Multiomics Molecular Research into the Recalcitrant and Orphan Quercus ilex Tree Species: Why, What for, and How

The holm oak (Quercus ilex L.) is the dominant tree species of the Mediterranean forest and the Spanish agrosilvopastoral ecosystem, “dehesa.” It has been, since the prehistoric period, an important part of the Iberian population from a social, cultural, and religious point of view, providing an ample variety of goods and services, and forming the basis of the economy in rural areas. Currently, there is renewed interest in its use for dietary diversification and sustainable food production. It is part of cultural richness, both economically (tangible) and environmentally (intangible), and must be preserved for future generations. However, a worrisome degradation of the species and associated ecosystems is occurring, observed in an increase in tree decline and mortality, which requires urgent action. Breeding programs based on the selection of elite genotypes by molecular markers is the only plausible biotechnological approach. To this end, the authors’ group started, in 2004, a research line aimed at characterizing the molecular biology of Q. ilex. It has been a challenging task due to its biological characteristics (long life cycle, allogamous, high phenotypic variability) and recalcitrant nature. The biology of this species has been characterized following the central dogma of molecular biology using the omics cascade. Molecular responses to biotic and abiotic stresses, as well as seed maturation and germination, are the two main objectives of our research. The contributions of the group to the knowledge of the species at the level of DNA-based markers, genomics, epigenomics, transcriptomics, proteomics, and metabolomics are discussed here. Moreover, data are compared with those reported for Quercus spp. All omics data generated, and the genome of Q. ilex available, will be integrated with morphological and physiological data in the systems biology direction. Thus, we will propose possible molecular markers related to resilient and productive genotypes to be used in reforestation programs. In addition, possible markers related to the nutritional value of acorn and derivate products, as well as bioactive compounds (peptides and phenolics) and allergens, will be suggested. Subsequently, the selected molecular markers will be validated by both genome-wide association and functional genomic analyses

Changes in orange juice (poly)phenol composition induced by controlled alcoholic fermentation

(Poly)phenols of fermented orange juice were characterized by UHPLC-MS/MS, and an increase of total content in relation to orange juice was evaluated.</p

Alcoholic fermentation induces melatonin synthesis in orange juice

FECYT -- Elaboración De Una Novedosa Bebida De Naranja De Baja Graduació...Melatonin (N-acetyl-5-methoxytryptamine) is a molecule implicated in multiple biological functions. Its level decreases with age, and the intake of foods rich in melatonin has been considered an exogenous source of this important agent. Orange is a natural source of melatonin. Melatonin synthesis occurs during alcoholic fermentation of grapes, malt and pomegranate. The amino acid tryptophan is the precursor of all 5-methoxytryptamines. Indeed, melatonin appears in a shorter time in wines when tryptophan is added before fermentation. The aim of the study was to measure melatonin content during alcoholic fermentation of orange juice and to evaluate the role of the precursor tryptophan. Identification and quantification of melatonin during the alcoholic fermentation of orange juice was carried out by UHPLC-QqQ-MS/MS. Melatonin significantly increased throughout fermentation from day 0 (3.15 ng/mL) until day 15 (21.80 ng/mL) reaching larger amounts with respect to other foods. Melatonin isomer was also analysed, but its content remained stable ranging from 11.59 to 14.18 ng/mL. The enhancement of melatonin occurred mainly in the soluble fraction. Tryptophan levels significantly dropped from 13.80 mg/L (day 0) up to 3.19 mg/L (day 15) during fermentation. Melatonin was inversely and significantly correlated with tryptophan (r = 0.907). Therefore, the enhancement in melatonin could be due to both the occurrence of tryptophan and the new synthesis by yeast. In summary, the enhancement of melatonin in novel fermented orange beverage would improve the health benefits of orange juice by increasing this bioactive compound.Área de Nutrición y Bromatología. Departamento de Biología Molecular e Ingeniería Bioquímica. Universidad Pablo de Olavid

Alcoholic fermentation induces melatonin synthesis in orange juice

Melatonin (N-acetyl-5-methoxytryptamine) is a molecule implicated in multiple biological functions. Its level decreases with age, and the intake of foods rich in melatonin has been considered an exogenous source of this important agent. Orange is a natural source of melatonin. Melatonin synthesis occurs during alcoholic fermentation of grapes, malt and pomegranate. The amino acid tryptophan is the precursor of all 5-methoxytryptamines. Indeed, melatonin appears in a shorter time in wines when tryptophan is added before fermentation. The aim of the study was to measure melatonin content during alcoholic fermentation of orange juice and to evaluate the role of the precursor tryptophan. Identification and quantification of melatonin during the alcoholic fermentation of orange juice was carried out by UHPLC-QqQ-MS/MS. Melatonin significantly increased throughout fermentation from day 0 (3.15 ng/mL) until day 15 (21.80 ng/mL) reaching larger amounts with respect to other foods. Melatonin isomer was also analysed, but its content remained stable ranging from 11.59 to 14.18 ng/mL. The enhancement of melatonin occurred mainly in the soluble fraction. Tryptophan levels significantly dropped from 13.80 mg/L (day 0) up to 3.19 mg/L (day 15) during fermentation. Melatonin was inversely and significantly correlated with tryptophan (r = 0.907). Therefore, the enhancement in melatonin could be due to both the occurrence of tryptophan and the new synthesis by yeast. In summary, the enhancement of melatonin in novel fermented orange beverage would improve the health benefits of orange juice by increasing this bioactive compoundThe authors are grateful to the company ‘Grupo Hespérides Biotech S.L.’ for providing the samples of fermented orange juice. The authors also gratefully acknowledge the support of Junta de Andalucía through the Project P09-AGR4814M and of National funding agencies through the Projects AGL2011-23690, CSD007-0063 (CONSOLIDER-INGENIO 2010 ‘Fun-C-Food’) and CSIC 201170E041. The authors are also grateful to the Fundación Séneca – CARM ‘Group of Excellence in Research’ 04486/GERM/06 and the Ibero-American Programme for Science, Technology and Development (CYTED) – Action 112RT0460 CORNUCOPIA. The Research Project grant of BEL is supported by Junta de AndalucíaPeer reviewe

Effect and Response of Quercus ilex subsp. ballota [Desf.] Samp. Seedlings From Three Contrasting Andalusian Populations to Individual and Combined Phytophthora cinnamomi and Drought Stresses

Quercus ilex L. is the dominant species in the Mediterranean forest and agrosilvopastoral ecosystem “dehesa.” Currently, this forest species is threatened by natural and anthropogenic agents, especially by the decline syndrome, which is caused by Phytophthora cinnamomi and drought periods. Although the morphological and physiological responses of Q. ilex to combined stress (P. cinnamomi and drought) have been examined already, little is known at the molecular level. In this study, we studied the effect and response of 8-month seedlings from three contrasting Andalusian populations (Seville [Se], Granada [Gr], and Almeria [Al]) to the individual and combined stresses of P. cinnamomi and drought from morphological, physiological, biochemical, and proteomics data. Whereas, seedling damage (leaf chlorosis and necrosis) and mortality were greater under the combined stresses in the three populations, the effect of each individual stress was population-dependent. Resilient individuals were found in all the populations at different percentages. The decrease in leaf chlorophyll fluorescence, photosynthetic activity, and stomatal conductance observed in undamaged seedlings was greater in the presence of both stresses, the three populations responding similarly to drought and P. cinnamomi. Biochemical and proteomic analyses of undamaged seedlings from the two most markedly contrasting populations (Se and Al) revealed the absence of significant differences in the contents in photosynthetic pigments, amino acids, and phenolics among treatments. The Se and Al populations exhibited changes in protein profile in response to the different treatments, with 83 variable proteins in the former population and 223 in the latter. Variable proteins belonged to 16 different functional groups, the best represented among which were protein folding, sorting and degradation, carbohydrate, amino acid, and secondary metabolism, photosynthesis, and ROS scavenging. While photosynthetic proteins were mainly downaccumulated, those of stress-responsive were upaccumulated. Although no treatment-specific response was observed in any functional group, differences in abundance were especially marked under the combined stresses. The following variable proteins are proposed as putative markers for resilience in Q. ilex, namely, aldehyde dehydrogenase, glucose-6-phosphate isomerase, 50S ribosomal protein L5, and α-1,4-glucan-protein synthase [UDP-forming].</jats:p

Data_Sheet_1_Effect and Response of Quercus ilex subsp. ballota [Desf.] Samp. Seedlings From Three Contrasting Andalusian Populations to Individual and Combined Phytophthora cinnamomi and Drought Stresses.docx

Quercus ilex L. is the dominant species in the Mediterranean forest and agrosilvopastoral ecosystem “dehesa.” Currently, this forest species is threatened by natural and anthropogenic agents, especially by the decline syndrome, which is caused by Phytophthora cinnamomi and drought periods. Although the morphological and physiological responses of Q. ilex to combined stress (P. cinnamomi and drought) have been examined already, little is known at the molecular level. In this study, we studied the effect and response of 8-month seedlings from three contrasting Andalusian populations (Seville [Se], Granada [Gr], and Almeria [Al]) to the individual and combined stresses of P. cinnamomi and drought from morphological, physiological, biochemical, and proteomics data. Whereas, seedling damage (leaf chlorosis and necrosis) and mortality were greater under the combined stresses in the three populations, the effect of each individual stress was population-dependent. Resilient individuals were found in all the populations at different percentages. The decrease in leaf chlorophyll fluorescence, photosynthetic activity, and stomatal conductance observed in undamaged seedlings was greater in the presence of both stresses, the three populations responding similarly to drought and P. cinnamomi. Biochemical and proteomic analyses of undamaged seedlings from the two most markedly contrasting populations (Se and Al) revealed the absence of significant differences in the contents in photosynthetic pigments, amino acids, and phenolics among treatments. The Se and Al populations exhibited changes in protein profile in response to the different treatments, with 83 variable proteins in the former population and 223 in the latter. Variable proteins belonged to 16 different functional groups, the best represented among which were protein folding, sorting and degradation, carbohydrate, amino acid, and secondary metabolism, photosynthesis, and ROS scavenging. While photosynthetic proteins were mainly downaccumulated, those of stress-responsive were upaccumulated. Although no treatment-specific response was observed in any functional group, differences in abundance were especially marked under the combined stresses. The following variable proteins are proposed as putative markers for resilience in Q. ilex, namely, aldehyde dehydrogenase, glucose-6-phosphate isomerase, 50S ribosomal protein L5, and α-1,4-glucan-protein synthase [UDP-forming].</p

Table_1_Effect and Response of Quercus ilex subsp. ballota [Desf.] Samp. Seedlings From Three Contrasting Andalusian Populations to Individual and Combined Phytophthora cinnamomi and Drought Stresses.xlsx

Quercus ilex L. is the dominant species in the Mediterranean forest and agrosilvopastoral ecosystem “dehesa.” Currently, this forest species is threatened by natural and anthropogenic agents, especially by the decline syndrome, which is caused by Phytophthora cinnamomi and drought periods. Although the morphological and physiological responses of Q. ilex to combined stress (P. cinnamomi and drought) have been examined already, little is known at the molecular level. In this study, we studied the effect and response of 8-month seedlings from three contrasting Andalusian populations (Seville [Se], Granada [Gr], and Almeria [Al]) to the individual and combined stresses of P. cinnamomi and drought from morphological, physiological, biochemical, and proteomics data. Whereas, seedling damage (leaf chlorosis and necrosis) and mortality were greater under the combined stresses in the three populations, the effect of each individual stress was population-dependent. Resilient individuals were found in all the populations at different percentages. The decrease in leaf chlorophyll fluorescence, photosynthetic activity, and stomatal conductance observed in undamaged seedlings was greater in the presence of both stresses, the three populations responding similarly to drought and P. cinnamomi. Biochemical and proteomic analyses of undamaged seedlings from the two most markedly contrasting populations (Se and Al) revealed the absence of significant differences in the contents in photosynthetic pigments, amino acids, and phenolics among treatments. The Se and Al populations exhibited changes in protein profile in response to the different treatments, with 83 variable proteins in the former population and 223 in the latter. Variable proteins belonged to 16 different functional groups, the best represented among which were protein folding, sorting and degradation, carbohydrate, amino acid, and secondary metabolism, photosynthesis, and ROS scavenging. While photosynthetic proteins were mainly downaccumulated, those of stress-responsive were upaccumulated. Although no treatment-specific response was observed in any functional group, differences in abundance were especially marked under the combined stresses. The following variable proteins are proposed as putative markers for resilience in Q. ilex, namely, aldehyde dehydrogenase, glucose-6-phosphate isomerase, 50S ribosomal protein L5, and α-1,4-glucan-protein synthase [UDP-forming].</p