Nitrogen starvation and nitrate or ammonium availability differently affect phenolic composition in green and purple basil

Basil (Ocimum basilicum L.) comprises green and purple cultivars, worldwide cultivated and appreciated for high contents of rosmarinic acid and anthocyanins, respectively. Although nitrogen (N) fertilization is needed for high yields, in basil it could have detrimental effects on the accumulation of phenolic compounds. In this study, plants of the cultivars 'Italiano Classico' (green) and 'Red Rubin' (purple) were grown in hydroponics and subjected to different nutritional treatments, consisting in N starvation, and nitrate (NO3 12) or ammonium (NH4+) nutrition. Plant growth and nutritional status, estimated by the contents of NO3 12, NH4+, and amino acids in roots and leaves, were evaluated and put in relation with quality traits of basil leaves, such as chlorophyll content, antioxidant capacity, total phenols, the activity of phenylalanine ammonia-lyase, and the concentrations of individual (poly)phenolic acids and flavonoids. This study reveals that N starvation, as well as the availability of the two inorganic N forms, differently affect the phenolic composition in the two cultivars. Compared to plants grown in NO3 12 availability, in NH4+ availability, green basil showed a higher content of (poly)phenolic acids, while in purple basil, an increase in the contents of anthocyanins was detected. Overall, the study suggests that the management of NH4+ supply could contribute to enhance crop quality in hydroponics, and provides new knowledge about the relationship between N nutrition and phenolic metabolism in basil

Insight into composition of bioactive phenolic compounds in leaves and flowers of green and purple basil

Basil (Ocimum basilicum L.) is a culinary, medicinal, and ornamental plant appreciated for its antioxidant properties, mainly attributed to high content of rosmarinic acid. This species also includes purple varieties, characterized by the accumulation of anthocyanins in leaves and flowers. In this work, we compared the main morphological characteristics, the antioxidant capacity and the chemical composition in leaves, flowers, and corollas of green (\u2018Italiano Classico\u2019) and purple (\u2018Red Rubin\u2019 and \u2018Dark Opal\u2019) basil varieties. The LC-ESI-MS/MS analysis of individual compounds allowed quantifying 17 (poly)phenolic acids and 18 flavonoids, differently accumulated in leaves and flowers of the three varieties. The study revealed that in addition to rosmarinic acid, basil contains several members of the salvianolic acid family, only scarcely descripted in this species, as well as, especially in flowers, simple phenolic acids, such as 4-hydroxybenzoic acid and salvianic acid A. Moreover, the study revealed that purple leaves mainly contain highly acylated anthocyanins, while purple flowers accumulate anthocyanins with low degree of decoration. Overall, this study provides new biochemical information about the presence of not yet characterized bioactive compounds in basil that could contribute to boosting the use of this crop and to gaining new knowledge about the roles of these compounds in plant physiology

Cinnamyl alcohol dehydrogenases in the mesocarp of ripening fruit of Prunus persica genotypes with different flesh characteristics: changes in activity and protein and transcript levels

Development of fruit flesh texture quality traits may involve the metabolism of phenolic compounds. This study presents molecular and biochemical results on the possible role played by cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) during ripening [S3, S4 I (pre-climacteric) and S4 III (climacteric) stages] of peach [Prunus persica (L.) Batsch] fruit with different flesh firmness [non-melting flesh (NMF) \u2018Oro A\u2019/melting flesh (MF) \u2018Springcrest\u2019 and \u2018Sanguinella\u2019] and color (blood-flesh Sanguinella). A total of 24 putative full-length PRUPE_CAD genes were identified (in silico analysis) in the peach genome. The most abundant CAD isoforms, encoded by genes located on scaffolds 8 and 6, were probed by specifically developed anti-PRUPE_CAD sc8 and by anti-FaCAD (PRUPE_CAD sc6) polyclonal antibodies, respectively. PRUPE_CAD sc8 proteins (SDS-PAGE and native-PAGE/western blot) appeared responsible for the CAD activity (in vitro/in-gel assays) that increased with ripening (parallel to PRUPE_ACO1 transcripts accumulation and ethylene evolution) only in the mesocarp of Oro A and blood-flesh Sanguinella. Accumulation of PRUPE_CAD sc8 transcripts (semi-quantitative RT-PCR) occurred in all three cultivars, but in Oro A and Springcrest it was not always accompanied by that of the related proteins, suggesting possible post-transcriptional regulation. Flesh firmness, as well as levels of lignin, total phenolics and, where present (Sanguinella), anthocyanins, declined with ripening, suggesting that, at least in the studied peach cultivars, CAD activity is related to neither lignification nor differences in flesh firmness (NMF/MF). Further studies are necessary to clarify whether the high levels of CAD activity/expression in Sanguinella play a role in determining the characteristics of this blood-flesh fruit

A possible role of the trehalose/trehalose-6-phosphate/SnRK1 system in rice response to salt stress

Among the abiotic stresses exacerbated by climate change, soil salinity is one of the most harmful for crops. Rice (Oryza sativa L.) is a very salt-sensitive cereal, with particular regard to the cultivars belonging to the subspecies japonica. In order to identify genomic traits conferring salt tolerance, a collection of 277 rice accessions (ssp. japonica) has been phenotypized under mild-salinity considering germination kinetic parameters and seedling emergence rate. Genotyping By Sequencing (GBS) followed by a Genome Wide Association Study (GWASs) were carried out. A total of 31.421 SNPs were used for the analysis. Significant genotypic-phenotypic associations were observed and a few Marker-Trait Associations (MTAs) were identified. By alignment with the rice reference genome (Os-Nipponbare-Reference-IRGSP-1.0), some of the genes putatively involved in salt stress were highlighted. Among them, our interest has been focused on OsTPP7 (chromosome 9) and OsTPP10 (chromosome 7), genes that belong to the OsTPP gene family encoding for Trehalose-6-Phosphate Phosphatase (TPP) enzymes catalyzing the dephosphorylation of Trehalose-6-Phosphate (T6P) to Trehalose (Tre). The level of T6P plays a central role in abiotic stress tolerance, acting as a signal in the cascade of events regulating sugar metabolism (sucrose) during seed germination and seedling growth. This activity is mediated by Snf1-Related protein Kinase-1 (SnRK1), a metabolic sensor fundamental in maintaining carbon homeostasis under stress condition. Biochemical and molecular analyses were performed on two rice accessions showing opposite behavior under salt stress (Olcenengo, tolerant, and SR113, sensitive). Results concerning T6P, Tre and sucrose levels in growing embryos, the evaluation of the time course of \u3b1-amylase enzyme (target of the SnRK1 system) activity and of OsTPP10-OsTPP7 gene expression appear to allow us to define a picture coherent with the different effects of salt stress in Olcenengo and SR113. Functional characterization of OsTPP10 gene and its allele mining analysis within the 277 rice accessions are in progress

The Trehalose-6-phosphate/SnRK1 system in the response to saline conditions during germination of two rice (O. sativa L., ssp. japonica) cultivars with different salt sensitivity

Soil salinity is extremely harmful for crops, and, among cereals, for rice (Oryza sativa L.), with particular regard to the ssp. japonica. Seed germination and seedling emergence are among the phenological stages particularly sensitive to this stress condition. A Genome Wide Association Study (GWAS) with a total of 31.421 SNPs was conducted on a collection of 277 japonica rice accessions phenotyped under mid-salinity considering germination kinetic parameters and seedling emergence rate. A few Marker-Trait Associations were identified on the basis of significant genotype-phenotype association analysis. Among the genes putatively involved in the salt response, two were particularly interesting: OsTPP7 (chromosome 9) and OsTPP10 (chromosome 7), both belonging to the family encoding Trehalose-6-Phosphate Phosphatase (TPP) catalyzing the dephosphorylation of Trehalose-6-Phosphate (T6P) to Trehalose. Salt stress affects carbohydrate production and the mobilization/use of C storage compounds altering the sink-source relationships, sugar allocation and energy metabolism. In this framework, the ratios T6P/Tre, regulated by TPP activity, acts as a signal in the cascade of events that regulate, through sugar metabolism, plant development with particular regard to seed germination and seedling growth. In turn, T6P regulates the activity of Snf1-Related protein Kinase-1 (SnRK1), a metabolic sensor able to regulate \u3b1-amylase and fundamental in maintaining C homeostasis under stress. Aim of the work was to establish the role of the SnRK1/Tre6P/Tre/Sucrose system and in particular of the OsTPP7 and OsTPP10 genes in the tolerance of japonica rice to salt stress during seed germination and early seedling development. In two model rice accessions (Olcenengo, tolerant, and SR113, sensitive) with opposite behavior in salt stress, biochemical and molecular analyses were conducted. In particular, have been considered: in the growing embryos, T6P, Tre, and sucrose levels, and OsTPP7-OsTPP10 gene expression; in the endosperm, the time-course of \u3b1-amylase activity. Te results define a picture coherent with the different effects of salt stress in Olcenengo and SR113. Functional characterization of the OsTPP10 gene and its allele mining analysis within the 277 rice accessions are in progress

Evaluation of borage extracts as potential biostimulant using a phenomic, agronomic, physiological and biochemical approach

Biostimulants are substances able to improve water and nutrient use efficiency and counteract stress factors by enhancing primary and secondary metabolism. Premise of the work was to exploit raw extracts from leaves (LE) or flowers (FE) of Borago officinalis L., to enhance yield and quality of Lactuca sativa ‘Longifolia,’ and to set up a protocol to assess their effects. To this aim, an integrated study on agronomic, physiological and biochemical aspects, including also a phenomic approach, has been adopted. Extracts were diluted to 1 or 10 mL L–1, sprayed onto lettuce plants at the middle of the growing cycle and 1 day before harvest. Control plants were treated with water. Non-destructive analyses were conducted to assess the effect of extracts on biomass with an innovative imaging technique, and on leaf photosynthetic efficiency (chlorophyll a fluorescence and leaf gas exchanges). At harvest, the levels of ethylene, photosynthetic pigments, nitrate, and primary (sucrose and total sugars) and secondary (total phenols and flavonoids) metabolites, including the activity and levels of phenylalanine ammonia lyase (PAL) were assessed. Moreover, a preliminary study of the effects during postharvest was performed. Borage extracts enhanced the primary metabolism by increasing leaf pigments and photosynthetic activity. Plant fresh weight increased upon treatments with 10 mL L–1 doses, as correctly estimated by multi-view angles images. Chlorophyll a fluorescence data showed that FEs were able to increase the number of active reaction centers per cross section; a similar trend was observed for the performance index. Ethylene was three-fold lower in FEs treatments. Nitrate and sugar levels did not change in response to the different treatments. Total flavonoids and phenols, as well as the total protein levels, the in vitro PAL specific activity, and the levels of PAL-like polypeptides were increased by all borage extracts, with particular regard to FEs. FEs also proved efficient in preventing degradation and inducing an increase in photosynthetic pigments during storage. In conclusion, borage extracts, with particular regard to the flower ones, appear to indeed exert biostimulant effects on lettuce; future work will be required to further investigate on their efficacy in different conditions and/or species

Changes in endopolygalacturonase levels and characterization of a putative endo-PG gene during fruit softening in peach genotypes with nonmelting and melting flesh fruit phenotypes

The changes in endopolygalacturonase (endo-PG) levels and endo-PG expression in nonmelting flesh (NMF) and melting flesh (MF) peach fruits (Prunus persica) during softening were studied. The endo-PG gene was analysed to identify polymorphisms exploitable for early marker-assisted selection (MAS) of flesh texture. The role of endo-PG in softening was assessed by western and northern blotting and by biochemical analyses. Polymorphisms in the endo-PG gene were revealed by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. An endo-PG protein was detected in both NMF and MF fruits. The levels of this endo-PG protein were higher and increased with softening in MF fruits, but remained lower and were constant in NMF fruits. The different levels of endo-PG appeared to be caused by the differential expression of an endo-PG gene, whose open-reading frame (ORF) showed five single nucleotide polymorphisms (SNPs) in NMF 'Oro A' compared with MF 'Bolero'. One of these SNPs allowed us to determine the allelic configuration at the melting flesh (M) locus and also seemed to be exploitable for early MAS in other NMF/MF phenotypes. The NMF phenotype does not seem to be caused by a large deletion of the endo-PG gene. (copyright) New Phytologist (2006)

Endopolygalacturonase Gene Polymorphisms : Asset of the Locus in Different Peach Accessions

Endopolygalacturonase (endoPG) plays a pivotal role in determining peach [Prunus persica L. (Batsch)] fruit characteristics. Different Pp-endoPG genes or allelic variants have been described, characterized by different polymorphisms: insertions-deletions (InDels) and single nucleotide polymorphisms (SNPs). Eighty-five peach accessions (comprising commercial cultivars, F1 progenies of selected crosses, and three haploid seedlings) with different flesh softening patterns (Non Melting: NM; Melting: M; Slow Softening: SS; Stony Hard: SH) were screened by exploiting specific polymorphisms, with the aim to characterize their asset at the endoPG locus and evaluate a potential relationship with fruit flesh texture phenotype. The results of InDel analysis allowed to distinguish, by a simple genotyping procedure, NM flesh phenotypes from the others. Further information arose from this analysis, showing that two Pp-endoPG genes, i.e., Pp-endoPGm (Ppa006839m), involved in the determination of the Melting/Non Melting trait, and Pp-endoPG_M (Ppa006857m), involved in the determination of the Clingstone/Freestone trait, always co-segregate, and that SS Big Top possesses a \u201cnull\u201d Pp-endoPG allele. Cleaved Amplified Polymorphic Sequence (CAPS) analysis allowed to preliminarily discriminate the Pp-endoPG variants of the SS and SH accessions considered. The integrated use of the considered polymorphisms in a high number of peach accessions proved useful, by individuating the different gene variants and their combinations, to describe the structure of the endoPG locus in different genotypes

Usher syndrome: An effective sequencing approach to establish a genetic and clinical diagnosis

12noUsher syndrome is an autosomal recessive disorder characterized by retinitis pigmentosa, sensorineural hearing loss and, in some cases, vestibular dysfunction. The disorder is clinically and genetically heterogeneous and, to date, mutations in 11 genes have been described. This finding makes difficult to get a precise molecular diagnosis and offer patients accurate genetic counselling. To overcome this problem and to increase our knowledge of the molecular basis of Usher syndrome, we designed a targeted resequencing custom panel. In a first validation step a series of 16 Italian patients with known molecular diagnosis were analysed and 31 out of 32 alleles were detected (97% of accuracy). After this step, 31 patients without a molecular diagnosis were enrolled in the study. Three out of them with an uncertain Usher diagnosis were excluded. One causative allele was detected in 24 out 28 patients (86%) while the presence of both causative alleles characterized 19 patients out 28 (68%). Sixteen novel and 27 known alleles were found in the following genes: USH2A (50%), MYO7A (7%), CDH23 (11%), PCDH15 (7%) and USH1G (2%). Overall, on the 44 patients the protocol was able to characterize 74 alleles out of 88 (84%). These results suggest that our panel is an effective approach for the genetic diagnosis of Usher syndrome leading to: 1) an accurate molecular diagnosis, 2) better genetic counselling, 3) more precise molecular epidemiology data fundamental for future interventional plans.partially_openembargoed_20160106Lenarduzzi, S.; Vozzi, D; Morgan, A.; Rubinato, E.; D'Eustacchio, A.; Osland, T.M.; Rossi, C.; Graziano, C.; Castorina, P.; Ambrosetti, U.; Morgutti, M.; Girotto, G.Lenarduzzi, Stefania; Vozzi, Diego; Morgan, Anna; Rubinato, Elisa; D'Eustacchio, A.; Osland, TERESA MARIA; Rossi, C.; Graziano, C.; Castorina, P.; Ambrosetti, U.; Morgutti, Marcello; Girotto, Giorgi

Continuous Flooding or Alternate Wetting and Drying Differently Affect the Accumulation of Health-Promoting Phytochemicals and Minerals in Rice Brown Grain

Climate changes impose adoption of water-saving techniques to improve the sustainability of irrigated rice systems. This study was aimed, by a two-years side-by-side comparison, at verifying the hypothesis whether \u201cAlternate Wetting and Drying\u201d (AWD) affects the concentrations of health-related compounds and minerals in brown grains of three japonica rice (Oryza sativa L.) cvs (\u2018Baldo\u2019, \u2018Gladio\u2019, and \u2018Loto\u2019) usually grown in temperate areas in continuous flooding (CF). Due to the rotational turns in water distribution imposed by local authorities and to the weather behavior, different AWD timing and severity occurred in the two years of the study. AWD induced in both seasons yield losses in \u2018Baldo\u2019 and \u2018Gladio\u2019 but not in \u2018Loto\u2019. In the brown grains of \u2018Loto\u2019, AWD increased the concentrations of total tocols, \u3b3-oryzanol, flavonoids, and the antioxidant activity. AWD affected the concentrations of minerals, particularly increasing copper, cadmium and nickel, and decreasing manganese, arsenic and zinc. In the sensitive cultivars, \u2018Baldo\u2019 and \u2018Gladio\u2019, AWD seems to affect plant yield, rather than for severity of the dry period, for prolonged absence of ponded water that exposes plants to cooler temperatures. The selection of suitable cultivars, like \u2018Loto\u2019, tolerant to AWD-related stresses, could combine environmental, yield-related, and nutritional benefits improving the product quality