We evaluated the effect of stocking density on the concentrations of oxy-hemocyanin and lactate in hemolymph, the antioxidant ability of the hepatopan

Lemon (Citrus limon L.), an economically important Citrus species, produces high levels of citric acid. However, the regulatory mechanisms underlying citric acid accumulation in lemon fruit are poorly understood. In this study, we generated a haplotype-resolved genome for ‘Eureka’, a widely cultivated commercial lemon cultivar. Based on the progenitor sequences, we elucidated the origin and identified some domesticated loci of the lemon haplomes, including those associated with citric acid metabolism. Comparative genomics analysis revealed that the gene families enriched in the pathways related to stress responses and soluble sugar biosynthesis were dramatically contracted in the lemon genome. Substantial allelic variations in sequences, gene expression and methylation levels were detected between the two haplotypes. Of note, transcript levels of vacuolar P-ATPases, PH5, encoding a proton pump involved in citric acid accumulation, were drastically higher in Eureka lemon relative to sweet lemon, which contains trace amount of citric acid in the fruit. In addition, whole-genome bisulfite sequencing revealed that the promoter of PH5 was highly methylated in sweet lemon but not in Eureka. Furthermore, demethylation of the PH5 promoter led to an increase in the citric acid content. Taken together, these findings demonstrate that low DNA methylation level in the promoter region of PH5 contributes to the abundant accumulation of citric acid in lemon fruit. Our study provides a valuable genetic resource for investigating the domestication mechanism in citrus and underpins genome-based genetic engineering to create either acid or acidless cultivars.

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.