Fifteen complete chloroplast genomes of Trapa species (Trapaceae): insight into genome structure, comparative analysis and phylogenetic relationships.

BACKGROUND: Trapa L. is a floating-leaved aquatic plant with important economic and ecological values. However, the species identification and phylogenetic relationship within Trapa are still controversial, which necessitates the need for plastid genome information of Trapa. In this study, complete chloroplast genomes of 13 Trapa species/taxa were sequenced and annotated. Combined with released sequences, comparative analyses of chloroplast genomes were performed on the 15 Trapa species/taxa for the first time. RESULTS: The Trapa chloroplast genomes exhibited typical quadripartite structures with lengths from 155,453 to 155,559 bp. The gene orders and contents within Trapa were conservative, but several changes were found in the microstructure. The intron loss of rpl2, also detected in Lythraceae, was found in all Trapa species/taxa, suggesting close genetic relationship between Lythraceae and Trapaceae. Notably, two small-seed species (T. incisa and T. maximowiczii) showed the smallest genome size with 155,453 and 155,477 bp, respectively. Each cp genome contained the same 130 genes consisting of 85 protein-coding genes, 37 tRNA genes and 8 rRNA genes. Trapa species/taxa showed 37 (T. incisa and T. maximowiczii) to 41 (T. sibirica) long repeats, including forward, palindromic, reversed and complementary repeats. There were 110 (T. quadrispinosa) to 123 (T. incisa and T. maximowiczii) SSR (simple sequence repeat) loci in Trapa chloroplast genomes. Comparative analyses revealed that two hotspot regions (atpA-atpF and rps2-rpoC2) in Trapa chloroplast genomes could be served as potential molecular markers. Three phylogenetic analyses (ML, MP and BI) consistently showed that there were two clusters within Trapa, including large- and small-seed species/taxa, respectively; for the large-seed Trapa, they clustered according to their geographical origin and tubercle morphology on the surface of seeds. CONCLUSION: In summary, we have acquired the sequences of 13 Trapa chloroplast genomes, and performed the comparative analyses within Trapa for the first time. The results have helped us better identify the Trapa species/taxa and deepen the understanding of genetic basis and phylogenetic relationship of Trapa, which will facilitate the effective management and utilization of the important genetic resources in the future.

Genome sequencing and transcriptome analyses provide insights into the origin and domestication of water caltrop (Trapa spp., Lythraceae).

Humans have domesticated diverse species from across the plant kingdom; however, our current understanding of plant domestication is largely founded on major cereal crops. Here, we examine the evolutionary processes and genetic basis underlying the domestication of water caltrop (Trapa spp., Lythraceae), a traditional, yet presently underutilized non-cereal crop that sustained early Chinese agriculturalists. We generated a chromosome-level genome assembly of tetraploid T. natans, and then divided the allotetraploid genome into two subgenomes. Based on resequencing data from 57 accessions, representing cultivated diploid T. natans, wild T. natans (2x and 4x) and diploid T. incisa, we showed that water caltrop was likely first domesticated in the Yangtze River Valley as early as 6300 yr BP, and experienced a second improvement c. 800 years ago. We also provided strong support for an allotetraploid origin of T. natans within the past 230 000-310 000 years. By integrating selective sweep and transcriptome profiling analyses, we identified a number of genes potentially selected and/or differentially expressed during domestication, some of which likely contributed not only to larger fruit sizes but also to a more vigorous root system, facilitating nutrient uptake, environmental stress response and underwater photosynthesis. Our results shed light on the evolutionary and domestication history of water caltrop, one of the earliest domesticated crops in China. This study has implications for genomic-assisted breeding of this presently underutilized aquatic plant, and improves our general understanding of plant domestication.

Identification, Analysis and Gene Cloning of the SWEET Gene Family Provide Insights into Sugar Transport in Pomegranate (Punica granatum).

Members of the sugars will eventually be exported transporter (SWEET) family regulate the transport of different sugars through the cell membrane and control the distribution of sugars inside and outside the cell. The SWEET gene family also plays important roles in plant growth and development and physiological processes. So far, there are no reports on the SWEET family in pomegranate. Meanwhile, pomegranate is rich in sugar, and three published pomegranate genome sequences provide resources for the study of the SWEET gene family. 20 PgSWEETs from pomegranate and the known Arabidopsis and grape SWEETs were divided into four clades (Ⅰ, Ⅱ, Ⅲ and Ⅳ) according to the phylogenetic relationships. PgSWEETs of the same clade share similar gene structures, predicting their similar biological functions. RNA-Seq data suggested that PgSWEET genes have a tissue-specific expression pattern. Foliar application of tripotassium phosphate significantly increased the total soluble sugar content of pomegranate fruits and leaves and significantly affected the expression levels of PgSWEETs. The plant growth hormone regulator assay also significantly affected the PgSWEETs expression both in buds of bisexual and functional male flowers. Among them, we selected PgSWEET17a as a candidate gene that plays a role in fructose transport in leaves. The 798 bp CDS sequence of PgSWEET17a was cloned, which encodes 265 amino acids. The subcellular localization of PgSWEET17a showed that it was localized to the cell membrane, indicating its involvement in sugar transport. Transient expression results showed that tobacco fructose content was significantly increased with the up-regulation of PgSWEET17a, while both sucrose and glucose contents were significantly down-regulated. The integration of the PgSWEET phylogenetic tree, gene structure and RNA-Seq data provide a genome-wide trait and expression pattern. Our findings suggest that tripotassium phosphate and plant exogenous hormone treatments could alter PgSWEET expression patterns. These provide a reference for further functional verification and sugar metabolism pathway regulation of PgSWEETs.

Chloroplast phylogenomics and divergence times of Lagerstroemia (Lythraceae).

BACKGROUND: Crape myrtles, belonging to the genus Lagerstroemia L., have beautiful paniculate inflorescences and are cultivated as important ornamental tree species for landscaping and gardening. However, the phylogenetic relationships within Lagerstroemia have remained unresolved likely caused by limited sampling and the insufficient number of informative sites used in previous studies. RESULTS: In this study, we sequenced 20 Lagerstroemia chloroplast genomes and combined with 15 existing chloroplast genomes from the genus to investigate the phylogenetic relationships and divergence times within Lagerstroemia. The phylogenetic results indicated that this genus is a monophyletic group containing four clades. Our dating analysis suggested that Lagerstroemia originated in the late Paleocene (~ 60 Ma) and started to diversify in the middle Miocene. The diversification of most species occurred during the Pleistocene. Four variable loci, trnD-trnY-trnE, rrn16-trnI, ndhF-rpl32-trnL and ycf1, were discovered in the Lagerstroemia chloroplast genomes. CONCLUSIONS: The chloroplast genome information was successfully utilized for molecular characterization of diverse crape myrtle samples. Our results are valuable for the global genetic diversity assessment, conservation and utilization of Lagerstroemia.

Molecular adaptation to salinity fluctuation in tropical intertidal environments of a mangrove tree Sonneratia alba.

BACKGROUND: Mangroves have adapted to intertidal zones - the interface between terrestrial and marine ecosystems. Various studies have shown adaptive evolution in mangroves at physiological, ecological, and genomic levels. However, these studies paid little attention to gene regulation of salt adaptation by transcriptome profiles. RESULTS: We sequenced the transcriptomes of Sonneratia alba under low (fresh water), medium (half the seawater salinity), and high salt (seawater salinity) conditions and investigated the underlying transcriptional regulation of salt adaptation. In leaf tissue, 64% potential salinity-related genes were not differentially expressed when salinity increased from freshwater to medium levels, but became up- or down-regulated when salt concentrations further increased to levels found in sea water, indicating that these genes are well adapted to the medium saline condition. We inferred that both maintenance and regulation of cellular environmental homeostasis are important adaptive processes in S. alba. i) The sulfur metabolism as well as flavone and flavonol biosynthesis KEGG pathways were significantly enriched among up-regulated genes in leaves. They are both involved in scavenging ROS or synthesis and accumulation of osmosis-related metabolites in plants. ii) There was a significantly increased percentage of transcription factor-encoding genes among up-regulated transcripts. High expressions of salt tolerance-related TF families were found under high salt conditions. iii) Some genes up-regulated in response to salt treatment showed signs of adaptive evolution at the amino acid level and might contribute to adaptation to fluctuating intertidal environments. CONCLUSIONS: This study first elucidates the mechanism of high-salt adaptation in mangroves at the whole-transcriptome level by salt gradient experimental treatments. It reveals that several candidate genes (including salt-related genes, TF-encoding genes, and PSGs) and major pathways are involved in adaptation to high-salt environments. Our study also provides a valuable resource for future investigation of adaptive evolution in extreme environments.

Punicic acid production in Brassica napus.

Punicic acid (PuA; 18:3Δ9cis,11trans,13cis), a conjugated linolenic acid isomer bearing three conjugated double bonds, is associated with various health benefits and has potential for industrial use. The major nature source of this unusual fatty acid is pomegranate (Punica granatum) seed oil, which contains up to 80% (w/w) of its fatty acids as PuA. Pomegranate seed oil, however, is low yielding with unstable production and thus limits the supply of PuA. Metabolic engineering of established temperate oil crops for PuA production, therefore, has the potential to be a feasible strategy to overcome the limitations associated with sourcing PuA from pomegranate. In this study, the cDNAs encoding a pomegranate fatty acid conjugase and a pomegranate oleate desaturase were co-expressed in canola-type Brassica napus. Transgenic B. napus lines accumulated up to 11% (w/w) of the total fatty acids as PuA in the seed oil, which is the highest level of PuA reported in metabolically engineered oilseed crops so far. Levels of seed oil PuA were stable over two generations and had no negative effects on seed germination. The transgenic B. napus lines with the highest PuA levels contained multiple transgene insertions and the PuA content of B. napus seed oil was correlated with efficiency of oleic acid desaturation and linoleic acid conjugation. In addition, PuA accumulated at lower levels in polar lipids (5.0-6.9%) than triacylglycerol (7.5-10.6%), and more than 60% of triacylglycerol-associated PuA was present at the sn-2 position. This study provides the basis for the commercial production of PuA in transgenic oilseed crops and thus would open new prospects for the application of this unusual fatty acid in health and industry.

Comparative analyses of chloroplast genomes from 22 Lythraceae species: inferences for phylogenetic relationships and genome evolution within Myrtales.

BACKGROUND: Lythraceae belongs to the order Myrtales, which is part of Archichlamydeae. The family has 31 genera containing approximately 620 species of herbs, shrubs and trees. Of these 31 genera, five large genera each possess 35 or more species. They are Lythrum, with 35; Rotala, with 45; Nesaea, with 50; Lagerstroemia, with 56; and Cuphea, with 275 species. RESULTS: We reported six newly sequenced chloroplast (cp) genomes (Duabanga grandiflora, Trapa natans, Lythrum salicaria, Lawsonia inermis, Woodfordia fruticosa and Rotala rotundifolia) and compared them with 16 other cp genomes of Lythraceae species. The cp genomes of the 22 Lythraceae species ranged in length from 152,049 bp to 160,769 bp. In each Lythraceae species, the cp genome contained 112 genes consisting of 78 protein coding genes, four ribosomal RNAs and 30 transfer RNAs. Furthermore, we detected 211-332 simple sequence repeats (SSRs) in six categories and 7-27 long repeats in four categories. We selected ten divergent hotspots (ndhF, matK, ycf1, rpl22, rpl32, trnK-rps16, trnR-atpA, rpl32-trnL, trnH-psbA and trnG-trnR) among the 22 Lythraceae species to be potential molecular markers. We constructed phylogenetic trees from 42 Myrtales plants with 8 Geraniales plants as out groups. The relationships among the Myrtales species were effectively distinguished by maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) trees constructed using 66 protein coding genes. Generally, the 22 Lythraceae species gathered into one clade, which was resolved as sister to the three Onagraceae species. Compared with Melastomataceae and Myrtaceae, Lythraceae and Onagraceae differentiated later within Myrtales. CONCLUSIONS: The study provided ten potential molecular markers as candidate DNA barcodes and contributed cp genome resources within Myrtales for further study.

The Complete Chloroplast Genomes of Punica granatum and a Comparison with Other Species in Lythraceae.

Pomegranates (Punica granatum L.) are one of the most popular fruit trees cultivated in arid and semi-arid tropics and subtropics. In this study, we determined and characterized three complete chloroplast (cp) genomes of P. granatum cultivars with different phenotypes using the genome skimming approach. The complete cp genomes of three pomegranate cultivars displayed the typical quadripartite structure of angiosperms, and their length ranged from 156,638 to 156,639 bp. They encoded 113 unique genes and 17 are duplicated in the inverted regions. We analyzed the sequence diversity of pomegranate cp genomes coupled with two previous reports. The results showed that the sequence diversity is extremely low and no informative sites were detected, which suggests that cp genome sequences may be not be suitable for investigating the genetic diversity of pomegranate genotypes. Further, we analyzed the codon usage pattern and identified the potential RNA editing sites. A comparative cp genome analysis with other species within Lythraceae revealed that the gene content and organization are highly conserved. Based on a site-specific model, 11 genes with positively selected sites were detected, and most of them were photosynthesis-related genes and genetic system-related genes. Together with previously released cp genomes of the order Myrtales, we determined the taxonomic position of P. granatum based on the complete chloroplast genomes. Phylogenetic analysis suggested that P. granatum form a single clade with other species from Lythraceae with a high support value. The complete cp genomes provides valuable information for understanding the phylogenetic position of P. gramatum in the order Myrtales.

Antioxidant activity and phenolic composition in pomegranate (Punica granatum L.) genotypes from south Italy by UHPLC-Orbitrap-MS approach.

BACKGROUND: Pomegranate fruits are a rich source of polyphenols with numerous health-promoting effects. Pomegranate juices of five genotypes ('Mollar', 'Kingdom', 'Dente di Cavallo', and two old populations 'Francofonte' and 'Santa Tecla') were evaluated regarding anthocyanin and non-anthocyanin phenolic contents using ultrahigh performance liquid chromatography (UHPLC)-Orbitrap-mass spectrometry (MS). Moreover, total antioxidant activity (TAA) was evaluated using a 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay. RESULTS: Twenty-three phenolic compounds were identified. Cyanidin-3,5-O-diglucoside and pelargonidin-3,5-O-diglucoside were the most representative anthocyanins in all genotypes; the Santa Tecla population had the highest content of these anthocyanins, 97.64 mg L-1 and 40.29 mg L-1 respectively. In the Francofonte population, ferulic acid hexoside was the most abundant compound (391.18 mg L-1 ). TAA values ranged between 221.5 and 36.73 µmol Trolox equivalents/100 mL of juice. A high TAA value was recorded for the Santa Tecla pomegranate population. CONCLUSION: The UHPLC-Orbitrap-MS approach was employed for the first time to identify the phenolic compound profiling in five pomegranate genotypes. TAA was analysed using an ABTS assay, and the results showed a significant variability in nutraceutical potential of the pomegranate genotypes studied. The inclusion of phenolic information in the linear discriminant analysis allowed very good discriminations among genotypes to be obtained. © 2018 Society of Chemical Industry.

Molecular mechanisms involved in postharvest chilling tolerance of pomegranate fruit.

Cold storage of pomegranates is essential for prolonging postharvest storage and for the implementation of cold-quarantine insect disinfestation treatments required for international trading. However, pomegranates are chilling sensitive; they may develop chilling injuries upon exposure to unfavorable low temperatures. In this mini-review, we summarize molecular data obtained from three different RNA Seq transcriptome analyses of responses of pomegranate fruits to cold storage. These experiments included comparisons among the transcriptomic responses following a 2-week exposure to 1 °C in three different model systems: 1) unconditioned chilling-sensitive fruits versus relatively chilling-tolerant low-temperature-conditioned fruits; 2) chilling-sensitive early harvested fruits versus relatively chilling-tolerant late-harvested ones; and 3) chilling-sensitive 'Ganesh' variety versus the relatively chilling-tolerant 'Wonderful' variety. Comparisons among differentially expressed transcripts that were exclusively and significantly up-regulated in the relatively chilling-tolerant fruits in all three model systems enabled identification of 573 common chilling tolerance-associated genes in pomegranates. Functional categorization and classification of the differentially expressed transcripts revealed several regulatory, metabolic, and stress-adaptation pathways that were uniquely activated in response to cold storage in relatively chilling-tolerant fruits. More specifically, we identified common up-regulation of transcripts involved in activation of jasmonic acid and ethylene hormone biosynthesis and signaling, stress-related transcription factors, calcium and MAPK signaling, starch degradation and galactinol and raffinose biosynthesis, phenol biosynthesis, lipid metabolism, and heat-shock proteins. We hypothesized these pathways to be involved in imparting chilling tolerance to pomegranate fruits. © 2019 Society of Chemical Industry.

Targeted metabolite profiling to gain chemometric insight into Indian pomegranate cultivars and elite germplasm.

BACKGROUND: Pomegranate fruit is an excellent source of bioactive polyphenolics, known to contribute significantly to human health. India is the largest producer of pomegranate in the world and produces the finest quality fruit with highly desirable consumer traits such as soft seeds, low acidity, and attractive fruit and aril color. Knowledge of the extent of variation in key metabolites (sugars, organic acids, phenolics, and anthocyanins) is key to selecting superior genotypes for germplasm improvement. Relevant information with respect to Indian genotypes is scarce. The present study therefore aims to evaluate quantitatively important metabolites in some cultivars and elite germplasm of pomegranate in India. RESULTS: Identification and quantification of primary and secondary metabolites such as sugars, organic acids, vitamin C, polyphenolics, and anthocyanins were conducted using a liquid chromatography - mass spectrometry (LC-MS) platform. Fructose and citric acid were the predominant sugar and organic acid, respectively. Wild genotypes had significantly higher concentrations of organic acids, antioxidant activity, and phenolics, namely punicalagin, ellagic acid, sinapic, and ferulic acid. CONCLUSION: Cyanidin and delphinidin derivatives of anthocyanins were more abundant in red aril commercial genotypes. Results suggest that wild-sour accessions represent a rich source of polyphenolics that can be utilized in future breeding programs to breed healthier varieties, food supplements, and pharmaceutical products. © 2019 Society of Chemical Industry.

The pomegranate (Punica granatum L.) genome and the genomics of punicalagin biosynthesis.

Pomegranate (Punica granatum L.) is a perennial fruit crop grown since ancient times that has been planted worldwide and is known for its functional metabolites, particularly punicalagins. We have sequenced and assembled the pomegranate genome with 328 Mb anchored into nine pseudo-chromosomes and annotated 29 229 gene models. A Myrtales lineage-specific whole-genome duplication event was detected that occurred in the common ancestor before the divergence of pomegranate and Eucalyptus. Repetitive sequences accounted for 46.1% of the assembled genome. We found that the integument development gene INNER NO OUTER (INO) was under positive selection and potentially contributed to the development of the fleshy outer layer of the seed coat, an edible part of pomegranate fruit. The genes encoding the enzymes for synthesis and degradation of lignin, hemicelluloses and cellulose were also differentially expressed between soft- and hard-seeded varieties, reflecting differences in their accumulation in cultivars differing in seed hardness. Candidate genes for punicalagin biosynthesis were identified and their expression patterns indicated that gallic acid synthesis in tissues could follow different biochemical pathways. The genome sequence of pomegranate provides a valuable resource for the dissection of many biological and biochemical traits and also provides important insights for the acceleration of breeding. Elucidation of the biochemical pathway(s) involved in punicalagin biosynthesis could assist breeding efforts to increase production of this bioactive compound.

The pomegranate (Punica granatum L.) genome provides insights into fruit quality and ovule developmental biology.

Pomegranate (Punica granatum L.) has an ancient cultivation history and has become an emerging profitable fruit crop due to its attractive features such as the bright red appearance and the high abundance of medicinally valuable ellagitannin-based compounds in its peel and aril. However, the limited genomic resources have restricted further elucidation of genetics and evolution of these interesting traits. Here, we report a 274-Mb high-quality draft pomegranate genome sequence, which covers approximately 81.5% of the estimated 336-Mb genome, consists of 2177 scaffolds with an N50 size of 1.7 Mb and contains 30 903 genes. Phylogenomic analysis supported that pomegranate belongs to the Lythraceae family rather than the monogeneric Punicaceae family, and comparative analyses showed that pomegranate and Eucalyptus grandis share the paleotetraploidy event. Integrated genomic and transcriptomic analyses provided insights into the molecular mechanisms underlying the biosynthesis of ellagitannin-based compounds, the colour formation in both peels and arils during pomegranate fruit development, and the unique ovule development processes that are characteristic of pomegranate. This genome sequence provides an important resource to expand our understanding of some unique biological processes and to facilitate both comparative biology studies and crop breeding.

First reported chloroplast genome sequence of Punica granatum (cultivar Helow) from Jabal Al-Akhdar, Oman: phylogenetic comparative assortment with Lagerstroemia.

Pomegranate (Punica granatum L.) is one of the oldest known edible fruits. It has grown in popularity and is a profitable fruit crop due to its attractive features including a bright red appearance and its biological activities. Scientific exploration of the genetics and evolution of these beneficial traits has been hampered by limited genomic information. In this study, we sequenced the complete chloroplast (cp) genome of the native P. granatum (cultivar Helow) cultivated in the mountains of Jabal Al-Akhdar, Oman. The results revealed a P. granatum cp genome length of 158,630 bp, characterized by a relatively conserved structure containing 2 inverted repeat regions of 25,466 bp, an 18,686 bp small single copy regions, and an 89,015 bp large single copy region. The 86 protein-coding genes included 37 transfer RNA genes and 8 ribosomal RNA genes. Comparison of the P. granatum whole cp genome with seven Lagerstroemia species revealed an overall high degree of sequence similarity with divergence among intergenic spacers. The location, distribution, and divergence of repeat sequences and shared genes of the Punica and Lagerstroemia species were highly similar. Analyses of nucleotide substitution, insertion/deletions, and highly variable regions in these cp genomes identified potential plastid markers for taxonomic and phylogenetic studies in Myrtales. A phylogenetic study of the cp genomes and 76 shared coding regions generated similar cladograms. The complete cp genome of P. granatum will aid in taxonomical studies of the family Lythraceae.

Bioactivity and biotechnological production of punicic acid.

Punicic acid (PuA; 18: 3Δ 9cis,11trans,13cis ) is an unusual 18-carbon fatty acid bearing three conjugated double bonds. It has been shown to exhibit a myriad of beneficial bioactivities including anti-cancer, anti-diabetes, anti-obesity, antioxidant, and anti-inflammatory properties. Pomegranate (Punica granatum) seed oil contains approximately 80% PuA and is currently the major natural source of this remarkable fatty acid. While both PuA and pomegranate seed oil have been used as functional ingredients in foods and cosmetics for some time, their value in pharmaceutical/medical and industrial applications are presently under further exploration. Unfortunately, the availability of PuA is severely limited by the low yield and unstable supply of pomegranate seeds. In addition, efforts to produce PuA in transgenic crops have been limited by a relatively low content of PuA in the resulting seed oil. The production of PuA in engineered microorganisms with modern fermentation technology is therefore a promising and emerging method with the potential to resolve this predicament. In this paper, we provide a comprehensive review of this unusual fatty acid, covering topics ranging from its natural sources, biosynthesis, extraction and analysis, bioactivity, health benefits, and industrial applications, to recent efforts and future perspectives on the production of PuA in engineered plants and microorganisms.

Xanthomonas axonopodis pv. punicae uses XopL effector to suppress pomegranate immunity.

Xanthomonas axonopodis pv. punicae (Xap) causing bacterial blight is an important pathogen that incurs significant losses to the exportability of pomegranate. Xap uses the Xop TTSS-effector, via the type three secretion system, to suppress pomegranate immunity. Here, we investigate the role of XopL during blight pathogenesis. We observed that XopL is essential for its in planta growth and full virulence. Leaves inoculated with Xap ΔxopL produced restricted water-soaked lesions compared to those inoculated with wild-type Xap. XopL supports Xap for its sustained multiplication in pomegranate by suppressing the plant cell death (PCD) event. We further demonstrated that XopL suppresses immune responses, such as callose deposition and production of reactive oxygen species (ROS). RT-qPCR analysis revealed that immune responsive genes were upregulated when challenged with Xap ΔxopL, whereas upregulation of such genes was compromised in the complemented strain containing the xopL gene. The transiently expressed XopL::EYFP fusion protein was localized to the plasma membrane, indicating the possible site of its action. Altogether, this study highlights that XopL is an important TTSS-effector of Xap that suppresses plant immune responses, including PCD, presumably to support the multiplication of Xap for a sufficient time-period during blight disease development.

Metabolic engineering of Schizosaccharomyces pombe to produce punicic acid, a conjugated fatty acid with nutraceutic properties.

Punicic acid (PuA) is a conjugated linolenic acid (C18:3Δ9c,11t,13c) with a wide range of nutraceutic effects with the potential to reduce the incidence of a number of health disorders including diabetes, obesity, and cancer. It is the main component of seed oil from Punica granatum and Trichosanthes kirilowii. Previously, production of relatively high levels of this unusual fatty acid in the seed oil of transgenic Arabidopsis thaliana plant was accomplished by the use of A. thaliana fad3/fae1 mutant high in linoleic acid (18:2∆9c,12c) and by co-expression of P. granatum FATTY ACID CONJUGASE (PgFADX) with Δ12-DESATURASE (FAD2). In the current study, P. granatum cDNAs governing PuA production were introduced into the yeast Schizosaccharomyces pombe. Expression of PgFADX alone resulted in production of PuA at the level of 19.6% of total fatty acids. Co-expression PgFADX with PgFAD2, however, further enhanced PuA content to 25.1% of total fatty acids, the highest level reported to date for heterologous expression. Therefore, microbial systems can be considered as a potential alternative to plant sources for a source of PuA for nutraceutic applications.

The linin promoter is highly effective in enhancing punicic acid production in Arabidopsis.

KEY MESSAGE: Enhanced levels of punicic acid were produced in the seed oil of Arabidopsis over-expressing pomegranate FATTY ACID CONJUGASE driven by heterologous promoters, among which the linin promoter was the most efficient. Fatty acids with conjugated double bonds play a special role in determining both the nutritional and industrial uses of plant oils. Punicic acid (18:3Δ9cis,11trans,13cis ), a conjugated fatty acid naturally enriched in the pomegranate (Punica granatum) seeds, has gained increasing attention from the biotechnology community toward its production in metabolically engineered oilseed crops because of its significant health benefits. The present study focused on selecting the best heterologous promoter to drive the expression of the P. granatum FATTY ACID CONJUGASE (PgFADX) cDNA as a means of producing punicic acid in Arabidopsis seed oil. Among the four promoters of genes encoding seed storage proteins from different crop species, the linin promoter led to the highest accumulation of punicic acid (13.2% of total fatty acids in the best homozygous line). Analysis of the relative expression level of PgFADX in developing seeds further confirmed that the linin promoter was most efficient in Arabidopsis. In addition, a conserved profile of cis-regulatory elements were identified in four heterologous promoters by bioinformatic analysis, and their possible roles in regulating gene expression during plant development were also discussed based on the results of this study in combination with the literature. This study contributes to metabolic engineering strategies aimed at enhancing the production of bioactive fatty acids in oilseed crops.

Effect of Genotype on the Sprouting of Pomegranate (Punica granatum L.) Seeds as a Source of Phenolic Compounds from Juice Industry by-Products.

Pomegranate (Punica granatum L.) fruits are used mainly by the juice industry, for which seeds are a by-product to be disposed of, though they could potentially be a source of bioactive compounds. In this work, germination (total germination percentage, G; mean germination time, MGT; time to reach 80% of germination, TG80; seedling shoot length, fresh weight and dry matter), and nutritional value (total phenolics, TP; total flavonoids, TF; total non-tannins, TNT; antioxidant activities) of pomegranate seeds and sprouts were determined on four commercial pomegranate cultivars (Akko, Dente di Cavallo, Mollar de Elche and Wonderful). Seeds were removed from ripe fruits and incubated in plastic trays containing sterile cotton wetted with distilled water. Sprout shoots were harvested when they reached the complete cotyledon expansion, i.e., the ready-to-eat stage. Akko showed the best germination performance (G = 98%; MGT = 14 days after sowing, DAS; TG80 = 16 DAS), followed by Mollar de Elche. Sprouting dramatically increased TP, TF, TNT and antioxidant activity in all genotypes, with the highest values recorded in Mollar de Elche and Dente di Cavallo. Overall, based on germination performance, Akko and Mollar de Elche would be the best cultivars for sprouting. Sprouting pomegranate seeds appears to be a suitable way of utilizing by-products of the juice industry to obtain bioactive compounds.

Genome-wide identification of microRNAs in pomegranate (Punica granatum L.) by high-throughput sequencing.

BACKGROUND: MicroRNAs (miRNAs), a class of small non-coding endogenous RNAs that regulate gene expression post-transcriptionally, play multiple key roles in plant growth and development and in biotic and abiotic stress response. Knowledge and roles of miRNAs in pomegranate fruit development have not been explored. RESULTS: Pomegranate, which accumulates a large amount of anthocyanins in skin and arils, is valuable to human health, mainly because of its antioxidant properties. In this study, we developed a small RNA library from pooled RNA samples from young seedlings to mature fruits and identified both conserved and pomegranate-specific miRNA from 29,948,480 high-quality reads. For the pool of 15- to 30-nt small RNAs, ~50 % were 24 nt. The miR157 family was the most abundant, followed by miR156, miR166, and miR168, with variants within each family. The base bias at the first position from the 5' end had a strong preference for U for most 18- to 26-nt sRNAs but a preference for A for 18-nt sRNAs. In addition, for all 24-nt sRNAs, the nucleotide U was preferred (97 %) in the first position. Stem-loop RT-qPCR was used to validate the expression of the predominant miRNAs and novel miRNAs in leaves, male and female flowers, and multiple fruit developmental stages; miR156, miR156a, miR159a, miR159b, and miR319b were upregulated during the later stages of fruit development. Higher expression of miR156 in later fruit developmental may positively regulate anthocyanin biosynthesis by reducing SPL transcription factor. Novel miRNAs showed variation in expression among different tissues. These novel miRNAs targeted different transcription factors and hormone related regulators. Gene ontology and KEGG pathway analyses revealed predominant metabolic processes and catalytic activities, important for fruit development. In addition, KEGG pathway analyses revealed the involvement of miRNAs in ascorbate and linolenic acid, starch and sucrose metabolism; RNA transport; plant hormone signaling pathways; and circadian clock. CONCLUSION: Our first and preliminary report of miRNAs will provide information on the synthesis of biochemical compounds of pomegranate for future research. The functions of the targets of the novel miRNAs need further investigation.