncovers divergent adaptive strategies employed by contrasting citrus species under combined B–Zn stress, and provides a mechanistic foundation for optimizing exogenous Zn application to mitigate B toxicity in citrus production.

Citron is a medicinal and edible plant. Terpenoids exhibit a wide range of biological activities. However, terpenoids in citron remain underexplored. In this study, a widely targeted metabolomics approach was employed to analyze the accumulation and variation patterns of terpenoid metabolites in citron flavedo at six developmental stages (50, 80, 110, 140, 170, and 210 d after flowering). The results showed that a total of 215 metabolites were detected, with monoterpenes (90) and triterpenes (51) being the major ones, accounting for 41.86% and 23.72% of the total, respectively. A total of 67 differential terpenoid metabolites were identified across different developmental stages. The number of up-regulated metabolites consistently exceeded that of down-regulated metabolites in each comparison group. Bitterness-related triterpenoid metabolites such as limonin, nomilin, obacunone, nomilinic acid, and obacunoic acid peaked at 170 d after flowering but exhibited an overall downward trend at 210 d. Most of the limonoid metabolites were significantly or extremely significantly positively correlated with one another; notably, nomilin displayed strong positive correlations with obacunone, deacetylnomilin, nomilinic acid, and obacunoic acid. This study delivers the first in-depth analysis of terpenoids in citron throughout development stages, laying a foundation for ongoing research and development of terpenoids in citron.