RNA-Seq analysis reveals the mechanism in response to cold stress of peach cv 'Dingjiaba'.

BACKGROUND: 'Dingjiaba' is an important Prunus persica cultivar (cv) mainly grown in the Hexi corridor in northwest China, which has an inherited strong cold tolerance. OBJECTIVE: To compare the transcriptome and physiology data of leaves of cvs 'Dingjiaba' (D) and 'Kanoiwa' (K) following cold treatment at different time periods, in order to gain new insights into the mechanisms of cold adaptation in 'Dingjiaba'. MATERIALS AND METHODS: We analyzed the transcriptomic and physiological data of leaves of D and K cvs exposed to 0 h (D0/K0), 2 h (D2/K2), 6 h (D6/K6) and 12 h (D12/K12) cold stress. RESULTS: Low temperature stress caused membrane damage and led to increased rate of electrolyte leakage and increased MDA content. Cold stress induced the accumulation of soluble sugars, soluble proteins and proline in leaves of both cvs, with a lower increase in K compared to D. Transcriptome analysis identified 4,631, 5,069, 5,662 and 3,886 differentially expressed genes (DEGs) between D0 and K0, D2 and K2, D6 and K6 and D12 and K12, respectively. The differentially expressed genes significantly enriched in metabolic pathways and biosynthesis of secondary metabolites. We further validated the reliability of sequencing data of the RNA-Seq with Real-Time Quantitative PCR, which suggested that the expression trend of the RNA-Seq were same as RT-PCR. CONCLUSIONS: These results provide novel insights into a series of molecular mechanisms underlying physiological metabolism and defense. https://doi.org/10.54680/fr24210110312.

Tandem transcription factors PpNAC1 and PpNAC5 synergistically activate the transcription of the PpPGF to regulate peach softening during fruit ripening.

Peach fruit rapidly soften after harvest, a significant challenge for producers and marketers as it results in rotting fruit and significantly reduces shelf life. In this study, we identified two tandem genes, PpNAC1 and PpNAC5, within the sr (slow ripening) locus. Phylogenetic analysis showed that NAC1 and NAC5 are highly conserved in dicots and that PpNAC1 is the orthologous gene of Non-ripening (NOR) in tomato. PpNAC1 and PpNAC5 were highly expressed in peach fruit, with their transcript levels up-regulated at the onset of ripening. Yeast two-hybrid and bimolecular fluorescence complementation assays showed PpNAC1 interacting with PpNAC5 and this interaction occurs with the tomato and apple orthologues. Transient gene silencing experiments showed that PpNAC1 and PpNAC5 positively regulate peach fruit softening. Yeast one-hybrid and dual luciferase assays and LUC bioluminescence imaging proved that PpNAC1 and PpNAC5 directly bind to the PpPGF promoter and activate its transcription. Co-expression of PpNAC1 and PpNAC5 showed higher levels of PpPGF activation than expression of PpNAC1 or PpNAC5 alone. In summary, our findings demonstrate that the tandem transcription factors PpNAC1 and PpNAC5 synergistically activate the transcription of PpPGF to regulate fruit softening during peach fruit ripening.

Characterisation of the MLP genes in peach postharvest cold storage and the regulatory role of PpMLP10 in the chilling stress response.

The major latex proteins/ripening-related proteins are a subfamily of the Bet v 1 protein superfamily and are commonly involved in plant development and responses to various stresses. However, the functions of MLPs in the postharvest cold storage of fruits remain uninvestigated. Herein, we identified 30 MLP genes in the peach (Prunus persica) genome that were clustered into three subgroups. Chromosomal location analysis revealed that the PpMLP genes were unevenly distributed on five of the eight peach chromosomes. Synteny analysis of the MLP genes between peach and seven other plant species (five dicotyledons and two monocotyledons) explored their evolutionary characteristics. Furthermore, the PpMLP promoters contained cis-elements for multiple hormones and stress responses. Gene expression analysis revealed that PpMLPs participated in chilling stress responses. Ectopic expression of PpMLP10 in Arabidopsis improved chilling stress tolerance by decreasing membrane damage and maintaining membrane stability. Additional research confirmed that PpWRKY2 participates in PpMLP10-mediated chilling stress by binding to its promoter. Collectively, these results suggest the role of PpMLP10 in enhancing chilling stress tolerance, which is significant for decreasing chilling injury during the postharvest cold storage of peaches.

Combined analysis of mRNA and miRNA transcriptomes reveals the regulatory mechanism of Xanthomonas arboricola pv pruni resistance in Prunus persica.

BACKGROUND: Peach bacterial shot hole, caused by Xanthomonas arboricola pv pruni (Xap), is a global bacterial disease that poses a threat to the yield and quality of cultivated peach trees (Prunus persica). RESULTS: This study compared the mRNA and miRNA profiles of two peach varieties, 'Yanbao' (resistant) and 'Yingzui' (susceptible), after inoculation with Xap to identify miRNAs and target genes associated with peach tree resistance. mRNA sequencing results revealed that in the S0-vs-S3 comparison group, 1574 genes were upregulated and 3975 genes were downregulated. In the R0-vs-R3 comparison group, 1575 genes were upregulated and 3726 genes were downregulated. Through miRNA sequencing, a total of 112 known miRNAs belonging to 70 miRNA families and 111 new miRNAs were identified. Notably, some miRNAs were exclusively expressed in either resistant or susceptible varieties. Additionally, 59 miRNAs were downregulated and 69 miRNAs were upregulated in the R0-vs-R3 comparison group, while 46 miRNAs were downregulated and 52 miRNAs were upregulated in the S0-vs-S3 comparison group. Joint analysis of mRNA and miRNA identified 79 relationship pairs in the S0-vs-S3 comparison group, consisting of 48 miRNAs and 51 target genes. In the R0-vs-R3 comparison group, there were 58 relationship pairs, comprising 28 miRNAs and 20 target genes. Several target genes related to resistance, such as SPL6, TIFY6B, and Prupe.4G041800_v2.0.a1 (PPO), were identified through literature reports and GO/KEGG enrichment analysis. CONCLUSION: In conclusion, this study discovered several candidate genes involved in peach tree resistance by analyzing differential expression of mRNA and miRNA. These findings provide valuable insights into the mechanisms underlying resistance to Xap in peach trees.

Transcription factor LBD16 targets cell wall modification/ion transport genes in peach lateral root formation.

LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKEs (LBDs/ASLs) are plant-specific transcription factors that function downstream of auxin-regulated lateral root (LR) formation. Our previous research found that PpLBD16 positively regulates peach (Prunus persica) LR formation. However, the downstream regulatory network and target genes of PpLBD16 are still largely unknown. Here, we constructed a PpLBD16 homologous overexpression line and a PpLBD16 silenced line. We found that overexpressing PpLBD16 promoted peach root initiation, while silencing PpLBD16 inhibited peach root formation. Through RNA sequencing (RNA-seq) analysis of roots from PpLBD16 overexpression and silenced lines, we discovered that genes positively regulated by PpLBD16 were closely related to cell wall synthesis and degradation, ion/substance transport, and ion binding and homeostasis. To further detect the binding motifs and potential target genes of PpLBD16, we performed DNA-affinity purification sequencing (DAP-seq) analysis in vitro. PpLBD16 preferentially bound to CCNGAAANNNNGG (MEME-1), [C/T]TTCT[C/T][T/C] (MEME-2), and GCGGCGG (ABR1) motifs. By combined analysis of RNA-seq and DAP-seq data, we screened candidate target genes for PpLBD16. We demonstrated that PpLBD16 bound and activated the cell wall modification-related genes EXPANSIN-B2 (PpEXPB2) and SUBTILISIN-LIKE PROTEASE 1.7 (PpSBT1.7), the ion transport-related gene CYCLIC NUCLEOTIDE-GATED ION CHANNEL 1 (PpCNGC1) and the polyphenol oxidase (PPO)-encoding gene PpPPO, thereby controlling peach root organogenesis and promoting LR formation. Moreover, our results displayed that PpLBD16 and its target genes are involved in peach LR primordia development. Overall, this work reveals the downstream regulatory network and target genes of PpLBD16, providing insights into the molecular network of LBD16-mediated LR development.

Differential Gene Expression Patterns in Peach Roots under Non-Uniform Soil Conditions in Response to Organic Matter.

Organic matter (OM) amendments are often encouraged in sustainable agriculture programs but can create heterogeneous soil environments when applied to perennial crops such as peaches (Prunus persica (L.) Batsch). To better understand the responses of peach roots to non-uniform soil conditions, transcriptomic analysis was performed in a split-root study using uniform soil (the same soil type for all roots) or non-uniform soil (different soil types for each half of the root system) from either (1) autoclaved sand (S), (2) autoclaved sand with autoclaved compost (A), or (3) autoclaved sand with compost which included inherent biological soil life (B). Each uniform soil type (S, A, and B) was grouped and compared by uniform and non-uniform soil comparisons for a total of nine treatments. Comparisons revealed peach roots had differentially expressed genes (DEGs) and gene ontology terms between soil groups, with the S and B groups having a range of 106-411 DEGs and the A group having a range of 19-94 DEGs. Additionally, six modules were identified and correlated (p > 0.69) for six of the nine treatment combinations. This study broadly highlights the complexity of how OM and biological life in the rhizosphere interact with immediate and distant roots and sheds light on how non-homogenous soil conditions can influence peach root gene expression.

Ppe.RPT/SSC-1: from QTL mapping to a predictive KASP test for ripening time and soluble solids concentration in peach.

Genomic regions associated with ripening time (RPT) and soluble solids concentration (SSC) were mapped using a pedigreed population including multiple F1 and F2 families from the Clemson University peach breeding program (CUPBP). RPT and SSC QTLs were consistently identified in two seasons (2011 and 2012) and the average datasets (average of two seasons). A target region spanning 10,981,971-11,298,736 bp on chromosome 4 of peach reference genome used for haplotype analysis revealed four haplotypes with significant differences in trait values among different diplotype combinations. Favorable alleles at the target region for both RPT and SSC were determined and a DNA test for predicting RPT and SSC was developed. Two Kompetitive Allele Specific PCR (KASP) assays were validated on 84 peach cultivars and 163 seedlings from the CUPBP, with only one assay (Ppe.RPT/SSC-1) needed to predict between early and late-season ripening cultivars and low and high SSC. These results advance our understanding of the genetic basis of RPT and SSC and facilitate selection of new peach cultivars with the desired RPT and SSC.

Characterization and Potential Action Mode Divergences of Homologous ACO1 Genes during the Organ Development and Ripening Process between Non-Climacteric Grape and Climacteric Peach.

Ethylene is one crucial phytohormone modulating plants' organ development and ripening process, especially in fruits, but its action modes and discrepancies in non-climacteric grape and climacteric peach in these processes remain elusive. This work is focused on the action mode divergences of ethylene during the modulation of the organ development and ripening process in climacteric/non-climacteric plants. We characterized the key enzyme genes in the ethylene synthesis pathway, VvACO1 and PpACO1, and uncovered that their sequence structures are highly conserved, although their promoters exhibit important divergences in the numbers and types of the cis-elements responsive to hormones, implying various responses to hormone signals. Subsequently, we found the two have similar expression modes in vegetative organ development but inverse patterns in reproductive ones, especially in fruits. Then, VvACO1 and PpACO1 were further validated in promoting fruit ripening functions through their transient over-expression/RNAi-expression in tomatoes, of which the former possesses a weaker role than the latter in the fruit ripening process. Our findings illuminated the divergence in the action patterns and function traits of the key VvACO1/PpACO1 genes in the tissue development of climacteric/non-climacteric plants, and they have implications for further gaining insight into the interaction mechanism of ethylene signaling during the modulation of the organ development and ripening process in climacteric/non-climacteric plants.

The PpMYB75-PpDFR module reveals the difference between 'SR' and its bud variant 'RMHC' in peach red flesh.

'Red Meat Honey Crisp (RMHC)' has been widely cultivated by growers in recent years due to its early maturity, and red meat type characteristics. As a bud variant of 'Super Red (SR)' peach, red flesh is the most distinctive characteristic of 'Red Meat Honey Crisp (RMHC)'. However, the mechanism of red flesh formation in 'RMHC' remains unclear. In this study, 79 differentially produced metabolites were identified by metabolomics analysis. The anthocyanin content in 'RMHC' was significantly higher than that in 'SR' during the same period, such as cyanidin O-syringic acid and cyanidin 3-O-glucoside. Other flavonoids also increased during the formation of red flesh, including flavonols (6-hydroxykaempferol-7-O-glucoside, hyperin), flavanols (protocatechuic acid, (+)-gallocatechin), and flavonoids (chrysoeriol 5-O-hexoside, tricetin). In addition, transcriptomic analysis and RT-qPCR showed that the expression levels of the flavonoid synthesis pathway transcription factor MYB75 and some structural genes, such as PpDFR, PpCHS, PpC4H, and PpLDOX increased significantly in 'RMHC'. Subcellular localization analysis revealed that MYB75 was localized to the nucleus. Yeast single hybridization assays showed that MYB75 bound to the cis-acting element CCGTTG of the PpDFR promoter region. The MYB75-PpDFR regulatory network was identified to be a key pathway in the reddening of 'RMHC' flesh. Moreover, this is the first study to describe the cause for red meat reddening in 'RMHC' compared to 'SR' peaches using transcriptomics, metabolomics and molecular methods. Our study identified a key transcription factor involved in the regulation of the flavonoid synthetic pathway and contributes to peach breeding-related efforts as well as the identification of genes involved in color formation in other species.

Transcription factor PpNAC1 and DNA demethylase PpDML1 synergistically regulate peach fruit ripening.

Fruit ripening is accompanied by dramatic changes in color, texture, and flavor and is regulated by transcription factors (TFs) and epigenetic factors. However, the detailed regulatory mechanism remains unclear. Gene expression patterns suggest that PpNAC1 (NAM/ATAF1/2/CUC) TF plays a major role in peach (Prunus persica) fruit ripening. DNA affinity purification (DAP)-seq combined with transactivation tests demonstrated that PpNAC1 can directly activate the expression of multiple ripening-related genes, including ACC synthase1 (PpACS1) and ACC oxidase1 (PpACO1) involved in ethylene biosynthesis, pectinesterase1 (PpPME1), pectate lyase1 (PpPL1), and polygalacturonase1 (PpPG1) related to cell wall modification, and lipase1 (PpLIP1), fatty acid desaturase (PpFAD3-1), and alcohol acyltransferase1 (PpAAT1) involved in volatiles synthesis. Overexpression of PpNAC1 in the tomato (Solanum lycopersicum) nor (nonripening) mutant restored fruit ripening, and its transient overexpression in peach fruit induced target gene expression, supporting a positive role of PpNAC1 in fruit ripening. The enhanced transcript levels of PpNAC1 and its target genes were associated with decreases in their promoter mCG methylation during ripening. Declining DNA methylation was negatively associated with increased transcripts of DNA demethylase1 (PpDML1), whose promoter is recognized and activated by PpNAC1. We propose that decreased methylation of the promoter region of PpNAC1 leads to a subsequent decrease in DNA methylation levels and enhanced transcription of ripening-related genes. These results indicate that positive feedback between PpNAC1 and PpDML1 plays an important role in directly regulating expression of multiple genes required for peach ripening and quality formation.

Two adjacent NAC transcription factors regulate fruit maturity date and flavor in peach.

Although maturity date (MD) is an essential factor affecting fresh fruit marketing and has a pleiotropic effect on fruit taste qualities, the underlying mechanisms remain largely unclear. In this study, we functionally characterized two adjacent NAM-ATAF1/2-CUC2 (NAC) transcription factors (TFs), PpNAC1 and PpNAC5, both of which were associated with fruit MD in peach. PpNAC1 and PpNAC5 were found capable of activating transcription of genes associated with cell elongation, cell wall degradation and ethylene biosynthesis, suggesting their regulatory roles in fruit enlargement and ripening. Furthermore, PpNAC1 and PpNAC5 had pleiotropic effects on fruit taste due to their ability to activate transcription of genes for sugar accumulation and organic acid degradation. Interestingly, both PpNAC1 and PpNAC5 orthologues were found in fruit-producing angiosperms and adjacently arranged in all 91 tested dicots but absent in fruitless gymnosperms, suggesting their important roles in fruit development. Our results provide insight into the regulatory roles of NAC TFs in MD and fruit taste.

Integration of metabolomics and transcriptomics analyses reveals the mechanism of nano-selenium treated to activate phenylpropanoid metabolism and enhance the antioxidant activity of peach.

Foliar spraying to improve the quality of fruits is a general approach nowadays. In this study, 10 ppm nano-selenium (nano-Se) diluted with distilled water was sprayed on peach leaves every 10 days for a total of 7 sprays during the fruit set period. And then their fruit quality was compared with that of control group. It was found that the firmness, soluble solid concentration, total phenol, and proanthocyanidin content of the peaches were raised after the nano-Se treatment. Moreover, the ascorbic acid glutathione loop (ASA-GSH loop) was fully activated in the nano-Se treated group, and the associated antioxidant capacity and enzyme activity were significantly increased. Metabolomics revealed that nano-Se could upregulate some metabolites, such as phenylalanine, naringenin, and pinocembrin, to fully activate the metabolism of phenylpropanoids. Further, based on transcriptomics, nano-Se treatment was found to affect fruit quality by regulating genes related to phenylpropanoid metabolism, such as arogenate/prephenate dehydratase (ADT), genes related to abscisic acid metabolism such as (+)-abscisic acid 8'-hydroxylase (CYP707A), and some transcription factors such as MYB. Based on the comprehensive analysis of physicochemical indicators, metabolomics, and transcriptomics, it was found that nano-Se improved fruit quality by activating phenylpropanoid metabolism and enhancing antioxidant capacity. This work provides insights into the mechanism of the effect of nano-Se fertilizer on peach fruit quality. PRACTICAL APPLICATION: The firmness and soluble solid concentration of peaches are higher after nano-Se treatment, which is more in line with people's demand for hard soluble peaches like "Yingzui." The antioxidant capacity, antioxidant substance content, and antioxidant enzyme activity of nano-Se-treated peaches are higher, with potential storage resistance and health effects on human body. The mechanism of nano-Se affecting peach quality was analyzed by metabolomics and transcriptomics, which is a reference and guide for the research and application of nano-Se.

PpBZR1, a BES/BZR transcription factor, enhances cold stress tolerance by suppressing sucrose degradation in peach fruit.

Brassinosteroids (BRs) are phytohormones that play numerous roles in a plant's response to environmental stress. While BES/BZR transcription factors are essential components in BR signaling, their role in regulating postharvest fruit responses to cold stress is largely unknown. In this study, the application of 24-epibrassinolide (EBR) to peaches alleviated chilling injury (CI) during postharvest cold storage. We further characterized a key BES/BZR gene, PpBZR1, which regulates peach cold resistance. Transient expression PpBZR1 in peaches showed that PpBZR1 inhibits PpVIN2 expression and VIN activity, resulting in an elevated level of sucrose, which protects fruit from CI. Arabidopsis thaliana expressing PpBZR1 that had a high germination and seedling survival rate at low temperatures, which may be due to higher level of sucrose and lower oxidative damage. Mechanistically, we confirmed that PpBZR1 directly binds to the PpVIN2 promoter and functions as a negative regulator for sucrose metabolism. In addition, PpCBF1/5/6 were induced by EBR treatment and AtCBFs were upregulated in PpBZR1 transgenic Arabidopsis thaliana. Combined with previous findings, we hypothesize that PpBZR1 regulates PpVIN2 and may also be mediated by CBF. In conclusion, PpBZR1 expression is induced by EBR treatment during cold storage, which futher inhibite sucrose degradation gene PpVIN2 transcription via direct binding its promoter and indirectly regulating PpVIN2, resulting in slower sucrose degradation and higher chilling tolerance of peach.

Identification of the Light-Harvesting Chlorophyll a/b Binding Protein Gene Family in Peach (Prunus persica L.) and Their Expression under Drought Stress.

In higher plants, light-harvesting chlorophyll a/b binding (Lhc) proteins play a vital role in photosynthetic processes and are widely involved in the regulation of plant growth, development, and response to abiotic stress. However, the Lhc gene family has not been well identified in peaches (Prunus persica L.). In this study, 19 PpLhc genes were identified in the peach genome database, which were unevenly distributed on all chromosomes. Phylogenetic analysis demonstrated that PpLhc proteins could be divided into three major subfamilies, each of whose members had different exon-intron structures but shared similar conserved motifs. A total of 17 different kinds of cis-regulatory elements were identified in the promoter regions of all PpLhc genes, which could be classified into three categories: plant growth and development, stress response, and phytohormone response. In addition, transcriptomic data analysis and RT-qPCR results revealed that the expression profiles of some PpLhc genes changed under drought treatment, suggesting the crucial roles of Lhc genes in the regulation of plant tolerance to drought stress. Taken together, these findings will provide valuable information for future functional studies of PpLhc genes, especially in response to drought stress.

PpMYB105 inhibits chilling injury by regulating PpMsrA1 in peach fruit.

KEY MESSAGE: MeJA supplementation enhanced the chilling tolerance and gene expression of PpMsrA1. PpMYB105 protein positively regulated the PpMsrA1 promoter. PpMYB105 mediated the MeJA-boosted chilling tolerance by regulating PpMsrA1. Cold storage can maintain the quality of postharvest fruit. However, peaches easily suffer from chilling injury (CI) during cold storage, leading to economic loss. Results showed that methyl jasmonate (MeJA) supplementation reduced the CI severity, and enhanced the gene expression of methionine sulfoxide reductase A1 (PpMsrA1). It was found that MeJA application elevated the MsrA activity and methionine (Met) content, and reduced the methionine-S-sulfoxide (Met-S-SO) content and reactive oxygen species (ROS) production afterwards. Moreover, PpMYB105 could activate the transcription of PpMsrA1 by binding to the MYB binding element in its promoter. The gene expression of PpMYB105 was up-regulated by MeJA application. Overexpression of PpMYB105 in tomatoes enhanced the chilling tolerance and gene expression of SlMsrA1. Virus-induced gene silencing of PpMYB105 in peaches resulted in the increase in CI severity and the decrease in gene expression of PpMsrA1. Thus, PpMYB105 was involved in the MeJA-boosted chilling tolerance by regulating PpMsrA1.

Genome-wide presence/absence variation discovery and its application in Peach (Prunus persica).

Gene presence/absence variation (PAV) is an important contributor to the studies of genetic diversity, gene identification, and molecular marker development in plants. In the present study, 100 peach (Prunus persica) accessions were used for genome resequencing to identify PAVs. Alignmentwith a reference genome yielded a total of 2.52 Mb non-reference sequences and 923 novel genes were identified. The dispensable PAVs were enriched in resistance, perhaps reflecting their roles in plant adaptation to various environments. Furthermore, selection sweeps associated with peach domestication and improvement were identified based on PAV data. Only 4.3% and 13.4% of domestication and improvement sweeps, respectively, were identified simultaneously using single nucleotide polymorphism (SNP) data, suggesting flexible identification between the different methods. To further verify the applicability of PAV identification, a genome-wide association study was conducted using 21 agronomic traits. Some of the identified loci were consistent with those reported in previous studies, while some were mapped for the first time; the latter included petiole length, petiole gland shape, and petiole gland number. Through tissue-specific expression analysis and gene transformation experiments, a novel gene, evm.model.Contig322_A94.1, was identified and found to be involved in chilling requirements. We speculated that this novel gene might regulate the trait by participating in the ABA signaling pathway. The PAVs identified in P. persica provide valuable resources for mapping the entire gene set and identifying optional markers for molecular selection in future studies.

Two interacting ethylene response factors negatively regulate peach resistance to Lasiodiplodia theobromae.

Gummosis is 1 of the most common and destructive diseases threatening global peach (Prunus persica) production. Our previous studies have revealed that ethylene and methyl jasmonate enhance peach susceptibility to Lasiodiplodia theobromae, a virulent pathogen inducing gummosis; however, the underlying molecular mechanisms remain obscure. Here, 2 ethylene response factors (ERFs), PpERF98 and PpERF1, were identified as negative regulators in peach response to L. theobromae infection. Expression of 2 putative paralogs, PpERF98-1/2, was dramatically induced by ethylene and L. theobromae treatments and accumulated highly in the gummosis-sensitive cultivar. Silencing of PpERF98-1/2 increased salicylic acid (SA) content and pathogenesis-related genes PpPR1 and PpPR2 transcripts, conferring peach resistance to L. theobromae, whereas peach and tomato (Solanum lycopersicum) plants overexpressing either of PpERF98-1/2 showed opposite changes. Also, jasmonic acid markedly accumulated in PpERF98-1/2-silenced plants, but reduction in PpPR3, PpPR4, and PpCHI (Chitinase) transcripts indicated a blocked signaling pathway. PpERF98-1 and 2 were further demonstrated to directly bind the promoters of 2 putative paralogous PpERF1 genes and to activate the ERF branch of the jasmonate/ethylene signaling pathway, thus attenuating SA-dependent defenses. The lesion phenotypes of peach seedlings overexpressing PpERF1-1/2 and PpERF98-1/2 were similar. Furthermore, PpERF98-1/2 formed homodimers/heterodimers and interacted with the 2 PpERF1 proteins to amplify the jasmonate/ethylene signaling pathway, as larger lesions were observed in peach plants cooverexpressing PpERF98 with PpERF1 relative to individual PpERF98 overexpression. Overall, our work deciphers an important regulatory network of ethylene-mediated peach susceptibility to L. theobromae based on a PpERF98-PpERF1 transcriptional cascade, which could be utilized as a potential target for genetic engineering to augment protection against L. theobromae-mediated diseases in crops and trees.

Discovery of a key gene associated with fruit maturity date and analysis of its regulatory pathway in peach.

Fruit maturity is an important agronomic trait of fruit crops. Although in previous studies, several molecular markers are developed for the trait, the knowledge about its candidate genes is particularly limited. In this study, a total of 357 peach accessions were re-sequenced to obtain 949,638 SNPs. Combing with 3-year fruit maturity dates, a genome-wide association analysis was performed, and 5, 8, and 9 association loci were identified. To screen the candidate genes for those year-stable loci on chromosomes 4 and 5, two maturity date mutants were used for transcriptome sequencing. Gene expression analysis indicated that Prupe.4G186800 and Prupe.4G187100 on chromosome 4 were essential to fruit ripening in peaches. However, the expression analysis of different tissues showed that the first gene has no tissue-specific character, but transgenic studies showed that the latter is more likely to be a key candidate gene than the first for the maturity date in peach. The yeast two-hybrid assay showed that the proteins encoded by the two genes interacted and then regulated fruit ripening. Moreover, the previously identified 9 bp insertion in Prupe.4G186800 may affect their interaction ability. This research is of great significance for understanding the molecular mechanism of peach fruit ripening and developing practical molecular markers in a breeding program.

MADS-box protein PpDAM6 regulates chilling requirement-mediated dormancy and bud break in peach.

Bud dormancy is crucial for winter survival and is characterized by the inability of the bud meristem to respond to growth-promotive signals before the chilling requirement (CR) is met. However, our understanding of the genetic mechanism regulating CR and bud dormancy remains limited. This study identified PpDAM6 (DORMANCY-ASSOCIATED MADS-box) as a key gene for CR using a genome-wide association study analysis based on structural variations in 345 peach (Prunus persica (L.) Batsch) accessions. The function of PpDAM6 in CR regulation was demonstrated by transiently silencing the gene in peach buds and stably overexpressing the gene in transgenic apple (Malus × domestica) plants. The results showed an evolutionarily conserved function of PpDAM6 in regulating bud dormancy release, followed by vegetative growth and flowering, in peach and apple. The 30-bp deletion in the PpDAM6 promoter was substantially associated with reducing PpDAM6 expression in low-CR accessions. A PCR marker based on the 30-bp indel was developed to distinguish peach plants with non-low and low CR. Modification of the H3K27me3 marker at the PpDAM6 locus showed no apparent change across the dormancy process in low- and non-low- CR cultivars. Additionally, H3K27me3 modification occurred earlier in low-CR cultivars on a genome-wide scale. PpDAM6 could mediate cell-cell communication by inducing the expression of the downstream genes PpNCED1 (9-cis-epoxycarotenoid dioxygenase 1), encoding a key enzyme for ABA biosynthesis, and CALS (CALLOSE SYNTHASE), encoding callose synthase. We shed light on a gene regulatory network formed by PpDAM6-containing complexes that mediate CR underlying dormancy and bud break in peach. A better understanding of the genetic basis for natural variations of CR can help breeders develop cultivars with different CR for growing in different geographical regions.