Characterization of Caleosin Genes in Carica papaya and Insights into Lineage-Specific Family Evolution in Brassicales

Caleosins (CLOs) or peroxygenases (PXGs), a class of structural proteins of lipid droplets (LDs), comprise a small family of multifunctional proteins widely involved in oil accumulation, organ development, and stress responses. Despite the proposal of two clades termed H and L in <i>Arabidopsis thaliana</i>, their evolution in the order Brassicales has not been well established. In this study, the first genome-wide analysis of the <i>caleosin</i> family was conducted in papaya (<i>Carica papaya</i>), a Caricaceae plant without any recent whole-genome duplication (WGD). A high number of five members representing both H and L clades were identified from the papaya genome. Further identification and comparison of 68 <i>caleosin</i> genes from 14 representative plant species revealed seven orthogroups, i.e., H1-4 and L1-3, where H1 and L1 have already appeared in the basal angiosperm <i>Amborella trichopoda</i>, supporting their early divergence before angiosperm radiation. Five <i>CpCLO</i> genes belong to H1 (1) and L1 (4), and extensive expansion of the L1 group was shown to be contributed to by species-specific tandem and transposed duplications, which may contribute to environmental adaptation. Orthologous and syntenic analyses uncovered that lineage-specific expansion of the <i>caleosin</i> family in Brassicales relative to <i>A. trichopoda</i> was largely contributed to by tandem duplication and recent WGDs, as well as the ancient γ whole-genome triplication (WGT) shared by all core eudicots. Independent gain or loss of certain introns and apparent expression divergence of <i>caleosin</i> genes were also observed. Tissue-specific expression analysis showed that <i>CpCLO2</i> and -<i>5</i> are constitutively expressed, whereas others appear to be tissue-specific, implying function divergence. Interestingly, the H-forms <i>CpCLO1</i> and <i>RcCLO1</i> were shown to exhibit similar expression profiles to most <i>oleosin</i> genes that are preferentially expressed oil-rich tissues such as seeds/endosperms, shoots, and calluses, implying their putative involvement in oil accumulation, as observed in <i>Arabidopsis</i>. The results obtained from this study provide a global view of <i>CpCLO</i> genes and insights into lineage-specific family evolution in Brassicales, which facilitates further functional studies in papaya and other non-model species.