Huping Zhang

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.

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.

Sucrose is an important factor affecting sweetness and flavor in pear fruits, but the molecular mechanism of sucrose synthesis regulation is relatively unknown. Here, we characterized a transcription factor gene from pear (Pyrus pyrifolia Nakai cv. “Hosui”) fruits, PpybZIP43, and found that the transient overexpression of PpybZIP43 in pear fruits significantly increased the sucrose content and the relative expression level of sucrose phosphate synthase genes (PpySPS3 and PpySPS8). Subcellular localization analysis in tobacco leaves showed that PpybZIP43 was localized in the nucleus. Yeast one‐hybrid, electrophoretic mobility shift assay (EMSA), and dual‐luciferase reporter assays indicated that PpybZIP43 was able to activate the expression of PpySPS3 by binding specifically to the G‐box (CACGTG) element in the promoter. The protein–protein interaction assays using yeast two‐hybrid, bimolecular fluorescence complementation (BiFC), firefly luciferase complementation imaging (LCI), and glutathione S‐transferase (GST) pull‐down demonstrated that PpybZIP43 could directly interact with PpySTOP1 to form a transcription complex. This study is helpful for understanding the molecular basis of sucrose synthesis and accumulation in pear fruits and provides candidate genes for breeding.

Main conclusion A total of 6763 proteins were identified in the developing pear flesh, which were further screened for differentially expressed proteins related to fruit quality and ATP-binding cassette transporters. Abstract To obtain further details on changes in protein levels during fruit ripening and to identify and evaluate changes in various metabolic pathways that affect fruit quality, a proteomic method using tandem mass tags was implemented at three developmental stages in Pyrus pyrifolia cv. “Hosui” that identified 6763 proteins. Subcellular localization and Gene Ontology enrichment analysis revealed major functions of all identified proteins. Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that all metabolic processes are reflected in the up- and downregulation of differentially expressed proteins during fruit development, which play predominant roles in cell division, cell expansion, and fruit ripening. Among the examined differentially expressed proteins, 160 related to fruit quality, and 14 ATP-binding cassette transporters related to fruit development were identified and analyzed. The quantitative data were validated by parallel reaction monitoring, which confirmed the reliability of the experimental results.

Vacuolar processing enzymes (VPEs) are important cysteine proteases and function in the processing and maturation of protein precursors, plant senescence and immunity, programmed cell death, as well as sugar accumulation. This paper investigated VPEs in pear and identified as a key finding eight members of the VPE family in the pear (Pyrus) genome, and their gene structure, protein conserved domains and phylogenetic relationship were analysed by bioinformatics methods. Further key findings were that two of these VPEs (PbrVPE2 and PbrVPE5) represented a vegetative and the others a seed-coat type VPE. Transcriptome data and quantitative real-time PCR were used separately to analyse the expression patterns of the pear VPE gene family. The PbrVPE2 and PbrVPE5 were, in comparison to the other VPEs, abundantly expressed by possibly playing a role during the development and ripening of the pear fruit. A highly expressed PbrVPE2 gene was isolated from a pear fruit and named PpyVPE2. The biological function of the newly PpyVPE2 genes was predicted by recombinant protein expression and subcellular localisation.

Tonoplast monosaccharide transporters (TMTs) play important roles in vacuolar sugar accumulation in plants. In this study, six TMT genes (PbTMT1–6) were identified in the Pyrus bretschneideri genome database, and their expression profiles were correlated with soluble sugar contents during the pear (P. bretschneideri cv. Ya Li) fruit development process. Subsequently, PbTMT4 was identified as a strong contributor to fructose, glucose and sucrose accumulation in fructescence of pears. Heterologous expression of PbTMT4, in the hexose transporter‐deficient yeast strain EBY.VW4000, facilitated growth in media containing low levels of glucose, fructose, sucrose or sorbitol. In addition, PbTMT4‐transformed tomato plants flowered and bore fruit significantly earlier than wild‐type (WT) plants, and glucose and fructose levels in mature tomatoes were increased by about 32 and 21% compared with those in WT plants. However, no obvious alterations in sucrose content, plant height and weight per fruit were observed. Finally, subcellular localization experiments in transformed Arabidopsis plants showed that PbTMT4 is localized to tonoplast vesicles of protoplasts. These preliminary results suggest that PbTMT4 participates in vacuolar accumulation of sugars, and thus affects plant growth and development.