An Efficient Chromatin Immunoprecipitation (ChIP) Protocol for Studying Histone Modifications in Peach Reproductive Tissues

BackgroundPerennial fruit trees display a perennial growth behaviour characterized by an annual cycling between growth and dormancy, with complex physiological features. Rosaceae fruit trees represent excellent models for studying not only the fruit growth/patterning, but also the progression of the reproductive cycle depending upon the impact of climate conditions. In addition, the current development of high‐throughput technologies is starting to have an important impact on Rosaceae tree research for investigating genome structure and function as well as (epi)genetic mechanisms involved in important developmental and environmental response processes during fruit tree growth. Among the epigenetic mechanisms, chromatin remodelling mediated by both histone modifications and other chromatin-related processes play a crucial role in gene modulation, controlling gene expression process. A very useful technique to investigate the chromatin states in plants and their dynamics is chromatin immunoprecipitation (ChIP), generally applied for studies on chromatin states and enrichment in post transcriptional modifications (PTMs) of histone proteins. Results Because peach is a model in Rosaceae family for studies in bud formation, dormancy and organ differentiation for climacteric fruits, in our work, we primarily established specific protocols for chromatin extraction and immunoprecipitation in reproductive tissues of peach Prunus persica. Subsequently focused our investigations on the role of two chromatin marks, namely trimethylation of histone H3 at lysine in position 4 (H3K4me3) and trimethylation of histone H3 at lysine 27 (H3K27me3) on modulating specific gene expression. Bud dormancy and fruit growth were investigated in a nectarine genotype called Fantasia as a model system. ConclusionsHere we presented general strategies to systematically optimize ChIP protocols for buds and mesocarp tissues and analyzed the correlation between gene expression and chromatin mark enrichment/depletion. Confirming like histone modifications are implicated in regulating bud dormancy progression and the core ripening genes.

Regulatory circuits involving bud dormancy factor PpeDAM6

DORMANCY-ASSOCIATED MADS-BOX (DAM) genes have recently emerged as key potential regulators of the dormancy cycle and climate adaptation in perennial species. Particularly, PpeDAM6 has been proposed to act as a major repressor of bud dormancy release and bud break in peach (Prunus persica). PpeDAM6 expression is downregulated concomitantly with the perception of a given genotype-dependent accumulation of winter chilling time, and the coincident enrichment in H3K27me3 chromatin modification at a specific genomic region. We have identified three peach BASIC PENTACYSTEINE PROTEINs (PpeBPCs) interacting with two GA-repeat motifs present in this H3K27me3-enriched region. Moreover, PpeBPC1 represses PpeDAM6 promoter activity by transient expression experiments. On the other hand, the heterologous overexpression of PpeDAM6 in European plum (Prunus domestica) alters plant vegetative growth, resulting in dwarf plants tending toward shoot meristem collapse. These alterations in vegetative growth of transgenic lines associate with impaired hormone homeostasis due to the modulation of genes involved in jasmonic acid, cytokinin, abscisic acid, and gibberellin pathways, and the downregulation of shoot meristem factors, specifically in transgenic leaf and apical tissues. The expression of many of these genes is also modified in flower buds of peach concomitantly with PpeDAM6 downregulation, which suggests a role of hormone homeostasis mechanisms in PpeDAM6-dependent maintenance of floral bud dormancy and growth repression.

Transcriptomic Analysis Reveals the Positive Role of Abscisic Acid in Endodormancy Maintenance of Leaf Buds of Magnolia wufengensis

Magnolia wufengensis (Magnoliaceae) is a deciduous landscape species, known for its ornamental value with uniquely shaped and coloured tepals. The species has been introduced to many cities in south China, but low temperatures limit the expansion of this species in cold regions. Bud dormancy is critical for plants to survive in cold environments during the winter. In this study, we performed transcriptomic analysis of leaf buds using RNA sequencing and compared their gene expression during endodormancy, endodormancy release, and ecodormancy. A total of 187,406 unigenes were generated with an average length of 621.82 bp (N50 = 895 bp). In the transcriptomic analysis, differentially expressed genes involved in metabolism and signal transduction of hormones especially abscisic acid (ABA) were substantially annotated during dormancy transition. Our results showed that ABA at a concentration of 100 μM promoted dormancy maintenance in buds of M. wufengensis. Furthermore, the expression of genes related to ABA biosynthesis, catabolism, and signalling pathway was analysed by qPCR. We found that the expression of MwCYP707A-1-2 was consistent with ABA content and the dormancy transition phase, indicating that MwCYP707A-1-2 played a role in endodormancy release. In addition, the upregulation of MwCBF1 during dormancy release highlighted the enhancement of cold resistance. This study provides new insights into the cold tolerance of M. wufengensis in the winter from bud dormancy based on RNA-sequencing and offers fundamental data for further research on breeding improvement of M. wufengensis.

ABA and Bud Dormancy in Perennials: Current Knowledge and Future Perspective

Bud dormancy is an evolved trait that confers adaptation to harsh environments, and affects flower differentiation, crop yield and vegetative growth in perennials. ABA is a stress hormone and a major regulator of dormancy. Although the physiology of bud dormancy is complex, several advancements have been achieved in this field recently by using genetics, omics and bioinformatics methods. Here, we review the current knowledge on the role of ABA and environmental signals, as well as the interplay of other hormones and sucrose, in the regulation of this process. We also discuss emerging potential mechanisms in this physiological process, including epigenetic regulation.

Endodormancy Release Can Be Modulated by the GA4-GID1c-DELLA2 Module in Peach Leaf Buds

Gibberellin (GA) plays a key role in the release of bud dormancy and the GA receptor GID1 (GIBBERELLIN INSENSITIVE DWARF1) and DELLA protein are the GA signaling parts, but the molecular mechanism of GA-GID1-DELLA module regulating leaf bud dormancy in peach (Prunus persica) is still not very clear. In this study, we isolated and characterized the GID1 gene PpGID1c from the peach cultivar “Zhong you No.4.” Overexpressing PpGID1c in Arabidopsis promoted seed germination, which indicated that PpGID1c has an important function in dormancy. The expression level of PpGID1c in peach leaf buds during endodormancy release was higher than that during ecodormancy and was positively correlated with GA4 levels. Our study also found that GA4 had the most obvious effect on promoting the bud break, indicating that GA4 may be the key gibberellin to promoting peach leaf bud endodormancy release. Moreover, a quantitative real-time PCR (qRT-PCR) found that GA4 could increase the expression of the gibberellin signaling gene PpDELLA2. A yeast two-hybrid (Y2H) assay suggested that the PpGID1c interaction with the PpDELLA1 protein was not dependent on gibberellin, while the PpGID1c interaction with PpDELLA2 required GA4 or another gibberellin. These findings suggested that the GA4-GID1c-DELLA2 module regulates peach leaf bud endodormancy release, with this finding significantly enhancing our comprehensive understanding of bud endodormancy release and revealing a new mechanism for regulating leaf bud endodormancy release in peach.

Analysis of Evolution, Expression and Genetic Transformation of TCP Transcription Factors in Blueberry Reveal That VcTCP18 Negatively Regulates the Release of Flower Bud Dormancy

Plant-specific TEOSINTE BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTORS (TCP) transcription factors have versatile functions in plant growth, development and response to environmental stress. Despite blueberry’s value as an important fruit crop, the TCP gene family has not been systematically studied in this plant. The current study identified blueberry TCP genes (VcTCPs) using genomic data from the tetraploid blueberry variety ‘Draper’; a total of 62 genes were obtained. Using multiple sequence alignment, conserved motif, and gene structure analyses, family members were divided into two subfamilies, of which class II was further divided into two subclasses, CIN and TB1. Synteny analysis showed that genome-wide or segment-based replication played an important role in the expansion of the blueberry TCP gene family. The expression patterns of VcTCP genes during fruit development, flower bud dormancy release, hormone treatment, and tissue-specific expression were analyzed using RNA-seq and qRT-PCR. The results showed that the TB1 subclass members exhibited a certain level of expression in the shoot, leaf, and bud; these genes were not expressed during fruit development, but transcript levels decreased uniformly during the release of flower bud dormancy by low-temperature accumulation. The further transgenic experiments showed the overexpression of VcTCP18 in Arabidopsis significantly decreased the seed germination rate in contrast to the wild type. The bud dormancy phenomena as late-flowering, fewer rosettes and main branches were also observed in transgenic plants. Overall, this study provides the first insight into the evolution, expression, and function of VcTCP genes, including the discovery that VcTCP18 negatively regulated bud dormancy release in blueberry. The results will deepen our understanding of the function of TCPs in plant growth and development.

Integrative Identification of Crucial Genes Associated With Plant Hormone-Mediated Bud Dormancy in Prunus mume

Prunus mume is an important ornamental woody plant with winter-flowering property, which is closely related to bud dormancy. Despite recent scientific headway in deciphering the mechanism of bud dormancy in P. mume, the overall picture of gene co-expression regulating P. mume bud dormancy is still unclear. Here a total of 23 modules were screened by weighted gene co-expression network analysis (WGCNA), of which 12 modules were significantly associated with heteroauxin, abscisic acid (ABA), and gibberellin (GA), including GA1, GA3, and GA4. The yellow module, which was positively correlated with the content of ABA and negatively correlated with the content of GA, was composed of 1,426 genes, among which 156 transcription factors (TFs) were annotated with transcriptional regulation function. An enrichment analysis revealed that these genes are related to the dormancy process and plant hormone signal transduction. Interestingly, the expression trends of PmABF2 and PmABF4 genes, the core members of ABA signal transduction, were positively correlated with P. mume bud dormancy. Additionally, the PmSVP gene had attracted lots of attention because of its co-expression, function enrichment, and expression level. PmABF2, PmABF4, and PmSVP were the genes with a high degree of expression in the co-expression network, which was upregulated by ABA treatment. Our results provide insights into the underlying molecular mechanism of plant hormone-regulated dormancy and screen the hub genes involved in bud dormancy in P. mume.

GA3 is superior to GA4 in promoting bud endodormancy release in tree peony (Paeonia suffruticosa) and their potential working mechanism

Background Sufficient low temperature accumulation is the key strategy to break bud dormancy and promote subsequent flowering in tree peony anti-season culturing production. Exogenous gibberellins (GAs) could partially replace chilling to accelerate dormancy release, and different kinds of GAs showed inconsistent effects in various plants. To understand the effects of exogenous GA3 and GA4 on dormancy release and subsequent growth, the morphological changes were observed after exogenous GAs applications, the differentially expressed genes (DEGs) were identified, and the contents of endogenous phytohormones, starch and sugar were measured, respectively. Results Morphological observation and photosynthesis measurements indicated that both GA3 and GA4 applications accelerated bud dormancy release, but GA3 feeding induced faster bud burst, higher shoot and more flowers per plant. Full-length transcriptome of dormant bud was used as the reference genome. Totally 124 110 459, 124 015 148 and 126 239 836 reads by illumina transcriptome sequencing were obtained in mock, GA3 and GA4 groups, respectively. Compared with the mock, there were 879 DEGs and 2 595 DEGs in GA3 and GA4 group, 1 179 DEGs in GA3 vs GA4, and 849 DEGs were common in these comparison groups. The significant enrichment KEGG pathways of 849 DEGs highlighted plant hormone signal transduction, starch and sucrose metabolism, cell cycle, DNA replication, etc. Interestingly, the contents of endogenous GA1, GA3, GA4, GA7 and IAA significantly increased, ABA decreased after GA3 and GA4 treatments by LC–MS/MS. Additionally, the soluble glucose, fructose and trehalose increased after exogenous GAs applications. Compared to GA4 treatment, GA3 induced higher GA1, GA3 and IAA level, more starch degradation to generate more monosaccharide for use, and promoted cell cycle and photosynthesis. Higher expression levels of dormancy-related genes, TFL, FT, EBB1, EBB3 and CYCD, and lower of SVP by GA3 treatment implied more efficiency of GA3. Conclusions Exogenous GA3 and GA4 significantly accelerated bud dormancy release and subsequent growth by increasing the contents of endogenous bioactive GAs, IAA, and soluble glucose such as fructose and trehalose, and accelerated cell cycle process, accompanied by decreasing ABA contents. GA3 was superior to GA4 in tree peony forcing culture, which might because tree peony was more sensitive to GA3 than GA4, and GA3 had a more effective ability to induce cell division and starch hydrolysis. These results provided the value data for understanding the mechanism of dormancy release in tree peony.

Ethylene-Mediated Modulation of Bud Phenology, Cold Hardiness, and Hormone Biosynthesis in Peach (Prunus persica)

Spring frosts exacerbated by global climate change have become a constant threat to temperate fruit production. Delaying the bloom date by plant growth regulators (PGRs) has been proposed as a practical frost avoidance strategy. Ethephon is an ethylene-releasing PGR found to delay bloom in several fruit species, yet its use is often coupled with harmful effects, limiting its applicability in commercial tree fruit production. Little information is available regarding the mechanisms by which ethephon influences blooming and bud dormancy. This study investigated the effects of fall-applied ethephon on bud phenology, cold hardiness, and hormonal balance throughout the bud dormancy cycle in peach. Our findings concluded that ethephon could alter several significant aspects of peach bud physiology, including accelerated leaf fall, extended chilling accumulation period, increased heat requirements, improved cold hardiness, and delayed bloom date. Ethephon effects on these traits were primarily dependent on its concentration and application timing, with a high concentration (500 ppm) and an early application timing (10% leaf fall) being the most effective. Endogenous ethylene levels were induced significantly in the buds when ethephon was applied at 10% versus 90% leaf fall, indicating that leaves are essential for ethephon uptake. The hormonal analysis of buds at regular intervals of chilling hours (CH) and growing degree hours (GDH) also indicated that ethephon might exert its effects through an abscisic acid (ABA)-independent way in dormant buds. Instead, our data signifies the role of jasmonic acid (JA) in mediating budburst and bloom in peach, which also appears to be influenced by ethephon treatment. Overall, this research presents a new perspective in interpreting horticultural traits in the light of biochemical and molecular data and sheds light on the potential role of JA in bud dormancy, which deserves further attention in future studies that aim at mitigating spring frosts.