Bud dormancy in perennial plants is indispensable to survival over winter and to regrowth and development in the following year. However, the molecular pathways of endo-dormancy induction, maintenance, and release are still unclear, especially in fruit crops. To identify genes with roles in regulating endo-dormancy, 30 MIKC(C)-type MADS-box genes were identified in the pear genome and characterized. The 30 genes were analysed to determine their phylogenetic relationships with homologous genes, genome locations, gene structure, tissue-specific transcript profiles, and transcriptional patterns during flower bud dormancy in 'Suli' pear (Pyrus pyrifolia white pear group). The roles in regulating bud dormancy varied among the MIKC gene family members. Yeast one-hybrid and transient assays showed that PpCBF enhanced PpDAM1 and PpDAM3 transcriptional activity during the induction of dormancy, probably by binding to the C-repeat/DRE binding site, while DAM proteins inhibited the transcriptional activity of PpFT2 during dormancy release. In the small RNA-seq analysis, 185 conserved, 24 less-conserved, and 32 pear-specific miRNAs with distinct expression patterns during bud dormancy were identified. Joint analyses of miRNAs and MIKC genes together with degradome data showed that miR6390 targeted PpDAM transcripts and degraded them to release PpFT2. Our data show that cross-talk among PpCBF, PpDAM, PpFT2, and miR6390 played important roles in regulating endo-dormancy. A model for the molecular mechanism of dormancy transition is proposed: short-term chilling in autumn activates the accumulation of CBF, which directly promotes DAM expression; DAM subsequently inhibits FT expression to induce endo-dormancy, and miR6390 degrades DAM genes to release endo-dormancy.

Plant hormones play a significant role in regulating growth and development during the entire life of a plant, and in response to biotic and abiotic stress. The determination of the concentrations of hormones in flowers and fruit is essential to understanding the role of hormones in the regulation of physiological and biochemical processes associated with flowering and fruit development. Based on high-performance liquid chromatography, coupled with tandem mass spectrometry, we developed and established a novel method to quantify four distinct endogenous hormones through ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/ESI-MS/MS), which achieves higher throughput screening and improved resolution than HPLC or HPLC/ESI-MS/MS. Crude plant extracts were prepared by extraction with extraction solvents I and II, then purified with a Sep-Pak™ C₁₈ reverse-phase extraction cartridge, and subsequently the purified extracts were analyzed by UPLC/ESI-MS/MS. Plant hormones, comprising indole-3-acetic acid, abscisic acid, gibberellin A₄, and trans-zeatin riboside, were separated and quantified in 6 min. The method was simple, rapid, and precise, and was applied for the determination of plant hormones in pear tissue, with recoveries ranging from 70.11% to 89.84% and relative standard deviations ranging from 4.25% to 14.96%. In conclusion, sample preparation, extraction, purification, and UPLC/ESI-MS/MS conditions were optimized for quantitative analysis of four major plant hormones in pear tissue.

Dormancy is an adaptive mechanism that allows temperate deciduous plants to survive unfavorable winter conditions. In the present work, we investigated the possible function of abscisic acid (ABA) on the endodormancy process in pear. The ABA content increased during pear flower bud endodormancy establishment and decreased towards endodormancy release. In total, 39 putative genes related to ABA metabolism and signal transductions were identified from pear genome. During the para- to endodormancy transition, PpNCED-2 and PpNCED-3 had high expression levels, while PpCYP707As expression levels were low. However, during endodormancy, the expression of PpCYP707A-3 sharply increased with increasing cold accumulation. At the same time, the ABA content of pear buds declined, and the percentage of bud breaks rapidly increased. On the other hand, the expression levels of PpPYLs, PpPP2Cs, PpSnRK2s, and PpABI4/ABI5s were also changed during the pear flower bud dormancy cycle. Furthermore, exogenous ABA application to para-dormant buds significantly reduced the bud breaks and accelerated the transition to endodormancy. During the whole treatment time, the expression level of PpPP2C-12 decreased to a greater extent in ABA-treated buds than in control. However, the expression levels of PpSnRK2-1, PpSnRK2-4, and PpABI5-1 were higher in ABA-treated buds. Our results indicated that PpCYP707A-3 and PpNCEDs play pivotal roles on the regulation of endodormancy release, while ABA signal transduction pathway also appears to be involved in the process. The present work provided the basic information about the function of ABA-related genes during pear flower bud dormancy process.

MiR156 is an evolutionarily highly conserved plant micro-RNA (miRNA) that controls an age-dependent flowering pathway. miR156 and its target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes regulate anthocyanin accumulation in plants, but it is unknown whether this process is affected by light. Red Chinese sand pear (Pyrus pyrifolia) fruits exhibit a unique coloration pattern in response to bagging treatments, which makes them appropriate for studying the molecular mechanism underlying light-induced anthocyanin accumulation in fruit. Based on high-throughput miRNA and degradome sequencing data, we determined that two miR156 members (i.e., MIR156a and MIR156ba) were expressed in pear fruit peels, and targeted four SPL genes. Light-responsive elements were detected in the promoter regions of the MIR156a and MIR156ba precursors. We identified 19 miR156-targeted SPL genes using the ‘Suli’ pear (Pyrus pyrifolia Chinese White Pear Group) genome database, of which seven members were miR156 targets. The upregulated expression of anthocyanin biosynthetic and regulatory genes and downregulated expression of PpSPL2, PpSPL5, PpSPL7, PpSPL9, PpSPL10, PpSPL13, PpSPL16, PpSPL17, and PpSPL18 were observed in pear fruits after bags were removed from plants during the anthocyanin accumulation period. Additionally, miR156a/ba abundance increased after bags were removed. Yeast two-hybrid results revealed that the MYB10/bHLH/WD40 protein complex exists in pear, likely to regulate anthocyanin biosynthesis. Additionally, PpSPL10 and PpSPL13 interacted with PyMYB10. We propose the following model for miRNA-mediated light-induced anthocyanin accumulation in pear fruits: miR156 positively regulates light-induced anthocyanin biosynthesis by cleaving PpSPL genes to stabilize the MYB10/bHLH/WD40 protein complex, which is essential for activating the expression of anthocyanin biosynthetic genes.

Fruit ripening is a complex process involving dynamic changes to metabolites and is controlled by multiple factors, including transcription factors (TFs). Several TFs are reportedly essential regulators of tomato (Solanum lycopersicum) fruit ripening. To evaluate the effects of specific TFs on metabolite accumulation during fruit ripening, we combined CRISPR/Cas9-mediated mutagenesis with metabolome and transcriptome analyses to explore regulatory mechanisms. Specifically, we generated various genetically engineered tomato lines that differed regarding metabolite contents and fruit colors. The metabolite and transcript profiles indicated that the selected TFs have distinct functions that control fruit metabolite contents, especially carotenoids and sugars. Moreover, a mutation to ELONGATED HYPOCOTYL5 (HY5) increased tomato fruit fructose and glucose contents by approximately 20% (relative to the wild-type levels). Our in vitro assay showed that HY5 can bind directly to the G-box cis-element in the Sugars Will Eventually be Exported Transporter (SWEET12c) promoter to activate expression, thereby modulating sugar transport. Our findings provide insights into the mechanisms regulating tomato fruit ripening and metabolic networks, providing the theoretical basis for breeding horticultural crops that produce fruit with diverse flavors and colors.

Morella rubra, red bayberry, is an economically important fruit tree in south China. Here, we assembled the first high‐quality genome for both a female and a male individual of red bayberry. The genome size was 313‐Mb, and 90% sequences were assembled into eight pseudo chromosome molecules, with 32 493 predicted genes. By whole‐genome comparison between the female and male and association analysis with sequences of bulked and individual DNA samples from female and male, a 59‐Kb region determining female was identified and located on distal end of pseudochromosome 8, which contains abundant transposable element and seven putative genes, four of them are related to sex floral development. This 59‐Kb female‐specific region was likely to be derived from duplication and rearrangement of paralogous genes and retained non‐recombinant in the female‐specific region. Sex‐specific molecular markers developed from candidate genes co‐segregated with sex in a genetically diverse female and male germplasm. We propose sex determination follow the ZW model of female heterogamety. The genome sequence of red bayberry provides a valuable resource for plant sex chromosome evolution and also provides important insights for molecular biology, genetics and modern breeding in Myricaceae family.