Metabolomics approach for phenolic compounds profiling of soursop (Annona muricata L.) fruit during postharvest storage.

INTRODUCTION: Soursop (Annona muricata L.) is a crop with medicinal properties and numerous bioactive compounds. Ripening is a complex process that regulates fruit quality and changes in metabolite content, such as flavonoids, polyphenols, and organic acids. OBJECTIVES: This study aimed to analyze the phenolic profiling of soursop fruit ripening. METHODS: The metabolic changes in different days of storage of soursop fruits were investigated using a semi-metabolomic approach based on ultra-performance liquid chromatography coupled to electrospray ionization quadrupole-time of flight mass spectrometry (UPLC-ESI-QTOF-MS). Further, multivariate analysis such as supervised partial least squares discriminant analysis (PLS-DA) was conducted to identify differential metabolites. RESULTS: A total of 68 metabolites were identified in soursop fruit during postharvest storage. A higher number of metabolites were identified in the Day zero (D0) compared to the Day one (D1), Day three (D3), and Day five (D5), belonging to flavonoids, other polyphenols, phenolic acids, and organic acids. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the pathways of flavone and flavonol biosynthesis, flavonoid biosynthesis, and biosynthesis of secondary metabolites were mostly enriched. Additionally, we included all the compounds and their postharvest storage in the public Phenolics profile database. CONCLUSIONS: Here, we show that the stage of ripening has a significant effect on the phenolic content, highlighting the point of cut (D0) and the onset of senescence (D5). The findings of this study provide new insights into the soursop fruit quality and may contribute to the identification of metabolic markers for its storage.

Bacillus mojavensis enhances the antioxidant defense mechanism of soursop (Annona muricata L.) fruits during postharvest storage.

Rapid softening of soursop (Annona muricata L.) fruit results in postharvest losses. Bacillus genus is one of the most studied antagonistic biological control agents against postharvest diseases. Nevertheless, information about how this bacterium acts on the fruits is still not understood. The objective of this study aims to gain an insight into the effect of Bacillus mojavensis on the activity and gene expression of antioxidant defense enzymes in soursop fruits during postharvest storage. Our findings indicate different responses in the fruits inoculated with B. mojavensis at biochemical and molecular levels. On day one, fruits inoculated with B. mojavensis presented a mean value of 79.09 GAE/100 gFW in total phenols, and higher superoxide dismutase (SOD) and catalase (CAT) activities (1.35 and 1.78-fold higher, respectively). On the other hand, on the third day of storage, the ferric reducing/antioxidant power (FRAP) reached its highest level, including an increase in the expression of SOD, and PPO genes by 18.7-fold and 4.5-fold in fruits inoculated with B. mojavensis. Finally, on the fifth day of storage, soursop fruits inoculated with B. mojavensis had the highest mean values for 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH·), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS· +), with values of 194.68 EAA/100 gFW, and 172.33 EAA/100 gFW, respectively. Indeed, higher polyphenol oxidase (PPO), and peroxidase (POD) activities (2.17-fold and 1.27-fold higher, respectively) were recorded compared to the control fruits. We show that depending on the stage of ripening, the antagonist bacteria B. mojavensis enhanced the antioxidant capacity, enzymatic activity, and gene expression of soursop fruits.

Transcriptome Analysis of Soursop (Annona muricata L.) Fruit under Postharvest Storage Identifies Genes Families Involved in Ripening.

Soursop (Annona muricata L.) is climacteric fruit with a short ripening period and postharvest shelf life, leading to a rapid softening. In this study, transcriptome analysis of soursop fruits was performed to identify key gene families involved in ripening under postharvest storage conditions (Day 0, Day 3 stored at 28 ± 2 °C, Day 6 at 28 ± 2 °C, Day 3 at 15 ± 2 °C, Day 6 at 15 ± 2 °C, Day 9 at 15 ± 2 °C). The transcriptome analysis showed 224,074 transcripts assembled clustering into 95, 832 unigenes, of which 21, 494 had ORF. RNA-seq analysis showed the highest number of differentially expressed genes on Day 9 at 15 ± 2 °C with 9291 genes (4772 up-regulated and 4519 down-regulated), recording the highest logarithmic fold change in pectin-related genes. Enrichment analysis presented significantly represented GO terms and KEGG pathways associated with molecular function, metabolic process, catalytic activity, biological process terms, as well as biosynthesis of secondary metabolites, plant hormone signal, starch, and sucrose metabolism, plant-pathogen interaction, plant-hormone signal transduction, and MAPK-signaling pathways, among others. Network analysis revealed that pectinesterase genes directly regulate the loss of firmness in fruits stored at 15 ± 2 °C.

Salpianthus macrodontus Extracts, a Novel Source of Phenolic Compounds with Antibacterial Activity against Potentially Pathogenic Bacteria Isolated from White Shrimp.

This study aimed to evaluate the antibacterial activity in vitro of Salpianthus macrodontus and Azadirachta indica extracts against potentially pathogenic bacteria for Pacific white shrimp. Furthermore, the extracts with higher inhibitory activity were analyzed to identify compounds responsible for bacterial inhibition and evaluate their effect on motility and biofilm formation. S. macrodontus and A. indica extracts were prepared using methanol, acetone, and hexane by ultrasound. The minimum inhibitory concentration (MIC) of the extracts was determined against Vibrio parahaemolyticus, V. harveyi, Photobacterium damselae and P. leiognathi. The polyphenol profile of those extracts showing the highest bacterial inhibition were determined. Besides, the bacterial swimming and swarming motility and biofilm formation were determined. The highest inhibitory activity against the four pathogens was found with the acetonic extract of S. macrodontus leaf (MIC of 50 mg/mL for Vibrio spp. and 25 mg/mL for Photobacterium spp.) and the methanol extract of S. macrodontus flower (MIC of 50 mg/mL for all pathogens tested). Both extracts affected the swarming and swimming motility and the biofilm formation of the tested bacteria. The main phenolic compounds related to Vibrio bacteria inhibition were naringin, vanillic acid, and rosmarinic acid, whilst hesperidin, kaempferol pentosyl-rutinoside, and rhamnetin were related to Photobacterium bacteria inhibition.

DIVERSIDAD GENÉTICA DE POBLACIONES DE GUANÁBANA (Annona muricata L.) EN NAYARIT, MÉXICO MEDIANTE MARCADORES SSR Y SRAP / Genetic diversity of soursop populations (Annona muricata L.) in Nayarit, Mexico using SSR and SRAP markers

RESUMEN La guanábana (Annona muricata L.) es un cultivo de importancia económica para Nayarit, México. Los frutos han tenido una excelente aceptación en el mercado regional, dificultando su comercialización a lugares lejanos porque la producción es altamente perecedera, aunado a que los árboles de los huertos de guanábana son en su mayoría ecotipos o fenotipos sin ningún plan de mejoramiento genético. Debido a la falta de variedades comerciales y de un banco de germoplasma, es importante conocer la diversidad genética para identificar y seleccionar genotipos; una de las herramientas para este propósito es el uso de marcadores moleculares. El objetivo de esta investigación fue analizar la diversidad genética de guanábana de las principales zonas productoras de Nayarit. Se extrajo ADN genómico de hojas de guanábana, las cuales fueron recolectadas de 11 huertos (poblaciones) de las siguientes zonas: Compostela (cinco poblaciones), Tepic (tres poblaciones) y San Blas (tres poblaciones). Posteriormente, se realizó un análisis mediante marcadores moleculares SSR y SRAP. Los resultados indicaron que los SSR no mostraron polimorfismo entre las poblaciones. Por otro lado, en los marcadores SRAP se obtuvieron 116 loci polimórficos con un promedio de porcentaje de loci polimórfico (P) entre las zonas productoras de 29,55 %. Asimismo, se realizó un AMOVA, el cual mostró que el mayor porcentaje de varianza se encuentra dentro de las poblaciones. Además, los análisis de agrupamiento demostraron la formación de tres grupos independientes. Por tanto, se obtuvo una alta homocigocidad y baja diversidad genética de guanábana entre las zonas y poblaciones estudiadas.

ABSTRACT Soursop (Annona muricata L.) is a crop of economic importance for Nayarit, Mexico. Soursop fruits have had an excellent acceptance in the regional market, making it difficult its commercialization to distant places because the production is highly perishable, in addition to the fact that the trees in the soursop orchards are mostly ecotypes or phenotypes without any genetic improvement plan. Due to the lack of commercial varieties and a germplasm bank, it is important to know the genetic diversity to identify and select genotypes; one of the tools for this purpose is the use of molecular markers. The objective of this research was to analyze the genetic diversity of soursop in the main producing areas of Nayarit. Genomic DNA was extracted from soursop leaves from 11 orchards (populations) in the following areas: Compostela (five populations), Tepic (three populations) and San Blas (three populations). Subsequently, we performed molecular analysis using SSR and SRAP molecular markers. The results indicated that the SSRs showed no polymorphism between the populations. On the other hand, we found 116 polymorphic loci in the SRAP markers with an average percentage of polymorphic loci (P) among the producing areas of 29.55 %. Likewise, an AMOVA was performed, showing that the highest percentage of variance is found within the populations. Furthermore, cluster analyzes demonstrated the formation of three independent groups. Therefore, a high homozygosity and low genetic diversity of soursop were obtained between the areas and populations studied.

Differential Responses of Antioxidative System during the Interaction of Soursop Fruits (Annona muricata L.) and Nectria haematococca at Postharvest Storage.

Soursop fruit (Annona muricata L.) production is diminished by the attack of pathogens such as Nectria haematococca. However, the fruit-pathogen interaction at the biochemical and molecular levels is still unknown. The objective of this study was to analyze the response of the soursop fruit to the presence of N. haematococca during postharvest storage. Soursop fruits were inoculated with the pathogen and total phenolic compounds, antioxidant capacity by Ferric reducing/antioxidant power (FRAP), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS•+), and 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH•), as well as enzymatic activity and transcript levels of polyphenol oxidase (PPO) and superoxide dismutase (SOD), were evaluated at 1, 3, and 5 days of storage. The noninoculated fruits were the controls of the experiment. The highest total phenol content was recorded on day one in the inoculated fruits. FRAP, ABTS, and DPPH activity presented the highest values on day three in the control fruits. Inoculated fruits recorded the highest PPO activity on day five and a five-fold induction in the PPO transcript on day three. SOD activity showed a decrease during the days of storage and 10-fold induction of SOD transcript on day three in the inoculated fruits. Principal component analysis showed that total phenols were the variable that contributed the most to the observed variations. Furthermore, a positive correlation between total phenols and SOD activity, PPO expression, and SOD expression, as well as between DPPH and FRAP, was recorded. The results showed a differential response in antioxidant capacity, enzymatic activity, and gene expression during the interaction of soursop fruits-N. haematococca at postharvest storage.

Functional analysis of tomato rhamnogalacturonan lyase gene Solyc11g011300 during fruit development and ripening.

Rhamnogalacturonan I (RG-I) is a domain of plant cell wall pectin. The rhamnogalacturonan lyase (RGL) enzyme (EC 4.2.2.23) degrades RG-I by cleaving the α-1,4 glycosidic bonds located between the l-rhamnose and d-galacturonic residues of the main chain. While RGL's biochemical mode of action is well known, its effects on plant physiology remain unclear. To investigate the role of the RGL enzyme in plants, we have expressed the Solyc11g011300 gene under a constitutive promoter (CaMV35S) in tomato cv. 'Ohio 8245' and evaluated the expression of this and other RGL genes, enzymatic activity and alterations in vegetative tissue, and tomato physiology in transformed lines compared to the positive control (plants harboring the pCAMBIA2301 vector) and the isogenic line. The highest expression levels of the Solyc11g011300, Solyc04g076630, and Solyc04g076660 genes were observed in leaves and roots and at 10 and 20 days after anthesis (DAA). Transgenic lines exhibited lower RGL activity in leaves and roots and during fruit ripening, whereas higher activity was observed at 10, 20, and 30 DAA than in the isogenic line and positive control. Both transgenic lines showed a lower number of seeds and fruits, higher root length, and less pollen germination percentage and viability. In red ripe tomatoes, transgenic fruits showed greater firmness, longer shelf life, and reduced shriveling than did the isogenic line. Additionally, a delay of one week in fruit ripening in transgenic fruits was also recorded. Altogether, our data demonstrate that the Solyc11g011300 gene participates in pollen tube germination, fruit firmness, and the fruit senescence phenomena that impact postharvest shelf life.

Functional analysis of a tomato (Solanum lycopersicum L.) rhamnogalacturonan lyase promoter.

The enzyme rhamnogalacturonan lyase (RGL) cleaves α-1,4 glycosidic bonds located between rhamnose and galacturonic acid residues in the main chain of rhamnogalacturonan-I (RG-I), a component of the plant cell wall polymer pectin. Although the mode of action of RGL is well known, its physiological functions associated with fruit biology are less understood. Here, we generated transgenic tomato plants expressing the ß-glucuronidase (GUS) reporter gene under the control of a -504 bp or a -776 bp fragment of the promoter of a tomato RGL gene, Solyc11g011300. GUS enzymatic activity and the expression levels of GUS and Solyc11g011300 were measured in a range of organs and fruit developmental stages. GUS staining was undetectable in leaves and roots, but high GUS enzymatic activity was detected in flowers and red ripe (RR) fruits. Maximal expression levels of Solyc11g011300 were detected at the RR developmental stage. GUS activity was 5-fold higher in flowers expressing GUS driven by the -504 bp RGL promoter fragment (RGFL3::GUS) than in the isogenic line, and 1.7-fold higher when GUS gene was driven by the -776 bp RGL promoter fragment (RGLF2::GUS) or the constitutive CaMV35S promoter. Quantitative real-time polymerase chain reaction analysis showed that the highest expression of GUS was in fruits at 40 days after anthesis, for both promoter fragments. The promoter of Solyc11g011300 is predicted to contain cis-acting elements, and to be active in pollen grains, pollen tubes, flowers and during tomato fruit ripening, suggesting that the Solyc11g011300 promoter is transcriptionally active and organ-specific.