Han Yang

Sepal doubling in Punica granatum (pomegranate), characterized by calyx splitting and bilayered structure formation, confers distinctive ornamental value but its developmental mechanisms remain poorly understood. Elucidating the molecular mechanisms of sepal doubling and screening for associated genes and proteins in pomegranate are of theoretical and practical significance. Herein, transcriptomic and proteomic analyses were performed on sepals from single, double, and proliferated flowers. Overall, 23,630 genes and 5803 proteins were identified, with 5634 quantified at both levels. Pairwise comparisons revealed stronger correlations between differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) than between the total gene and protein sets. Because SeSi and SeDo/SePr were obtained from different cultivars, SeSi comparisons are partly confounded by genotype, whereas SeDo vs. SePr is the least cultivar-affected comparison. Cell wall-related terms were detected in the transcriptomic, proteomic and integrated datasets, alongside dominant photosynthesis-related terms in the transcriptomic comparisons. Eleven cell wall metabolism-related DEGs and three cell wall metabolism-related DAPs were identified, including ten concordant DEG–DAP pairs. These candidates included both concordant DEG–DAP pairs and those supported at a single omics level. Among them, cellulose synthase A (CESA), extensin-2 (EXT2), endo-1,4-β-D-glucanase (EG), and xyloglucan endotransglucosylase/hydrolase 16 (XTH16) were associated with bilayered calyx formation, whereas pectinesterase (PE), alpha-galactosidase (α-Gal), xylan α-glucuronyltransferase (α-GLU), and polygalacturonase (PG) were associated with calyx splitting. Two protein-modifying enzymes, serine/threonine-protein kinase (STPK) and serine carboxypeptidase (SCP), were associated with calyx splitting. Additionally, two transcription factors, basic helix–loop–helix (bHLH) and cold-shock domain protein (CSD), were associated with sepal doubling. These findings provide a molecular basis for further studies of floral development and type improvement in pomegranate.

Double-petal ornamental pomegranate presents for its enhanced ornamental value. Thus, cultivation techniques that promote petaloidy while modulating related gene expression are desired. To screen out the efficient treatments of plant growth regulator and key genes that enhance petaloidy, this study treated the flower buds of double- and single-petal ornamental pomegranate varieties with different concentrations of plant growth regulators naphthaleneacetic acid (NAA), methyl jasmonate (MeJA), abscisic acid (ABA), and ethephon (ETH) and quantified the number of petalized stamens (NOPSs) and the number of petals (NOPs) in both varieties. Furthermore, we investigated the expression levels of the genes flavin-containing monooxygenase (YUC), IAA-amino acid hydrolase (ILR1),indole-3-acetic acid-amido synthetase (GH3.17), auxin transporter (LAX2), auxin response factor (ARF), auxin-induced in root cultures protein (AIR12), jasmonic acid-amido synthetase (JAR1), and ABA stress ripening-induced protein (ASR) under the different treatments and analyzed their role in regulating relevant phenotypic traits. Plant growth regulator experiments demonstrated that NAA (10 mg/L) significantly increased the number of petalized stamens (NOPSs) and petals (NOPs), MeJA (100 mg/L) significantly increased the number of petalized stamens, while neither ABA nor ETH induced this morphological shift. qRT-PCR analysis confirmed that NAA upregulated ILR1, LAX2, ARF, and JAR1 in the stamens of single-petal flowers (StSi) and double-petal flowers (StDo) and petals of single-petal flowers (PeSi) and double-petal flowers (PeDo), with their expression levels strongly positively correlated with NOPS in both single- and double-petal flowers and NOP in double-petal flowers. MeJA upregulated ILR1, GH3.17, LAX2, ARF, and JAR1 in StDo and PeDo and was strongly positively correlated with NOPS and NOP in double-petal flowers. Consequently, NAA (10 mg/L) and MeJA (100 mg/L) were efficient treatments, and ILR1, GH3.17, LAX2, ARF, and JAR1 were identified as key genes in NAA- and MeJA-mediated petaloidy in ornamental pomegranates. Our results provide theoretical support for identifying the formation mechanism and improving industrial cultivation techniques for double-petal pomegranates.