Comparative transcriptomic and metabolomic profiles reveal fruit peel color variation in two red pomegranate cultivars.

Pomegranate (Punica granatum L.) which belongs to family Lythraceae, is one of the most important fruit crops of many tropical and subtropical regions. A high variability in fruit color is observed among different pomegranate accessions, which arises from the qualitative and quantitative differences in anthocyanins. However, the mechanism of fruit color variation is still not fully elucidated. In the present study, we investigated the red color mutation between a red-skinned pomegranate 'Hongbaoshi' and a purple-red-skinned cultivar 'Moshiliu', by using transcriptomic and metabolomic approaches. A total of 51 anthocyanins were identified from fruit peels, among which 3-glucoside and 3,5-diglucoside of cyanidin (Cy), delphinidin (Dp), and pelargonidin (Pg) were dominant. High proportion of Pg in early stages of 'Hongbaoshi' but high Dp in late stages of 'Moshiliu' were characterized. The unique high levels of Cy and Dp anthocyanins accumulating from early developmental stages accounted for the purple-red phenotype of 'Moshiliu'. Transcriptomic analysis revealed an early down-regulated and late up-regulated of anthocyanin-related structure genes in 'Moshiliu' compared with 'Hongbaoshi'. Alao, ANR was specially expressed in 'Hongbaoshi', with extremely low expression levels in 'Moshiliu'. For transcription factors R2R3-MYB, the profiles demonstrated a much higher transcription levels of three subgroup (SG) 5 MYBs and a sharp decrease in expression of SG6 MYB LOC116202527 in high-anthocyanin 'Moshiliu'. SG4 MYBs exhibited two entirely different patterns, LOC116203744 and LOC116212505 were down-regulated whereas LOC116205515 and LOC116212778 were up-regulated in 'Moshiliu' pomegranate. The results indicate that specific SG members of the MYB family might promote the peel coloration in different manners and play important roles in color mutation in pomegranate.

Identification of Candidate Expansin Genes Associated with Seed Weight in Pomegranate (Punica granatum L.).

Seed weight is an important target trait in pomegranate breeding and culture. Expansins act by loosening plant cell walls and cellulosic materials, permitting turgor-driven cell enlargement. However, the role of expansin genes (EXPs) in pomegranate seed weight remains elusive. A total of 29 PgrEXPs were identified in the 'Dabenzi' genome. These genes were classified into four subfamilies and 14 subgroups, including 22 PgrEXPAs, 5 PgrEXPBs, 1 PgrEXPLA, and 1 PgrEXPLB. Transcriptome analysis of PgrEXPs in different tissues (root, leaf, flower, peel, and seed testa) in 'Dabenzi', and the seed testa of the hard-seeded pomegranate cultivar 'Dabenzi' and soft-seeded cultivar 'Tunisia' at three development stages showed that three PgrEXPs (PgrEXPA11, PgrEXPA22, PgrEXPA6) were highly expressed throughout seed development, especially in the sarcotesta. SNP/Indel markers of these PgrEXPs were developed and used to genotype 101 pomegranate accessions. The association of polymorphic PgrEXPs with seed weight-related traits (100-seed weight, 100-kernel weight, 100-sarcotesta weight, and the percentage of 100-sarcotesta to 100-seed weight) were analyzed. PgrEXP22 was significantly associated with 100-seed weight and 100-sarcotesta weight and is a likely candidate for regulating seed weight and sarcotesta development in particular. This study provides an effective tool for the genetic improvement of seed weight in pomegranate breeding programs.

Multichromosomal mitochondrial genome of Punica granatum: comparative evolutionary analysis and gene transformation from chloroplast genomes.

BACKGROUND: Punica granatum is a fundamentally important fruit tree that has important economic, medicinal and ornamental properties. At present, there are few reports on the mitochondrial genome of pomegranate. Hence, in this study the P. granatum mitogenome was sequenced and assembled to further understanding of organization, variation, and evolution of mitogenomes of this tree species. RESULTS: The genome structure was multi-chromosomes with seven circular contigs, measuring 382,774 bp in length with a 45.91% GC content. It contained 74 genes, including 46 protein-coding genes, 25 tRNA genes, and three rRNA genes. There were 188 pairs of dispersed repeats with lengths of 30 or greater, primarily consisting of reverse complementary repeats. The mitogenome analysis identified 114SSRs and 466 RNA editing sites. Analyses of codon usage, nucleotide diversity and gene migration from chloroplast to mitochondrial were also conducted. The collinear and comparative analysis of mitochondrial structures between P. granatum and its proximal species indicated that P. granatum 'Taishanhong' was closely related to P. granatum 'Qingpitian' and Lagerstroemia indica. Phylogenetic examination based on the mitogenome also confirmed the evolutionary relationship. CONCLUSION: The results offered crucial information on the evolutionary biology of pomegranate and highlighted ways to promote the utilization of the species' germplasm.

Elucidating the physiological and molecular characteristics of bacterial blight incitant Xanthomonas auxonopodis pv. punicae; a life threatening phytopathogen of pomegranate (Punica granatum. L) and assessment of H2O2 accumulation during host-pathogen interaction.

Bacterial blight of pomegranate caused by Xanthomonas auxonopodis pv.punicae (Xap) threaten the existence of a group of farmers for the past few decades who rely on pomegranate cultivation for their livelihood since it will cause huge yield loss. The primary focus of this study was to conduct a thorough analysis of the characterization of this blight incitant Xap. Physiological, biochemical, and molecular characteristics of six phytopathogenic strains of Xap, designated as PBF1 (PBF: Pomegranate Blight Fruit), PBF2, PBF3, PBF4, PBF5, and PBF6, isolated from the infected fruits were examined. Bacterial colonies were featured as gram-negative, yellow-pigmented circular with a glistening appearance. An attempt to determine the best culture medium, favouring bacterial proliferation was successfully done with four distinct medium, Nutrient Glucose Agar (NGA), Nutrient sucrose Agar (NSA), Yeast Dextrose Calcium Carbonate Agar (YDCA) and Yeast Glucose Calcium Carbonate Agar (YGCA) and comparatively, significant growth was found in NGA (66.66%) followed by YDCA (33%). According to the antibiotic susceptibility results, both ampicillin and streptomycin were determined as potentially effective drugs in preventing the proliferation of Xap (P 0.05). The reactive oxygen species-mediated plant immune response during host-pathogen interaction was confirmed by accessing the presence of H2O2 accumulation in infected leaves via 3,3 - diaminobenzidine (DAB) -staining technique. Bacterial isolates from this study were confirmed by two universal constitutive genes such as gyrB and 16S rRNA. From the BLAST analysis, the isolates were identified as Xap with base pair lengths of 1408bp, 1180bp, and 1159bp, which correspond to PBF1, PBF2, and PBF3, respectively. A neighbor-joining phylogenetic tree study explaining a strong phylogenetic relationship between the query sequence and closely related bacterial species.

Inter-species competition of surface bacterial flora of pomegranate and their role in spoilage.

The surface of fruits is heterogenous in term of its microenvironment hence dictate the kind of microflora that develops during storage. A better understanding of spoilage organisms would lead to better preservation methods. The pomegranate was chosen, since its sturdy and spoils slow at room temperature and is ideal for studying fruit spoilage in-situ. In the current study we isolated organisms from fruit surface and study the spoilage and competition amongst microbial species. Total 17 unique bacterial isolates from pomegranate were identified. The 16S rRNA gene identification placed them in 8 major genera (Acinetobacter, Micrococcus, Pantoea, Microbacterium, Strenotrophomonas, Bacillus, Staphylococcus and Exiguobacterium). Competition assay among isolate suggested that Exiguobacterium is dominant species followed by Micrococcus, Pantoea and Bacillus. The consortium of 3 different combinations (5 bacteria each) of isolated bacteria showed the spoilage phenotype on pomegranate. Except for 3 bacterial isolates, the rest of the isolates produced any one or multiple enzymes associated with the food spoilage (cellulase, amylase, lactase, pectinase and protease). The isolates were checked for the presence of genes associated with antibiotic resistance and 78.9% of the tested micro-organisms were blaTEM positive. Aminoglycoside resistance genes were present in 10% of the tested microbes. This study demonstrated interspecies competition amongst spoilage organisms. This understanding of surface flora of fruit would give better insights to preserve fruits.

PgMYB1 Positively Regulates Anthocyanin Accumulation by Activating PgGSTF6 in Pomegranate.

The peel color of pomegranates is an important exterior quality that determines market value. Anthocyanins are biosynthesized in the cytosol and then transported to the vacuole for storage. However, the molecular mechanism that determines the color variation between red and white pomegranates remains unclear. In this study, we identified an R2R3-MYB protein (PgMYB1) that interacts with the PgGSTF6 promoter and regulates its transcriptional expression, thus promoting the accumulation of anthocyanins in pomegranate. The expression of PgMYB1 and PgGSTF6 was positively correlated with the anthocyanin content in red and white pomegranates. Further investigation showed that the knockdown of PgMYB1 in red pomegranate 'Taishanhong' (TSH), by the virus-induced gene-silencing system, inhibited anthocyanin accumulation. Together, our results indicate that PgMYB1 controls the transport of anthocyanin via PgGSTF6 and thus promotes anthocyanin accumulation in red pomegranates. Our results have a certain reference value for further clarifying the regulation of anthocyanin synthesis and transport in pomegranate fruits.

BELL1 interacts with CRABS CLAW and INNER NO OUTER to regulate ovule and seed development in pomegranate.

Pomegranate (Punica granatum) flowers are classified as bisexual flowers and functional male flowers. Functional male flowers have sterile pistils that show abnormal ovule development. In previous studies, we identified INNER NO OUTER (INO), CRABS CLAW (CRC), and BELL1 (BEL1), which were specifically expressed in bisexual and functional male flowers. However, the functions of ovule identity genes and the mechanism underlying ovule sterility in pomegranate remain unknown. Here, we found that the integument primordia formed and then ceased developing in the ovules of functional male flowers with a vertical diameter of 8.1-13.0 mm. Megaspore mother cells were observed in bisexual flowers when the vertical diameters of flowers were 10.1-13.0 mm, but not in functional male flowers. We analyzed the expression patterns of ovule-related genes in pomegranate ovule sterility and found that PgCRC mRNA was highly expressed at a critical stage of ovule development in bisexual flowers. Ectopic expression of PgCRC and PgINO was sufficient to increase seed number in transgenic lines. PgCRC partially complemented the Arabidopsis (Arabidopsis thaliana) crc mutant, and PgINO successfully rescued the seeds set in the Arabidopsis ino mutant. The results of yeast two-hybrid assays, bimolecular fluorescence complementation assays, and genetic data analyses showed that PgCRC and PgINO directly interact with PgBEL1. Our results also showed that PgCRC and PgINO could not interact directly with MADS-box proteins and that PgBEL1 interacted with SEPALLATA proteins. We report the function of PgCRC and PgINO in ovule and seed development and show that PgCRC and PgINO interact with PgBEL1. Thus, our results provide understanding of the genetic regulatory networks underlying ovule development in pomegranate.

Overexpression of PgCBF3 and PgCBF7 Transcription Factors from Pomegranate Enhances Freezing Tolerance in Arabidopsis under the Promoter Activity Positively Regulated by PgICE1.

Cold stress limits plant growth, development and yields, and the C-repeat binding factors (CBFs) function in the cold resistance in plants. However, how pomegranate CBF transcription factors respond to cold signal remains unclear. Considering the significantly up-regulated expression of PgCBF3 and PgCBF7 in cold-tolerant Punica granatum 'Yudazi' in comparison with cold-sensitive 'Tunisia' under 4 °C, the present study focused on the two CBF genes. PgCBF3 was localized in the nucleus, while PgCBF7 was localized in the cell membrane, cytoplasm, and nucleus, both owning transcriptional activation activity in yeast. Yeast one-hybrid and dual-luciferase reporter assay further confirmed that PgICE1 could specifically bind to and significantly enhance the activation activity of the promoters of PgCBF3 and PgCBF7. Compared with the wild-type plants, the PgCBF3 and PgCBF7 transgenic Arabidopsis thaliana lines had the higher survival rate after cold treatment; exhibited increased the contents of soluble sugar and proline, while lower electrolyte leakage, malondialdehyde content, and reactive oxygen species production, accompanying with elevated enzyme activity of catalase, peroxidase, and superoxide dismutase; and upregulated the expression of AtCOR15A, AtCOR47, AtRD29A, and AtKIN1. Collectively, PgCBFs were positively regulated by the upstream PgICE1 and mediated the downstream COR genes expression, thereby enhancing freezing tolerance.

Genome-Wide Identification and Comprehensive Analysis of the AP2/ERF Gene Family in Pomegranate Fruit Development and Postharvest Preservation.

Pomegranate (Punica granatum L.) is a kind of fruit with significant economic, ecological and health values. AP2/ERF transcription factors belong to a large group of factors mainly found in plants and play key roles in plant growth and development. However, AP2/ERF genes in pomegranate and their implication in development and postharvest preservation have been little described. In this study, 116 PgAP2/ERF genes in pomegranate were identified and renamed based on their chromosomal distributions. Phylogenetic relationship with genes from other species, structures, duplications, annotations, cis-elements in promoter sequences, and protein-protein interaction networks among PgAP2/ERF proteins were comprehensively explored. Expression analysis revealed several PgAP2/ERFs associated with the phenotypes of pomegranate seed hardness, including PgAP2/ERF5, PgAP2/ERF36, PgAP2/ERF58, and PgAP2/ERF86. Subsequent analysis indicated that many differentially expressed PgAP2/ERF genes are potentially important regulators of pomegranate fruit development. Furthermore, expression of more than one-half of PgAP2/ERFs was repressed in 'Tunisian soft seed' pomegranate fruit under low-temperature cold storage. The results showed that 1-MCP implicated in promoting postharvest preservation of 'Tunisian soft seed' pomegranate upregulated the PgAP2/ERF4, PgAP2/ERF15, PgAP2/ERF26, PgAP2/ERF30, PgAP2/ERF35 and PgAP2/ERF45 genes compared to those under low-temperature cold storage. This indicates that these genes are important candidate genes involved in pomegranate postharvest preservation. In summary, the findings of the present study provide an important basis for characterizing the PgAP2/ERF family genes and provide information on the candidate genes involved in pomegranate fruit development and postharvest preservation.

Systematic Analysis and Expression Profiles of the 4-Coumarate: CoA Ligase (4CL) Gene Family in Pomegranate (Punica granatum L.).

4-Coumarate:CoA ligase (4CL, EC6.2.1.12), located at the end of the phenylpropanoid metabolic pathway, regulates the metabolic direction of phenylpropanoid derivatives and plays a pivotal role in the biosynthesis of flavonoids, lignin, and other secondary metabolites. In order to understand the molecular characteristics and potential biological functions of the 4CL gene family in the pomegranate, a bioinformatics analysis was carried out on the identified 4CLs. In this study, 12 Pg4CLs were identified in the pomegranate genome, which contained two conserved amino acid domains: AMP-binding domain Box I (SSGTTGLPKGV) and Box II (GEICIRG). During the identification, it was found that Pg4CL2 was missing Box II. The gene cloning and sequencing verified that this partial amino acid deletion was caused by genome sequencing and splicing errors, and the gene cloning results corrected the Pg4CL2 sequence information in the 'Taishanhong' genome. According to the phylogenetic tree, Pg4CLs were divided into three subfamilies, and each subfamily had 1, 1, and 10 members, respectively. Analysis of cis-acting elements found that all the upstream sequences of Pg4CLs contained at least one phytohormone response element. An RNA-seq and protein interaction network analysis suggested that Pg4CL5 was highly expressed in different tissues and may participate in lignin synthesis of pomegranate. The expression of Pg4CL in developing pomegranate fruits was analyzed by quantitative real-time PCR (qRT-PCR), and the expression level of Pg4CL2 demonstrated a decreasing trend, similar to the trend of flavonoid content, indicating Pg4CL2 may involve in flavonoid synthesis and pigment accumulation. Pg4CL3, Pg4CL7, Pg4CL8, and Pg4CL10 were almost not expressed or lowly expressed, the expression level of Pg4CL4 was higher in the later stage of fruit development, suggesting that Pg4CL4 played a crucial role in fruit ripening. The expression levels of 4CL genes were significantly different in various fruit development stages. The results laid the foundation for an in-depth analysis of pomegranate 4CL gene functions.

Genome-wide identification and characterization of bZIP gene family and cloning of candidate genes for anthocyanin biosynthesis in pomegranate (Punica granatum).

BACKGROUND: The basic leucine zipper (bZIP) transcription factor is one of the most abundant and conserved gene families in eukaryotes. In addition to participating in plant development and growth, bZIP transcription factors play crucial roles in various abiotic stress responses and anthocyanin accumulation. Up to now, analysis of bZIP gene family members in pomegranate (Punica granatum) has not been reported. Three published pomegranate genome sequences provide valuable resources for further gene function analysis. RESULTS: Using bioinformatics analysis, 65 PgbZIPs were identified and analyzed from the 'Taishanhong' pomegranate genome. We divided them into 13 groups (A, B, C, D, E, F, G, H, I, J, K, M, and S) according to the phylogenetic relationship with those of Arabidopsis, each containing a different number of genes. The regularity of exon/intron number and distribution was consistent with the classification of groups in the evolutionary tree. Transcriptome analysis of different tissues showed that members of the PgbZIP gene family were differentially expressed in different developmental stages and tissues of pomegranate. Among them, we selected PgbZIP16 and PgbZIP34 as candidate genes which affect anthocyanin accumulation. The full-length CDS region of PgbZIP16 and PgbZIP34 were cloned from pomegranate petals by homologous cloning technique, encoding 170 and 174 amino acids, which were 510 bp and 522 bp, respectively. Subcellular localization assays suggested that both PgbZIP16 and PgbZIP34 were nucleus-localized. Real-time quantitative PCR (qPCR) was used to explore the expression of PgbZIP16 and PgbZIP34 in the petals of three kinds of ornamental pomegranates at the full flowering stage. The results demonstrated that the expression of PgbZIP16 in red petals was 5.83 times of that in white petals, while PgbZIP34 was 3.9 times. The results of transient expression in tobacco showed that consistent trends were observed in anthocyanin concentration and expression levels of related genes, which both increased and then decreased. Both PgbZIP16 and PgbZIP34 could promote anthocyanin accumulation in tobacco leaves. We obtained transgenic strains overexpressing PgbZIP16, and the histochemical staining for GUS activity showed that overexpressed PgbZIP16 seedlings were expressed in the stem. Transgenic experiments indicated that overexpression of PgbZIP16 significantly upregulated UF3GT, ANS and DFR genes in Arabidopsis and enhanced anthocyanin accumulation. CONCLUSIONS: The whole genome identification, gene structure, phylogeny, gene cloning, subcellular location and functional verification of the pomegranate bZIP gene family provide a theoretical foundation for the functional study of the PgbZIP gene family and candidate genes for anthocyanin biosynthesis.

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.

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.

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.

Identification and Functional Analysis of CAD Gene Family in Pomegranate (Punica granatum).

[Objective] Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. The aim of this study was to identify CAD gene family members in pomegranate and its expression correlation with seed hardness. [Methods] Based on the reported CAD sequence of Arabidopsis, the CAD gene family of pomegranate was identified by homologous comparison, and then phylogenetic, molecular characterization, and expression profile analysis were performed. [Results] Pomegranate CAD gene family has 25 members, distributed on seven chromosomes of pomegranate. All pomegranate CAD proteins have similar physical and chemical properties. We divide the family into four groups based on evolutionary relationships. The member of group I, called bona fide CAD, was involved in lignin synthesis. Most of the members of group II were involved in stress resistance. The functions of groups III and IV need to be explored. We found four duplicated modes (whole genome duplication or segmental (WGD), tandem duplication (TD), dispersed duplication (DSD), proximal duplication (PD) in this family; TD (36%) had the largest number of them. We predicted that 20 cis-acting elements were involved in lignin synthesis, stress resistance, and response to various hormones. Gene expression profiles further demonstrated that the PgCAD gene family had multiple functions. [Conclusions] Pomegranate CAD gene family is involved in lignin synthesis of hard-seeded cultivar Hongyushizi and Baiyushizi, but its role in seed hardness of soft-seeded cultivar Tunisia needs to be further studied.

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.

Identification of suitable reference genes for expression studies in pomegranate under different biotic and abiotic stress conditions.

Pomegranate (Punica granatum L.) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis. Eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ∆Ct, geNorm, BestKeeper, NormFinder, and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. For normalizing target gene expression under wilt, nematode, bacterial blight, and abiotic stress conditions both GAPDH and CYPFreference genes are adequate for qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate.

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.

Genome-wide identification and expression analysis of the CLC gene family in pomegranate (Punica granatum) reveals its roles in salt resistance.

BACKGROUNDS: Pomegranate (Punica granatum L.) is an important commercial fruit tree, with moderate tolerance to salinity. The balance of Cl- and other anions in pomegranate tissues are affected by salinity, however, the accumulation patterns of anions are poorly understood. The chloride channel (CLC) gene family is involved in conducting Cl-, NO3-, HCO3- and I-, but its characteristics have not been reported on pomegranate. RESULTS: In this study, we identified seven PgCLC genes, consisting of four antiporters and three channels, based on the presence of the gating glutamate (E) and the proton glutamate (E). Phylogenetic analysis revealed that seven PgCLCs were divided into two clades, with clade I containing the typical conserved regions GxGIPE (I), GKxGPxxH (II) and PxxGxLF (III), whereas clade II not. Multiple sequence alignment revealed that PgCLC-B had a P [proline, Pro] residue in region I, which was suspected to be a NO3-/H+ exchanger, while PgCLC-C1, PgCLC-C2, PgCLC-D and PgCLC-G contained a S [serine, Ser] residue, with a high affinity to Cl-. We determined the content of Cl-, NO3-, H2PO4-, and SO42- in pomegranate tissues after 18 days of salt treatments (0, 100, 200 and 300 mM NaCl). Compared with control, the Cl- content increased sharply in pomegranate tissues. Salinity inhibited the uptake of NO3- and SO42-, but accelerated H2PO4- uptake. The results of real-time reverse transcription PCR (qRT-PCR) revealed that PgCLC genes had tissue-specific expression patterns. The high expression levels of three antiporters PgCLC-C1, PgCLC-C2 and PgCLC-D in leaves might be contributed to sequestrating Cl- into the vacuoles. However, the low expression levels of PgCLCs in roots might be associated with the exclusion of Cl- from root cells. Also, the up-regulated PgCLC-B in leaves indicated that more NO3- was transported into leaves to mitigate the nitrogen deficiency. CONCLUSIONS: Our findings suggested that the PgCLC genes played important roles in balancing of Cl- and NO3- in pomegranate tissues under salt stress. This study established a theoretical foundation for the further functional characterization of the CLC genes in pomegranate.

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.