Metabolic and gene-expression analyses reveal developmental dynamics of cutin deposition in pomegranate fruit grown under different environmental conditions.

The chemical and transcriptional changes in the cuticle of pomegranate (Punica granatum L.) fruit grown under different environmental conditions were studied. We collected fruit from three orchards located in different regions in Israel, each with a distinct microclimate. Fruit were collected at six phenological stages, and cutin monomers in the fruit cuticle were profiled by gas chromatography-mass spectrometry (GC-MS), along with qPCR transcript-expression analyses of selected cutin-related genes. While fruit phenotypes were comparable along development in all three orchards, principal component analyses of cutin monomer profiles suggested clear separation between cuticle samples of young green fruit to those of maturing fruit. Moreover, total cutin contents in green fruit were lower in the orchard characterized by a hot and dry climate compared to orchards with moderate temperatures. The variances detected in total cutin contents between orchards corresponded well with the expression patterns of BODYGUARD, a key biosynthetic gene operating in the cutin biosynthetic pathway. Based on our extraction protocols, we found that the cutin polyester that builds the pomegranate fruit cuticle accumulates some levels of gallic acid-the precursor of punicalagin, a well-known potent antioxidant metabolite in pomegranate fruit. The gallic acid was also one of the predominant metabolites contributing to the variability between developmental stages and orchards, and its accumulation levels were opposite to the expression patterns of the UGT73AL1 gene which glycosylates gallic acid to synthesize punicalagin. To the best of our knowledge, this is the first detailed composition of the cutin polyester that forms the pomegranate fruit cuticle.

Pomegranate Rhizosphere Microbial Diversity Revealed by Metagenomics: Toward Organic Farming, Plant Growth Promotion and Biocontrol?

Food production must undergo systems change to meet the sustainable development goals (SDGs). For example, organic farming can be empowered by soil microorganisms with plant growth promotion (PGP) and biocontrol features. In this context, there have been limited studies on pomegranate. We investigated microbial diversity in rhizosphere of the pomegranate "Bhagwa" variety and its potential role in PGP and biocontrol. Both bulk and rhizosphere soil samples were analyzed for their physicochemical properties. Whole metagenome sequencing was conducted using the Illumina NovaSeq6000 platform. Surprisingly, we found that bulk and rhizosphere soil samples had comparable microbial diversity. Metagenome sequencing revealed the abundance of Streptomyces indicus, Bradyrhizobium kalamazoonesis, and Pseudomonas cellulosum in the rhizosphere that are reported here for the first time in agricultural literature. Pathway prediction analysis using KEGG (Kyoto Encyclopedia for Genes and Genomes) and COG (clusters of orthologous genes) databases identified metabolic pathways associated with biocontrol properties against pathogens. We confirmed the metagenome data in vitro, which demonstrated their PGP potential and antimicrobial properties. For instance, S. indicus produced high concentration of indole-3-acetic acid, a PGP phytohormone, that can stimulate plant growth. In addition, an antimicrobial susceptibility assay suggested that bacterial extracts displayed activity against Xanthomonas, a primary pathogen causing the pomegranate wilt disease. In conclusion, this study suggests that S. indicus, B. kalamazoonesis, and P. cellulosum can potentially be PGP and biocontrol agents that may contribute to increased crop productivity in pomegranate cultivation. These agents and their combinations warrant future research with an eye on SDGs and so as to enable and innovate organic farming and pomegranate agricultural practices.

Integrated transcriptomic, metabolomic, and functional analyses unravel the mechanism of bagging delaying fruit cracking of pomegranate (Punica granatum L.).

The economic impact of fruit cracking in pomegranate products is substantial. In this study, we present the inaugural comprehensive analysis of transcriptome and metabolome in the outermost pericarp of pomegranate fruit in bagging conditions. Our investigation revealed a notable upregulation of differentially expressed genes (DEGs) associated with the calcium signaling pathway (76.92%) and xyloglucan endotransglucosylase/hydrolase (XTH) genes (87.50%) in the fruit peel of non-cracking fruit under bagging. Metabolomic analysis revealed that multiple phenolics, flavonoids, and tannins were identified in pomegranate. Among these, calmodulin-like 23 (PgCML23) exhibited a significant correlation with triterpenoids and demonstrated a marked upregulation under bagging treatment. The transgenic tomatoes overexpressing PgCML23 exhibited significantly higher cellulose content and xyloglucan endotransglucosylase (XET) enzyme activity in the pericarp at the red ripening stage compared to the wild type. Conversely, water-soluble pectin content, polygalacturonase (PG), and ß-galactosidase (ß-GAL) enzyme activities were significantly lower in the transgenic tomatoes. Importantly, the heterologous expression of PgCML23 led to a substantial reduction in the fruit cracking rate in tomatoes. Our findings highlight the reduction of fruit cracking in bagging conditions through the manipulation of PgCML23 expression.

Understanding the role of SWEET genes in fruit development and abiotic stress in pomegranate (Punica granatum L.).

BACKGROUND: The Sugar Will Eventually Be Exported Transporters (SWEET), consisting of the MtN3 and salvia domain, are sugar transporters having an active role in diverse activities in plants such as pollen nutrition, phloem loading, nectar secretion, reproductive tissue development, and plant-pathogen interaction. The SWEET genes have been characterized only in a few fruit crop species. METHODS AND RESULTS: In this study, a total of 15 SWEET genes were identified in the pomegranate (Punica granatum) genome. The gene structure, transmembrane (TM) helices, domain architecture, and phylogenetic relationships of these genes were evaluated using computational approaches. Genes were further classified as Semi-SWEETs or SWEETs based on the TM domains. Similarly, pomegranate, Arabidopsis, rice, and soybean SWEETs were studied together to classify into major groups. In addition, analysis of RNAseq transcriptome data was performed to study SWEEET gene expression dynamics in different tissue. The expression suggests that SWEETs are mostly expressed in pomegranate peel. In addition, PgSWEET13 was found to be differentially expressed under high salinity stress in pomegranate. Further, quantitative PCR analysis confirmed the expression of four candidate genes in leaf and stem tissues. CONCLUSION: The information provided here will help to understand the role of SWEET genes in fruit development and under abiotic stress conditions in pomegranate.

Comparative effects of gibberellin A3 , glycine betaine, and Si, Ca, and K fertilizers on physiological disorders and yield of pomegranate cv. Wonderful.

BACKGROUND: Damage from cracking, russeting, and sunscalds causes significant yield losses in pomegranate worldwide and may result from stressful environmental conditions. Although foliar sprays with minerals or growth regulators could be an important orchard management, little is known on the effectiveness of glycine betaine, silicon (Si)-based fertilizers or the response of cv. Wonderful to gibberellin A3 (GA3 ). RESULTS: During a 2-year study, foliar spraying with GA3 at 75 or 150 mg L-1 applied in July substantially reduced cracking, russeting, and sunscald symptoms and increased fruit size, yield, and 100-aril weight, without affecting the % edible portion or % juice, suggesting that arils and skin increased similarly. Nevertheless, they reduced the skin red coloration, especially at the higher dose. GA3 at 75 mg L-1 applied in September resulted in a low number of harvested fruit as a result of delayed maturation. Spraying with glycine betaine at seven repeated times at biweekly intervals starting in July, reduced sunscald symptoms, red coloration, and maturity index only in the year with high damage. Foliar sprays with calcium chloride or Si-based fertilizer containing potassium, applied as in the glycine betaine treatment, did not affect the occurrence of physiological disorders, whereas Si-based fertilizer containing potassium and calcium increased cracking and decreased sunscald only in the year with high damage. CONCLUSION: Spraying with GA3 at 75 mg L-1 in July could have a significant impact on a grower's income by reducing damage from physiological disorders, improving yield with a minimum decrease in red skin coloration. The efficacy of nutrient-related fertilizers and glycine betaine were not constant, and this would be useful to evaluate at earlier application times and under stress conditions. © 2021 Society of Chemical Industry.

Fresh pomegranate juices from cultivars and local ecotypes grown in southeastern Italy: comparison of physicochemical properties, antioxidant activity and bioactive compounds.

BACKGROUND: Pomegranate juice has gained attention for its health properties, becoming consequently a highly demanded product. The revival of the pomegranate in Italy, as in other Mediterranean countries, starts with the planting of new intensive orchards characterized by both the new cultivation technique and new varieties. As a result of growing demand and high productivity, pomegranate could become an interesting crop to diversify farm income. This study seeks to determine the aril juice quality attributes and bioactive compounds of six pomegranate cultivars ('Mollar', 'Dente di cavallo', 'Acco', 'Jolly red', 'Wonderful' and 'Wonderful Super') and two local ecotypes ('Eco BA' and 'Eco FG') grown in Apulia region, southern Italy. RESULTS: The aril juices were evaluated for their main physicochemical properties (yield, color, pH, total soluble solids content, titratable acidity, sugar-acid ratio), chemical and bioactive compounds (vitamin C, phenolics, anthocyanins and antioxidant activities). 'Eco BA', 'Mollar' and 'Jolly red' genotypes were characterized by the highest maturity index, and then could be considered to be sweet-sour in taste. Total phenols and antioxidant activity were higher in 'Dente di cavallo' and 'Eco FG' genotypes. 'Eco FG' was also the richest in vitamin C, punicalagin and ellagic acids, while 'Dente di cavallo', 'Acco' and 'Wonderful' showed the highest content of the detected anthocyanin compounds. CONCLUSION: These results contribute to current knowledge about chemical composition, phenolic contents, anthocyanin profiles and antioxidant activity of pomegranate juice from different genotypes, showing in most cases an appreciable juice quality and bioactive profile, although significant differences among them were detected. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genome-wide identification, gene cloning, subcellular location and expression analysis of SPL gene family in P. granatum L.

BACKGROUNDS: Pomegranate is an excellent tree species with nutritional, medicinal, ornamental and ecological values. Studies have confirmed that SPL factors play an important role in floral transition and flower development. RESULTS: Used bioinformatics methods, 15 SPL (SQUAMOSA promoter-binding protein-like) genes were identified and analyzed from the 'Taishanhong' pomegranate (P. granatum L.) genome. Phylogenetic analysis showed that PgSPLs were divided into six subfamilies (G1 ~ G6). PgSPL promoter sequences contained multiple cis-acting elements associated with abiotic stress or hormonal response. Based on the transcriptome data, expression profiles of different tissues and different developmental stages showed that PgSPL genes had distinct temporal and spatial expression characteristics. The expression analysis of miR156 in small RNA sequencing results showed that miR156 negatively regulated the expression of target genes. qRT-PCR analysis showed that the expression levels of PgSPL2, PgSPL3, PgSPL6, PgSPL11 and PgSPL14 in leaves were significantly higher than those in buds and stems (p < 0.05). The expression levels of PgSPL5, PgSPL12 and PgSPL13 in flower buds were significantly higher than that in leaves and stems (p < 0.05). The full-length of coding sequence of PgSPL5 and PgSPL13 were obtained by homologous cloning technology. The full length of PgSPL5 is 1020 bp, and PgSPL13 is 489 bp, which encodes 339 and 162 amino acids, respectively. Further investigation revealed that PgSPL5 and PgSPL13 proteins were located in the nucleus. Exogenous plant growth regulator induction experiments showed that PgSPL5 was up-regulated in leaves and stems. PgSPL13 was up-regulated in leaves and down-regulated in stems. When sprayed with 6-BA, IBA and PP333 respectively, PgSPL5 and PgSPL13 were up-regulated most significantly at P2 (bud vertical diameter was 5.1 ~ 12.0 mm) stage of bisexual and functional male flowers. CONCLUSIONS: Our findings suggested that PgSPL2, PgSPL3, PgSPL6, PgSPL11 and PgSPL14 played roles in leaves development of pomegranate. PgSPL5, PgSPL12 and PgSPL13 played roles in pomegranate flower development. PgSPL5 and PgSPL13 were involved in the response process of different plant hormone signal transduction in pomegranate development. This study provided a robust basis for further functional analyses of SPL genes in pomegranate.

Mitigation of the effect of drought on growth and yield of pomegranates by foliar spraying of different sizes of selenium nanoparticles.

BACKGROUND: Drought is a very important environmental stressor, which has negative effects on the growth of trees, decreasing their yield. The role of different-sized selenium nanoparticles (Se-NPs) in the mitigation of environmental stresses such as drought in crops has not yet been investigated. RESULTS: Trees treated with Se-NPs displayed higher levels of photosynthetic pigments, a better nutrient status, better physical parameters (especially fruit cracking) and chemical parameters, a higher phenolic content, and higher concentrations of osmolytes, antioxidant enzymes, and abscisic acid than untreated trees under drought stress. Foliar spraying of 10 and 50 nm Se-NPs alleviated many of the deleterious effects of drought in pomegranate leaves and fruits and this was achieved by reducing stress-induced lipid peroxidation and H2 O2 content by enhancing the activity of antioxidant enzymes. Furthermore, the 10 nm Se-NPs treatment produced more noticeable effects than the treatment with 50 nm Se-NPs. CONCLUSION: Results confirm the positive effects of nanoparticle spraying, especially the role of 10 nm Se-NPs in the management of negative effects of drought not only for pomegranates but potentially also for other crops. © 2021 Society of Chemical Industry.

Chemical and physical attributes of fruit juice and peel of pomegranate genotypes grown in Florida, USA.

The global demand for pomegranate has led to increasing research and improvement of cultivars that produce higher antioxidant compounds. The current study was carried out to evaluate the bioactive constituents and physical properties of fourteen pomegranate genotypes grown in the subtropical region of Florida. There were differences in aril color among genotypes. The highest total anthocyanin content was found in 'Ariana', 'Molla Nepes', and 'Parfianka' genotypes. Furthermore, total anthocyanin content in peel ranged from 2.14 to 10.86 mg/100 g dry weight. Total phenolic content in the pomegranate fruit juice differed significantly among genotypes, varying from 365.71 to 1167.40 mg/L. Moreover, total phenolic content in the fruit peel ranged from 1313.08 to 1700.07 mg/L. Total phenolic compounds and reducing power activity in peel tissues were greater than in pomegranate juice. Reducing power activity and titratable acidity were positively and significantly correlated with total anthocyanin content.

Identification of the SUT Gene Family in Pomegranate (Punica granatum L.) and Functional Analysis of PgL0145810.1.

Sucrose, an important sugar, is transported from source to sink tissues through the phloem, and plays important role in the development of important traits in plants. However, the SUT gene family is still not well characterized in pomegranate. In this study, we first identified the pomegranate sucrose transporter (SUT) gene family from the whole genome. Then, the phylogenetic relationship of SUT genes, gene structure and their promoters were analyzed. Additionally, their expression patterns were detected during the development of the seed. Lastly, genetic transformation and cytological observation were used to study the function of PgL0145810.1. A total of ten pomegranate SUT genes were identified from the whole genome of pomegranate 'Tunisia'. The promoter region of all the pomegranate SUT genes contained myeloblastosis (MYB) elements. Four of the SUT genes, PgL0328370.1, PgL0099690.1, PgL0145810.1 and PgL0145770.1, were differentially expressed during seed development. We further noticed that PgL0145810.1 was expressed most prominently in the stem parts in transgenic plants compared to other tissue parts (leaves, flowers and silique). The cells in the xylem vessels were small and lignin content was lower in the transgenic plants as compared to wild Arabidopsis plants. In general, our result suggests that the MYB cis-elements in the promoter region might regulate PgL0145810.1 expression to control the structure of xylem, thereby affecting seed hardness in pomegranate.

Bioactive compounds and quality evaluation of 'Wonderful' pomegranate fruit and juice as affected by deficit irrigation.

BACKGROUND: Persistent water shortage, typical of Mediterranean agricultural systems, has driven production methods towards the use of regulated deficit irrigation (DI) strategies, which may allow important water savings. To evaluate the extent to which pomegranate (cv. 'Wonderful') attributes may differ depending on the irrigation application rates, we investigated the effect of four different irrigation regimes (100, 75, 50, and 25% of maximum crop evapotranspiration - ETC ) on their morpho-pomological, physico-chemical properties, and bioactive compounds. RESULTS: The weight and the size of whole fruits, and the number of arils and their weight per fruit, decreased significantly with the restriction of ETC water volumes applied to the crop. Minor differences were observed in soluble solids, pH, color of fruit skins, arils, and juices, although total acidity, maturity index, vitamin C, glucose, and fructose were not affected. However, total phenol content and antioxidant activity in juices were higher in the samples subjected to the DI treatments than in those submitted to full irrigation, showing the highest value at 50% ETC . A similar trend was found for anthocyanins delphinidin-3,5-diglucoside and delphinidin-3-glucoside, reaching the highest concentration at 50% ETC . Among non-anthocyanin compounds, punicalagins and pedunculagin were more concentrated in the 75% ETc samples. CONCLUSION: The results of the present study indicate that the DI strategy throughout all the growing season is likely to have important implication for the pomegranate's physical, chemical, and phytochemical properties. Knowledge of them is useful in view of their processing and nutritional properties. © 2020 Society of Chemical Industry.

DNA Fingerprinting of in vitro Micropropagated Pomegranate Genotypes.

BACKGROUND AND OBJECTIVE: Pomegranate is grown for its rich flavour in numerous tropical and subtropical areas, like Egypt and the Kingdom of Saudi Arabia (KSA). Assessing the genetic background of the pomegranate is the key to its expansion through the Middle East, where tissue culture reproduction strategies could be used to solve environmental and economic problems. This study aimed at studying the genetic stability of 2 pomegranate genotypes in vitro micro-propagated in the Kingdom of Saudi Arabia by using the random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and inter simple sequence repeats (ISSR) tools. MATERIALS AND METHODS: The two above mentioned molecular tools were used to evaluate the DNA fingerprints of Taify and Yemeni pomegranate genotypes 12 weeks post in vitro propagation in Taif, Kingdom of Saudi Arabia compared to the mother plant. Shoot tip explants of 4-5 cm long were grown on Murashige and Skoog (MS) medium supplemented by 1.0 mg L-1 NAA, 2.00 mg L-1 IBA and 2 g L-1 activated carbon for 4 weeks for rooting. On 12 weeks DNA extracts were prepared from the acquired plantlets obtained and used as templates for each of RAPD-PCR and ISSR tools. RESULTS: The RAPD-PCR and ISSR assays generated a total of 79-94 and 57-72 DNA fragments, respectively. In case of RAPD-PCR 80 and 90% of the primers used and developed monomorphic fragments of the Yemeni and Taify genotypes, respectively, particularly OPG08 primer for Taify genotype and OPA04 and OPD07 primers for the Yemeni genotype. Regarding ISSR, no DNA polymorphic for the micropropagated clones were recorded compared to the mother plant. CONCLUSION: The ISSR assay's findings indicated the genetic homogeneity between the in vitro micropropagated clones of both pomegranate genotypes and the mother plants.

Pattern recognition-based Raman spectroscopy for non-destructive detection of pomegranates during maturity.

In this study, the feasibility of utilizing Fourier transform Raman spectroscopy, combined with supervised and unsupervised pattern recognition methods was considered, to distinguish the maturity stage of pomegranate "Ashraf variety" during four distinct maturity stages between 88 and 143 days after full bloom. Principal component analysis (PCA) as an unsupervised pattern recognition method was performed to verify the possibility of clustering of the pomegranate samples into four groups. Two supervised pattern recognition techniques namely, partial least squares Discriminant Analysis (PLS-DA) and Soft Independent Modeling of Class Analogy (SIMCA) were also used as powerful supervised pattern recognition methods to classify the samples. The results showed that in all groups of samples, the Raman spectra of the samples were correctly clustered using PCA. The accuracy of the SIMCA classification for differentiation of four pomegranate groups was 82%. Also, the overall discriminant power of PLS-DA classes was about 96%, and 95% for calibration and validation sample sets, respectively. Due to the misclassification among different classes of immature pomegranates, that was lower than the expected, it was not possible to discriminate all the immature samples in individual classes. However, when considering only the two main categories of "immature" and "mature", a reasonable separation between the classes were obtained using supervised pattern recognition methods of SIMCA and PLS-DA. The SIMCA based on PCA modeling could correctly categorize the samples in two classes of immature and mature with classification accuracy of 100%.

A low cost alternative, using mycorrhiza and organic fertilizer, to optimize the production of foliar bioactive compounds in pomegranates.

AIM: To select the best combination of arbuscular mycorrhizal fungi and efficient vermicompost dose in maximizing the production of leaf metabolites in Punica granatum seedlings. METHODS AND RESULTS: The experimental design was in a 3 × 3 factorial arrangement: three inoculation treatments (inoculated with Gigaspora albida, inoculated with Acaulospora longula and control not inoculated) × 3 doses of vermicompost (0, 5 and 7·5%). After 120 days of inoculation, biomolecules, plant growth parameters and mycorrhizal colonization were evaluated. The combination of 7·5% of vermicompost and A. longula was favourable to the accumulation of leaf phenols, with an increase of 116·11% in relation to the non-inoculated control. The total tannins was optimized/enhanced when G. albida and 7·5% of fertilizer were used, registering an increase of 276·71%. CONCLUSIONS: The application of 7·5% of vermicompost associated with A. longula and G. albida is a low cost alternative to increase the levels of bioactive compounds in pomegranate leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first published report of optimization of bioactive compound production in P. granatum by the combined use of mycorrhiza and vermicompost doses.

Effects of nitrogen fertilization on pomegranate fruit, aril and juice quality.

BACKGROUND: Nitrogen (N) fertilization influences plant growth and yield, and may also affect fruit quality. For two consecutive seasons, we examined the effects of various N fertilization levels - 5 to 200 mg L-1  - on pomegranate fruit, aril and juice quality. Evaluations included fruit and aril weight, size and color, appearance of peel blemishes, internal black rot and nutritional composition of extracted juices. RESULTS: Nitrogen fertilization affected pomegranate fruit, aril and juice quality. The most pronounced effects were observed in trees grown under the lowest N fertilization level, which bore smaller fruit and arils, the latter with lighter color; the fruit suffered from sunburn, and the juice had lower total soluble solid, acidity and anthocyanin contents. The proportion of edible aril weight per total fruit weight gradually increased with an increase in N fertilization concentration. In contrast, N fertilization did not affect peel color, roughness or cracking incidence. Black rot incidence increased with increasing N concentration. CONCLUSIONS: Nitrogen fertilization affected pomegranate fruit, aril and juice quality, and the total number of marketable fruits per tree. The optimal N fertilization levels, which were most beneficial for achieving high-quality marketable fruit, were between 40 and 100 mg L-1 . © 2019 Society of Chemical Industry.

Molecular cloning and characterization of genes related to the ethylene signal transduction pathway in pomegranate (Punica granatum L.) under different temperature treatments.

Low temperature storage is a common method for storing pomegranates post-harvest; however, unsuitable low temperatures can cause fruit chilling injuries, the molecular mechanism of which is as yet unclear. Ethylene is a major factor affecting the post-harvest storage quality of pomegranates, and functions mainly through the ethylene signal transduction pathway. ERF1, ERF2 and ETR are key genes in the ethylene signal transduction pathway. Here, we used RACE and homologous cloning techniques to obtain PgERF1 (KU058889), PgERF2 (KU058890) and PgETR (KU058891) from Punica granatum cv. Yushizi. Sequence alignment and functional domain analysis revealed that both PgERF1 and PgERF2 contained a DNA-binding-site at the 120th to 177th amino acids of the N-terminus, which is a typical AP2/ERF center structure domain. Analysis of changes in expression of PgERF1, PgERF2 and PgETR following storage for different lengths of time (0, 14, 28, 42 and 56 days) at different temperatures (0°C, 5°C, 10°C and 15°C) revealed that the expression levels of PgERF1 and PgERF2 had a significant positive correlation. At the same time, the expression of both PgERF1 and PgERF2 increased continuously with time when seeds were stored at 0°C. However, there was no obvious linear relationship between time stored and the levels of expression of PgETR. Therefore, we inferred that at 0°C, the ethylene signal transduction pathway might play an important role in fruit chilling injuries during post-harvest storage.