Physicochemical properties and transcriptional changes underlying the quality of 'Gala' apples (Malus × domestica Borkh.) under atmosphere manipulation in long-term storage.

BACKGROUND: The year-round availability of apples (Malus × domestica Borkh.) depends on post-harvest technologies, which are essential for the retention of fruit sensory and chemical properties by delaying senescence. The effectiveness of strategies for preserving the quality of apples depends on complex interactions between the storage environment and endogenous biological factors. In the current work, we integrated instrumental, sensory, and transcriptional data to determine the role of conservation technologies cold storage, controlled atmosphere, and 1-methylcyclopropene-mediated ethylene blockage on the long-term conservation of apples. RESULTS: The results demonstrated that inhibition of the consumer's perception of the apples' ethylene content is essential for long-term cold storage, and such quality conservation can be achieved by reducing oxygen pressure. Overall appreciation of apples after storage was determined mainly by their texture, with crispness and juiciness contributing favorably, and mealiness contributing negatively. Reduced oxygen pressure and inhibition of ethylene perception exerted distinct effects on the transcription of candidate genes associated with ripening in apple. Hexose and cell-wall carbohydrate metabolism genes exhibit distinct expression patterns under storage. CONCLUSION: Inhibition of ethylene perception and reduction of relative oxygen pressure under cold storage both promote similar conservation of apple sensory traits under long-term cold storage. Texture was the main contributor to global appreciation of apples subjected to long-term storage. The conditions that were investigated were able to delay, but not fully prevent, senescence, as evidenced by physicochemical and gene expression analyses. The expression of gene-encoding enzymes involved in hexose metabolism was mainly developmentally regulated, whereas storage conditions exerted a stronger effect on the expression of genes associated with cell-wall metabolism. © 2022 Society of Chemical Industry.

Technical benefit on apple fruit of controlled atmosphere influenced by 1-MCP at molecular levels.

The apple is a highly perishable fruit after harvesting and, therefore, several storage technologies have been studied to provide the consumer market with a quality product with a longer shelf life. However, little is known about the apple genome that is submitted to the storage, and even less with the application of ripening inhibitors. Due to these factors, this study sought to elucidate the transcriptional profile of apple cultivate Gala stored in a controlled atmosphere (AC) treated and not treated with 1-methyl cyclopropene (1-MCP). Through the genetic mapping of the apple, applying the microarray technique, it was possible to verify the action of treatments on transcripts related to photosynthesis, carbohydrate metabolism, response to hormonal stimuli, nucleic acid metabolism, reduction of oxidation, regulation of transcription and metabolism of cell wall and lipids. The results showed that the transcriptional profile in the entire genome of the fruit showed significant differences in the relative expression of the gene, this in response to CA in the presence and absence of 1-MCP. It should be noted that the transcription genes involved in the anabolic pathway were only maintained after six months in fruits treated with 1-MCP. The data in this work suggests that the apple in the absence of 1-MCP begins to prepare its metabolism to mature, even during the storage period in AC. Meanwhile, in the presence of the inhibitor, the transcriptional profile of the fruit is similar to that at the time of harvest. It was also found that a set of genes that code for ethylene receptors, auxin homeostasis, MADS Box, and NAC transcription factors may be involved in the regulation of post-harvest ripening after storage and in the absence of 1-MCP.

Apple Phenolic Extracts Strongly Inhibit α-Glucosidase Activity.

The beneficial health effects of apple consumption are well known, however, little is known about the potential of its phenolic fractions to inhibit α-glucosidases and thereafter to treat diseases related to the carbohydrate metabolism, such as postprandial hyperglycemia and diabetes. In the present study, the α-glucosidase inhibition and antioxidant activity of different phenolic fractions of apple were evaluated using the 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radical scavenging activity. Moreover, the phenolic fractions were chemically characterized by LC-MS in order to identify the compounds responsible for the biological properties. The purified extract (not fractionated) had the highest α-glucosidase and hydroxyl radical inhibitions. The purified extract and fractions III and IV were more active against the enzyme activity than the positive control acarbose, the drug used by diabetic patients to treat postprandial hyperglycaemia. Our results show that apple phenolic extracts strongly inhibit α-glucosidase acitivity, validating their potential to be used in the management of type 2 diabetes.

Untargeted Metabolomic Analysis of Capsicum spp. by GC-MS.

INTRODUCTION: In order to conserve the biodiversity of Capsicum species and find genotypes with potential to be utilised commercially, Embrapa Clima Temperado maintains an active germplasm collection (AGC) that requires characterisation, enabling genotype selection and support for breeding programmes. OBJECTIVE: The objective of this study was to characterise pepper accessions from the Embrapa Clima Temperado AGC and differentiate species based on their metabolic profile using an untargeted metabolomics approach. MATERIAL AND METHODS: Cold (-20°C) methanol extraction residue of freeze-dried fruit samples was partitioned into water/methanol (A) and chloroform (B) fractions. The polar fraction (A) was derivatised and both fractions (A and B) were analysed by gas chromatography coupled to mass spectrometry (GC-MS). Data from each fraction was analysed using a multivariate principal component analysis (PCA) with XCMS software. RESULTS: Amino acids, sugars, organic acids, capsaicinoids, and hydrocarbons were identified. Outlying accessions including P116 (C. chinense), P46, and P76 (C. annuum) were observed in a PCA plot mainly due to their high sucrose and fructose contents. PCA also indicated a separation of P221 (C. annuum) and P200 (C. chinense), because of their high dihydrocapsaicin content. CONCLUSIONS: Although the metabolic profiling did not allow for grouping by species, it permitted the simultaneous identification and quantification of several compounds complementing and expanding the metabolic database of the studied Capsicum spp. in the AGC. Copyright © 2017 John Wiley & Sons, Ltd.

Transcriptional regulatory networks controlling woolliness in peach in response to preharvest gibberellin application and cold storage.

BACKGROUND: Postharvest fruit conservation relies on low temperatures and manipulations of hormone metabolism to maintain sensory properties. Peaches are susceptible to chilling injuries, such as 'woolliness' that is caused by juice loss leading to a 'wooly' fruit texture. Application of gibberellic acid at the initial stages of pit hardening impairs woolliness incidence, however the mechanisms controlling the response remain unknown. We have employed genome wide transcriptional profiling to investigate the effects of gibberellic acid application and cold storage on harvested peaches. RESULTS: Approximately half of the investigated genes exhibited significant differential expression in response to the treatments. Cellular and developmental process gene ontologies were overrepresented among the differentially regulated genes, whereas sequences in cell death and immune response categories were underrepresented. Gene set enrichment demonstrated a predominant role of cold storage in repressing the transcription of genes associated to cell wall metabolism. In contrast, genes involved in hormone responses exhibited a more complex transcriptional response, indicating an extensive network of crosstalk between hormone signaling and low temperatures. Time course transcriptional analyses demonstrate the large contribution of gene expression regulation on the biochemical changes leading to woolliness in peach. CONCLUSION: Overall, our results provide insights on the mechanisms controlling the complex phenotypes associated to postharvest textural changes in peach and suggest that hormone mediated reprogramming previous to pit hardening affects the onset of chilling injuries.

Micronutrient and functional compounds biofortification of maize grains.

Maize, in addition to being the main staple food in many countries, is used in the production of hundreds of products. It is rich in compounds with potential benefits to health, such as carotenoids, phenolic compounds, vitamin E, and minerals that act as cofactors for antioxidant enzymes. Many of these compounds have been neglected thus far in the scientific literature. Nevertheless, deficiencies in the precursors of vitamin A and some minerals, such as iron and zinc, in maize, in association with the great genetic variability in its cultivars and our genomic, transcriptomic, and metabolomic knowledge of this species make targeted biofortification strategies for maize promising. This review discusses the potential of the main microconstituents found in maize with a focus on studies aimed at biofortification.

Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses.

The process of selection and validation of reference genes is the first step in studies of gene expression by real-time quantitative polymerase chain reaction (RT-qPCR). The genome of lettuce, the most popular leaf vegetable cultivated worldwide, has recently been sequenced; therefore, suitable reference genes for reliable results in RT-qPCR analyses are required. In the present study, 17 candidate reference genes were selected, and their expression stability in lettuce leaves under drought, salt, heavy metal, and UV-C irradiation conditions and under the application of abscisic acid (ABA) was evaluated using geNorm and NormFinder software. The candidate reference genes included protein-coding traditional and novel reference genes and microRNAs (miRNAs). The results indicate that the expression stability is dependent on the experimental conditions. The novel protein-coding reference genes were more suitable than the traditional reference genes under drought, UV-C irradiation, and heavy metal conditions and under the application of ABA. Only under salinity conditions were the traditional protein-coding reference genes more stable than the novel genes. In addition, the miRNAs, mainly MIR169, MIR171/170 and MIR172, were stably expressed under the abiotic stresses evaluated, representing a suitable alternative approach for gene expression data normalization. The expression of phenylalanine ammonia lyase (PAL) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) was used to further confirm the validated protein-coding reference genes, and the expression of MIR172 and MIR398 was used to confirm the validated miRNA genes, showing that the use of an inappropriate reference gene induces erroneous results. This work is the first survey of the stability of reference genes in lettuce and provides guidelines to obtain more accurate RT-qPCR results in lettuce studies.

Isolation of high-quality RNA from grains of different maize varieties.

The study of gene expression in maize varieties represents a powerful tool aiming to increase vitamin A precursors. However, the isolation of RNA from different maize varieties is challenging because these varieties show different levels of polysaccharides, and most methods available for RNA isolation are inappropriate for grain samples. The polysaccharides co-purify and co-precipitate with RNA during isolation, resulting in low-quality RNA, compromising the use of RNA in subsequent applications. Thus, a cetyltrimethylammonium bromide (CTAB)-based method was adapted in this study and compared with six methods for RNA isolation, including commercial reagents and RNA and DNA isolation kits, in order to identify the most appropriate for maize grains from different varieties. Most of the methods evaluated were considered inadequate due to limitations in terms of purity and/or quantity of the isolated RNA, which affected the efficiency of subsequent RT-qPCR analysis, resulting in nonamplification of ß-carotene hydroxylase gene (HYD3) or high deviation among replicates. However, the CTAB modified method allowed the study to obtain intact RNA, with high quality and quantity, from 25 maize varieties. Furthermore, this RNA was successfully used to evaluate the expression of HYD3 gene by real-time qualitative polymerase chain reaction (RT-qPCR), and thus represents a simple, efficient, and low-cost strategy.

Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains.

KEY MESSAGE: The stability of candidate reference genes was evaluated in maize landrace varieties and during multiple grain developmental stages to evaluate the expression of carotenoid-related genes by RT-qPCR for application to maize biofortification. Vitamin A deficiency affects millions of children worldwide; therefore, increasing the content of vitamin A precursors in maize grains is of interest. The study of the expression of genes involved in the carotenoid biosynthetic pathway in maize grains has provided useful information for metabolic engineering approaches. However, reliable results using real-time quantitative polymerase chain reaction (RT-qPCR) experiments are dependent on the use of the appropriate reference genes. In this study, we utilized geNorm and NormFinder softwares to identify the most stably expressed candidate reference genes in samples from seven stages of grain development and from eight landrace varieties. The results of the analysis performed using geNorm indicated that tubulin (TUB) and actin (ACT) were the most suitable reference genes among all experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) showed the least stability. The same result was obtained with the NormFinder software. The minimum number of genes required in each experimental condition to normalize the gene expression data was also determined by geNorm. The expression of phytoene synthase gene (PSY1), the first enzyme in the carotenoid biosynthetic pathway, was overestimated when the least stable candidate gene (GAPDH) was used as the internal control instead of the most stable gene pair (ACT + TUB), thus highlighting the importance of validating reference genes before conducting a RT-qPCR experiment to obtain accurate results. This study is the first survey of the stability of genes for use as reference genes to normalize RT-qPCR data from maize landraces during multiple stages of grain development.

Approximate entropy: a promising tool to understand the hidden electrical activity of fruit.

Fruits, like other parts of the plant, appear to have a rich electrical activity that may contain information. Here, we present data showing differences in the electrome complexity of tomato fruits through ripening and discuss possible physiological processes involved. The complexity of the signals, measured through approximate entropy, varied along the fruit ripening process. When analyzing the fruits individually, a decrease in entropy values was observed when they entered the breaker stage, followed by a tendency to increase again when they entered the light red stage. Consequently, the data obtained showed a decrease in signal complexity in the breaker stage, probably due to some physiological process that ends up predominating to the detriment of others. This result may be linked to processes involved in ripening, such as climacteric. Electrophysiological studies in the reproductive stage of the plant are still scarce, and research in this direction is of paramount importance to understand whether the electrical signals observed can transmit information from reproductive structures to other modules of plants. This work opens the possibility of studying the relationship between the electrical activity and fruit ripening through the analysis of approximate entropy. More studies are necessary to understand whether there is a correlation or a cause-response relationship in the phenomena involved. There is a myriad of possibilities for the applicability of this knowledge to different areas, from understanding the cognitive processes of plants to achieving more accurate and sustainable agriculture.

Red onion skin extract rich in flavonoids encapsulated in ultrafine fibers of sweet potato starch by electrospinning.

Electrospinning encapsulation is a highly viable method to protect bioactive compounds and prevent their degradation. Hence, this study produced ultrafine fibers based on yellow and white sweet potato starches and a red onion skin extract (ROSE; 0, 3, 6, and 9 %, w/w) using electrospinning. The fibers were evaluated for morphology, thermogravimetric properties, antioxidant, in vitro release simulation, thermal resistance (100 and 180 °C), and wettability. The fibers with ROSE presented 251-611 nm diameters, 67-78 % loading capacity, and 51.6-95.4 and 13.4-99.4 % thermal resistance (100 and 180 °C, respectively); apigenin presented the highest thermal protection. The phenolic compounds showed low release using 10 % ethanol and greater release with 50 % ethanol. The fibers with 9 % ROSE showed 2,2'-azino-bis(3-ethylbenzothiazoline) 6-sulfonic acid radical inhibition above 92 %. The ultrafine fibers and the unencapsulated ROSE showed inhibitory action against Escherichia coli and Staphylococcus aureus; only unencapsulated ROSE showed bactericidal activity.

Extraction of hops pelletized (Humulus lupulus) with subcritical CO2 and hydrodistillation: Chemical composition identification, kinetic model, and evaluation of antioxidant and antimicrobial activity.

Hop essential oil and hop extract using carbon dioxide (CO2) are products with high added value because they have bioactive and sensory properties. In this context, the objective of this study was to obtain and characterize essential oil and extracts from pelleted hops of El Dorado, Polaris, Hallertau Blanc and Callista varieties using hydrodistillation and subcritical CO2 extraction methods. Extraction yield ranged from 0.38 % to 1.97 % (m/m) for essential oils and from 8.76 % to 15.35 % (m/m) for extracts using subcritical CO2. The chemical compositions of the essential oils were mainly monoterpene (18.14 % to 29.91 %) and sesquiterpene (46.01 % to 59.03 %) hydrocarbons and for the extracts were sesquiterpene hydrocarbons (33.05 % to 71.90 %) and oxygenated sesquiterpenes (14.80 % to 34.89 %). The extracts showed better antioxidant activity than essential oils due to the presence of phenolic compounds and flavonoids. Hop extracts showed some antimicrobial activity, but essential oils did not demonstrate antimicrobial potential. Hop extracts obtained with subCO2 have the potential to be used in the brewing industry as a flavoring and as natural antioxidants.

The postharvest ripening of strawberry fruits induced by abscisic acid and sucrose differs from their in vivo ripening.

In this study, we compared the chemical composition of strawberry (Fragaria × ananassa) fruits that were ripened in vivo (attached to plant) to those ripened during postharvest storage. The effects of the application of abscisic acid (ABA) and sucrose on the postharvest ripening were also evaluated. The results suggested that the postharvest ripening process was dependent on the signal triggered by ABA and differed from in vivo ripening, resulting in fruits with altered chemical composition and firmness. The application of sucrose in unripe strawberries resulted in the induction of ripening, which is dependent on ABA and its derivatives. This induction was more pronounced during the first days of storage and associated with the application of mannitol rather than water, suggesting that mannitol negatively regulated the postharvest strawberry ripening. These results provide further insights into the role of ABA and sucrose in the regulation of postharvest ripening of strawberry.

Extraction and characterization of phytochemical compounds from araçazeiro (Psidium cattleianum) leaf: Putative antioxidant and antimicrobial properties.

Underexplored species have phytochemical potential for pharmacological and nutraceutical applications. The fruits of such species, including aracá (Psidium cattleianum Sabine), are rich in specialized metabolites with putative antioxidant and antimicrobial activity; therefore, the leaves of these species are also a potential source of bioactive compounds. In this study, araçazeiro leaves were extracted using an aqueous infusion (Al) and a pressurized liquid extraction system with water (PLE-W), ethanol (PLE-E), and 1:1 water:ethanol ratio combination (PLE-W:E). PLE-W:E yielded a greater diversity of extracted compounds. Nonetheless, all extracts showed inhibitory activity against pathogenic Gram-positive and Gram-negative bacteria and antioxidant activity in the in vitro thiobarbituric acid reactive substances (TBARS) and reactive oxygen species (ROS) assays with rat brain and yeast model systems. Thus, araçazeiro leaves can be exploited as a promising source of bioactive compounds.

Genome-wide identification, and characterization of the CDPK gene family reveal their involvement in abiotic stress response in Fragaria x ananassa.

Calcium-dependent protein kinases (CDPKs) are encoded by a large gene family and play important roles against biotic and abiotic stresses and in plant growth and development. To date, little is known about the CDPK genes in strawberry (Fragaria x ananassa). In this study, analysis of Fragaria x ananassa CDPK gene family was performed, including gene structures, phylogeny, interactome and expression profiles. Nine new CDPK genes in Fragaria x ananassa were identified based on RNA-seq data. These identified strawberry FaCDPK genes were classified into four main groups, based on the phylogenetic analysis and structural features. FaCDPK genes were differentially expressed during fruit development and ripening, as well as in response to abiotic stress (salt and drought), and hormone (abscisic acid) treatment. In addition, the interaction network analysis pointed out proteins involved in the ABA-dependent response to plant stress via Ca2+ signaling, especially RBOHs. To our knowledge, this is the first report on CDPK families in Fragaria x ananassa, and it will provide valuable information for development of biofortified fruits and stress tolerant plants.

Discrimination of genotype and geographical origin of black rice grown in Brazil by LC-MS analysis of phenolics.

Physicochemical properties, cooking time, and phenolics profile of two black rice genotypes grown at six different locations in Brazil were determined. The cultivar IAC 600 and the elite-line AE 153045 were used. The main growing locations for black rice were considered, as follows: Alegrete (ALG), Capão do Leão (CPL), Guaratinguetá (GUA), Roseira (ROS), Santa Vitória do Palmar (SVP), and Taubaté (TBT). Principal component analysis (PCA) and supervised partial least squares-discriminant analysis (PLS-DA) from liquid chromatography-mass spectrometry (LC-MS) data sets showed distinction among genotypes and locations. Quercetin-3-O-glucoside and vanillic acid were the most relevant compounds for discriminating genotypes. SVP location provided the most distinctive black rice, with greater total phenolics content. Characteristics of black rice from SVP location were associated to effects of latitude and wind conditions. Hesperetin, vanillic acid, quercetion-3-O-glucoside, and p-coumaric acid were the most relevant compounds for discriminating locations.

ABA-dependent salt and drought stress improve strawberry fruit quality.

Strawberry crop is very sensitive to osmotic stress conditions. We investigated the effect of the stress induced by mild drought (DS) and salt (SS) stresses, on molecular, physiological, and metabolic processes in the strawberry crop (Fragaria ananassa), cv. Camarosa. The results showed that the fruit yield was not affected. Mild DS and SS also resulted in an increased content of phenolics, anthocyanins, and l-ascorbic acid, and an increased antioxidant activity. These effects were accompanied by increased levels of ABA and its derivatives (phaseic and dehydrophasic acids), alongside the upregulation of several genes involved on their synthesis. Therefore, the results obtained in this study suggest that mild DS and SS improve the functional quality of strawberry fruits through and ABA-dependent mechanism.

Butia spp. (Arecaceae) LC-MS-Based Metabolomics for Species and Geographical Origin Discrimination.

The metabolic variability of fruit from Butia spp. (Arecaceae) genotypes from different geographical locations was characterized using untargeted metabolomics by liquid chromatography-mass spectrometry (LC-MS) followed by multivariate data analyses. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) from LC-MS data sets showed a clear distinction among Butia catarinensis, Butia odorata, Butia paraguayensis, and Butia yatay. The major metabolites that contributed to species discrimination were primary metabolites including sugars and organic acids and specialized metabolites such as tetrahydroxy-trans-stilbene and rutin. B. odorata fruit from Tapes, RS, Brazil, showed a high content of organic acids and flavonoids, whereas B. odorata fruits from Capão do Leão, RS, Brazil, showed a high sugar content. The results demonstrate that LC-ESI-qToF-MS-based metabolic profiling coupled with chemometric analysis can be used to discriminate among Butia species and between geographical origins of B. odorata and to identify primary and specialized metabolites responsible for the discrimination.

Stability of bioactive compounds in butiá (Butia odorata) fruit pulp and nectar.

Butia odorata is a palm tree native to southern Brazil whose fruit (known as butiá) and leaves are used to make many food products and crafts. Butiá contain several biologically active compounds with potential health benefits. However, processing conditions can alter quality attributes including bioactive compound content. This study evaluated the stability of bioactive compounds in butiá pulp upon pasteurization, during 12months of frozen storage, and in butiá nectar after a 3-month storage period. Pulp pasteurization resulted in a reduction in phenolic, flavonoid, carotenoid, and ascorbic acid contents. After a 12-month frozen storage period, flavonoid, phenolic, and ascorbic acid contents decreased while carotenoid content remained unaltered. Carotenoid, ascorbic acid, and phenolic contents were unaffected by the 3-month storage of butiá nectar; however, flavonoid content and antioxidant potential were reduced. Despite bioactive compound degradation upon heat treatment and storage, butiá nectar remained rich in phenolics, especially (-)-epicatechin, rutin, and (+)-catechin.

Contrasting Transcriptional Programs Control Postharvest Development of Apples (Malus x domestica Borkh.) Submitted to Cold Storage and Ethylene Blockage.

Apple is commercially important worldwide. Favorable genomic contexts and postharvest technologies allow year-round availability. Although ripening is considered a unidirectional developmental process toward senescence, storage at low temperatures, alone or in combination with ethylene blockage, is effective in preserving apple properties. Quality traits and genome wide expression were integrated to investigate the mechanisms underlying postharvest changes. Development and conservation techniques were responsible for transcriptional reprogramming and distinct programs associated with quality traits. A large portion of the differentially regulated genes constitutes a program involved in ripening and senescence, whereas a smaller module consists of genes associated with reestablishment and maintenance of juvenile traits after harvest. Ethylene inhibition was associated with a reversal of ripening by transcriptional induction of anabolic pathways. Our results demonstrate that the blockage of ethylene perception and signaling leads to upregulation of genes in anabolic pathways. We also associated complex phenotypes to subsets of differentially regulated genes.