Zhen Cao

Fruit aroma is a key factor shaping the commercial value of pears. MYB and ERF transcription factors (TFs) are involved in regulating the biosynthesis of fruit aroma compounds, yet the mechanisms underlying their synergistic regulation of pear aroma formation remain elusive. Calcium signaling exerts an important role in fruit aroma biosynthesis; nevertheless, how calcium signaling mediates MYB-ERF transcription factors to participate in pear aroma biosynthesis remains unclear. Here, transcriptomic profiling of calcium-treated pear fruits screened two significantly upregulated TFs, PuMYB44 and PuERF118, as candidate genes. Phylogenetic analysis and conserved domain characterization further suggested that these two genes may be involved in regulating fatty acid biosynthesis. Transient overexpression experiments in pear fruits revealed that the co-expression of PuMYB44 and PuERF118 synergistically activated a set of fatty acid biosynthesis genes, including PuFAD2, PuFAD3, PuFAD8, PuLOX5, PuLOX21, and PuSCD. Meanwhile, the co-expression markedly increased the contents of key aroma volatiles, such as ethyl acetate, 1-hexanol, ethyl 2-methylbutyrate and hexadecane. Yeast two-hybrid and luciferase complementation assays confirmed an interaction between PuMYB44 and PuERF118. Analysis of the PuFAD8 promoter sequence revealed a conserved MYB-binding motif and an ERF-responsive cis-acting element. Dual-luciferase reporter assays further confirmed that both PuMYB44 and PuERF118 can independently activate PuFAD8 transcription, and that the synergistic activation effect resulting from the co-expression of these two factors is significantly stronger than that observed when either factor is expressed alone. In summary, this study elucidated a calcium-responsive transcriptional module underlying the biosynthesis of pear aroma and provided potential molecular targets for improving the flavor quality of pear fruit.

Pomegranate (Punica granatum L.) is widely valued for its rich nutritional profile and distinctive sensory characteristics. As one of the oldest cultivated fruits, it has an extensive history of cultivation in China and possesses abundant germplasm resources. Nevertheless, systematic evaluation of these resources remains inadequate, limiting progress in germplasm innovation and utilization. In this study, we analyzed 16 fruit quality traits across 142 pomegranate accessions. Most traits showed wide phenotypic variation, with coefficients of variation (CVs) ranging from 2.10% to 108.83%. Notably, titratable acidity (TA) and anthocyanin content showed high variability (coefficient of variation, CV > 78%), while fruit shape index and total soluble solids (TSS) showed relatively low variability (CV < 10%). Cluster analysis delineated three distinct phenotypic groups. The first group comprised accessions characterized by large fruit size, thick peel, high acidity, and soft seeds. The second group exhibited high seed hardness, low acidity, and elevated sugar-acid and TSS–acid ratios. The third group displayed reduced levels of bioactive compounds such as tannins, phenols, and anthocyanins, combined with high seed hardness. Correlation analysis followed by principal component analysis (PCA) extracted six principal components, and based on comprehensive scoring, SXXA23, AHHB13, SD41, SXXA27, AHHB40, HS1, AHHB8, HY22, HN4, and AH27 were identified as priority accessions for further evaluation within this repository panel.

Seed weight is an important target trait in pomegranate breeding and culture. Expansins act by loosening plant cell walls and cellulosic materials, permitting turgor-driven cell enlargement. However, the role of expansin genes (EXPs) in pomegranate seed weight remains elusive. A total of 29 PgrEXPs were identified in the ‘Dabenzi’ genome. These genes were classified into four subfamilies and 14 subgroups, including 22 PgrEXPAs, 5 PgrEXPBs, 1 PgrEXPLA, and 1 PgrEXPLB. Transcriptome analysis of PgrEXPs in different tissues (root, leaf, flower, peel, and seed testa) in ‘Dabenzi’, and the seed testa of the hard-seeded pomegranate cultivar ‘Dabenzi’ and soft-seeded cultivar ‘Tunisia’ at three development stages showed that three PgrEXPs (PgrEXPA11, PgrEXPA22, PgrEXPA6) were highly expressed throughout seed development, especially in the sarcotesta. SNP/Indel markers of these PgrEXPs were developed and used to genotype 101 pomegranate accessions. The association of polymorphic PgrEXPs with seed weight-related traits (100-seed weight, 100-kernel weight, 100-sarcotesta weight, and the percentage of 100-sarcotesta to 100-seed weight) were analyzed. PgrEXP22 was significantly associated with 100-seed weight and 100-sarcotesta weight and is a likely candidate for regulating seed weight and sarcotesta development in particular. This study provides an effective tool for the genetic improvement of seed weight in pomegranate breeding programs.

The pomegranate (Punica granatum L.) fruit is favorable for its nutrient-rich benefits to human health. However, the global metabolic profiles of pomegranate juice and the metabolic mechanisms of its essential metabolites are poorly understood. In this study, we conducted a widely targeted metabolome, integrated with the transcriptome of juices (edible parts) of pomegranate fruits at 50, 95, and 140 days after flowering (DAF) to comprehensively investigate the metabolic profiles and potential metabolism of essential metabolites. Five hundred and nine metabolites, including 11 sugar and sugar alcohols, 17 common organic acids, 20 essential amino acids, and a variety of flavonoids, were detected in pomegranate juices. Among them, metabolites in the flavonoid biosynthesis pathway greatly changed during fruit development. Notably, the redirection of metabolite flux from catechin and its derivative synthesis to anthocyanin synthesis occurred at the later developmental stages. The increased expression of Pgr021399.1 encoding dihydroflavonol 4-reductase (DFR), Pgr017842.1 encoding anthocyanidin synthesis (ANS), Pgr015322.1 encoding anthocyanidin 3-O-glucosyltransferase (BZ1), Pgr000447.1 encoding UTG75C1, and the decreased expression of Pgr024128.1 encoding leucoanthocyanidin reductase (LAR) may trigger redirection. The results of this study provide a global view of the metabolic profiles of pomegranate juices and valuable information on the molecular mechanisms underlying the redirection of flavonoid metabolism. It also sheds light on the genetic regulation of flavonoid metabolism in pomegranate juices.

UDP glycosyltransferases (UGTs) play an indispensable role in regulating signaling pathways and intracellular homeostasis in plants by catalyzing the glycosylation of metabolites. To date, the molecular characteristics and potential biological functions of the UGT gene family in pomegranate (Punica granatum L.) remain elusive. In this study, a total of 120 PgUGT genes were identified in the pomegranate genome. Phylogenetic analysis revealed that these PgUGTs were clustered into 15 groups: 13 conserved groups (A–J and L–N) and two newly discovered groups (P and R). Structural analysis showed that most members in the same evolutionary branch shared similar motifs and gene structures. Gene duplication analysis demonstrated that tandem duplication and fragment duplication were the primary driving force for the expansion of the PgUGT family. Expression analysis based on RNA-seq data indicated that PgUGTs exhibited various expression profiles in different pomegranate tissues. We further analyzed the expression patterns of the PgUGTs of groups E and L in the seed coat of the hard-seeded cultivar ‘Dabenzi’ and the soft-seeded cultivar ‘Tunisia’ at different developmental stages. There were eight PgUGTs with high expression levels in the seed coat of both cultivars: PgUGTE10 was highly expressed in inner and outer seed coats; PgUGTE20, PgUGTE21, PgUGTL6, PgUGTL11, and PgUGTL12 were mainly expressed in the inner seed coat; and PgUGTE12 and PgUGTL13 were mainly expressed in the outer seed coat. Interestingly, the relative expression levels of PgUGTE10 and PgUGTL11 in ‘Tunisia’ were higher than in ‘Dabenzi’. In the seedlings, quantitative real-time PCR analysis showed that the expression level of PgUGTE10 was induced by brassinolide treatment, while the expression of PgUGTL11 was up-regulated both by indole-3-acetic acid and the brassinolide treatment. In addition, the expressions of PgUGTE10 and PgUGTL11 were highly correlated with the expression of genes involved in hormone signaling and lignin biosynthesis pathways. These results suggested that PgUGTE10 and PgUGTL11 are potential candidate genes involved in seed hardness development by catalyzing the glycosylation of specific substrates.

Pericarp browning occurs widely in postharvest pomegranate fruit, but little is known about its mechanism. In this study, ‘Baiyushizi’, a browning-sensitive cultivar, was used to investigate the physiological and genetic changes during pericarp browning. The pericarp browning index (BI) increased greatly after 3 d storage at room temperature, and, consequently, the fruit appearance became unattractive after 6 d. The increasing concentration of malondialdehyde (MDA) and electrolyte leakage were detected in browning pericarp. Polyphenol oxidase (PPO) activity increased significantly after 3 d, while ascorbate peroxidase (APX) and catalase (CAT) activity decreased steadily during storage. Total phenolics content decreased gradually during storage, while ascorbic acid (AsA) and glutathione (GSH) contents increased firstly, then declined. Differentially expressed genes (DEGs) of pericarp under different browning degrees were identified in the metabolic processes of phenolic compounds, lipids, ascorbic acid, glutathione, sugar, starch, energy-related, ethylene biosynthesis and signal transduction pathways. Accordingly, the potential metabolic pathways and related processes involved in pericarp browning were described. The results not only provided insights on the genetic mechanism of pericarp browning in pomegranate but also guidance on controlling postharvest fruit browning in general.

ATP-binding cassette subfamily G (ABCG) proteins play important roles in plant growth and development by transporting metabolites across cell membranes. To date, the genetic characteristics and potential functions of pomegranate ABCG proteins (PgrABCGs) have remained largely unknown. In this study, we found that 47 PgrABCGs were divided into five groups according to a phylogenetic analysis; groups I, II, III, and IV members are half-size proteins, and group V members are full-size proteins. PgrABCG14, PgrABCG21, and PgrABCG47 were highly expressed in the inner seed coat but had very low expression levels in the outer seed coat, and the expression levels of these three PgrABCG genes in the inner seed coats of hard-seeded pomegranate ‘Dabenzi’ were higher than those of soft-seeded pomegranate ‘Tunisia’. In addition, the expression of these three PgrABCG genes was highly correlated with the expression of genes involved in lignin biosynthesis and hormone signaling pathways. The evolution of PgrABCG14 presents a highly similar trend to the origin and evolution of lignin biosynthesis during land plant evolution. Ectopic expression of PgrABCG14 in Arabidopsis promoted plant growth and lignin accumulation compared to wild type plants; meanwhile, the expression levels of lignin biosynthesis-related genes (CAD5, C4H, and Prx71) and cytokinin response marker genes (ARR5 and ARR15) were significantly upregulated in transgenic plants, which suggests the potential role of PgrABCG14 in promoting plant growth and lignin accumulation. Taken together, these findings not only provide insight into the characteristics and evolution of PgrABCGs, but also shed a light on the potential functions of PgrABCGs in seed hardness development.