Revealing the Genetic Components Responsible for the Unique Photosynthetic Stem Capability of the Wild Almond Prunus arabica (Olivier) Meikle.

Almond [Prunus dulcis (Mill.) D. A. Webb] is a major deciduous fruit tree crop worldwide. During dormancy, under warmer temperatures and inadequate chilling hours, the plant metabolic activity increases and may lead to carbohydrate deficiency. Prunus arabica (Olivier) Meikle is a bushy wild almond species known for its green, unbarked stem, which stays green even during the dormancy period. Our study revealed that P. arabica green stems assimilate significantly high rates of CO2 during the winter as compared to P. dulcis cv. Um el Fahem (U.E.F.) and may improve carbohydrate status throughout dormancy. To uncover the genetic inheritance and mechanism behind the P. arabica stem photosynthetic capability (SPC), a segregated F1 population was generated by crossing P. arabica to U.E.F. Both parent's whole genome was sequenced, and SNP calling identified 4,887 informative SNPs for genotyping. A robust genetic map for U.E.F. and P. arabica was constructed (971 and 571 markers, respectively). QTL mapping and association study for the SPC phenotype revealed major QTL [log of odd (LOD) = 20.8] on chromosome 7 and another minor but significant QTL on chromosome 1 (LOD = 3.9). As expected, the P. arabica allele in the current loci significantly increased the SPC phenotype. Finally, a list of 64 candidate genes was generated. This work sets the stage for future research to investigate the mechanism regulating the SPC trait, how it affects the tree's physiology, and its importance for breeding new cultivars better adapted to high winter temperatures.

Fine Mapping of the "black" Peel Color in Pomegranate (Punica granatum L.) Strongly Suggests That a Mutation in the Anthocyanidin Reductase (ANR) Gene Is Responsible for the Trait.

Anthocyanins are important dietary and health-promoting substances present in high quantities in the peel and arils of the pomegranate (Punica granatum L.) fruit. Yet, there is a high variation in the content of anthocyanin among different pomegranate varieties. The 'Black' pomegranate variety (P.G.127-28) found in Israel contains exceptionally high levels of anthocyanins in its fruit peel which can reach up to two orders of magnitude higher content as compared to that of other pomegranate varieties' peel anthocyanins. Biochemical analysis reveals that delphinidin is highly abundant in the peel of 'Black' variety. The pattern of anthocyanin accumulation in the fruit peel during fruit development of 'Black' variety differs from that of other pomegranates. High anthocyanin levels are maintained during all developmental stages. Moreover, the accumulation of anthocyanin in the fruit peel of 'Black' variety is not dependent on light. Genetic analysis of an F2 population segregating for the "black" phenotype reveals that it is determined by a single recessive gene. Genetic mapping of the F2 population using single nucleotide polymorphism (SNP) markers identified few markers tightly linked to the "black" phenotype. Recombination analysis of the F2 population and F3 populations narrowed the "black" trait to an area of 178.5 kb on the draft genome sequence of pomegranate cv. 'Dabenzi.' A putative anthocyanidin reductase (ANR) gene is located in this area. Only pomegranate varieties displaying the "black" trait carry a base pair deletion toward the end of the gene, causing a frame shift resulting in a shorter protein. We propose that this mutation in the ANR gene is responsible for the different anthocyanin composition and high anthocyanin levels of the "black" trait in pomegranate.

Primary Metabolites, Anthocyanins, and Hydrolyzable Tannins in the Pomegranate Fruit.

Pomegranate (Punica granatum L.) is an important and interesting fruit tree that is cultivated in many parts of the world. In recent years, along with the increase in its cultivation and consumption there has been a dramatic increase in the scientific interest in its biology, methods of cultivation, adaptation to environmental cues and its health-promoting properties. Quite a large proportion of the various metabolites produced in the pomegranate were determined and their content in the bark, roots, leaves, and fruit was reported. Many reviews on polyphenolic compound content, antioxidant activity and health-promoting compounds were published recently. However, only very few recent reports were dedicated to primary metabolites, despite the fact that much work was done on organic acids, sugars, proteins, lipids, and amino acids of the pomegranate fruit. In this review, a special effort was made to present these recent studies and the review is devoted to primary metabolites. The reported data show high variation in the content of primary metabolites within the pomegranate fruit; therefore the data is presented (whenever possible) according to fruit tissues (peel, arils, and seeds), developmental stages of the fruit, environmental and climatic conditions, and genetic background. Most of the data on pomegranate is based on metabolic content and contains no genetic or molecular analysis except for work done on anthocyanins and hydrolyzable tannins. In those cases, gene assignment and genetic control studies were pointed out in the review. The recent publication of the genome sequences from several pomegranate varieties and transcriptomic data from fruits, flowers, and leaves is expected to facilitate the understanding of genetic control of metabolites in pomegranate.

Diversity among Pomegranate Varieties in Chilling Tolerance and Transcriptome Responses to Cold Storage.

We found great variability in chilling tolerance among 84 pomegranate varieties from the Newe Ya'ar collection; among them, 'Ganesh' was chilling-sensitive, whereas 'Wonderful' was relatively chilling-tolerant. To evaluate the different molecular responses of these varieties to cold storage, we analyzed the transcriptomic changes in the inner membrane tissues of 'Ganesh' and 'Wonderful' fruit after 2 weeks of cold storage at 1 °C. By functional categorization of the differentially expressed transcripts using MapMan, we found that many transcripts related to various pathways, such as jasmonic acid biosynthesis and signaling, galactinol, raffinose, phenol, and phenylpropanoid biosynthesis, calcium and mitogen-activated protein kinase signaling, lipid metabolism, and various transcription factors and heat-shock proteins, have been massively upregulated in 'Wonderful' but not in 'Ganesh' fruit. Thus, it is suggested that these pathways most likely participate in imparting chilling tolerance in pomegranate fruit.

Grafting on resistant interstocks reduces scion susceptibility to pear psylla, Cacopsylla bidens.

BACKGROUND: Pear psylla is a major obstacle to efficient integrated pest management in pear orchards in Israel and around the world. We used two accessions with natural resistance to pear psylla Cacopsylla bidens (Sulc) - Py.760-261 (760) and Py.701-202 (701), both apparently of Pyrus communis L. origin - as interstock grafts to confer psylla resistance to the commercially important 'Spadona Estiva' (Pyrus communis) scion (Spadona) cultivar. The interaction of the interstocks with quince (Cydonia oblong Mill.) and Pyrus betulifolia Bunge rootstocks was also tested. RESULTS: Usage of Py.760-261 (760) and Py.701-202 (701) as interstocks for the psylla-sensitive Spadona resulted in a five-fold decrease in the C. bidens population, apparently as a consequence of antibiosis affecting nymph survival. Additionally, psylla survival was negatively correlated with the interstock length and amount of foliage. The yield and fruit quality of Spadona grafted on the '701' interstock equaled or even exceeded those of the control in fruit quantity, fruit size and soluble solids content, especially on P. betulifolia rootstock. CONCLUSION: Susceptibility to pear psylla decreased significantly following grafting of commercial Spadona on resistant interstock. This is the first demonstration of increased resistance to pear psylla conferred by the use of resistant interstock in pear trees and among the few examples demonstrating transfer of resistance to insects from the interstock in fruit trees. © 2017 Society of Chemical Industry.

A "White" Anthocyanin-less Pomegranate (Punica granatum L.) Caused by an Insertion in the Coding Region of the Leucoanthocyanidin Dioxygenase (LDOX; ANS) Gene.

Color is an important determinant of pomegranate fruit quality and commercial value. To understand the genetic factors controlling color in pomegranate, chemical, molecular and genetic characterization of a "white" pomegranate was performed. This unique accession is lacking the typical pomegranate color rendered by anthocyanins in all tissues of the plant, including flowers, fruit (skin and arils) and leaves. Steady-state gene-expression analysis indicated that none of the analyzed "white" pomegranate tissues are able to synthesize mRNA corresponding to the PgLDOX gene (leucoanthocyanidin dioxygenase, also called ANS, anthocyanidin synthase), which is one of the central structural genes in the anthocyanin-biosynthesis pathway. HPLC analysis revealed that none of the "white" pomegranate tissues accumulate anthocyanins, whereas other flavonoids, corresponding to biochemical reactions upstream of LDOX, were present. Molecular analysis of the "white" pomegranate revealed the presence of an insertion and an SNP within the coding region of PgLDOX. It was found that the SNP does not change amino acid sequence and is not fully linked with the "white" phenotype in all pomegranate accessions from the collection. On the other hand, genotyping of pomegranate accessions from the collection and segregating populations for the "white" phenotype demonstrated its complete linkage with the insertion, inherited as a recessive single-gene trait. Taken together, the results indicate that the insertion in PgLDOX is responsible for the "white" anthocyanin-less phenotype. These data provide the first direct molecular, genetic and chemical evidence for the effect of a natural modification in the LDOX gene on color accumulation in a fruit-bearing woody perennial deciduous tree. This modification can be further utilized to elucidate the physiological role of anthocyanins in protecting the tree organs from harmful environmental conditions, such as temperature and UV radiation.

Single-nucleotide polymorphism markers from de-novo assembly of the pomegranate transcriptome reveal germplasm genetic diversity.

Pomegranate is a valuable crop that is grown commercially in many parts of the world. Wild species have been reported from India, Turkmenistan and Socotra. Pomegranate fruit has a variety of health-beneficial qualities. However, despite this crop's importance, only moderate effort has been invested in studying its biochemical or physiological properties or in establishing genomic and genetic infrastructures. In this study, we reconstructed a transcriptome from two phenotypically different accessions using 454-GS-FLX Titanium technology. These data were used to explore the functional annotation of 45,187 fully annotated contigs. We further compiled a genetic-variation resource of 7,155 simple-sequence repeats (SSRs) and 6,500 single-nucleotide polymorphisms (SNPs). A subset of 480 SNPs was sampled to investigate the genetic structure of the broad pomegranate germplasm collection at the Agricultural Research Organization (ARO), which includes accessions from different geographical areas worldwide. This subset of SNPs was found to be polymorphic, with 10.7% loci with minor allele frequencies of (MAF<0.05). These SNPs were successfully used to classify the ARO pomegranate collection into two major groups of accessions: one from India, China and Iran, composed of mainly unknown country origin and which was more of an admixture than the other major group, composed of accessions mainly from the Mediterranean basin, Central Asia and California. This study establishes a high-throughput transcriptome and genetic-marker infrastructure. Moreover, it sheds new light on the genetic interrelations between pomegranate species worldwide and more accurately defines their genetic nature.

Two pear accessions evaluated for susceptibility to pear psylla Cacopsylla bidens (Sulc) in Israel.

BACKGROUND: The pear psylla, Cacopsylla bidens (Sulc), is one of the most damaging pests of commercial pear orchards in Israel. Psylla control is a major obstacle to efficient integrated pest management, necessitating research on cultivars with natural resistance to pear psylla. Recently, two pear accessions (Py.760-261 and Py.701-202) from the local Newe Ya'ar fruit tree live collection were identified as having apparent resistance to pear psylla. Our goal was to evaluate the resistance of these two accessions relative to the commercial cultivar Spadona Estiva, and to identify whether the resistance mechanisms in the former interfere with insect colonisation of the plant (antixenosis) or inhibit insect growth, development, reproduction and survival (antibiosis). RESULTS: Settlement and development of C. bidens was evaluated under natural conditions (pear orchard), semi-natural conditions (potted plants), and on detached branches and leaves (laboratory). Our results indicate that the selection Py.760-261 is 10 times more resistant than Spadona while Py.701-202 is five times more resistant. CONCLUSIONS: The resistance mechanism in both accessions appears to be antibiosis affecting nymph survival. These resistant accessions may be used as rootstock or as a source of resistant genes in breeding programmes.

A pomegranate (Punica granatum L.) WD40-repeat gene is a functional homologue of Arabidopsis TTG1 and is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit development.

Anthocyanins are the major pigments responsible for the pomegranate (Punica granatum L.) fruit skin color. The high variability in fruit external color in pomegranate cultivars reflects variations in anthocyanin composition. To identify genes involved in the regulation of anthocyanin biosynthesis pathway in the pomegranate fruit skin we have isolated, expressed and characterized the pomegranate homologue of the Arabidopsis thaliana TRANSPARENT TESTA GLABRA1 (TTG1), encoding a WD40-repeat protein. The TTG1 protein is a regulator of anthocyanins and proanthocyanidins (PAs) biosynthesis in Arabidopsis, and acts by the formation of a transcriptional regulatory complex with two other regulatory proteins: bHLH and MYB. Our results reveal that the pomegranate gene, designated PgWD40, recovered the anthocyanin, PAs, trichome and seed coat mucilage phenotype in Arabidopsis ttg1 mutant. PgWD40 expression and anthocyanin composition in the skin were analyzed during pomegranate fruit development, in two accessions that differ in skin color intensity and timing of appearance. The results indicate high positive correlation between the total cyanidin derivatives quantity (red pigments) and the expression level of PgWD40. Furthermore, strong correlation was found between the steady state levels of PgWD40 transcripts and the transcripts of pomegranate homologues of the structural genes PgDFR and PgLDOX. PgWD40, PgDFR and PgLDOX expression also correlated with the expression of pomegranate homologues of the regulatory genes PgAn1 (bHLH) and PgAn2 (MYB). On the basis of our results we propose that PgWD40 is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit development and that expression of PgWD40, PgAn1 and PgAn2 in the pomegranate fruit skin is required to regulate the expression of downstream structural genes involved in the anthocyanin biosynthesis.

Climate effects on anthocyanin accumulation and composition in the pomegranate (Punica granatum L.) fruit arils.

Worldwide pomegranate (Punica granatum L.) production has expanded greatly due to recent evidence on the fruit health attributes. The fruit's unique red color, conferred by anthocyanins, is an imperative sensory quality. Climate effects on the fruit's internal color were reported earlier. The present study investigated the influence of a wide range of temperature regimes (∼7-40 °C) on pomegranates' aril anthocyanins. The study included two deciduous and two evergreen accessions as well as desert and Mediterranean orchards. RP-HPLC analysis of the arils' anthocyanins revealed mono- and diglucosylated delphinidins and cyanidins as the major anthocyanins and pelargonidins as minor components. Anthocyanin accumulation changed inversely to the season's temperatures. Cyanidins were generally more abundant but delphinidin accumulation was enhanced in cooler season. Monoglucosylated anthocyanins prevailed at cooler temperatures and subsided during seasonal warming with a concomitant increase in diglucoside proportion. The findings can benefit breeding and agricultural efforts to enhance pomegranate quality, especially in the face of "global warming".

Environmental conditions affect the color, taste, and antioxidant capacity of 11 pomegranate accessions' fruits.

The well-established health beneficial value of pomegranate juice is leading to increased demand for pomegranate products and to the expansion of pomegranate orchards worldwide. The current study describes differences in the chemical composition of major ingredients of the arils and peels of 11 accessions grown in Mediterranean and desert climates in Israel. In most of the accessions, the levels of antioxidant activity and content of total phenolics, total anthocyanins, total soluble solids, glucose, fructose, and acidity were higher in the aril juice of fruit grown in the Mediterranean climate compared to those grown in the desert climate. However, the peels of fruit grown in the desert climate exhibited higher antioxidant activity, and the levels of total phenolics, including the two hydrolyzable tannins, punicalagin and punicalin, were higher compared to those in the peels of fruit grown in the Mediterranean climate. The results indicate that environmental conditions significantly affect pomegranate fruit quality and health beneficial compounds.

Developmental and varietal differences in volatile ester formation and acetyl-CoA: alcohol acetyl transferase activities in apple (Malus domestica Borkh.) fruit.

Apple (Malus domestica Borkh.) cultivars differ in their aroma and composition of volatile acetates in their fruit flesh and peel. Cv. Fuji flesh contains substantial levels of 2-methyl butyl acetate (fruity banana-like odor), while the flesh of cv. Granny Smith apples lacks this compound. Granny Smith apples accumulate mainly hexyl acetate (apple-pear odor) in their peel. Feeding experiments indicated that Fuji apples were able to convert hexanol and 2-methyl butanol to their respective acetate derivatives in vivo, while Granny Smith apples could only convert exogenous hexanol to hexyl acetate. Differential substrate specificities of the in vitro acetyl-CoA:alcohol acetyl transferase (AAT) activities were also detected among cultivars. In Granny Smith apples, the AAT activity was detected only in the peel, and its specificity was almost exclusively restricted to hexanol and cis-3-hexenol. In Fuji apples, the AAT activity was detected in both peel and flesh and apparently accepted a broader range of alcohols as substrates than the Granny Smith enzyme activity. Our data strongly suggest that different AAT activities are operational in apple tissues and cultivars and that these differences contribute to the variation observed in the accumulation of volatile acetates.