Genome-Wide Analysis of ATP Binding Cassette (ABC) Transporters in Peach (Prunus persica) and Identification of a Gene PpABCC1 Involved in Anthocyanin Accumulation.

The ATP-binding cassette (ABC) transporter family is a large and diverse protein superfamily that plays various roles in plant growth and development. Although the ABC transporters are known to aid in the transport of a wide range of substrates across biological membranes, their role in anthocyanin transport remains elusive. In this study, we identified a total of 132 putative ABC genes in the peach genome, and they were phylogenetically classified into eight subfamilies. Variations in spatial and temporal gene expression levels resulted in differential expression patterns of PpABC family members in various tissues of peach. PpABCC1 was identified as the most likely candidate gene essential for anthocyanin accumulation in peach. Transient overexpression of PpABCC1 caused a significant increase in anthocyanin accumulation in tobacco leaves and peach fruit, whereas virus-induced gene silencing of PpABCC1 in the blood-fleshed peach resulted in a significant decrease in anthocyanin accumulation. The PpABCC1 promoter contained an MYB binding cis-element, and it could be activated by anthocyanin-activator PpMYB10.1 based on yeast one-hybrid and dual luciferase assays. Thus, it seems that PpABCC1 plays a crucial role in anthocyanin accumulation in peach. Our results provide a new insight into the vacuolar transport of anthocyanins in peach.

Epigenetic reprogramming of H3K27me3 and DNA methylation during leaf-to-callus transition in peach.

Plant tissues are capable of developing unorganized cell masses termed calluses in response to the appropriate combination of auxin and cytokinin. Revealing the potential epigenetic mechanisms involved in callus development can improve our understanding of the regeneration process of plant cells, which will be beneficial for overcoming regeneration recalcitrance in peach. In this study, we report on single-base resolution mapping of DNA methylation and reprogramming of the pattern of trimethylation of histone H3 at lysine 27 (H3K27me3) at the genome-wide level during the leaf-to-callus transition in peach. Overall, mCG and mCHH were predominant at the genome-wide level and mCG was predominant in genic regions. H3K27me3 deposition was mainly detected in the gene body and at the TSS site, and GAGA repetitive sequences were prone to recruit H3K27me3 modification. H3K27me3 methylation was negatively correlated with gene expression. In vitro culture of leaf explants was accompanied by DNA hypomethylation and H3K27me3 demethylation, which could activate auxin- and cytokinin-related regulators to induce callus development. The DNA methylation inhibitor 5-azacytidine could significantly increase callus development, while the H3K27me3 demethylase inhibitor GSK-J4 dramatically reduced callus development. These results demonstrate the roles of DNA methylation and H3K27me3 modification in mediating chromatin status during callus development. Our study provides new insights into the epigenetic mechanisms through which differentiated cells acquire proliferative competence to induce callus development in plants.

Comprehensive analysis of quality characteristics in main commercial coffee varieties and wild Arabica in Kenya.

Coffee flavor is a complex commercial trait and its generation mechanisms remain largely unclear. Here, we investigated non-volatile and volatile compounds in the AA grade coffee beans of cultivated and wild Coffea arabica accessions in Kenya. An increased accumulation of trigonelline and sucrose along with a decreased accumulation of caffeine and 5-caffeoylquinic acid or chlorogenic acid (CGA) relative to wild Arabica contribute to the improved flavor of commercial varieties. Trigonelline was strongly associated with attributes of coffee brews such as pH, aroma intensity and antioxidant activity, suggesting that it is one of the main flavor precursors. Partial least squares discriminant analysis (PLS-DA) identified 18 volatiles that could potentially define flavor quality of coffee brews, with pyrazines and thiols as the major coffee flavor determinants.

PpHYH is responsible for light-induced anthocyanin accumulation in fruit peel of Prunus persica.

Peach Prunus persica is an economically important fruit tree crop worldwide. Although the external color of fruit is an important aspect of fruit quality, the mechanisms underlying its formation remain elusive in peach. Here, we report an elongated hypocotyl 5-homolog gene PpHYH involved in the regulation of anthocyanin pigmentation in peach fruit peel. Anthocyanin accumulation in fruit peel is light-dependent in peach. PpHYH had no auto-activation activity and its transcription was induced by sunlight. PpHYH activated transcription of a cluster of three PpMYB10 genes in the present of a cofactor PpBBX4 encoding a B-BOX protein, leading to anthocyanin accumulation in the sun-exposed peel. However, the PpHYH activity was repressed by a negative regulator of PpCOP1 encoding constitutive photomorphogenesis protein 1 which accumulated in the nucleus under dark condition, resulting in failure of anthocyanin accumulation in the shaded peel. PpCOP1 was re-localized into the cytosol under light condition, in accordance with fruit peel pigmentation. Additionally, transport of anthocyanins from the cytoplasm to the vacuole was a rate-limiting step for anthocyanin accumulation in peach fruit peel. Our results reveal for the first time the HYH gene involved in the regulation of anthocyanin accumulation in fruits, and provide target genes for genetic manipulation of fruit coloration.

Two R2R3-MYB genes cooperatively control trichome development and cuticular wax biosynthesis in Prunus persica.

The fruit surface has an enormous impact on the external appearance and postharvest shelf-life of fruit. Here, we report two functionally redundant genes, PpMYB25 and PpMYB26, involved in regulation of fruit skin texture in peach. PpMYB25 can activate transcription of PpMYB26 and they both induce trichome development and cuticular wax accumulation, resulting in peach fruit with a fuzzy and dull appearance. By contrast, nonfunctional mutation of PpMYB25 caused by an insertional retrotransposon in the last exon in nectarine fails to activate transcription of PpMYB26, resulting in nectarine fruit with a smooth and shiny appearance due to loss of trichome initiation and decreased cuticular wax accumulation. Secondary cell wall biosynthesis in peach fruit pubescence is controlled by a transcriptional regulatory network, including the master regulator PpNAC43 and its downstream MYB transcription factors such as PpMYB42, PpMYB46 and PpMYB83. Our results show that PpMYB25 and PpMYB26 coordinately regulate fruit pubescence and cuticular wax accumulation and their simultaneous perturbation results in the origin of nectarine, which is botanically classified as a subspecies of peach.

Two vacuolar invertase inhibitors PpINHa and PpINH3 display opposite effects on fruit sugar accumulation in peach.

Soluble sugars are an important determinant of fruit taste, but their accumulation mechanisms remain elusive. In this study, we report two vacuolar invertase inhibitor genes involved in sugar accumulation in peach, PpINHa and PpINH3. Transient overexpression of PpINH3 in peach fruits resulted in an increase in sugar content, while the opposite trend was detected for PpINHa. Unexpectedly, PpINH3 and PpINHa both had no physical interaction with vacuolar invertase (VIN). Moreover, the PpVIN genes had no or extremely low expression in fruits at the ripening stage. These results suggested that the regulatory role of PpINHa and PpINH3 in sugar accumulation is unlikely due to their interaction with PpVINs. Additionally, overexpression of PpINHa and PpINH3 had an impact on transcription of genes related to fruit sugar metabolism and transport, which is likely responsible for their regulatory role in fruit sugar accumulation. Altogether, these results indicated an important role of PpINHs in fruit accumulation in peach. Our study provides new insights into molecular mechanisms underlying sugar accumulation, which could be useful for genetic improvement of fruit taste in breeding programs of peach and other fruit crops.

The MADS-box gene PpPI is a key regulator of the double-flower trait in peach.

Double flower is an invaluable trait in ornamental peach, but the mechanism underlying its development remains largely unknown. Here, we report the roles of ABCE model genes in double flower development in peach. A total of nine ABCE regulatory genes, including eight MADS-box genes and one AP2/EREBP gene, were identified in the peach genome. Subcellular localization assay showed that all the ABCE proteins were localized in the nucleus. Four genes, PpAP1, PpAP3, PpSEP3, and PpPI, showed a difference in expression levels between single and double flowers. Ectopic overexpression of PpPI increased petal number in Arabidopsis, while transgenic lines overexpressing PpAP3 or PpSEP3 were morphologically similar to wild-type. Ectopic overexpression of PpAP1 resulted in a significant decrease in the number of basal leaves and caused early flowering. These results suggest that PpPI is likely crucial for double flower development in peach. In addition, double flowers have petaloid sepals and stamens, and single flower could occasionally change to be double flower by converting stamens to petals in peach, suggesting that the double-flower trait is likely to have evolved from an ancestral single-flower structure. Our results provide new insights into mechanisms underlying the double-flower trait in peach.

Unraveling a genetic roadmap for improved taste in the domesticated apple.

Although taste is an important aspect of fruit quality, an understanding of its genetic control remains elusive in apple and other fruit crops. In this study, we conducted genomic sequence analysis of 497 Malus accessions and revealed erosion of genetic diversity caused by apple breeding and possible independent domestication events of dessert and cider apples. Signatures of selection for fruit acidity and size, but not for fruit sugar content, were detected during the processes of both domestication and improvement. Furthermore, we found that single mutations in major genes affecting fruit taste, including Ma1, MdTDT, and MdSOT2, dramatically decrease malate, citrate, and sorbitol accumulation, respectively, and correspond to important domestication events. Interestingly, Ma1 was identified to have pleiotropic effects on both organic acid content and sugar:acid ratio, suggesting that it plays a vital role in determining fruit taste. Fruit taste is unlikely to have been negatively affected by linkage drag associated with selection for larger fruit that resulted from the pyramiding of multiple genes with minor effects on fruit size. Collectively, our study provides new insights into the genetic basis of fruit quality and its evolutionary roadmap during apple domestication, pinpointing several candidate genes for genetic manipulation of fruit taste in apple.

Potential Association of Reactive Oxygen Species With Male Sterility in Peach.

Male sterility is an important agronomic trait for hybrid vigor utilization and hybrid seed production, but its underlying mechanisms remain to be uncovered. Here, we investigated the mechanisms of male sterility in peach using a combined cytology, physiology, and molecular approach. Cytological features of male sterility include deformed microspores and tapetum cells along with absence of pollen grains. Microspores had smaller nucleus at the mononuclear stage and were compressed into belts and subsequently disappeared in the anther cavity, whereas tapetum cells were swollen and vacuolated, with a delayed degradation to flowering time. Male sterile anthers had an ROS burst and lower levels of major antioxidants, which may cause abnormal development of microspores and tapetum, leading to male sterility in peach. In addition, the male sterility appears to be cytoplasmic in peach, which could be due to sequence variation in the mitochondrial genome. Our results are helpful for further investigation of the genetic mechanisms underlying male sterility in peach.

Transcriptome analysis and molecular mechanism of linseed (Linum usitatissimum L.) drought tolerance under repeated drought using single-molecule long-read sequencing.

BACKGROUND: Oil flax (linseed, Linum usitatissimum L.) is one of the most important oil crops., However, the increases in drought resulting from climate change have dramatically reduces linseed yield and quality, but very little is known about how linseed coordinates the expression of drought resistance gene in response to different level of drought stress (DS) on the genome-wide level. RESULTS: To explore the linseed transcriptional response of DS and repeated drought (RD) stress, we determined the drought tolerance of different linseed varieties. Then we performed full-length transcriptome sequencing of drought-resistant variety (Z141) and drought-sensitive variety (NY-17) under DS and RD stress at the seedling stage using single-molecule real-time sequencing and RNA-sequencing. Gene Ontology (GO) and reduce and visualize GO (REVIGO) enrichment analysis showed that upregulated genes of Z141 were enriched in more functional pathways related to plant drought tolerance than those of NY-17 were under DS. In addition, 4436 linseed transcription factors were identified, and 1190 were responsive to stress treatments. Moreover, protein-protein interaction (PPI) network analysis showed that the proline biosynthesis pathway interacts with stress response genes through RAD50 (DNA repair protein 50) interacting protein 1 (RIN-1). Finally, proline biosynthesis and DNA repair structural gene expression patterns were verified by RT- PCR. CONCLUSIONS: The drought tolerance of Z141 may be related to its upregulation of drought tolerance genes under DS. Proline may play an important role in linseed drought tolerance by maintaining cell osmotic and protecting DNA from ROS damage. In summary, this study provides a new perspective to understand the drought adaptability of linseed.

A 1.7-Mb chromosomal inversion downstream of a PpOFP1 gene is responsible for flat fruit shape in peach.

Flat peaches have become popular worldwide due to their novelty and convenience. The peach flat fruit trait is genetically controlled by a single gene at the S locus, but its genetic basis remains unclear. Here, we report a 1.7-Mb chromosomal inversion downstream of a candidate gene encoding OVATE Family Protein, designated PpOFP1, as the causal mutation for the peach flat fruit trait. Genotyping of 727 peach cultivars revealed an occurrence of this large inversion in flat peaches, but absent in round peaches. Ectopic overexpression of PpOFP1 resulted in oval-shaped leaves and shortened siliques in Arabidopsis, suggesting its role in repressing cell elongation. Transcriptional activation of PpOFP1 by the chromosomal inversion may repress vertical elongation in flat-shaped fruits at early stages of development, resulting in the flat fruit shape. Moreover, PpOFP1 can interact with fruit elongation activator PpTRM17, suggesting a regulatory network controlling fruit shape in peach. Additionally, screening of peach wild relatives revealed an exclusive presence of the chromosomal inversion in P. ferganensis, supporting that this species is the ancestor of the domesticated peach. This study provides new insights into mechanisms underlying fruit shape evolution and molecular tools for genetic improvement of fruit shape trait in peach breeding programmes.

Assessment of organic acid accumulation and its related genes in peach.

Fruit acidity is an important determinant of peach organoleptic quality, but its regulatory mechanism remains elusive. Measurement of organic acids in ripe fruits of seventy-five peach cultivars revealed the predominant components malate and citrate, accompanied by quinate. Organic acid accumulation increased at early stages of fruit growth, but exhibited a more dramatic reduction in low-acid cultivar during later stages of fruit development compared to high-acid cultivars. Low-acid cultivars showed citrate degradation and less transport of malate into the vacuole due to up- and down-regulation of a GABA pathway gene GAD and a malate transporter gene ALMT9, respectively. The NAD-MDH1 gene might control the rate-limiting step in malate synthesis, while three genes, PDK, PK, and ADH, could affect citrate synthesis through the pyruvate-to-acetyl-CoA-to-citrate pathway. Altogether, these results suggested that malate accumulation is controlled at the level of metabolism and vacuolar storage, while metabolism is crucial for citrate accumulation in peach.

The sucrose transporter MdSUT4.1 participates in the regulation of fruit sugar accumulation in apple.

BACKGROUND: Sugar content is an important determinant of fruit sweetness, but details on the complex molecular mechanism underlying fruit sugar accumulation remain scarce. Here, we report the role of sucrose transporter (SUT) family in regulating fruit sugar accumulation in apple. RESULTS: Gene-tagged markers were developed to conduct candidate gene-based association study, and an SUT4 member MdSUT4.1 was found to be significantly associated with fruit sugar accumulation. MdSUT4.1 encodes a tonoplast localized protein and its expression level had a negative correlation with fruit sugar content. Overexpression of MdSUT4.1 in strawberry and apple callus had an overall negative impact on sugar accumulation, suggesting that it functions to remobilize sugar out of the vacuole. In addition, MdSUT4.1 is located on chromosomal region harboring a previously reported QTL for sugar content, suggesting that it is a candidate gene for fruit sugar accumulation in apple. CONCLUSIONS: MdSUT4.1 is involved in the regulation of fruit sugar accumulation in apple. This study is not only helpful for understanding the complex mechanism of fruit sugar accumulation, but it also provides molecular tools for genetic improvement of fruit quality in breeding programs of apple.

Identification of EIL and ERF Genes Related to Fruit Ripening in Peach.

Peach (Prunus persica) is a climacteric fruit with a relatively short shelf life due to its fast ripening or softening process. Here, we report the association of gene families encoding ethylene insensitive-3 like (EIL) and ethylene response factor (ERF) with fruit ripening in peach. In total, 3 PpEILs and 12 PpERFs were highly expressed in fruit, with the majority showing a peak of expression at different stages. All three EILs could activate ethylene biosynthesis genes PpACS1 and PpACO1. One out of the 12 PpERFs, termed PpERF.E2, is a homolog of ripening-associated ERFs in tomato, with a consistently high expression throughout fruit development and an ability to activate PpACS1 and PpACO1. Additionally, four subgroup F PpERFs harboring the EAR repressive motif were able to repress the PpACO1 promoter but could also activate the PpACS1 promoter. Promoter deletion assay revealed that PpEILs and PpERFs could participate in transcriptional regulation of PpACS1 through either direct or indirect interaction with various cis-elements. Taken together, these results suggested that all three PpEILs and PpERF.E2 are candidates involved in ethylene biosynthesis, and EAR motif-containing PpERFs may function as activator or repressor of ethylene biosynthesis genes in peach. Our study provides an insight into the roles of EILs and ERFs in the fruit ripening process.

Evolutionary rate variation among genes involved in galactomannan biosynthesis in Coffea canephora.

The endosperm cell walls of mature coffee seeds accumulate large amounts of mannan storage polysaccharides, which serve as nutrient reserve for embryo and contribute to beverage quality. Our study investigated the evolutionary patterns of key galactomannan (GM) biosynthesis genes using d N/d S ratio, synteny, and phylogenetic analysis and detected heterogeneity in rate of evolution among gene copies. Selection ratio index revealed evidence of positive selection in the branch editing gene Coffea canephora alpha (α) galactosidase (Cc-alpha Gal) at Cc11_g15950 copy (ω = 1.12), whereas strong purifying selection on deleterious mutations was observed in the Coffea canephora uridine diphosphate (UDP)-glucose 4'-epimerase (Cc-UG4E) and Coffea canephora mannose-1P guanylytransferase (Cc-MGT) genes controlling the crucial nucleotide carbon sugar building blocks flux in the pathway. Relatively low sequence diversity and strong syntenic linkages were detected in all GM pathway genes except in Cc-alpha Gal, which suggests a correlation between selection pressure and nucleotide diversity or synteny analysis. In addition, phylogenetic analysis revealed independent evolution or expansion of GM pathway genes in different plant species, with no obvious inferable clustering patterns according to either gene family or congruent with evolutionary plants lineages tested due to high dynamic nature and specific biochemical cell wall modification requirements. Altogether, our study shows a significant high rate of evolutionary variation among GM pathway genes in the diploid C. canephora and demonstrates the inherent variation in evolution of gene copies and their potential role in understanding selection rates in a homogenously connected metabolic pathway.

Development of a fast and efficient root transgenic system for functional genomics and genetic engineering in peach.

Peach is an economically import fruit crop worldwide, and serves as a model species of the Rosaceae family as well. However, peach functional genomics studies are severely hampered due to its recalcitrance to regeneration and stable transformation. Here, we report a fast and efficient Agrobacterium rhizogenes-mediated transformation system in peach. Various explants, including leaf, hypocotyl and shoot, were all able to induce transgenic hairy roots, with a transformation efficiency of over 50% for hypocotyl. Composite plants were generated by infecting shoots with A. rhizogenes to induce transgenic adventitious hairy roots. The composite plant system was successfully used to validate function of an anthocyanin-related regulatory gene PpMYB10.1 in transgenic hairy roots, and two downstream genes, PpUFGT and PpGST, were strongly activated. Our stable and reproductive A. rhizogenes-mediated transformation system provides an avenue for gene function assay, genetic engineering, and investigation of root-rhizosphere microorganism interaction in peach.

Functional Analysis Reveals the Regulatory Role of PpTST1 Encoding Tonoplast Sugar Transporter in Sugar Accumulation of Peach Fruit.

Sugar content is related to fruit sweetness, and the complex mechanisms underlying fruit sugar accumulation still remain elusive. Here, we report a peach PpTST1 gene encoding tonoplast sugar transporter that is located in the quantitative trait loci (QTL) interval on Chr5 controlling fruit sucrose content. One derived Cleaved Amplified Polymorphic Sequence (dCAPS) marker was developed based on a nonsynonymous G/T variant in the third exon of PpTST1. Genotyping of peach cultivars with the dCAPS marker revealed a significant difference in fruit sugar content among genotypes. PpTST1 is located in the tonoplast, and substitution of glutamine by histidine caused by the G/T variation has no impact on subcellular location. The expression profile of PpTST1 exhibited a consistency with the sugar accumulation pattern, and its transient silencing significantly inhibited sugar accumulation in peach fruits. All of these results demonstrated the role of PpTST1 in regulating sugar accumulation in peach fruit. In addition, cis-elements for binding of MYB and WRKY transcript factors were found in the promoter sequence of PpTST1, suggesting a gene regulatory network of fruit sugar accumulation. Our results are not only helpful for understanding the mechanisms underlying fruit sugar accumulation, but will also be useful for the genetic improvement of fruit sweetness in peach breeding programs.

Analysis of sorbitol content variation in wild and cultivated apples.

BACKGROUND: Sorbitol is the major sugar alcohol in apple and its accumulation in fruit is associated with fruit sweetness. However, little is known about variation in sorbitol content in fruits of apple germplasm. In this study, we investigated sorbitol content in mature fruits of 243 apple cultivars and 20 wild relatives using high-performance liquid chromatography (HPLC). RESULTS: Sorbitol accumulation showed a significant variation in apple germplasm. Overall, cultivated fruits had significantly lower content of sorbitol than wild fruits. Fruit sorbitol concentration was significantly correlated with fruit size and acidity that are extensively domesticated traits. Hence, the variation in sorbitol accumulation between cultivated and wild fruits may be the indirect result of fruit size and acidity selection during domestication. Moreover, sorbitol content was maintained at low levels throughout fruit development, with a dramatic decrease at the middle stage. The SDH1 gene was highly expressed throughout fruit development, and its expression showed a significant correlation with fruit sorbitol concentration, suggesting its potential role in apple fruit sorbitol accumulation. CONCLUSIONS: The finding that there is a great variation in fruit sorbitol content among apple germplasm will be helpful for genetic improvement of fruit sorbitol content in apple breeding programs. © 2019 Society of Chemical Industry.

A Ma10 gene encoding P-type ATPase is involved in fruit organic acid accumulation in apple.

Acidity is one of the main determinants of fruit organoleptic quality. Here, comparative transcriptome analysis was conducted between two cultivars that showed a significant difference in fruit acidity, but contained homozygous non-functional alleles at the major gene Ma1 locus controlling apple fruit acidity. A candidate gene for fruit acidity, designated M10, was identified. The M10 gene encodes a P-type proton pump, P3A -ATPase, which facilitates malate uptake into the vacuole. The Ma10 gene is significantly associated with fruit malate content, accounting for ~7.5% of the observed phenotypic variation in apple germplasm. Subcellular localization assay showed that the Ma10 is targeted to the tonoplast. Overexpression of the Ma10 gene can complement the defect in proton transport of the mutant YAK2 yeast strain and enhance the accumulation of malic acid in apple callus. Moreover, its ectopic expression in tomato induces a decrease in fruit pH. These results suggest that the Ma10 gene has the capacity for proton pumping and plays an important role in fruit vacuolar acidification in apple. Our study provides useful knowledge towards comprehensive understanding of the complex mechanism regulating apple fruit acidity.

Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation.

Anthocyanin and proanthocyanidin (PA) accumulation is regulated by both myeloblastosis (MYB) activators and repressors, but little information is available on hierarchical interactions between the positive and negative regulators. Here, we report on a R2R3-MYB repressor in peach, designated PpMYB18, which acts as a negative regulator of anthocyanin and PA accumulation. PpMYB18 can be activated by both anthocyanin- and PA-related MYB activators, and is expressed both at fruit ripening and juvenile stages when anthocyanins or PAs, respectively, are being synthesized. The PpMYB18 protein competes with MYB activators for binding to basic Helix Loop Helixes (bHLHs), which develops a fine-tuning regulatory loop to balance PA and anthocyanin accumulation. In addition, the bHLH binding motif in the R3 domain and the C1 and C2 repression motifs in the C-terminus of PpMYB18 both confer repressive activity of PpMYB18. Our study also demonstrates a modifying negative feedback loop, which prevents cells from excess accumulation of anthocyanin and PAs, and serves as a model for balancing secondary metabolite accumulation at the transcriptional level.