Phenolic compounds in peach (Prunus persica) cultivars at harvest and during fruit maturation

Six peach and six nectarine cultivars were evaluated for the phenolic content in their pulp and peel tissues. Chlorogenic acid, catechin, epicatechin, rutin and cyanidin-3-glucoside were detected as the main phenolic compounds of ripened fruits. The concentration was always higher in peel tissue, with average values ranging from 1 to 8 mg g⁻¹ dry weight (DW) depending on cultivar. Of the tested varieties, the white-flesh nectarine 'Silver Rome' emerged as the cultivar with the highest amount of total phenolics. Phenolic compounds were also profiled during fruit growth and ripening in the yellow nectarine cv. 'Stark Red Gold', which showed a decreasing concentration during fruit development in both peel and pulp tissues. Average amounts of total phenolics were approximately 25 mg g⁻¹ DW 60 days after full bloom and decreased to 3 mg g⁻¹ DW at ripening in pulp tissue. Differences among peel and pulp composition show the different dietetic and antioxidant potential of fruits consumed unpeeled and peeled.     

Molecular cloning, identification, and chromosomal localization of two MADS box genes in peach （Prunus persica）

MADS box proteins play an important role in floral development. To find genes involved in the floral transition of Prunus species, cDNAs for two MADS box genes, PpMADS1 and PpMADS10, were cloned using degenerate primers and 5'- and 3'- RACE based on the sequence database of P. persica and P. dulcis. The full length of PpMADS1 eDNA is 1, 071bp containing an open reading frame (ORF) of 717bp and coding for a polypeptide of 238 amino acid residues. The full length of PpMADS10 cDNA is 937bp containing an ORF of 633bp and coding for a polypeptide of 210 amino acid residues. Sequence comparison revealed that PpMADS1 and PpMADS10 were highly homologous to genes AP1 and PI in Arabidopsis, respectively. Phylogenetic analysis indicated that PpMADS1 belongs to the euAP1 clade of class A, and PpMADS10 is a member of GLO/PI clade of class B. RT-PCR analysis showed that PpMADS1 was expressed in sepal, petal, carpel, and fruit, which was slightly different from the expression pattern of AP1; PpMADS10 was expressed in petal and stamen, which shared the same expression pattern as PI. Using selective mapping strategy, PpMADS1 was assigned onto the Bin 1:50 on the G1 linkage group between the markers MCO44 and TSA2, and PpMADS10 onto the Bin 1:73 on the same linkage group between the markers Lap-1 and FGA8. Our results provided the basis for further dissection of the two MADS box gene function.     

Soil insects alter fine root demography in peach (Prunus persica)

Minirhizotrons were used to assess the effects of soil insect suppression on the demography of peach fine roots (<1 mm diameter) over two growing seasons. The experiment was conducted at the USDA–ARS Appalachian Fruit Research Station in Kearneysville, WV, USA using six 15‐year‐old peach trees. Clear butyrate minirhizotrons were installed beneath each tree in April 1996. Soil drench treatments were applied around individual minirhizotron tubes at monthly intervals and consisted of 1 L of water or 250 µL of a broad‐spectrum insecticide in 1 L of water. Roots were videotaped at 2‐ to 4‐week intervals during the 1996 and 1997 growing seasons. Insecticide application was associated with a significant increase in fine root longevity: the median lifespans of insecticide‐treated roots were 46–125 d longer than those of control roots. In addition, the development of brown pigmentation was significantly delayed in insecticide‐treated roots. Insecticide application did not appear to increase soil fertility, as accumulation of NO₃^–, NH₄⁺, and PO₄²‐ on mixed bed ion‐exchange resin was similar in treated and untreated soil. These results suggest that interactions with below‐ground insects can significantly influence root longevity and may alter the rate at which roots undergo developmental changes in anatomy and physiology.     

Purification and characterization of a beta-galactosidase from peach (Prunus persica)

A beta-galactosidase (EC 3.2.1.23) from peach (Prunus persica cv Mibackdo) was purified and characterized. The purified peach beta-galactosidase was 42 kDa in molecular mass and showed high enzyme activity against a the beta-galactosidase substrate, rho-nitrophenyl-beta-D-galactopyranoside. The Km and Vmax values of the enzyme activity of the peach beta-galactosidase were 5.16 and 0.19 mM for rho-nitrophenyl-beta-D-galactopyranoside mM/h, respectively. The optimum pH of the enzyme activity was pH 3.0, but it was relatively stable from pH 3.0-10.0. The temperature optimum was 50 degrees C. The enzyme activities were not improved in the buffers that contained Ca2+, Cu2+, Zn2+, and Mg2+, which indicates that the purified peach beta-galactosidase did not require these cations as co-factors. However, the enzyme was completely inhibited by Hg2+. The purified protein was cross-reacted with an antibody against the persimmon fruit beta-galactosidase. A further comparison of the N-terminal amino acid sequence of the purified protein showed high homologies to those of beta-galactosidase in apple (87%), persimmon (80%), and tomato (87%). Therefore, enzymatic, immunological, and molecular evidences in this study indicate that the purified 42-kDa protein is a peach beta-galactosidase.     

Identification of QTLs controlling seed dormancy in peach (Prunus persica)

Dormancy is a condition that delays or inhibits growth in seed, vegetative buds, and floral buds. In peach, seed germination occurs when seed accumulate sufficient stratification and growing degree hours to break dormancy and begin growing. Correlations have been reported between mean seed stratification requirements and mean bud chilling requirements among Prunus families, but an individual seed’s germination date and subsequent vegetative and floral bud break date are not correlated. Prior to this study, the genetic factors involved in regulating seed dormancy and their location on the peach genomic map were unknown. Segregating F₂ seed were collected from a high?×?low chill F₁ peach hybrid in 2005, 2006, and 2008. Germination date and growth habit was measured after the stratification requirement of the 2005 seed was fully met. The seed collected in 2006 and 2008 received varying amounts of stratification, which enabled data on stratification requirement, heat requirement, and growth habit to be collected. Genomic DNA was extracted from seedling leaf tissue and screened with SSR markers selected from the Prunus reference map at an average resolution of 20 cM. Seed dormancy quantitative trait loci (QTLs) were detected on G1, G4, G6/8, and G7. The QTLs detected on G6/8 and G7 were discovered in the same region as QTLs associated with floral bud chilling requirement and bloom time in peach.     

Dormancy in peach (Prunus persica L.) flower buds

Morphological studies were carried out with peach flower buds collected monthly in 1989 and 1990, from two months before leaf fall (7 March) until two to three weeks before bloom (7/8 August). Chilled (2–4°C for 30 days) and unchilled buds were exposed to 20 to 25°C, 100% RH and continuous light. Gibberellin A₃ (3 ng or 30 ng) was applied to some of the non-chilled cuttings at three days intervals. Then, 12, 19, and 26 days after they were planted, the buds were sampled and processed for histological studies. Cultured flower buds (chilled or unchilled) had accelerated anther and gynoecium morphogenesis after 12 days under controlled conditions, compared to buds processed immediately after collection from the field. Chilling treatment augmented the bud culture effect, while Gibberellin A₃ applications to the excised buds retarded bud morphogenesis to a stage comparable to that of buds collected directly from the field. This, suggests that the comparatively high levels of Gibberellin A_1/3 we previously found in mid winter [15, 18] could be at least one of the factors that controls floral bud dormancy by retarding anther and gynoecium development.     

Dormancy in peach (Prunus persica L.) flower buds

Summary Peach buds (floral and vegetative) were periodically collected from midsummer until the spring flowering and sprouted under continuous light, 100% relative humidity and 20–25°C. Treatments with 200 ppm gibberellin A₃ (GA₃) or chilling (2–4°C for 30 days before planting) were applied. Vegetative buds showed well-defined phenological stages: pre-dormancy, true dormancy, and end of dormancy. Both GA₃ and chilling treatments shortened the sprouting times of vegetative dormant buds close to those in predormancy. Isolated floral buds were irresponsive under all conditions and did not sprout even with the GA₃ or chilling treatments. In a comparative study with buds immediately after collection anatomical analysis demonstrated that vegetative buds were almost completely developed by midsummer/early automn and remained in a resting state until the end of winter. Floral buds developed continuously over the same period. Both types of verticils began to differentiate in midsummer. Sepals and petals developed mainly in late summer, androecious floral parts developed throughout the resting period, while gynoecious floral parts showed differentiation in late winter. The flower was completely formed a few days prior to blossoming. Thus, in isolated peach buds fertile verticils are not sufficiently developed during the resting time to allow sprouting.     

Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes

To determine the optimum conditions for Agrobacterium-mediated gene transfer, peach explants including cotyledons, embryonic axes and hypocotyl slices from non-germinated seeds and epicotyl internode slices from germinating seeds were exposed to Agrobacterium-mediated transformation treatments. The GUS (uidA) marker gene was tested using two different A. tumefaciens strains, three plasmids and four promoters [CaMV35s, (Aocs)3AmasPmas (“super-promoter”), mas-CaMV35s, and CAB]. GFP was tested with six A.␣tumefaciens strains, one plasmid (pLC101) and the doubleCaMV35s (dCaMV35s) promoter. The CaMV35s promoter produced more GUS expression than the CAB promoter. A. tumefaciens strains EHA105 and LBA4404 harboring the same plasmid (pBIN19) differed in their effects on GUS expression suggesting an interaction between A. tumefaciens strain and plasmid. A combination of A. tumefaciens EHA105, plasmid pBIN19 and the CaMV35s promoter produced the highest rates of transformation in peach epicotyl internodes (56.8%), cotyledons (52.7%), leaves (20%), and embryonic axes (46.7%) as evaluated by the percentage of explants expressing GUS 14 days after co-cultivation. GFP expression under the control of the dCaMV35s promoter was highest for internode explants but only reached levels of 18–19%. When GFP-containing plasmid pCL101 was combined with each of five A. tumefaciens strains the highest levels of transformation were 20–21% (internode and cotyledons, respectively). When nine peach genotypes were co-cultivated with A. tumefaciens strain EHA105 and GFP-containing plasmid pCL101 the highest levels of transformation were 26–28% (cotyledons and internodes, respectively). While GFP represents a potentially useful transformation marker that allows the non-destructive evaluation of transformation, rates of GFP transformation under the conditions of this study were low. It will be necessary to optimize expression of this marker gene in peach.     

High-throughput targeted SSR marker development in peach (Prunus persica).

Simple sequence repeats (SSRs) have proven to be highly polymorphic, easily reproducible, codominant markers. However, developing an SSR map is very time consuming and expensive, and most SSRs are not specifically linked to gene loci of immediate interest. The ideal situation would be to combine a high-throughput, relatively inexpensive mapping technique with rapid identification of SSR loci in mapped regions of interest. For this reason, we coupled the high-throughput technique of AFLP mapping with subsequent direct targeting of SSRs identified in AFLP-marked regions of interest. This approach relied on the availability of peach bacterial artificial chromosome (BAC) library resources. We present examples of using this strategy to rapidly identify SSR loci tightly linked to two important, simply inherited traits in peach (Prunus persica (L.) Batsch): root-knot nematode resistance and control of the evergrowing trait. SSRs developed in this study were also tested for their transportability in other Prunus species and in apricots.     

Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission

Using theArabidopsis ethylene receptorETR1 as a probe, we have isolated a tomato homologue (tETR) from a ripening cDNA library. The predicted amino acid sequence is 70% identical toETR1 and homologous to a variety of bacterial two component response regulators over the histidine kinase domain. Sequencing of four separate cDNAs indicates that tETR lacks the carboxyl terminal response domain and is identical to that encoded by the tomatoNever ripe gene. Ribonuclease protection showed tETR mRNA was undetectable in unripe fruit or pre-senescent flowers, increased in abundance during the early stages of ripening, flower senescence, and in abscission zones, and was greatly reduced in fruit of ripening mutants deficient in ethylene synthesis or response. These results suggest that changes in ethylene sensitivity are mediated by modulation of receptor levels during development.     

Mapping QTLs controlling fruit quality in peach (Prunus persica (L.) Batsch)

 The organoleptic quality of fleshy fruits is in a large part defined by their composition of soluble sugars and organic acids. An F₂ population issuing from a cross between two peach varieties, ‘Ferjalou Jalousia’, a non-acid peach, and ‘Fantasia’, an acid nectarine, was analysed over 2 successive years for agronomic characters and for molecular-marker (isoenzymes, RFLPs, RAPDs, IMAs and AFLPs) segregations. Blooming and maturity dates, as well as productivity, were noted for each tree. Four fruits per tree were analysed at maturity for fresh weight, colour, pH, titratable acidity, soluble-solids content (SSC), acid (malic, citric and quinic acids) and sugar (sucrose, glucose, fructose, sorbitol) contents. QTLs were detected for all fruit components analysed, except for fruit colour. The QTLs for nearly all components were present on two linkage groups. For productivity, fresh weight, pH, quinic acid, sucrose and sorbitol content, all the detected QTLs displayed the same effect as the parental phenotypes. By contrast, for maturity date, titratable acidity, malic and citric acids and fructose, some QTLs displayed the same effect as the parental phenotypes while others displayed the opposite effect. The fraction of the total variation in each trait throughout the population explained by the QTLs was very high and reached more than 90% for some characters. For most of the characters analysed, epistasis was observed between QTLs. Received: 10 October 1997 / Accepted: 18 August 1998     

Genetic diversity, linkage disequilibrium, and association mapping analyses of peach (Prunus persica) landraces in China

The genetic diversity, population structure, and linkage disequilibrium (LD) of peaches are greatly important in genome-wide association mapping. In the current study, 104 peach landrace accessions from six Chinese geographical regions were evaluated for fruit and phenological period. The accessions were genotyped with 53 genome-wide simple sequence repeat (SSR) markers. All SSR markers were highly polymorphic across the accessions, and a total of 340 alleles were detected, including 59 private alleles. Of the six regions studied, the northern part of China as well as the middle and lower reaches of the Changjiang River were found to be the most highly diverse genetically. Based on population structure analysis, the peaches were divided into five groups, which well agreed with the geographical distribution. Of the SSR pairs in these accessions, 18.07% (P?<?0.05) were in LD. The mean r ² value for all intrachromosomal loci pairs was 0.0149, and LD decayed at 6.01?cM. The general linear model was used to calculate the genome-wide marker-trait associations of 10 complex traits. The traits include flesh color around the stone, red pigment in the flesh, flesh texture, flesh adhesion, flesh firmness, fruit weight, chilling requirement, flowering time, ripening time, and fruit development period. These traits were estimated by analyzing the 104 landraces. Many of the associated markers were located in regions where quantitative trait loci (QTLs) were previously identified. Peach association mapping is an effective approach for identifying QTLs and may be an alternative to QTL mapping based on crosses between different lines.     

Oxidative stress induced by long-term plum pox virus infection in peach (Prunus persica)

In this study, the effect of long-term plum pox virus (PPV) infection on the response of certain antioxidant enzymes at the subcellular level was studied in peach plants ( Prunus persica (L.) Batch) (cv. GF305), which are characterized by great susceptibility to the virus. In infected plants, a decrease in the efficiency of excitation energy capture by PSII ( F _v'/ F _m') was observed, which was accompanied by a decrease in non-photochemical quenching (NPQ). p -Hydroxy-mercury benzoic acid (pHMB)-insensitive ascorbate peroxidase (APX) activity (class III peroxidase) was detected in both chloroplast and soluble fractions. In soluble fractions from inoculated peaches, a significant increase in pHMB-sensitive APX activity and a significant decrease in superoxide dismutase (SOD) activity were observed. These changes were correlated with the observations in isolated chloroplasts, where an increase in both pHMB-sensitive and pHMB-insensitive APX activities was observed, whereas significant decreases in SOD, monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) activities were produced. According to these results, as a consequence of PPV infection, an oxidative stress, indicated by an increase in lipid peroxidation and protein oxidation, was produced in peach leaves, which was monitored by the diaminobenzidine (DAB) peroxidase-coupled H₂O₂ probe. PPV infection produced an alteration in chloroplast ultrastructure, giving rise to dilated thylakoid membranes. PPV-infected peach leaves showed a decreased amount of starch in chloroplasts from palisade parenchyma, as well as an increase in the number and size of plastoglobuli, in relation to control plants. The results suggest that long-term PPV infection produces an oxidative stress, and that an antioxidative metabolism imbalance may be related to the progress of PPV infection and symptoms in peach plants.