Combined transcriptome and metabolome analysis identifies triterpenoid-induced defense responses in Myzus persicae Sülzer-infested peach.

The defense response of peach (Prunus persica) to insect attack involves changes in gene expression and metabolites. Piercing/sucking insects such as green peach aphid cause direct damage by obtaining phloem nutrients and indirect damage by spreading plant viruses. To investigate the response of peach trees to aphids, the leaf transcriptome and metabolome of two genotypes with different sensitivities to green peach aphid (GPA, Myzus persicae) were studied. The transcriptome analysis of infected peach leaves showed two different response patterns. The gene expression of aphid-susceptible peach plants infected by aphids was more similar to that of the control plants, while the gene expression of aphid-resistant peach plants infected by aphids showed strongly induced changes in gene expression compared with the response in the control plants. Furthermore, gene transcripts in defense-related pathways, including plant-pathogen interaction, MAPK signaling, and several metabolic pathways, were more strongly enriched upon aphid infestation. Untargeted secondary metabolite profiling confirmed that aphid treatment induced larger changes in aphid-resistant peaches than in aphid-susceptible peaches. Consistent with transcriptomic alterations, nine triterpenoids showed extremely significant GPA-induced accumulation in aphid-resistant peaches, whereas triterpenoid abundance remained predominantly unchanged or undetected in aphid- susceptible peaches. Furthermore, some types of transcription factors (including WRKYs, ERFs, NACs, etc.) were more strongly induced upon GPA infestation in aphid-resistant peaches but not in aphid-susceptible peaches. Aphid feeding-dependent transcriptome and metabolite profiles provide the foundation for understanding the molecular mechanisms underlying the response of peach to aphid infestation. These results suggested that accumulation of specialized triterpenoids and the corresponding pathway transcripts may play a key role in peach GPA resistance.

Characterization and expression analysis of basic leucine zipper (bZIP) transcription factors responsive to chilling injury in peach fruit.

BACKGROUND: Peach (Prunus persica L.) is prone to chilling injury as exhibited by inhibition of the ethylene production, failure in softening, and the manifestation of internal browning. The basic leucine zipper (bZIP) transcription factors play an essential role in regulatory networks that control many processes associated with physiological, abiotic and biotic stress responses in fruits. Formerly, the underlying molecular and regulatory mechanism of (bZIP) transcription factors responsive to chilling injury in peach fruit is still elusive. METHODS AND RESULTS: In the current experiment, the solute peach 'Zhongyou Peach No. 13' was used as the test material and cold storage at low temperature (4 °C). It was found that long-term low-temperature storage induced the production of ethylene, the hardness of the pulp decreased, and the low temperature also induced ABA accumulation. The changes of ABA and ethylene in peach fruits during low-temperature storage were clarified. Since the bZIP transcription factor is involved in the regulation of downstream pathways of ABA signals, 47 peach bZIP transcription factor family genes were identified through bioinformatics analysis. Further based on RT-qPCR analysis, 18 PpbZIP genes were discovered to be expressed in refrigerated peach fruits. Among them, the expression of PpbZIP23 and PpbZIP25 was significantly reduced during the refrigeration process, the promoter analysis of these genes found that this region contains the MYC/MYB/ABRES binding element, but not the DRES/CBFS element, indicating that the expression may be regulated by the ABA-dependent cold induction pathway, thereby responding to chilling injury in peach fruit. CONCLUSIONS: Over investigation will provide new insights for further postharvest protocols related to molecular changes during cold storage and will prove a better cope for chilling injury.

Two loss-of-function alleles of the glutathione S-transferase (GST) gene cause anthocyanin deficiency in flower and fruit skin of peach (Prunus persica).

Flower and fruit colors are important agronomic traits. To date, there is no forward genetic evidence that the glutathione S-transferase (GST) gene is responsible for the white flower color in peach (Prunus persica). In this study, genetic analysis indicated that the white-flower trait is monogenetic, is recessive to the non-white allele, and shows pleiotropic effects with non-white-flowered types. The genetic locus underpinning this trait was mapped onto chromosome 3 between 0.421951 and 3.227115 Mb by using bulked segregant analysis in conjunction with whole-genome sequencing, and was further mapped between 0 and 1.178149 Mb by using the backcross 1 (BC1 ) population. Finally, the locus was fine-mapped within 535.974- and 552.027-kb intervals by using 151 F2 individuals and 75 individuals from a BC1 self-pollinated (BC1 S1 ) population, respectively. Pp3G013600, encoding a GST that is known to transport anthocyanin, was identified within the mapping interval. The analysis of genome sequence data showed Pp3G013600 in white flowers has a 2-bp insertion or a 5-bp deletion in the third exon. These variants likely render the GST non-functional because of early stop codons that reduce the protein length from 215 amino acids to 167 and 175 amino acids, respectively. Genetic markers based on these variants validated a complete correlation between the GST loss-of-function alleles and white flower in 128 peach accessions. This correlation was further confirmed by silencing of Pp3G013600 using virus-induced gene silencing technology, which reduced anthocyanin accumulation in peach fruit. The new knowledge from this study is useful for designing peach breeding programs to generate cultivars with white flower and fruit skin.

Fine mapping of the gene controlling the weeping trait of Prunus persica and its uses for MAS in progenies.

BACKGROUND: Fruit tree yield and fruit quality are affected by the tree's growth type, and branching angle is an important agronomic trait of fruit trees, which largely determines the crown structure. The weeping type of peach tree shows good ventilation and light transmission; therefore, it is commonly cultivated. However, there is no molecular marker closely linked with peach weeping traits for target gene screening and assisted breeding. RESULTS: First, we confirmed that the peach weeping trait is a recessive trait controlled by a single gene by constructing segregating populations. Based on BSA-seq, we mapped the gene controlling this trait within 159 kb of physical distance on chromosome 3. We found a 35 bp deletion in the candidate area in standard type, which was not lacking in weeping type. For histological assessments, different types of branches were sliced and examined, showing fiber bundles in the secondary xylem of ordinary branches but not in weeping branches. CONCLUSIONS: This study established a molecular marker that is firmly linked to weeping trait. This marker can be used for the selection of parents in the breeding process and the early screening of hybrid offspring to shorten the breeding cycle. Moreover, we preliminary explored histological differences between growth types. These results lay the groundwork for a better understanding of the weeping growth habit of peach trees.

NLR1 is a strong candidate for the Rm3 dominant green peach aphid (Myzus persicae) resistance trait in peach.

The green peach aphid (GPA), Myzus persicae, is a polyphagous, sap-sucking aphid and a vector of many plant viruses. In peach, Prunus persica, three individual dominant GPA resistance loci have been genetically defined (Rm1-3), but knowledge of the underlying genes is limited. In this study, we focused on the Rm3 locus. Bulk segregant analysis (BSA) mapping in segregating progeny populations delimited Rm3 to an interval spanning 160 kb containing 21 genes on chromosome 1. RNA-seq data provided no evidence of candidate genes, but chromosomal structural variations were predicted around a nucleotide-binding site-leucine-rich repeat (NLR) gene (ppa000596m) within the Rm3 fine-mapping interval. Following bacterial artificial chromosome (BAC) library construction for a GPA-resistant peach cultivar and the sequencing of three target BAC clones, a chromosomal structural variation encompassing two novel TIR-NLR-class disease resistance (R) protein-coding genes was identified, and the expressed NLR gene (NLR1) was identified as a candidate for M. persicae resistance. Consistent with its proposed role in controlling GPA resistance, NLR1 was only expressed in the leaves of resistant peach phenotypes. A molecular marker that was designed based on the NLR1 sequence co-segregated with the GPA-resistant phenotype in four segregating populations, 162 peach cultivars, and 14 wild relatives, demonstrating the dominant inheritance of the Rm3 locus. Our findings can be exploited to facilitate future breeding for GPA-resistance in peach.

Transcriptomic and Metabolic Analyses Reveal the Mechanism of Ethylene Production in Stony Hard Peach Fruit during Cold Storage.

Stony hard (SH) peach (Prunus persica L. Batsch) fruit does not release ethylene and has very firm and crisp flesh at ripening, both on- and off-tree. Long-term cold storage can induce ethylene production and a serious risk of chilling injury in SH peach fruit; however, the regulatory mechanism underlying ethylene production in stony hard peach is relatively unclear. In this study, we analyzed the phytohormone levels, fruit firmness, transcriptome, and lipidome changes in SH peach 'Zhongtao 9' (CP9) during cold storage (4 °C). The expression level of the ethylene biosynthesis gene PpACS1 and the content of ethylene in SH peach fruit were found to be upregulated during cold storage. A peak in ABA release was observed before the release of ethylene and the genes involved in ABA biosynthesis and degradation, such as zeaxanthin epoxidase (ZEP) and 8'-hydroxylase (CYP707A) genes, were specifically induced in response to low temperatures. Fruit firmness decreased fairly slowly during the first 20 d of refrigeration, followed by a sharp decline. Furthermore, the expression level of genes encoding cell wall metabolic enzymes, such as polygalacturonase, pectin methylesterase, expansin, galactosidase, and ß-galactosidase, were upregulated only upon refrigeration, as correlated with the decrease in fruit firmness. Lipids belonging to 23 sub-classes underwent differential rearrangement during cold storage, especially ceramide (Cer), monoglycosylceramide (CerG1), phosphatidic acid (PA), and diacyglyceride (DG), which may eventually lead to ethylene production. Exogenous PC treatment provoked a higher rate of ethylene production. We suspected that the abnormal metabolism of ABA and cell membrane lipids promotes the production of ethylene under low temperature conditions, causing the fruit to soften. In addition, ERF transcription factors also play an important role in regulating lipid, hormone, and cell wall metabolism during long-term cold storage. Overall, the results of this study give us a deeper understanding of the molecular mechanism of ethylene biosynthesis during the postharvest storage of SH peach fruit under low-temperature conditions.

Dynamic transcriptomes of resistant and susceptible peach lines after infestation by green peach aphids (Myzus persicae Sülzer) reveal defence responses controlled by the Rm3 locus.

BACKGROUND: The green peach aphid (GPA), Myzus persicae (Sülzer), is a widespread phloem-feeding insect that significantly influences the yield and visual quality of peach [Prunus persica (L.) Batsch]. Single dominant gene (Rm3)-based resistance provides effective management of this invasive pest, although little is known about the molecular responses of plants to GPA feeding. RESULTS: To illustrate the molecular mechanisms of monogenic resistance in peach to young tissue-infecting GPAs, aphid-resistant/aphid-susceptible peach lines from a segregating population with Rm3/rm3 and rm3/rm3 genotypes were infested with GPAs for 3 to 72 h. Transcriptome analysis of the infested tissues identified 3854 differentially expressed genes (DEGs). Although the majority of the DEGs in the resistant line also responded to aphid attack in the susceptible line, the overall magnitude of change was greater in the resistant line than in the susceptible line. The enriched gene ontology of the 3854 DEGs involved in plant defence responses included redox situation, calcium-mediated signalling, transcription factor (e.g., WRKY, MYB, and ERF), MAPK signalling cascade, phytohormone signalling, pathogenesis-related protein, and secondary metabolite terms. Of the 53 genes annotated in a 460 kb interval of the rm3 locus, seven genes were differentially expressed between the aphid-resistant and aphid-susceptible peach lines following aphid infestation. CONCLUSIONS: Together, these results suggest that the Rm3-dependent resistance relies mainly on the inducible expression of defence-related pathways and signalling elements within hours after the initiation of aphid feeding and that the production of specific secondary metabolites from phenylpropanoid/flavonoid pathways can have major effects on peach-aphid interactions.

PpYUC11, a strong candidate gene for the stony hard phenotype in peach (Prunus persica L. Batsch), participates in IAA biosynthesis during fruit ripening.

High concentrations of indole-3-acetic acid (IAA) are required for climacteric ethylene biosynthesis to cause fruit softening in melting flesh peaches at the late ripening stage. By contrast, the fruits of stony hard peach cultivars do not soften and produce little ethylene due to the low IAA concentrations. To investigate the regulation of IAA accumulation during peach ripening [the transition from stage S3 to stage S4 III (climacteric)], a digital gene expression (DGE) analysis was performed. The expression patterns of auxin-homeostasis-related genes were compared in fruits of the melting flesh peach 'Goldhoney 3' and the stony hard flesh peach 'Yumyeong' during the ripening stage. It is revealed here that a YUCCA flavin mono-oxygenase gene (PpYUC11, ppa008176m), a key gene in auxin biosynthesis, displayed an identical differential expression profile to the profiles of IAA accumulation and PpACS1 transcription: the mRNA transcripts increased at the late ripening stage in melting flesh peaches but were below the limit of detection in mature fruits of stony hard peaches. In addition, the strong association between intron TC microsatellite genotypes of PpYUC11 and the flesh texture (normal or stony hard) is described in 43 peach varieties, indicating that this locus may be responsible for the stony hard phenotype in peach. These findings support the hypothesis that PpYUC11 may play an essential role in auxin biosynthesis during peach fruit ripening and is a candidate gene for the control of the stony hard phenotype in peach.

Interaction between PpERF5 and PpERF7 enhances peach fruit aroma by upregulating PpLOX4 expression.

Ethylene plays a critical role in peach (Prunus persica) fruit ripening; however, the molecular mechanism underlying ethylene-mediated aroma biosynthesis remains unclear. Here, we compared the difference in aroma-related volatiles and gene expression levels between melting-flesh (MF) and stony hard (SH) peach cultivars at S3, S4 I, S4 II, S4 III stages, and explored the relation between volatile biosynthesis related genes and ethylene response factor (ERF) genes. The concentration of fruity aromatic compounds such as lactones and terpenes increased significantly in MF peach during fruit ripening, while it was nearly undetectable in SH peach. LOX4 and FAD1 genes expressed concomitantly with ethylene emission and significantly downregulated by 1-MCP. Besides, 1-MCP treatment could sharply influence the fruity aromatic compounds, suggesting that these genes play key roles in volatile biosynthesis during fruit ripening. Furthermore, PpERF5 and PpERF7 could bind together to form a protein complex that enhanced the transcription of LOX4 more than each transcription factor individually. Overall, this work provides new insights into the transcriptional regulatory mechanisms associated with aroma formation during peach fruit ripening.

Fine Mapping of the Gene Controlling the Fruit Skin Hairiness of Prunus persica and Its Uses for MAS in Progenies.

The fruit skin pubescence of Prunus persica is an economically important characteristic and comprises the classification criteria. The mapping and identification of a complete linkage marker to the fruit skin trichome trait locus of peach fruit are critical for the molecular marker-assisted selection for peach/nectarine. In this study, the BC1 population was constructed from the parents "Zhongyou No. 4", the recurrent parent, and "Baihuashanbitao", the non-recurrent parent. Based on the 38 BC1 individuals' phenotypes and their genotyping using next-generation sequencing, the G (Glabrous skin) locus of the gene was first identified between 14.099 and 16.721 Mb on chromosome 5. Using other individuals of this population, the gene was fine-mapped in the range of 481 kb with SNP markers. Based on the resequencing data of other cultivars (lines), the candidate SNP in the gene Prupe.5G196400 was obtained. Subsequently, the SNP marker was designed and applied to natural and hybrid peach populations. Via genotyping analysis, we confirmed co-segregation between the peach/nectarine phenotype, which was used in the identification of peach or nectarine with 100% accuracy.

PpIAA1 and PpERF4 form a positive feedback loop to regulate peach fruit ripening by integrating auxin and ethylene signals.

The signaling pathways of both auxin and ethylene regulate peach fruit ripening via the Aux/IAA and ERF transcription factors, respectively. However, the molecular mechanisms that coordinate both auxin and ethylene signals during peach fruit ripening remain unclear. In this study, we show that PpIAA1 and PpERF4 act as key players in a positive feedback loop, and promote peach fruit ripening by directly binding to and enhancing the activity of target gene promoters. PpIAA1 increased the expression of the ethylene biosynthesis gene PpACS1. Furthermore, PpERF4 enhanced the transcription of PpACO1 and PpIAA1 genes by binding to their promoters. Additionally, PpIAA1 and PpERF4 bound to each other to form a complex, which then enhanced the transcription of abscisic acid biosynthesis genes (PpNCED2 and PpNCED3) and the fruit softening gene (PpPG1) to levels higher than those achieved by each transcription factor individually. Moreover, overexpression of PpIAA1 in tomato accelerated fruit ripening and shortened the fruit shelf-life by increasing the production of ethylene and the expression levels of ripening regulator genes. Collectively, these results advance our understanding of the molecular mechanisms underlying peach fruit ripening and softening via auxin and ethylene signaling pathways.

Application of an antibody chip for screening differentially expressed proteins during peach ripening and identification of a metabolon in the SAM cycle to generate a peach ethylene biosynthesis model.

Peach (Prunus persica) is a typical climacteric fruit that produces ethylene rapidly during ripening, and its fruit softens quickly. Stony hard peach cultivars, however, do not produce large amounts of ethylene, and the fruit remains firm until fully ripe, thus differing from melting flesh peach cultivars. To identify the key proteins involved in peach fruit ripening, an antibody-based proteomic analysis was conducted. A mega-monoclonal antibody (mAb) library was generated and arrayed on a chip (mAbArray) at a high density, covering ~4950 different proteins of peach. Through the screening of peach fruit proteins with the mAbArray chip, differentially expressed proteins recognized by 1587 mAbs were identified, and 33 corresponding antigens were ultimately identified by immunoprecipitation and mass spectrometry. These proteins included not only important enzymes involved in ethylene biosynthesis, such as ACO1, SAHH, SAMS, and MetE, but also novel factors such as NUDT2. Furthermore, protein-protein interaction analysis identified a metabolon containing SAHH and MetE. By combining the antibody-based proteomic data with the transcriptomic and metabolic data, a mathematical model of ethylene biosynthesis in peach was constructed. Simulation results showed that MetE is an important regulator during peach ripening, partially through interaction with SAHH.

Peach ethylene response factor PpeERF2 represses the expression of ABA biosynthesis and cell wall degradation genes during fruit ripening.

Ethylene response factors (ERFs) are known to regulate fruit ripening. However, the ERF regulatory networks are not clear. In this study, we have shown that peach (Prunus persica) PpeERF2 regulates fruit ripening through suppressing the expression of two ABA biosynthesis genes (PpeNCED2, PpeNCED3) and a cell wall degradation gene (PpePG1). The transcript levels of PpeERF2 in fruit were opposite to that of PpeNCED2, PpeNCED3 and PpePG1 during ripening and in response to various ripening treatments. PpeERF2 was found to bind to the PpeNCED2, PpeNCED3 and PpePG1 promotors as demonstrated by yeast one-hybrid (Y1H) and EMSA assays; and further found to repress the promoter activities of the three genes in tobacco leaf tissues after Agrobacterium infiltration. Taken together, these results provide new information for a better understanding of the crosstalk network between ethylene signaling, cell wall degradation and ABA biosynthesis during fruit ripening.

PpERF3 positively regulates ABA biosynthesis by activating PpNCED2/3 transcription during fruit ripening in peach.

The plant hormone ethylene regulates ripening in climacteric fruits. The phytohormone abscisic acid (ABA) affects ethylene biosynthesis, but whether ethylene influences ABA biosynthesis is unknown. To explore this possibility, we investigated the interactions between the ABA biosynthesis genes PpNCED2/3 and the ethylene response transcription factor PpERF3 in peach fruit. The ABA content increased during fruit maturation and reached a peak at stage S4 III. The increase was greatly inhibited by the ethylene inhibitor 1-MCP, which also suppressed PpERF3 expression. PpERF3 shared a similar expression profile with PpNCED2/3, encoding a rate-limiting enzyme involved in ABA biosynthesis, during fruit ripening. A yeast one-hybrid assay suggested that the nuclear-localized PpERF3 might bind to the promoters of PpNCED2/3. PpERF3 increased the expression of PpNCED2/3 as shown by dual-luciferase reporters, promoter-GUS assays and transient expression analyses in peach fruit. Collectively, these results suggest that ethylene promotes ABA biosynthesis through PpERF3's regulation of the expression of ABA biosynthesis genes PpNCED2/3.