Qingfeng Niu

Dormancy-associated MADS-box genes and microRNAs jointly control dormancy transition in pear (Pyrus pyrifolia white pear group) flower bud

Highlight Short-term chilling in autumn activates the accumulation of CBF, which directly promotes DAM expression. DAMs subsequently inhibit FT2 expression to induce endo-dormancy; miR6390 might degrade DAM genes to release endo-dormancy.

Simultaneous quantitative determination of major plant hormones in pear flowers and fruit by UPLC/ESI-MS/MS

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.

Genome-wide identification, characterization, and expression analysis of the dehydrin gene family in Asian pear (Pyrus pyrifolia)

Dehydrins (DHNs) are a complex family of plant proteins that play an important role in protection of higher plant cells from dehydration and desiccation damage during environmental stresses, such as drought, high salinity, and low temperature. However, information on DHN genes of Asian pear (Pyrus pyrifolia), an economically important fruit crop grown in temperate regions in East Asia, e.g., China and Japan, is limited. To gain insights into this gene family in pear and to elucidate their roles in floral buds under low-temperature conditions, we performed a genome-wide identification, characterization, and expression analysis of DHN genes. Seven PpDHN genes were identified. Sequence alignment analysis of all putative proteins from these genes showed that all of the proteins contained a typical K-domain. These genes were categorized into SKn, YnSKn, YKn, and Kn groups based on gene characterization and phylogenetic relationships. Hierarchical cluster analyses showed that in non-stressed pear, PpDHN genes were expressed in all vegetative tissues except young leaves and shoot tips, in which PpDHN1, PpDHN2, and PpDHN4 were not expressed. Transcript levels of four PpDHN genes increased significantly in floral buds in response to low-temperature treatment, which indicated that they play important roles during stress adaptation. This study provides evidence that the family of pear DHN genes may function in tissue development and stress responses. The data will be valuable for further studies of the functions of DHN genes under different stress conditions in pear.

Response of miR156-SPL Module during the Red Peel Coloration of Bagging-Treated Chinese Sand Pear (Pyrus pyrifolia Nakai)

MicroRNA156 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 miR156 was 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 SPL genes using the “Suli” pear (Pyrus pyrifolia Chinese White Pear Group) genome database, of which seven members were putative 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/g/s/sa abundance increased after bags were removed. Yeast two-hybrid results suggested that PpMYB10, PpbHLH, and PpWD40 could form a protein complex, probably involved in anthocyanin biosynthesis. Additionally, PpSPL10 and PpSPL13 interacted with PpMYB10. The results obtained in this study are helpful in understanding the possible role of miR156 and its target PpSPL genes in regulating light-induced red peel coloration and anthocyanin accumulation in pear.

The possible role of chilling in floral and vegetative bud dormancy release in Pyrus pyrifolia

The role of chilling in bud dormancy release and biochemical changes in different organs were evaluated in stem cuttings of pear (Pyrus pyrifolia) cv. Cuiguan selected at the leaf fall. The cuttings were exposed to 5 °C for 0, 100, 200, 300, 400, 500, 600, and 700 chilling hours (named positive chill units; PCU). A 50 % bud break was observed in floral and vegetative bud cuttings at 300 and 600 PCU, respectively. A mean time to bud break was inversely proportional to the chilling treatment. The low-temperature stimulated starch hydrolysis accompanied with sucrose accumulation in all organs. Sucrose and sorbitol content increased substantially peaking at 100, 400, and 100 PCU in floral buds, vegetative buds, and bark, respectively, thereafter decreased when buds approached chilling satisfaction (300 and 600 PCU for the floral and vegetative buds, respectively), and then increased again up to 700 PCU. Hexoses (glucose and fructose) accumulated constantly in the buds from 0 to 700 PCU. In bark, glucose and fructose content increased up to 400 PCU, and then gradually decreased. Total amylolytic and α-amylase activities increased in all organs, especially in the floral and vegetative buds up to 100 PCU and then decreased in the floral and vegetative buds before increasing again after endo-dormancy release. Invertase activity remained high in the buds during chilling satisfaction possibly because of translocation of sucrose to the buds which functioned as a strong sink. The results suggest that a low availability of hexoses may be the cause of limited bud breaks due to lack of chilling. Chilling satisfaction of the buds may increase the content of soluble sugars and acid invertase activity, and decrease the starch content, which may correlate with improved bud breaks.

Abscisic Acid (ABA ) Promotes the Induction and Maintenance of Pear (Pyrus pyrifolia White Pear Group) Flower Bud Endodormancy

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.

PpHB22, a member of HD-Zip proteins, activates PpDAM1 to regulate bud dormancy transition in ‘Suli’ pear (Pyrus pyrifolia White Pear Group)

Homeodomain-leucine zipper (HD-Zip) proteins, which form one of the largest and most diverse families, regulate many biological processes in plants, including differentiation, flowering, vascular development, and stress signaling. Abscisic acid (ABA) has been proved to be one of the key regulators of bud dormancy and to influence several HD-Zip genes expression. However, the role of HD-Zip genes in regulating bud dormancy remains unclear. We identified 47 pear (P. pyrifolia White Pear Group) HD-Zip genes, which were classified into four subfamilies (HD-Zip I-IV). We further revealed that gene expression levels of some HD-Zip members were closely related to ABA concentrations in flower buds during dormancy transition. Exogenous ABA treatment confirmed that PpHB22 and several other HD-Zip genes responded to ABA. Yeast one-hybrid and dual luciferase assay results combining subcellular localization showed that PpHB22 was present in nucleus and directly induced PpDAM1 (dormancy associated MADS-box 1) expression. Thus, PpHB22 is a negative regulator of plant growth associated with the ABA response pathway and functions upstream of PpDAM1. These findings enrich our understanding of the function of HD-Zip genes related to the bud dormancy transition.