Anita Solar

Novel Roles for the Polyphenol Oxidase Enzyme in Secondary Metabolism and the Regulation of Cell Death in Walnut

The polyphenol oxidase enzyme functions in the metabolism of the amino acid tyrosine in walnut. The enzyme polyphenol oxidase (PPO) catalyzes the oxidation of phenolic compounds into highly reactive quinones. Polymerization of PPO-derived quinones causes the postharvest browning of cut or bruised fruit, but the native physiological functions of PPOs in undamaged, intact plant cells are not well understood. Walnut (Juglans regia) produces a rich array of phenolic compounds and possesses a single PPO enzyme, rendering it an ideal model to study PPO. We generated a series of PPO-silenced transgenic walnut lines that display less than 5% of wild-type PPO activity. Strikingly, the PPO-silenced plants developed spontaneous necrotic lesions on their leaves in the absence of pathogen challenge (i.e. a lesion mimic phenotype). To gain a clearer perspective on the potential functions of PPO and its possible connection to cell death, we compared the leaf transcriptomes and metabolomes of wild-type and PPO-silenced plants. Silencing of PPO caused major alterations in the metabolism of phenolic compounds and their derivatives (e.g. coumaric acid and catechin) and in the expression of phenylpropanoid pathway genes. Several observed metabolic changes point to a direct role for PPO in the metabolism of tyrosine and in the biosynthesis of the hydroxycoumarin esculetin in vivo. In addition, PPO-silenced plants displayed massive (9-fold) increases in the tyrosine-derived metabolite tyramine, whose exogenous application elicits cell death in walnut and several other plant species. Overall, these results suggest that PPO plays a novel and fundamental role in secondary metabolism and acts as an indirect regulator of cell death in walnut.

Characterisation of selected hazelnut cultivars: phenology, growing and yielding capacity, market quality and nutraceutical value.

BACKGROUND Sixteen hazelnut cultivars growing in the continental climate of Slovenia were analysed over 15 years for their phenology, growth habit, yield potential, susceptibility to hazelnut weevil and the pomological traits and phenolic content of their nuts in order to obtain a complex value of these cultivars for growers, the confectionary industry and consumers. RESULTS Blooming occurred over an interval of 10-23 days for female (pistillate) flowers and 11-22 days for male (staminate) flowers. Nocchione, Romai, Pauetet, ID and Daria were the most productive cultivars, with a nine-year cumulative yield ranging from 31.8 to 44.7 kg per plant. Pauetet, F. Coutard, Nocchione and Segorbe were less susceptible to unfavourable weather conditions during blooming and fertilisation, in terms of maintaining acceptable yields with limited blank production. Under integrated pest management, less than 2% of the nuts of Romai, Daria, TGDL and Nocchione were affected by hazelnut weevil, compared with an average of 5.5% for the other cultivars. Daria, Pauetet and T. Giffoni performed best with regard to kernel percentage and blanching ratio. The results suggest that raw kernels are a good source of the natural antioxidants gallic acid and epicatechin. CONCLUSION The results of this study provide direction when choosing hazelnut cultivars for planting, consuming or processing. They can be applied not only in Slovenia and nearby countries but also in other parts of central and northern European countries with similar climates and growing conditions.

Flavonols of leaves in relation to apple scab resistance

Phenolic compounds of three scab-resistant apple cultivars ‘Topaz’, ‘Goldstar’ and ‘Gold Rush’ and two susceptible ones ‘Golden Delicious Weinsberg’ and ‘Golden Delicious Clone B’ were studied. The leaves were sampled four times in the period from 12 June to 10 September 2001. Flavonols rutin, quercitrin and ‘phenol 173’ (phenol with retention time 173,38 min) were analyzed using the Hplc system. Between the resistant and susceptible cultivars, statistically significant differences were established in the relative content of ‘phenol 173’, which was higher in the resistant cultivars during the growing season. It appears that the content is not affected either by different environmental conditions or by different stressors, but only by the genotype. Differences in the content of rutin and quercitrin were not significant. During the growth period, the content of phenolic compounds changed and was related to the physiological stage of the tissue and cultivar used.ZusammenfassungDie phenolischen Verbindungen in den drei gegen Schorf resistenten Apfelsorten ‘Topaz’, ‘Goldstar’ und ‘Gold Rush’ und in den zwei für Schorf anfälligen Sorten ‘Golden Delicious Weinsberg’ und ‘Golden Delicious Clone B’ wurden untersucht. Die Blätter für die Analyse wurden an vier verschiedenen Terminen zwischen dem 12. Juni und 10. September 2001 gesammelt. Die Flavonole Rutin und Quercitrin und ‘Phenole 173’ (Phenole mit der Retentionszeit 173,38 min) wurden mit Hilfe des Hplc bestimmt. Bei ‘Phenole 173’ konnten statistische Unterschiede gefunden werden. Die Gehalte dieser Substanz waren bei den resistenten Sorten während der ganzen Messperiode höher als bei den Standards. Sie wurden weder von verschiedenen Klimaverhältnissen noch von unterschiedlichen Stressfaktoren beeinflusst, sondern nur vom Genotyp. Die Gehaltsunterschiede bei den anderen Flavonolen ließen sich nicht absichern. Die Gehalte der phenol ischen Verbindungen veränderten sich in der Wachstumsperiode und waren vom physiologischen Status des Gewebes und von der Sorte abhängig.

Molecular and morphological diversity of on-farm hazelnut (Corylus avellana L.) landraces from southern Europe and their role in the origin and diffusion of cultivated germplasm

Hazelnut (Corylus avellana L.) is a traditional nut crop in southern Europe. Germplasm exploration conducted on-farm in five countries (Portugal, Spain, Italy, Slovenia, and Greece) identified 77 landraces. The present work describes phenotypic variation in nut and husk traits and investigates genetic relationships using ten simple sequence repeat (SSR) markers among these landraces, 57 well-known references cultivars, and 19 wild accessions. Among the 77 landraces, 42 had unique fingerprints while 35 showed a SSR profile identical to a known cultivar. Among the 42 unique landraces, morphological observations revealed high phenotypic diversity, and some had characteristics appreciated by the market such as nut round and caliber. Analysis of genetic relationships and population structure allowed investigation of the origin and spread of the cultivated germplasm in southern Europe. Our results indicate the existence of three primary centers of diversity in the Mediterranean basin: northwestern Spain (Tarragona) and southern Italy (Campania) in the West and Black Sea (Turkey) in the East. Moreover, the data suggest the existence of secondary gene pools in the Iberian (Asturias) and Italian (Liguria and Latium) Peninsulas, where local varieties were recently domesticated from wild forms and/or from introduced ancient domesticated varieties.

HPLC-MSn identification and quantification of phenolic compounds in hazelnut kernels, oil and bagasse pellets

The paper reports the phenolic content of kernels, bagasse pellets (residue of oil pressing) and oils from five different cultivars and a mix of cultivars. Phenolic compounds were identified by high-performance liquid chromatography coupled with mass spectrometry. Two compounds, glansreginin A and glansreginin B, were detected for the first time in hazelnuts. The main polyphenolic subclass comprised of mono- and oligomeric flavan 3-ols, which accounted between 34.2 and 58.3% in hazelnut kernels and between 36.7 and 48.6% in pellets of the total phenolics detected. In hazelnut oils four compounds have been detected, their content levels ranged from 0.97 to 0.01μgg-1. Total phenolic content ranged from 491.2 to 1700.4mgGAEkg-1 in kernels, from 848.4 to 1148.5mgGAEkg-1 in pellets and only from 0.14 to 0.25mgGAEg-1 in oils. The percentage of radical scavenging activity ranged from 60.0 to 96.4% for kernels, 63.0 to 73.2% for pellets and from 17.7 to 29.9% for oil. The study provides clear evidence on high phenolic contents and similarly high antioxidant potential of hazelnut kernels and bagasse pellets. The latter could be used as ingredients in cooking and baking or even for production of plant-based pharmaceuticals.

Stability of the annual shoot diameter in Persian walnut : a case study of different morphotypes and years

The basal diameter of the annual shoot (1YD) affects vegetative growth and fruiting of the walnut trees. In order to determine interdependency between the 1YD and the older parent wood, 64 walnut genotypes belonging to four different branching and fruiting habits (morphotypes M-I, M-II, M-III and M-IV) were investigated. Year-to-year stability of 1YD was tested with the architectural analysis of a 3-year-old fruiting branch and its constituents (a 3-year-old bearer + corresponding 2-year-old + annual shoots) during 3 successive years. Based on Pearsons correlation coefficients and the multiple regression analysis of 12 quantitative traits, 12 models (four morphotype in 3 successive years) of 1YD were formed. They were compared with the standard model which was calculated on the basis of 1-year measurements of 1Y with no respect to the branching and fruiting type and comprises three quantitative traits, i.e. basal diameter of a 2-year-old parent shoot (2YD), the length of 2Y shoot (2YL), and the length of annual shoot (1YL). In a single year, the 1YD was influenced by two–five parameters. Five out of 12 models agreed with the standard model: in the lateral fruiting genotypes (M-IV), 1YD was always under the influence of the 2Y diameter, and the 1Y length. In addition, the number of nodes of the 2Y parent shoot had an important influence on 1Y diameter. In the terminal bearers (M-I), the impact of 2YD on the 1YD slightly increased with the tree age, and some other parameters, like 1Ynumber and 1Ynodes, which became to be important for 1YD. In the intermediate genotypes with mezotonic ramification (M-II), the number of vegetative buds per 1Y and angles of 1Y had significant effects on 1YD. In the intermediate bearers with acrotonic ramification (M-III), one to four other parameters were included into the model each year beside the 1Y number. Since the traits of a 2-year-old parent shoot have a great influence on the 1YD, the information from the year N can be used for the prediction of the annual shoot development in the year N+1. Such a prediction is more reliable in M-I and M-IV than in M-II and M-III. When we deal with the intermediate fruiting cultivars, 1Y number has to be considered in prediction of 1Y diameter beside 2YD and 1YL.

Shifts in walnut (Juglans regia L.) phenology due to increasing temperatures in Slovenia.

Summary Bud-breaking date (BBD) is one of the most important traits in walnut cultivars for commercial cultivation. In regions with frequent late-Spring frost injuries, BBD affects the stability, quantity, and quality of walnut yield. Due to global warming, shifts in walnut phenology are expected in the future, and the suitability of some cultivars for certain climatic regions may be affected. In the present study, the influence of increasing Winter and Spring air temperatures on BBD was analysed. The late BBD cultivar ‘Franquette’ and the medium BBD cultivar ‘G-139’ were observed over two timeperiods: 1984 – 1990 (P1), and 2000 – 2006 (P2). During P1, the mean air temperature from January to April was 3.7ºC, and the average BBDs were 23 April and 5 May for ‘G-139’ and ‘Franquette’, respectively. During P2, the mean air temperature from January to April increased by 0.9ºC and the BBDs were 3 days (d) earlier for ‘G-139’ and 7 d earlier for ‘Franquette’. Phenological models showed that the mean air temperatures in the preceding 1 – 2 months (TMar and TApr) were important for predicting BBD and, due to increasing temperatures in Winter and early-Spring, BBDs will be shifted to earlier in the year. Depending on walnut cultivar, the average BBD could be advanced by as much as 4 weeks by 2060. Consequently, the frost hazard will probably increase.

Non-destructive determination of moisture content in hazelnut (Corylus avellana L.)

MC control is essential in hazelnut production from an orchard to a consumer.A special probe for non-destructive determination of hazelnut MC was constructed.Dielectric properties of in-shell hazelnut were tested at low frequencies.Regression model made of conductance and capacitance reliable predicts hazelnut MC.Theoretical basis for practical realisation of the in-shell hazelnut MC-metre was provided. There is a need in commercial hazelnut production for a rapid, nondestructive and reliable moisture content (MC) sensing in order to obtain quality nuts from the orchard to the consumer. In the presented study, the MC of in-shell hazelnuts were determined on the base of their dielectric properties, which have never been described in hazelnuts to date. The measurement system from a probe with parallel, vertically placed electrodes, a generator of sine wave signals and oscilloscope was completed for measuring voltage on the input (V1) and output (V2) of the probe, and the phase angle between V1 and V2 within a frequency range from 1kHz to 1MHz. Eight dielectric properties (impedance, admittance, resistance, capacitance, dielectric constant, dielectric loss factor, dissipation factor and phase angle) were calculated and graphically presented for hazelnuts with various MC and compared with the reference MC values determined using the standard air-oven method. All dielectric properties except the resistance and phase angles decreased with decreased hazelnut MC. Measurement frequencies were negatively correlated regarding all properties except for the phase angle. Six statistical MC-models were calculated by conducting stepwise multiple regression analysis of the dielectric properties. Model 5, which comprised conductance and capacitance of the nuts, was proved to be the more predictive for MC determination, showing the lowest standard error of estimation (0.39), the highest coefficient of determination (99.78) and the lowest mean absolute error (0.23). Good agreement was found between the MC simulated by model 5 and the observed data in both mixed hazelnut sample as well as in cultivars with nuts of different shapes and sizes. Performing conductance and capacitance measurements at frequencies between 3 and 500kHz, the moisture content of the in-shell hazelnuts could be efficiently determined, using a low-cost and non-invasive technique.

Fruiting behaviour of walnut trees influences the relationship between the morphometric traits of the parent wood and nut weight

Summary Relationships between nut weight and the morphometric characteristics of annual shoots (1Y) and current year shoots (cY) in walnut (Juglans regia L.) were investigated to test the hypothesis that these traits are linked, and influenced by fruiting behaviour. The terminal fruiting cultivar, ‘Franquette’, the intermediate bearer ‘Zdole-59’, and two cultivars, ‘Lara’ and ‘Fernor’, with lateral bearing habits but different lateral branching densities, were studied over 3 years. Regression analysis was conducted for modelling the relationship between nut weight and 1Y shoot diameter, 1Y shoot length, cY shoot diameter, cY shoot length, cY shoot number, cY leaves, and the number of nuts per cY shoot. In ‘Franquette’, nut weight was significantly correlated with 1Y shoot diameter, 1Y shoot length, cY shoot diameter, cY leaves, and nuts per cY shoot. Nut weight was expected to increase by 0.7 g when the nuts per cY shoot increased by 1. When 1Y shoot diameter (as well as cY shoot diameter) increased by 4 mm, nuts were heavier by 1 g. In the vigorous and densely-branched, lateral-bearing cultivar ‘Lara’, nut weight was negatively correlated with nuts per cY shoot, and with cY shoot length. Each nut was lighter by 0.6 g when one additional nut was inserted on each cY shoot. In ‘Zdole-59’, nut weight was closely correlated with 1Y shoot diameter and cY shoot number. An increase in cY shoot number by 1, caused an increase in nut weight by 0.4 g. In the less vigorous, sparsely-branched and lateral fruiting cultivar ‘Fernor’, none of the measured shoot traits correlated with nut weight and, consequently, none could be used to predict nut weight. In the terminal bearers, nut weight could be reliably predicted, based on 1Y shoot diameter measurements. In the vigorous, lateral-bearing cultivars, the number of nuts per cY shoot was shown to be the most useful parameter for nut weight prediction, whereas in intermediate bearers it was cY shoot number.

A multidisciplinary approach to enhance the conservation and use of hazelnut Corylus avellana L. genetic resources

During recent years, there has been increasing awareness of the importance of adopting a holistic view of biodiversity, including agricultural biodiversity, conservation for sustainable utilization and development. These principles have been underlined in the Convention on Biological Diversity and the European efficiency resources towards 2050. Thus critical issues are now to understand the distribution and extent of genetic diversity available to breeders and stakeholders, the kind and range of characterization, how to face the problem of continuous expanding of germplasm to be conserved. Focusing on the case study of hazelnut which is a crop of great importance for European countries, the paper describes a resourceful strategy for re-organizing and sharing hazelnut genetic resources through an upgrading of knowledge on their value and uses. The paper summarizes the progresses so far and provides a ‘launching pad’ for future researches. The brief review discusses also the recent progresses in recovery, characterization conservation and uses of European hazelnut germplasm achieved by 068 AGRI GEN RES SAFENUT which was one of the 17 Action financed by the European Commission—Directorate General for Agriculture and Rural Development. The current status on the morphological and molecular characterization of the in situ and ex situ of the most important European collections, the rescue and preservation of new accessions recovered on farm were discussed underling critical aspects. A better understanding of hazelnut genetic diversity and its distribution is essential for its conservation and use as well as the harmonization of the morphological and biochemical descriptors. The importance of traditional knowledge is also considered as integrated part of the multidisciplinary approach useful to rationalize genetic resources maintained in the collections. Thus improving the characterization on cultivated and wild forms through the development of a core collection, is the further step to achieve a more effective management and use of European nuts germplasm.