In this work, a recombinant plum pox virus (PPV, Sharka) encoding green fluorescent protein is used to study its effect on antioxidant enzymes and protein expression at the subcellular level in pea plants (cv. Alaska). PPV had produced chlorotic spots as well as necrotic spots in the oldest leaves at 13–15 d post-inoculation. At 15 d post-inoculation, PPV was present in the chlorotic and necrotic areas, as shown by the fluorescence signal produced by the presence of the green fluorescent protein. In the same areas, an accumulation of reactive oxygen species was noticed. Studies with laser confocal and electron microscopy demonstrated that PPV accumulated in the cytosol of infected cells. In addition, PPV infection produced an alteration in the chloroplast ultrastructure, giving rise to dilated thylakoids, an increase in the number of plastoglobuli, and a decreased amount of starch content. At 3 d post-inoculation, although no changes in the oxidative stress parameters were observed, an increase in the chloroplastic hydrogen peroxide levels was observed that correlated with a decrease in the enzymatic mechanisms involved in its elimination (ascorbate peroxidase and peroxidase) in this cell compartment. These results indicate that an alteration in the chloroplastic metabolism is produced in the early response to PPV. This oxidative stress is more pronounced during the development of the disease (15 d post-inoculation) judging from the increase in oxidative stress parameters as well as the imbalance in the antioxidative systems, mainly at the chloroplastic level. Finally, proteomic analyses showed that most of the changes produced by PPV infection with regard to protein expression at the subcellular level were related mainly to photosynthesis and carbohydrate metabolism. It seems that PPV infection has some effect on PSII, directly or indirectly, by decreasing the amount of Rubisco, oxygen-evolving enhancer, and PSII stability factor proteins. The results indicate that Sharka symptoms observed in pea leaves could be due to an imbalance in antioxidant systems as well as to an increased generation of reactive oxygen species in chloroplasts, induced probably by a disturbance of the electron transport chain, suggesting that chloroplasts can be a source of oxidative stress during viral disease development.

This work describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxymercuribenzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (class I APX) and pHMB-insensitive APX (class III APX), superoxide dismutase (SOD), peroxidase (POX), NADH-POX, and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in class I APX, POX, NADH-POX, and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H2O2 contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H2O2 levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H2O2 levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4-6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. It is concluded that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plants.

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 (Fv′/Fm′) 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.

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The effect of plum pox virus (PPV) infection on the response of some antioxidant enzymes was studied in two apricot cultivars, which behaved differently against PPV infection: cultivar Real Fino (susceptible) and cultivar Stark Early Orange (cv. SEO, resistant). In the susceptible cultivar, PPV produced a decrease in F PSII , F 0 v /F 0 m and Q p . PPV infection produced a drop in p-hydroxy mercury benzoic acid (pHMB)-sensitive ascorbate peroxidase, dehydroascorbate reductase and peroxidase in the soluble fraction from susceptible plants, whereas in the resistant apricot cultivar, pHMB-insensitive ascorbate peroxidase, monodehydroascorbate reductase, glutathione reductase and superoxide dismutase increased. However, catalase decreased in the soluble fractions from both infected cultivars. Long-term PPV infection also produced a decrease in the chloroplastic ascorbate-glutathione cycle enzymes only in the susceptible plants. As a consequence of PPV infection, an oxidative stress, indicated by an increase in lipid peroxidation and in protein oxidation, was produced only in the leaves from the susceptible cultivar which was also monitored by the diaminobenzidine peroxidasecoupled H 2 O 2 probe. The loss of F PSII , indicative of activated oxygen species production, and the decrease in the levels of antioxidant enzymes in chloroplasts from susceptible plants could be responsible for the chlorosis symptoms observed. The results suggest that the higher antioxidant capacity showed by cv. SEO could be a consequence of a systemic acquired resistance induced by PPV penetration in stem tissue at the graft site and could be related, among other factors, to their resistance to PPV.

Marker-assisted selection (MAS) in stone fruit (Prunus species) breeding is currently difficult to achieve due to the polygenic nature of the most relevant agronomic traits linked to fruit quality. Genotyping by sequencing (GBS), however, provides a large quantity of useful data suitable for fine mapping using Single Nucleotide Polymorphisms (SNPs) from a reference genome. In this study, GBS was used to genotype 272 seedlings of three F1 Japanese plum (Prunus salicina Lindl) progenies derived from crossing “98–99” (as a common female parent) with “Angeleno,” “September King,” and “September Queen” as male parents. Raw sequences were aligned to the Peach genome v1, and 42,909 filtered SNPs were obtained after sequence alignment. In addition, 153 seedlings from the “98–99” × “Angeleno” cross were used to develop a genetic map for each parent. A total of 981 SNPs were mapped (479 for “98–99” and 502 for “Angeleno”), covering a genetic distance of 688.8 and 647.03 cM, respectively. Fifty five seedlings from this progeny were phenotyped for different fruit quality traits including ripening time, fruit weight, fruit shape, chlorophyll index, skin color, flesh color, over color, firmness, and soluble solids content in the years 2015 and 2016. Linkage-based QTL analysis allowed the identification of genomic regions significantly associated with ripening time (LG4 of both parents and both phenotyping years), fruit skin color (LG3 and LG4 of both parents and both years), chlorophyll degradation index (LG3 of both parents in 2015) and fruit weight (LG7 of both parents in 2016). These results represent a promising situation for GBS in the identification of SNP variants associated to fruit quality traits, potentially applicable in breeding programs through MAS, in a highly heterozygous crop species such as Japanese plum.

A total of 137 accessions from 18 wild almond species were collected from Iran and leaf and fruit traits were characterized. Also evaluated were flowering and ripening date, self-incompatibility and kernel bitterness. An extensive phenotypic diversity was found both among and within species. Differences in average leaf dimensions among and within species were associated with average rainfall but not altitude of collection site. Adjacent accessions located in drier areas had smaller leaf dimensions than those located in semi-humid or humid regions. No relation was found between average fruit dimensions and collection site conditions. Principal component analysis revealed that the nut weight and width, and the kernel weight had highest loading in the first component accounting for 45.8% of total variation. In contrast, leaf traits in the second component accounted for 22.3% of total variation. No significant correlations were detected between leaf dimensions and fruit traits in all species evaluated. Results document a rich source of new germplasm for almond improvement programs. Small fruit size, pollen-pistil self-incompatibility, and bitter kernel flavour are the most common obstacles to the utilization of this wild germplasm in breeding.