Genome-Wide Identification of GAST Family Members and Their Potential Roles in Epicotyl Dormancy in Chinese Cork Oak (Quercus variabilis).

Chinese cork oak (Quercus variabilis Blume) is a widespread tree species with high economic and ecological values. Chinese cork oak exhibits epicotyl dormancy, causing emergence heterogeneity and affecting the quality of seedling cultivation. Gibberellic acid-stimulated transcript (GAST) is a plant-specific protein family that plays a crucial regulatory role in plant growth, development, and seed germination. However, their evolution in Chinese cork oak and roles in epicotyl dormancy are still unclear. Here, a genome-wide identification of the GAST gene family was conducted in Chinese cork oak. Ten QvGAST genes were identified, and nine of them were expressed in seed. The physicochemical properties and promoter cis-acting elements of the selected Chinese cork oak GAST family genes indicated that the cis-acting elements in the GAST promoter are involved in plant development, hormone response, and stress response. Germinated seeds were subjected to gibberellins (GAs), abscisic acid (ABA), and fluridone treatments to show their response during epicotyl dormancy release. Significant changes in the expression of certain QvGAST genes were observed under different hormone treatments. QvGAST1, QvGAST2, QvGAST3, and QvGAST6 exhibited upregulation in response to gibberellin. QvGAST2 was markedly upregulated during the release of epicotyl dormancy in response to GA. These findings suggested that QvGAST2 might play an important role in epicotyl dormancy release. This study provides a basis for further analysis of the mechanisms underlying the alleviation of epicotyl dormancy in Chinese cork oak by QvGASTs genes.

Catechin promotes the germination of Pistacia chinensis seeds via GA biosynthesis.

BACKGROUND AND AIMS: Chinese pistachio (Pistacia chinensis Bunge), an important horticultural plant species, holds great ornamental value with beautiful leaves and fruits. Seedling propagation of this tree species is restricted by its erratic seed germination, however, the germination mechanism is ambiguous yet. The aim of this study was to figure out the germination mechanism from the novel perspective based on the multi-omics data. METHODS: The multi-omics technique combined with hormone content measurement was first applied in seed germination of Chinese pistachio. KEY RESULTS: Due to the great accumulation during seed germination, catechin stood out from the identified metabolites by broadly targeted metabolomic analysis. Exogenous catechin of 10 mg/L significantly improved the germination of Chinese pistachio seeds. An interesting result of hormone analysis showed that the improving effect of catechin could be attributed to increase of the gibberellic acid 3 (GA3) content rather than decrease of the abscisic acid (ABA) content before germination. The paclobutrazol (PAC, a GA biosynthesis inhibitor) and PAC + catechin treatments also showed that the promoting effect of catechin on seed germination depends on GA biosynthesis. Transcriptome analysis and qRT‒PCR further revealed that catechin induced the expression of PcGA20ox5 to activate GA biosynthesis. Several transcription factors were induced by catechin and GA treatments, such as TCP, bZIP and C3H, which may play an important regulatory role in GA biosynthesis in a catechin-mediated way. CONCLUSIONS: Catechin promotes seed germination via GA biosynthesis in Chinese pistachios. This study proposes a novel mechanism by which catechin promotes seed germination via the GA pathway, which provides new insight into a comprehensive understanding of seed dormancy and germination.

Genomic basis of the distinct biosynthesis of ß-glucogallin, a biochemical marker for hydrolyzable tannin production, in three oak species.

Hydrolyzable tannins (HTs), predominant polyphenols in oaks, are widely used in grape wine aging, feed additives, and human healthcare. However, the limited availability of a high-quality reference genome of oaks greatly hampered the recognition of the mechanism of HT biosynthesis. Here, high-quality reference genomes of three Asian oak species (Quercus variabilis, Quercus aliena, and Quercus dentata) that have different HT contents were generated. Multi-omics studies were carried out to identify key genes regulating HT biosynthesis. In vitro enzyme activity assay was also conducted. Dual-luciferase and yeast one-hybrid assays were used to reveal the transcriptional regulation. Our results revealed that ß-glucogallin was a biochemical marker for HT production in the cupules of the three Asian oaks. UGT84A13 was confirmed as the key enzyme for ß-glucogallin biosynthesis. The differential expression of UGT84A13, rather than enzyme activity, was the main reason for different ß-glucogallin and HT accumulation. Notably, sequence variations in UGT84A13 promoters led to different trans-activating activities of WRKY32/59, explaining the different expression patterns of UGT84A13 among the three species. Our findings provide three high-quality new reference genomes for oak trees and give new insights into different transcriptional regulation for understanding ß-glucogallin and HT biosynthesis in closely related oak species.

PpyABF3 recruits the COMPASS-like complex to regulate bud dormancy maintenance via integrating ABA signaling and GA catabolism.

Bud dormancy is essential for perennial trees that survive the cold winters and to flower on time in the following spring. Histone modifications have been reported to be involved in the control of the dormancy cycle and DAM/SVPs are considered targets. However, how the histone modification marks are added to the specific gene loci during bud dormancy cycle is still unknown. Using yeast-two hybrid library screening and co-immunoprecipitation assays, we found that PpyABF3, a key protein regulating bud dormancy, recruits Complex of Proteins Associated with Set1-like complex via interacting with PpyWDR5a, which increases the H3K4me3 deposition at DAM4 locus. Chromatin immunoprecipitation-quantitative polymerase chain reaction showed that PpyGA2OX1 was downstream gene of PpyABF3 and it was also activated by H3K4me3 deposition. Silencing of GA2OX1 in pear calli and pear buds resulted in a similar phenotype with silencing of ABF3. Furthermore, overexpression of PpyWDR5a increased H3K4me3 levels at DAM4 and GA2OX1 loci and inhibited the growth of pear calli, whereas silencing of PpyWDR5a in pear buds resulted in a higher bud-break percentage. Our findings provide new insights into how H3K4me3 marks are added to dormancy-related genes in perennial woody plants and reveal a novel mechanism by which ABF3 integrates abscisic acid signaling and gibberellic acid catabolism during bud dormancy maintenance.

Blue Light Simultaneously Induces Peel Anthocyanin Biosynthesis and Flesh Carotenoid/Sucrose Biosynthesis in Mango Fruit.

Previous studies focused on the effects of light on fruit appearance, especially the peel color. However, the effect of light on fruit internal quality and the underlying mechanisms are unclear. In this study, we analyzed the effects of blue light on the appearance and internal quality of mango fruit (Mangifera indica L.). Blue light simultaneously induced peel anthocyanin and flesh sucrose/carotenoid biosynthesis in mango fruit. Analyses of co-expression networks and gene expression trends in mango fruit peel and flesh identified candidate genes, including transcription factor genes, involved in blue light-regulated anthocyanin, carotenoid, and sucrose biosynthesis pathways. Key blue light signaling-related genes (MiCRY and MiHY5) and blue light-triggered phytohormones were involved in these pathways. Additionally, there were common and tissue-specific pathways for the blue light-promoted accumulation of anthocyanins, carotenoids, and sucrose. Our results provide new insights into the regulatory effects of light on the appearance and internal quality of mango fruit.

Early defoliation induces auxin redistribution, promoting paradormancy release in pear buds.

Paradormancy of fruit trees occurs in summer and autumn when signals from adjacent organs stimulate buds to develop slowly. This stage has received less attention that the other stages of dormancy, and the underlying mechanism remains uncharacterized. Early defoliation in late summer and early autumn is usually followed by out-of-season blooming in pear (Pyrus spp.), which substantially decreases the number of buds the following spring and negatively affects fruit production. This early bud flush is an example of paradormancy release. Here, we determined that flower bud auxin content is stable after defoliation; however, polar distribution of the pear (Pyrus pyrifolia) PIN-FORMED auxin efflux carrier 1b (PpyPIN1b) implied that auxin tends to be exported from buds. Transcriptome analysis of floral buds after artificial defoliation revealed changes in auxin metabolism, transport, and signal transduction pathways. Exogenous application of a high concentration of the auxin analog 1-naphthaleneacetic acid (300 mg/L) suppressed PpyPIN1b expression and its protein accumulation in the cell membrane, likely leading to decreased auxin efflux from buds, which hindered flower bud sprouting. Furthermore, carbohydrates and additional hormones also influenced out-of-season flowering. Our results indicate that defoliation-induced auxin efflux from buds accelerates bud paradormancy release. This differs from release of apical-dominance-related lateral bud paradormancy after the apex is removed. Our findings and proposed model further elucidate the mechanism underlying paradormancy and will help researchers to develop methods for inhibiting early defoliation-induced out-of-season bud sprouting.

Anthocyanin Biosynthesis Associated with Natural Variation in Autumn Leaf Coloration in Quercus aliena Accessions.

Quercus aliena is an economically important tree species and one of the dominant native oak species in China. Although its leaves typically turn yellow in autumn, we observed natural variants with red leaves. It is important to understand the mechanisms involved in leaf color variation in this species. Therefore, we compared a Q. aliena tree with yellow leaves and three variants with red leaves at different stages of senescence in order to determine the causes of natural variation. We found that the accumulation of anthocyanins such as cyanidin 3-O-glucoside and cyanidin 3-O-sambubiglycoside had a significant effect on leaf coloration. Gene expression analysis showed upregulation of almost all genes encoding enzymes involved in anthocyanin synthesis in the red-leaved variants during the early and main discoloration stages of senescence. These findings are consistent with the accumulation of anthocyanin in red variants. Furthermore, the variants showed significantly higher expression of transcription factors associated with anthocyanin synthesis, such as those encoded by genes QaMYB1 and QaMYB3. Our findings provide new insights into the physiological and molecular mechanisms involved in autumn leaf coloration in Q. aliena, as well as provide genetic resources for further development and cultivation of valuable ornamental variants of this species.

Genome-wide identification of Pistacia R2R3-MYB gene family and function characterization of PcMYB113 during autumn leaf coloration in Pistacia chinensis.

Pistacia chinensis is known for its biodiesel production. Several varieties of this plant have leaves that produce anthocyanin, which is responsible for their reddish coloration in autumn. This reddish hue is what makes them useful as ornamental plants. However, the mechanism of anthocyanin accumulation during autumn leaf coloration remains unclear. R2R3-MYB proteins reportedly regulated anthocyanin biosynthesis in many plant species. Here, we performed a genome-wide analysis and expression profiles of R2R3-MYB transcription factor in Pistacia. A total of 158 R2R3-MYB proteins were identified and grouped into 32 clades. Combining the data from RNA-seq and qRT-PCR, one key gene, EVM0016534, was screened and identified to have the highest correlation with anthocyanin accumulation. It was named PcMYB113 due to its sequence similarity to AtMYB113 and it could bind to the promoter of PcF3H. Furthermore, ectopic expression of PcMYB113 in Arabidopsis promoted the accumulation of anthocyanin in the seed coat, cotyledon, and mature leaves, thus confirming the function of PcMYB113 in anthocyanin biosynthesis. In addition, PcMYB113 had a specifically higher expression in senesced red leaves than in mature green leaves and young red leaves in P. chinensis, thereby suggesting the potential role of PcMYB113 in promoting anthocyanin biosynthesis during autumn leaf coloration. These findings enrich our understanding of the function of R2R3-MYB genes in anthocyanin biosynthesis and autumn leaf coloration.

High-quality genome assembly of 'Cuiguan' pear (Pyrus pyrifolia) as a reference genome for identifying regulatory genes and epigenetic modifications responsible for bud dormancy.

Dormancy-associated MADS-box (DAM) genes serve as crucial regulators of the endodormancy cycle in rosaceous plants. Although pear DAM genes have been identified previously, the lack of a high-quality reference genome and techniques to study gene function have prevented accurate genome-wide analysis and functional verification of such genes. Additionally, the contribution of other genes to the regulation of endodormancy release remains poorly understood. In this study, a high-quality genome assembly for 'Cuiguan' pear (Pyrus pyrifolia), which is a leading cultivar with a low chilling requirement cultivated in China, was constructed using PacBio and Hi-C technologies. Using this genome sequence, we revealed that pear DAM genes were tandemly clustered on Chr8 and Chr15 and were differentially expressed in the buds between 'Cuiguan' and the high-chilling-requirement cultivar 'Suli' during the dormancy cycle. Using a virus-induced gene silencing system, we determined the repressive effects of DAM genes on bud break. Several novel genes potentially involved in the regulation of endodormancy release were identified by RNA sequencing and H3K4me3 chromatin immunoprecipitation sequencing analyses of 'Suli' buds during artificial chilling using the new reference genome. Our findings enrich the knowledge of the regulatory mechanism underlying endodormancy release and chilling requirements and provide a foundation for the practical regulation of dormancy release in fruit trees as an adaptation to climate change.

Alternative splicing of the dormancy-associated MADS-box transcription factor gene PpDAM1 is associated with flower bud dormancy in 'Dangshansu' pear (Pyrus pyrifolia white pear group).

Alternative splicing (AS) plays a crucial role in plant growth, development and response to various environmental changes. However, whether alternative splicing of MADS-box transcription factors contributes to the flower bud dormancy process in fruit trees still remains unknown. In this work, the AS profile of genes in the dormant flower buds of 'Dangshansu' pear tree were examined. A total number of 3661 alternatively spliced genes were identified, and three mRNA isoforms of the dormancy associated MADS box (DAM) gene, PpDAM1, derived by alternative splicing, designated as PpDAM1.1, PpDAM1.2 and PpDAM1.3, were characterized. Bimolecular fluorescence complementation (BiFC) analysis indicated that AS of PpDAM1 didn't affect the nucleus localization and homo-/heterodimerization of PpDAM1.1, PpDAM1.2 and PpDAM1.3 proteins, but disturbed the translocation of PpDAM1.1/PpDAM1.1, PpDAM1.3/PpDAM1.3, PpDAM1.1/PpDAM1.3, and PpDAM1.2/PpDAM1.3 dimers to the nucleus. Constitutive expression of PpDAM1.2, but not PpDAM1.1 and PpDAM1.3, in Arabidopsis retarded the growth and development of transgenic plants. Further comparative expression analyses of PpDAM1.1, PpDAM1.2 and PpDAM1.3 in the flower buds of 'Dangshansu' and a less dormant pear cultivar, 'Cuiguan', exhibited that the expression of all the three isoforms in 'Dangshansu' were significantly higher than in 'Cuiguan', especially PpDAM1.2, which showed a predominantly higher expression than PpDAM1.1 and PpDAM1.3 in both cultivars. Our results suggest that alternative splicing of PpDAM1 could play a crucial role in pear flower bud dormancy process.

Bud endodormancy in deciduous fruit trees: advances and prospects.

Bud endodormancy is a complex physiological process that is indispensable for the survival, growth, and development of deciduous perennial plants. The timely release of endodormancy is essential for flowering and fruit production of deciduous fruit trees. A better understanding of the mechanism of endodormancy will be of great help in the artificial regulation of endodormancy to cope with climate change and in creating new cultivars with different chilling requirements. Studies in poplar have clarified the mechanism of vegetative bud endodormancy, but the endodormancy of floral buds in fruit trees needs further study. In this review, we focus on the molecular regulation of endodormancy induction, maintenance and release in floral buds of deciduous fruit trees. We also describe recent advances in quantitative trait loci analysis of chilling requirements in fruit trees. We discuss phytohormones, epigenetic regulation, and the detailed molecular network controlling endodormancy, centered on SHORT VEGETATIVE PHASE (SVP) and Dormancy-associated MADS-box (DAM) genes during endodormancy maintenance and release. Combining previous studies and our observations, we propose a regulatory model for bud endodormancy and offer some perspectives for the future.

MrTPS3 and MrTPS20 Are Responsible for ß-Caryophyllene and α-Pinene Production, Respectively, in Red Bayberry (Morella rubra).

Red bayberry is a sweet, tart fruit native to China and grown widely in the south. The key organic compounds forming the distinctive aroma in red bayberry, are terpenoids, mainly ß-caryophyllene and α-pinene. However, the key genes responsible for different terpenoids are still unknown. Here, transcriptome analysis on samples from four cultivars, during fruit development, with different terpenoid production, provided candidate genes for volatile organic compound (VOC) production. Terpene synthases (TPS) are key enzymes regulating terpenoid biosynthesis, and 34 TPS family members were identified in the red bayberry genome. MrTPS3 in chromosome 2 and MrTPS20 in chromosome 7 were identified as key genes regulating ß-caryophyllene and α-pinene synthesis, respectively, by qRT-PCR. Subcellular localization and enzyme activity assay showed that MrTPS3 was responsible for ß-caryophyllene (sesquiterpenes) production and MrTPS20 for α-pinene (monoterpenes). Notably, one amino acid substitution between dark color cultivars and light color cultivars resulted in the loss of function of MrTPS3, causing the different ß-caryophyllene production. Our results lay the foundation to study volatile organic compounds (VOCs) in red bayberry and provide potential genes for molecular breeding.

Construction of an anchoring SSR marker genetic linkage map and detection of a sex-linked region in two dioecious populations of red bayberry.

Red bayberry (Morella rubra) is an evergreen fruit tree found in southern China whose whole-genome sequence has recently been published. We updated the linkage map of the species by adding 118 SSR markers and the female-specific marker MrFT2_BD-SEX. The integrated map included eight linkage groups and spanned 491 cM. Eleven sex-associated markers were identified, six of which were located in linkage group 8, in agreement with the previously reported location of the sex-determining region. The MrFT2_BD-SEX marker was genotyped in 203 cultivated accessions. Among the females of the accessions, we found two female-specific alleles, designated W-b (151 bp) and W-d (129 bp). We previously found that 'Dongkui', a female cultivar, could produce viable pollen (we refer to such plants 'Dongkui-male') and serve as the paternal parent in crosses. The genotypes of the MrFT2_BD-SEX marker were W-b/Z in 'Biqi' and W-d/Z in 'Dongkui-male'. The progeny of a cross between these parents produced a 3:1 female (W-) to male (ZZ) ratio and the expected 1:1:1:1 ratio of W-b/W-d: W-b/Z: W-d/Z: Z/Z. In addition, the flowering and fruiting phenotypes of all the F1 progeny fit their genotypes. Our results confirm the existence of ZW sex determination and show that the female phenotype is controlled by a single dominant locus (W) in a small genomic region (59 kb and less than 3.3 cM). Furthermore, we have produced a homozygous "super female" (WW) that should produce all-female offspring in the F2 generation, providing a foundation for commercial use and presenting great potential for use in modern breeding programs.

ABA-responsive ABRE-BINDING FACTOR3 activates DAM3 expression to promote bud dormancy in Asian pear.

Bud dormancy is indispensable for the survival of perennial plants in cold winters. Abscisic acid (ABA) has essential functions influencing the endo-dormancy status. Dormancy-associated MADS-box/SHORT VEGETATIVE PHASE-like genes function downstream of the ABA signalling pathway to regulate bud dormancy. However, the regulation of DAM/SVP expression remains largely uncharacterized. In this study, we confirmed that endo-dormancy maintenance and PpyDAM3 expression are controlled by the ABA content in pear (Pyrus pyrifolia) buds. The expression of pear ABRE-BINDING FACTOR3 (PpyABF3) was positively correlated with PpyDAM3 expression. Furthermore, PpyABF3 directly bound to the second ABRE in the PpyDAM3 promoter to activate its expression. Interestingly, both PpyABF3 and PpyDAM3 repressed the cell division and growth of transgenic pear calli. Another ABA-induced ABF protein, PpyABF2, physically interacted with PpyABF3 and disrupted the activation of the PpyDAM3 promoter by PpyABF3, indicating DAM expression was precisely controlled. Additionally, our results suggested that the differences in the PpyDAM3 promoter in two pear cultivars might be responsible for the diversity in the chilling requirements. In summary, our data clarify the finely tuned regulatory mechanism underlying the effect of ABA on DAM gene expression and provide new insights into ABA-related bud dormancy regulation.

Two B-box proteins, PpBBX18 and PpBBX21, antagonistically regulate anthocyanin biosynthesis via competitive association with Pyrus pyrifolia ELONGATED HYPOCOTYL 5 in the peel of pear fruit.

Light is indispensable for the accumulation of anthocyanin in the peel of red pear fruit (Pyrus pyrifolia Nakai). ELONGATED HYPOCOTYL 5 (HY5) is considered to be a critical regulator for induction of anthocyanin biosynthesis, but detailed characterization of its regulatory mechanism is needed. In this study, multiple genetic and biochemical approaches were applied to identify the roles of P. pyrifolia HY5 (PpHY5) and two B-box (BBX) proteins, PpBBX18 and PpBBX21, in the transcriptional regulation of PpMYB10. The functions of the two BBX proteins were analyzed in overexpression lines using pear calli-based approaches. On its own PpHY5 was unable to activate downstream genes. The two BBX proteins, PpBBX18 and PpBBX21, physically interacted with PpHY5 and antagonistically regulated anthocyanin biosynthesis in Arabidopsis and pear. PpBBX18 formed a heterodimer with PpHY5 via two B-box domains, in which PpHY5 bound to the G-box motif of PpMYB10 and PpBBX18 provided the trans-acting activity, thus inducing transcription of PpMYB10. PpBBX21 interacted with PpHY5 and PpBBX18 and hampered formation of the PpHY5-PpBBX18 active transcription activator complex, and subsequently repressed anthocyanin biosynthesis. The present results demonstrate the fine-tuned regulation of anthocyanin biosynthesis via transcriptional regulation of PpMYB10 by PpHY5-associated proteins and provide insights into light-induced anthocyanin biosynthesis.

Phylogenetic, Molecular, and Functional Characterization of PpyCBF Proteins in Asian Pears (Pyrus pyrifolia).

C-repeat binding factor/dehydration-responsive element (CBF/DRE) transcription factors (TFs) participate in a variety of adaptive mechanisms, and are involved in molecular signaling and abiotic stress tolerance in plants. In pear (Pyrus pyrifolia) and other rosaceous crops, the independent evolution of CBF subfamily members requires investigation to understand the possible divergent functions of these proteins. In this study, phylogenetic analysis divided six PpyCBFs from the Asian pear genome into three clades/subtypes, and collinearity and phylogenetic analyses suggested that PpyCBF3 was the mother CBF. All PpyCBFs were found to be highly expressed in response to low temperature, salt, drought, and abscisic acid (ABA) as well as bud endodormancy, similar to PpyCORs (PpyCOR47, PpyCOR15A, PpyRD29A, and PpyKIN). Transcript levels of clade II PpyCBFs during low temperature and ABA treatments were higher than those of clades I and III. Ectopic expression of PpyCBF2 and PpyCBF3 in Arabidopsis enhanced its tolerance against abiotic stresses, especially to low temperature in the first case and salt and drought stresses in the latter, and resulted in lower reactive oxygen species (ROS) and antioxidant gene activities compared with the wild type. The increased expression of endogenous ABA-dependent and -independent genes during normal conditions in PpyCBF2- and PpyCBF3-overexpressing Arabidopsis lines suggested that PpyCBFs were involved in both ABA-dependent and -independent pathways. All PpyCBFs, especially the mother CBF, had high transactivation activities with 6XCCGAC binding elements. Luciferase and Y1H assays revealed the existence of phylogenetically and promoter-dependent conserved CBF-COR cascades in the pear. The presence of a previously identified CCGA binding site, combined with the results of mutagenesis of the CGACA binding site of the PpyCOR15A promoter, indicated that CGA was a core binding element of PpyCBFs. In conclusion, PpyCBF TFs might operate redundantly via both ABA-dependent and -independent pathways, and are strongly linked to abiotic stress signaling and responses in the Asian pear.

BBX16, a B-box protein, positively regulates light-induced anthocyanin accumulation by activating MYB10 in red pear.

The red coloration of pear (Pyrus pyrifolia) results from anthocyanin accumulation in the fruit peel. Light is required for anthocyanin biosynthesis in pear. A pear homolog of Arabidopsis thaliana BBX22, PpBBX16, was differentially expressed after fruits were removed from bags and may be involved in anthocyanin biosynthesis. Here, the expression and function of PpBBX16 were analysed. PpBBX16's expression was highly induced by white-light irradiation, as was anthocyanin accumulation. PpBBX16's ectopic expression in Arabidopsis increased anthocyanin biosynthesis in the hypocotyls and tops of flower stalks. PpBBX16 was localized in the nucleus and showed trans-activity in yeast cells. Although PpBBX16 could not directly bind to the promoter of PpMYB10 or PpCHS in yeast one-hybrid assays, the complex of PpBBX16/PpHY5 strongly trans-activated anthocyanin pathway genes in tobacco. PpBBX16's overexpression in pear calli enhanced the red coloration during light treatments. Additionally, PpBBX16's transient overexpression in pear peel increased anthocyanin accumulation, while virus-induced gene silencing of PpBBX16 decreased anthocyanin accumulation. The expression patterns of pear BBX family members were analysed, and six additional BBX genes, which were differentially expressed during light-induced anthocyanin biosynthesis, were identified. Thus, PpBBX16 is a positive regulator of light-induced anthocyanin accumulation, but it could not directly induce the expression of the anthocyanin biosynthesis-related genes by itself but needed PpHY5 to gain full function. Our work uncovered regulatory modes for PpBBX16 and suggested the potential functions of other pear BBX genes in the regulation of anthocyanin accumulation, thereby providing target genes for further studies on anthocyanin biosynthesis.

PpCBFs selectively regulate PpDAMs and contribute to the pear bud endodormancy process.

KEY MESSAGE: PpCBF2 directly binds to the promoters of PpCBF3 and PpCBF4 to activate their expressions and selectively regulates PpDAMs during the leaf bud endodormancy process of 'Wonhwang' pear (Pyrus pyrifolia). Endodormancy is critical for temperate plant survival under freezing winter conditions, and low temperature is a vital environmental factor in endodormancy regulation. A C-repeat binding factor (CBF) has been found to regulate important DAM transcription factors during endodormancy in pear (Pyrus pyrifolia). In this study, we analyzed the regulation of pear DAM genes by CBFs in further detail. Four CBF and three DAM genes were identified in the pear cultivar 'Wonhwang'. Under natural conditions, PpDAM1 expression decreased from the start of chilling accumulation, while the other two DAM and three CBF genes peaked during endodormancy release. Under chilling treatment, the expressions of PpDAM1, PpDAM2 and PpCBF1 genes were similar to those under natural conditions. Different biochemical methods revealed that PpCBF2/4 can bind to the promoter of PpDAM1 and activate its expression and that PpCBF1/4 can activate PpDAM3. Interestingly, we found that PpCBF2 can activate PpCBF3/4 transcription by directly binding to their promoters. The ICE-CBF regulon is conserved in some plants; three ICE genes were identified in pear, but their expressions did not obviously change under natural and artificial chilling conditions. On the contrary, the selective transcriptional induction of PpCBFs by PpICE1s was observed in a dual-luciferase assay. Considering all these results, we propose that the PpCBF1-PpDAM2 regulon mainly responds to low temperature during endodormancy regulation, with further post-translational regulation by PpICE3. Our results provide basic information on CBF genes functional redundancy and differentiation and demonstrate that the CBF-DAM signaling pathway is involved in the pear bud endodormancy process.

Genome wide identification and predicted functional analyses of NAC transcription factors in Asian pears.

BACKGROUND: NAC proteins contribute to diverse plant developmental processes as well as tolerances to biotic and abiotic stresses. The pear genome had been decoded and provided the basis for the genome-wide analysis to find the evolution, duplication, gene structures and predicted functions of PpNAC transcription factors. RESULTS: A total of 185 PpNAC genes were found in pear, of which 148 were mapped on chromosomes while 37 were on unanchored scaffolds. Phylogeny split the NAC genes into 6 clades (Group1- Group6) with their sub clades (~ subgroup A to subgroup H) and each group displayed common motifs with no/minor change. The numbers of exons in each group varied from 1 to 12 with an average of 3 while 44 pairs from all groups showed their duplication events. qPCR and RNA-Seq data analyses in different pear cultivars/species revealed some predicted functions of PpNAC genes i.e. PpNACs 37, 61, 70 (2A), 53, 151(2D), 10, 92, 130 and 154 (3D) were potentially involved in bud endodormancy, PpNACs 61, 70 (2A), 172, 176 and 23 (4E) were associated with fruit pigmentations in blue light, PpNACs 127 (1E), 46 (1G) and 56 (5A) might be related to early, middle and late fruit developments respectively. Besides, all genes from subgroups 2D and 3D were found to be related with abiotic stress (cold, salt and drought) tolerances by targeting the stress responsive genes in pear. CONCLUSIONS: The present genome-wide analysis provided valuable information for understanding the classification, motif and gene structure, evolution and predicted functions of NAC gene family in pear as well as in higher plants. NAC TFs play diverse and multifunctional roles in biotic and abiotic stresses, growth and development and fruit ripening and pigmentation through multiple pathways in pear.

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.