María Ángeles Moreno

Evaluation of the Antioxidant Capacity, Phenolic Compounds, and Vitamin C Content of Different Peach and Nectarine [Prunus persica (L.) Batsch] Breeding Progenies

Antioxidant capacity and contents of total phenolics, anthocyanins, flavonoids, and vitamin C were evaluated in 218 genotypes from 15 peach and nectarine breeding progenies. Significant differences were found among progenies on the fruit antioxidant profile, corroborated by the high contribution showed by cross to the phenotypic variance of each phytochemical trait analyzed (16-45%). Phytochemical profile varied depending on peach/nectarine and yellow/white flesh color qualitative traits. On the other hand, no significant effect of year was found on the bioactive profile of peaches and nectarines. Antioxidant capacity was linearly correlated to total phenolic content, but correlation varied depending on the progeny. No correlation was found for vitamin C versus any other phytochemical trait. The results suggest the importance of genetic background on the antioxidant profile of peaches and nectarines and stress its relevance for the ultimate objective of this work: selecting new peach and nectarine genotypes rich in bioactive compounds to benefit consumers health.

Genetic diversity of Prunus rootstocks analyzed by RAPD markers

We have used RAPD markers to characterize Prunus rootstocks from different species, both commercial, and selected clones from the breeding program at Aula Dei Experimental Station (Zaragoza, Spain). Molecular markers were used to study the genetic variation among different species, and within species. Forty one genotypes were used in this study. They included P. amygdalo-persica, and P. persica × P. davidiana hybrids; P. cerasifera, P. domestica, and P. insititia clones, and other diverse interspecific hybrids, which were divided in three groups according to postulated taxonomic classification. Diversity patterns obtained from 80 RAPD primers were evaluated in a representative subset of genotypes. This screening helped to identify 7 RAPD primers that were selected to produce a combined classification of the whole set of rootstock clones. This analysis successfully clustered rootstocks according to the classification scheme widely used to characterize Prunus clones, mainly based on morphological descriptors. Further than that, it supported the alleged origin of some interspecific materials, and confirmed a case of possible misclassification (‘Myrobalan 29 C’). A more thorough diversity analysis was conducted within each group of materials, using larger sets of primers (12–14). After this analysis, disjointed clusters were formed for P. amygdalo-persica and P. persica × P. davidiana hybrids in one group, and for Myrobalan (P. cerasifera) and Marianna (P. cerasifera × P. munsoniana) plums in another group. P. insititia and P. domestica clones, however, formed a jumbled cluster, possibly due to genetic interchange among them during their domestication and breeding history.

Phenotypic diversity and relationships of fruit quality traits in peach and nectarine (Prunus persica (L.) Batsch) breeding progenies

Agronomic and fruit quality traits were evaluated and compared for three consecutive years on 1,111 seedlings coming from 15 peach and nectarine breeding crosses, grown under a Mediterranean climate. Significant differences among and within the different progenies were found for most of the traits analyzed. The breeding population segregated for several Mendelian characters such as peach or nectarine fruit, round or flat fruit, yellow or white flesh and freestone or clingstone. In addition, aborting fruit and flat fruit trees were found in our progeny, and our data seem to support multi-allelic control of both flat shape and aborting fruit. The variation within the progenies of some traits such as blooming and harvesting date, yield, fruit weight and SSC was continuous, suggesting a polygenic inheritance. Relationships between qualitative pomological traits and these agronomic and fruit quality parameters were also found. Valuable correlations among agronomic and fruit quality parameters were found, although coefficients of variation depending on the progeny should be considered. In addition, principal component analysis (PCA) revealed several relationships among quality traits in the evaluated progenies. Based on this evaluation, 26 outstanding genotypes were pre-selected from the initial breeding population for further studies.

Metabolic response in roots of Prunus rootstocks submitted to iron chlorosis

Iron deficiency induces several responses to iron shortage in plants. Metabolic changes occur to sustain the increased iron uptake capacity of Fe-deficient plants. We evaluated the metabolic changes of three Prunus rootstocks submitted to iron chlorosis and their different responses for tolerance using measurements of metabolites and enzymatic activities. The more tolerant rootstocks Adesoto (Prunus insititia) and GF 677 (Prunus amygdalus×Prunus persica), and the more sensitive Barrier (P. persica×Prunus davidiana) were grown hydroponically in iron-sufficient and -deficient conditions over two weeks. Sugar, organic and amino acid concentrations of root tips were determined after two weeks of iron shortage by proton nuclear magnetic resonance spectroscopy of extracts. Complementary analyses of organic acids were performed by liquid chromatography coupled to mass spectrometry. The major soluble sugars found were glucose and sucrose. The major organic acids were malic and citric acids, and the major amino acid was asparagine. Iron deficiency increased root sucrose, total organic and amino acid concentrations and phosphoenolpyruvate carboxylase activity. After two weeks of iron deficiency, the malic, citric and succinic acid concentrations increased in the three rootstocks, although no significant differences were found among genotypes with different tolerance to iron chlorosis. The tolerant rootstock Adesoto showed higher total organic and amino acid concentrations. In contrast, the susceptible rootstock Barrier showed lower total amino acid concentration and phosphoenolpyruvate carboxylase activity values. These results suggest that the induction of this enzyme activity under iron deficiency, as previously shown in herbaceous plants, indicates the tolerance level of rootstocks to iron chlorosis. The analysis of other metabolic parameters, such as organic and amino acid concentrations, provides complementary information for selection of genotypes tolerant to iron chlorosis.

Structural Basis of the Regulatory Mechanism of the Plant Cipk Family of Protein Kinases Controlling Ion Homeostasis and Abiotic Stress

Significance The transport of ions through the plant cell membrane establishes the key physicochemical parameters for cell function. Stress situations such as those created by soil salinity or low potassium conditions alter the ion transport across the membrane producing dramatic changes in the cell turgor, the membrane potential, and the intracellular pH and concentrations of toxic cations such as sodium and lithium. As a consequence, fundamental metabolic routes are inhibited. The CIPK family of 26 protein kinases regulates the function of several ion transporters at the cell membrane to restore ion homeostasis under stress situations. Our analyses provide an explanation on how the CIPKs are differentially activated to coordinate the adequate cell response to a particular stress. Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.

Elemental 2-D mapping and changes in leaf iron and chlorophyll in response to iron re-supply in iron-deficient GF 677 peach-almond hybrid

Iron is an essential micronutrient for plant growth and development, involved in key cellular processes. However, the distribution of Fe in plant tissues is still not well known. In the so-called Fe chlorosis paradox, leaves of fruit trees grown in the field usually have high concentrations of Fe but still are Fe-deficient. Leaves of the Prunus rootstock GF 677 (P. dulcis × P. persica) grown in hydroponics have been used to carry out two-dimensional (2-D) nutrient mapping by synchrotron radiation-induced X-ray fluorescence. Iron-deficient leaves accumulated more Fe in the midrib and veins, with Fe concentration being markedly lower in mesophyll leaf areas. The effects of Fe deficiency and Fe re-supply on leaf chlorophyll concentration and on the distribution of Fe and other nutrients within different plant tissues have been investigated in the same plants. After Fe re-supply, leaf Fe concentrations increased largely in all leaf types. However, whereas re-greening was almost completely achieved in apical leaves, in some expanded leaves the increase in chlorophyll concentration was only moderate. Therefore, after Fe re-supply Fe-deficient expanded leaves of the Prunus rootstock GF 677 had significant increases in Fe concentration but were still chlorotic. This is similar to what occurs in leaves of peach trees in field conditions, opening the possibility that this system could be used as a model to study the Fe chlorosis paradox.

Phenotypic diversity among local Spanish and foreign peach and nectarine [Prunus persica (L.) Batsch] accessions

Phenotypic data for tree and fruit characteristics was collected over three consecutive years from a germplasm collection of 94 peach and nectarine accessions representing both traditional Spanish as well as foreign cultivars with widespread global plantings. All accessions were grown at the Experimental Station of Aula Dei located in the Ebro Valley (Northern Spain, Zaragoza) under a Mediterranean climate. Tree traits evaluated included bloom and harvest date, vigor, yield, yield efficiency and flower and leaf characteristics. Fruit traits included fresh weight, firmness, soluble solids, titratable acidity, levels of individual soluble sugars (sucrose, glucose, fructose and sorbitol), vitamin C, total phenolics, flavonoids, anthocyanins, relative antioxidant capacity and ripening index. Extensive variability was observed for most qualitative and quantitative traits with significant correlations identified between many traits. While the traditional Spanish accessions demonstrated good adaptability to the northern Spain evaluation site, opportunities for continued improvement in tree and fruit quality traits were demonstrated by an extensive phenotypic variability within the germplasm collection.

Influence of antioxidant compounds, total sugars and genetic background on the chilling injury susceptibility of a non-melting peach (Prunus persica (L.) Batsch) progeny.

BACKGROUND To identify genotypes with good organoleptic properties, antioxidant-rich content and low susceptibility to chilling injury (CI), fruits from 130 peach cultivars were studied over three consecutive years. Pomological traits, l-ascorbic acid, flavonoids, total phenolics, relative antioxidant capacity (RAC) and sugars were determined. Major symptoms of CI developed at 5 °C, such as leatheriness, flesh browning, bleeding and loss of flavor, were evaluated. RESULTS The population exhibited wide phenotypic variation in agronomic and biochemical traits. Six genotypes with high total phenolics, RAC, flavonoids and total sugars were selected. The progeny also showed variability for all evaluated CI symptoms, and 16 genotypes showed considerably lower susceptibility to CI. After 2 weeks of cold storage, leatheriness and bleeding were the main CI symptoms observed, whereas flesh browning was predominant after 4 weeks. CONCLUSION It was possible to find varieties with high phenolic concentration and relatively low or intermediate CI susceptibility (22, 33, 68, 80, 81, 96 and 120). However, the correlations observed between CI and phenolic contents highlight their potential influence on susceptibility to internal browning. This relationship should be considered in the current breeding programs to select cultivars with high bioactive compound contents, health-enhancing properties and good postharvest performance.

Genetic variability of introduced and local Spanish peach cultivars determined by SSR markers

A set of 94 peach cultivars including Spanish native peach and foreign commercial cultivars were analyzed using 15 SSR markers, selected for their high level of polymorphism. The number of alleles obtained varied from two to 11 with an average of 6.73 giving 185 different genotypes. All the cultivars showed a unique genetic profile, each one using different genotypic combination of all loci. BPPCT001 was the most informative locus showing also the highest discrimination power. Only six loci allowed the unambiguous separation of all the Spanish native cultivars studied, and the genotypic combination of only eight loci permitted the total differentiation of the 94 peach cultivars analyzed. The six selected loci (BPPCT001, BPPCT006, BPPCT008, PS9f8, UDP98-022, and UDP98-412) seem to be very useful for future Spanish peach identification works, and they will help to establish a molecular data base for native peach cultivars. UPGMA analysis was performed from the genetic distance matrix, and allowed the arrangement of all genotypes according to their genetic diversity. The genetic diversity among cultivars, observed in this work, led to their separation according to their regional origin, their morphological characteristics, and especially according to their fruit traits. Analysis of molecular variance was performed for seven populations from different regions of Spain and USA to examine the distribution of genetic variation of the studied accessions, showing that the major variation occurred within populations in each geographic site. The results reveal the existence of two diversity regions in Spain for peach germplasm.

Physiological responses and differential gene expression in Prunus rootstocks under iron deficiency conditions.

Two Prunus rootstocks, the Myrobalan plum P 2175 and the interspecific peach-almond hybrid, Felinem, were studied to characterize their biochemical and molecular responses induced under iron-Deficient conditions. Plants of both genotypes were submitted to different treatments using a hydroponic system that permitted removal of Fe from the nutrient solution. Control plants were grown in 90 μM Fe (III)-EDTA, Deficient plants were grown in an iron free solution, and plants submitted to an Inductor treatment were resupplied with 180 μM Fe (III)-EDTA over 1 and 2 days after a period of 4 or 15 days of growth on an iron-free solution. Felinem increased the activity of the iron chelate reductase (FC-R) in the Inductor treatment after 4 days of iron deprivation. In contrast, P 2175 did not show any response after at least 15 days without iron. The induction of the FC-R activity in this genotype was coincident in time with the medium acidification. These results suggest two different mechanisms of iron chlorosis tolerance in both Strategy I genotypes. Felinem would use the iron reduction as the main mechanism to capture the iron from the soil, and in P 2175, the mechanism of response would be slower and start with the acidification of the medium synchronized with the gradual loss of chlorophyll in leaves. To better understand the control of these responses at the molecular level, the differential expression of PFRO2, PIRT1 and PAHA2 genes involved in the reductase activity, the iron transport in roots, and the proton release, respectively, were analyzed. The expression of these genes, estimated by quantitative real-time PCR, was different between genotypes and among treatments. The results were in agreement with the physiological responses observed.