Elucidating the role of shikimate dehydrogenase in controlling the production of anthocyanins and hydrolysable tannins in the outer peels of pomegranate
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The outer peels of pomegranate (Punica granatum L.) possess two groups of polyphenols that have health beneficial properties: anthocyanins (ATs, which also affect peel color); and hydrolysable tannins (HTs). Their biosynthesis intersects at 3-dehydroshikimate (3-DHS) in the shikimate pathway by the activity of shikimate dehydrogenase (SDH), which converts 3-DHS to shikimate (providing the precursor for AT biosynthesis) or to gallic acid (the precursor for HTs biosynthesis) using NADPH or NADP+ as a cofactor. The aim of this study is to gain more knowledge about the factors that regulate the levels of HTs and ATs, and the role of SDH.
The results have shown that the levels of ATs and HTs are negatively correlated in the outer fruit peels of 33 pomegranate accessions, in the outer peels of two fruits exposed to sunlight, and in those covered by paper bags. When calli obtained from the outer fruit peel were subjected to light/dark treatment and osmotic stresses (imposed by different sucrose concentrations), it was shown that light with high sucrose promotes the synthesis of ATs, while dark at the same sucrose concentration promotes the synthesis of HTs. To verify the role of SDH, six PgSDHs (PgSDH1, PgSDH3–1,2, PgSDH3a-1,2 and PgSDH4) were identified in pomegranate. The expression of PgSDH1, which presumably contributes to shikimate biosynthesis, was relatively constant at different sucrose concentrations. However, the transcript levels of PgSDH3s and PgSDH4 increased with the accumulation of gallic acid and HTs under osmotic stress, which apparently accumulates to protect the cells from the stress.
The results strongly suggest that the biosynthesis of HTs and ATs competes for the same substrate, 3-DHS, and that SDH activity is regulated not only by the NADPH/NADP+ ratio, but also by the expression of the PgSDHs. Since the outer peel affects the customer’s decision regarding fruit consumption, such knowledge could be utilized for the development of new genetic markers for breeding pomegranates having higher levels of both ATs and HTs.
KeywordsAnthocyanins Hydrolysable tannins Osmotic stress Outer peel Peel-tissue culture Pomegranate Shikimate dehydrogenase
high performance liquid chromatography-diode array detector
Murashige and Skoog
oxidative pentose phosphate pathway
principal component analysis
reactive oxygen species
reverse transcription-quantitative polymerase chain reaction
tricarboxylic acid cycle
total phenols content
Fruits of pomegranate (Punica granatum L.) are well known for their health beneficial properties . The health-promoting activities are attributed to the high concentrations of polyphenols found mainly in the fruit’s peels, which possess about 300-fold more polyphenols and 40-fold higher antioxidant activities than arils, the edible section [2, 3, 4, 5]. These polyphenols act as scavengers of reactive oxygen species (ROS), and are associated with a reduction in stress-related chronic diseases and age-related disorders [2, 3, 4, 5, 6]. Previous studies showed that hydrolysable tannins (HTs) and anthocyanins (ATs) are the predominant polyphenols present in outer fruit peels [3, 4, 5, 7]. ATs are water-soluble pigments that are generally responsible for the red/violet/blue color of many fruits and flowers, including pomegranate arils and peels [3, 8]. These compounds are also produced in vegetative tissues and protect plants from environmental stresses, such as osmotic stress, drought, UV irradiation and low temperatures . HTs are polyphenols in which some or all of the hydroxyl groups of the core carbohydrate moiety are esterified with phenolic acids, such as gallic acid (GA) or ellagic acid. HTs reportedly possess antioxidant, antimicrobial and anticancer activities in humans . In pomegranates, HTs contain several unique compounds, including the α and β isomers of punicalagins and punicalins [11, 12]. Punicalagin isomers are most abundant in pomegranate peels and are responsible for more than 50% of the juice’s potent antioxidant activity [3, 13]. They also have health benefits such as induced growth inhibition and apoptosis in human prostate and papillary thyroid carcinoma cancer cells (e.g., [14, 15]).
Therefore, the redox state of the cell (NADPH/NADP+ ratio) is suggested to control the activity of SDH and the competitive partitioning of 3-DHS to shikimate and its associated metabolites (e.g., ATs), or GA and its derivatives (e.g., HTs). Consistent with this hypothesis, it was reported that a high level of ATs was correlated to a low amount of punicalagins (and vice versa) in the aril juice of most of the 29 pomegranate accessions analyzed . However, such a correlation was not observed for ATs and HTs in fruit peels , apparently because both the inner and outer peels of these pomegranate accessions were analyzed together. Considering that ATs are present in the outer peels but not in the inner peels, it remains unclear whether an inverse correlation of ATs and HTs concentrations can be found in the outer peels of pomegranates where both compounds are abundant.
The objectives of the current study were to: (i) understand the distribution of the ATs and HTs in the outer peels of pomegranate; (ii) investigate the role of DQD/SDH in the accumulation of HTs and ATs in this fruit section; and (iii) reveal how environmental conditions could affect the expression of DQD/SDH genes and metabolite levels in the outer peels of pomegranate. Addressing these goals is important since high levels of ATs regulate the color of peels that attract consumers to purchase the fruit, while high levels of HTs are related to a reduction in some of the defects of the peels such as husk scald [23, 24]. Such data could also assist the breeder in producing fruits having high levels of both HTs and ATs.
A negative correlation was detected between ATs and HTs in the outer peels of 33 pomegranate accessions
An inverse relationship between HTs and ATs in the outer fruit peel was observed when the ATs biosynthesis pathway was physically suppressed
It was observed that ATs accumulation in fruits is induced by sunlight to protect them from photoinhibition and photobleaching . Indeed, pomegranate fruits developed in the tree shade have a less intense red color than those produced at the edge of the tree, which is exposed to sunlight. To examine the accumulation of ATs and HTs when AT biosynthesis is induced by sunlight, ATs and HTs profiles were determined in the fruits of PG100–1 and UG3, two accessions having a red-peel color, with and without exposure to sunlight. To prevent exposure to sunlight, seven fruits from PG100–1 and UG3 were covered with paper bags for about two months (August 1 to October 2). An additional seven fruits from each accession of similar size were marked as sun-exposed controls. All fruits selected were located in the outer canopy of pomegranate trees at similar positions on the tree.
An inverse correlation between ATs and HTs was observed in peel calli cultures under different growth conditions
To further investigate the inverse relationship between HTs and ATs, and also to understand how abiotic stresses and different growth conditions affect the contents of ATs and HTs, we initiated an outer fruit peel culture from accession UG28, which has a red peel color . The advantage of this culture is that it is an isolated system that does not receive molecular signals and metabolites from other tissues located outside the cells of the outer peel. To obtain sufficient tissues for the experiment, the peel cultures were grown for three months in MS medium containing 1% sucrose under dark conditions . Subsequently, about one gram of calli was transferred to MS plates supplemented with different sucrose concentrations (1, 2.5, 5%, or 7.5%) that, at high concentrations, lead to osmotic stress, and the plates were placed in light or dark. After biweekly transfers for 75 days, calli grown in 1% sucrose in the dark appeared gray, while those that developed in the light had a green color resulting from chlorophyll accumulation (Additional file 3: Figure S1). Red spots and patches were observed in calli grown under light in 2.5% sucrose, which intensified in 5%, but decreased in 7.5% sucrose (Additional file 3: Figure S1). Calli grown in the dark showed a range of light gray to dark gray in media containing 1–7.5% sucrose (Additional file 3: Figure S1).
Five different types of ATs were identified in calli grown in the light using HPLC-DAD- TOF-MS, as we had detected in the fruit peels (Additional file 3: Figure S4). Except for pelargonidin 3,5-diglucoside, the four other ATs have the highest levels in 5% sucrose, which fits with the total ATs level. Cyanidin 3-glucoside and cyanidin 3,5-glucoside are the two main ATs in cultures. These results suggest that light/dark and sucrose concentrations significantly affect SDH activity, and thus determine the pathways that lead to HTs and ATs.
Changes in levels of amino acids and the metabolic profiling of tissue culture cells
To understand the broader impact of varied growth conditions on metabolic profiles of peel calli, levels of aromatic amino acids (phenylalanine, tyrosine and tryptophan) derived from the shikimate pathway and other amino acids were determined using GC-MS. Phenylalanine and tryptophan levels were the highest in 1% sucrose in the dark and the light, and gradually decreased with an increase in sucrose concentrations. In contrast, the level of tyrosine tended to increase up to the highest level of 5% sucrose, mainly in the light (Additional file 3: Figure S5; Additional file 2: Table S1). The reduction in levels of phenylalanine and tryptophan is opposite to the elevations in TPC levels, suggesting that these two amino acids are used for the synthesis of various polyphenols compounds under osmotic stress.
Similar to these two latter amino acids, the levels of threonine, methionine and isoleucine decreased at 5 and 7.5% sucrose concentrations compared to 1–2.5% sucrose in the medium in the dark and the light, while valine and leucine decreased similarly but only in the dark. The levels of proline, lysine, serine and alanine increased with sucrose concentration in the medium. The levels of the latter two amino acids tend to increase in the light compared to the dark. The total soluble amino acids increased significantly at 2.5 and 5% sucrose concentrations in the medium, whose levels in the light were higher than in the dark (Additional file 2: Table S1).
A correlation analysis with all the metabolites in the entire dataset showed that metabolites that tend to increase under osmotic stress (sugars and several amino acids, such as proline, GABA, lysine, serine, alanine, mannitol and inositol) were positively correlated (Additional file 3: Figure S6). However, they were negatively related to other metabolites such as aromatic and aspartate family amino acids, as well as to several amino acids whose levels were higher under low osmotic stress (Additional file 3: Figure S6).
Identification and characterization of PgSDHs in pomegranate
The phylogenetic tree for these six sequences shows that the proteins encoded from these genes are grouped as expected into three clusters (Fig. 6b), as previously reported for V. vinifera’s SDHs . Since the two PgSDHs having the PgSDH3 and PgSDH5 motifs are grouped in the same cluster of PgSDH3, we called these two sequences PgSDH3a (− 1 and − 2) (Fig. 6b). Blast and alignment analysis to their protein sequences indeed shows that they have high similarities (Additional file 1). Example is the comparison between PgSDH3–1 to PgHDS3a-1 that has 89.4% identity, and similarity of 92.7%.
The protein sequences of PgSDH1, PgSDH3, PgSDH3a and PgSDH4 have 65–75% similarity to AtSDH1, the only DQD/SDH in A. thaliana. The three PgSDHs are similar to their corresponding VvSDHs . The VvSDH4 is 82.2% identical and has 90.7% similarity to PgSDH4; the VvSDH3 is 83.1% identical and has 91.9% similarity to PgSDH3–2, while VvSDH1 is 76.4% identical and has 87.8% similarity to PgSDH3 (Additional file 1). All of the PgSDHs, except for PgSDH3–2, do not contain transit peptide sequences at their N-terminus, suggesting that they function in the cytosol, whereas PgSDH3–2 is expected to be located in plastids (Additional file 1).
This study suggests that an inverse relationship exists between the levels of HTs and total ATs in the outer peels of most of the accessions of pomegranate fruits. This supported by three lines of evidence: (i) the inverse correlation between HTs and ATs in the outer peels of 33 pomegranate accessions (Fig. 2); (ii) the inverse correlation between punicalagins and ATs in fruits with and without sunlight exposure (Fig. 3); and (iii) increased ATs and decreased GA, ellagic acid and punicalagins in fruit peel calli grown in the light compared to the dark under osmotic stress (Fig. 4). In addition to the negative correlation between HTs and ATs, an opposite trend was found between the GA and shikimate in fruit peel calli grown in the dark on MS medium supplemented with 2.5–7.5% sucrose. These observations suggest a critical role of DQD/SDH in partitioning the common precursor 3-DHS between the synthesis of GA (precursor for HTs biosynthesis) and shikimate (providing precursors for ATs biosynthesis). It also suggests that the biosynthesis pathways leading to ATs and HTs competed for their common precursor, 3-DHS. However, there are many other shikimate pathway-derived metabolites besides ATs, such as flavonoids, condensed tannins and lignins (reviewed by [18, 21]), which may at least partially explain why the correlation between HTs to ATs in the outer peels of the 33 accessions (although it is significant) is only p > − 0.53.
Although our results are in accordance with previous studies indicating that GA derived from the shikimate is the main precursor for HTs synthesis [16, 18, 22], we cannot exclude the possibility that other pathways also contribute to GA synthesis. It was suggested that GA could be produced from syringate, intermediate metabolites of polyphenols such as lignin and ATs [36, 37]. Indeed, it was proposed that GA is produced from syringate in pomegranate leaves at early stages of development in the inner seed coat and pericarp, while in the other organs and in late stages of these latter organs, GA is mostly produced from shikimate . The finding that there are at least two pathways that leads to the synthesis of GA in pomegranates hints to its important roles. GA is produced in plants that do not produce HTs, also implying its additional role in plants in general. Indeed, GA is reported to be the most potent antioxidant among simple phenols , and it has been identified as an allelopathic compound secreted from roots inhibiting the root growth of neighboring competing plants . Furthermore, GA can convert to compounds such as gallic acid-glucoside (used as a signal during development), to epitheaflagallin and to epitheaflagallin 3-O-gallate . Its levels are also dependent on its degradation, since GA is used to produce pyruvate and oxaloacetate, both of which are critical components involved in multiple pathways, including the TCA cycle . The finding that the levels of GA remain relatively stable in covered and non-covered pomegranate fruits (Fig. 3) while the levels of punicalagins changed suggests that GA plays additional roles also in the pomegranate’ outer peels, and therefore an inverse relationship between its contents and punicalagin is not trivial.
The putative source of NADPH/NADP+ for PgSDH activity
It was previously suggested that the ratio between NADPH/NADP+, the cofactors of SDH, regulates its activities to GA (when NADP+ is abundant) or shikimate (when the level of NADPH is high) . NADPH can be formed in plant cells from several different pathways in the chloroplast, cytosol and mitochondrion 39, 40]. The major pathways that maintain the cytosolic NADPH are the oxidative pentose phosphate pathway (OPPP), in which NADPH is produced in the conversion from glucose-6-phosphate to 6-phosphogluconolactone, and the conversion from 6-phosphogluconate to ribulose-5-phosphate. In mitochondria, NADPH can be formed by the activity of the cytosolic NADP-isocitrate dehydrogenase using carbon derived from mitochondrial citrate, from a triose-phosphate shuttle suggested to export reductant from the chloroplast in the light, and from the citrate valve that exports NADPH from mitochondria under photo-respiratory conditions ( and references therein). In chloroplasts, NADPH is derived from the reaction of ferredoxin-NADP+ reductase in the last step of the electron transport chain of photosynthesis [34, 38].
Among the above-mentioned pathways for the generation of NADPH, OPPP is found to play a major role during stress responses in plants . OPPP can operate in the plastids and the cytosol. In plastids, both the OPPP (in the dark) and the Calvin cycle (in the light) produce erythrose-4-phosphate, which, together with glycolysis-derived phosphoenolpyruvate, act as precursors for the shikimate pathway . Moreover, it was strongly suggested that the increased NADP+/NADPH ratio that occurs mainly in stress enhances OPPP activity, providing precursors required for the increased demand of phenolic metabolites that are essential during stress to protect the plants [42, 43]. Our results are in accordance with those studies. The observation that the TPC and HTs levels increased in calli that are grown in the dark under osmotic stress further implies that the NADP+/NADPH ratio increased, which eventually led to enhanced synthesis of polyphenols and specifically HTs from GA to protect the calli from the osmotic stress.
The role of PgSDH in the branch point leading to GA and shikimate
In accordance with the pomegranate dataset, six PgSDHs were identified, two of which that are called PgSDH3a have new combinations of amino acids in the sequences that are defined at the active site of this enzyme (based on [22, 31]), and, as far as we know, they have not yet been reported in other plants’ SDHs. Youan et al. (2018) also reported on the existence of six PgSDHs isozymes in the pomegranate genome . Although they did not publish the sequences, they showed that three are in tandem and are duplicated and located in a 100-kb region, and two showed decreased expression during fruit development, consistent with the observation that the levels of HTs also decreased during pomegranate fruit development . Two other SDH genes are also found in tandem . These findings are in accordance with the high identity between PgSDH3 and PgSDH3a, which are most probably those that are in tandem. Further studies could clarify this issue.
The study of VvSDHs showed that VvSDH1 exhibited the highest SDH activity forming shikimate, but did not convert 3-DHS to GA . VvSDH1 was highly expressed both before and after the beginning of the ripening stage, showing its relative stability in contrast to VvSDH3 and VvSDH4, whose expression levels were significantly higher in immature berry tissues . Similar to VvSDH1, PgSDH1 expression is relatively stable (except at 7.5% sucrose in the dark when its level increased significantly), suggesting that PgSDH1 could be active to form shikimate and possibly use NADPH formed by the OPPP as it is predicted to be located in the cytosol.
It was proposed that the shikimate pathway in plants is regulated primarily at the transcriptional level . It was also reported that the expression of shikimate pathway genes responded to abiotic and biotic stresses . Indeed, the transcripts of PgSDH3–2 and PgSDH4 increased 15- and 35-fold, respectively, in calli grown in 7.5% sucrose medium compared to calli placed in 1% sucrose (Fig. 7). This elevation occurs in the dark, where significantly higher concentrations of GA and HTs accumulated, suggesting that the activity of PgSDH3, PgSDH3a and PgSDH4 may lead to the synthesis of phenols required for protecting the cells from osmotic stress. Since PgSDH3–2 has a plastid transit peptide, it may mainly use the NADP+ that accumulates in the dark in plastids, while PgSDH3–1, PgSDH3a and PgSDH4 used this co-factor that mainly accumulates during stress .
The results obtained in the current study are in accordance with other studies showing that members from SDH groups 3 or 4 are highly expressed in plants having high amounts of HTs such as strawberry and eucalyptus, or galloylated flavan-3-ols like in tea plants, grape and persimmon [22, 44]. By contrast, species without SDH3 and SDH4, such as Arabidopsis, tobacco, tomato and orange, do not produce HTs and galloylated tannins . Measurements of their activities in vitro in grape berry indeed showed that VvSDH3 and VvSDH4 exhibited high activity in forming GA and lower ‘classical’ SDH activity forming shikimate . In addition, the formation of GA and gallotannins such as galloylated flavan-3-ols in grape berry during fruit development is synchronized with the relative expression patterns of VvSDH3 and VvSDH4. Furthermore, over-expression of VvSDH3 in grapevine hairy roots resulted in an increase in GA and galloylated tannins . The role of SDH3 in the synthesis of HTs was also recently verified in a study carried out on Quercus ilex . The observation that the protein sequences of PgSDH are about 82% identical to V. vinifera SDHs, and that no differences were observed in the amino acid residues in the active site of the enzymes, suggests that the PgSDHs have similar activity as VvSDHs.
Taken together, the findings of the current study are in line with those described in grapes, whereby PgSDH1 is a more constantly expressed enzyme that is active with NADPH to form shikimate, while PgSDH3, PgSDH3a and PgSDH4 can be induced by stresses and are operated more with NADP+ to form GA and thus HTs. Moreover, the current results also point to the observation that the expression levels of PgSDH3, PgSDH3a and PgSDH4, which significantly increased under osmotic stress in the dark, are major factors regulating the accumulation of GA and HTs, and not only the NADPH/NADP+ ratio.
The effect of light, dark and sucrose concentration on the formation of HTs and ATs
Previous studies showed that abiotic stresses can affect the production and accumulation of ATs and HTs. The levels of ATs increase, for example, in stresses such as high light, salt, osmotic and drought [45, 46, 47]. The levels of HTs increased significantly in the peels of pomegranate fruits of 11 accessions grown under hot and dry environmental conditions compared to those grown in a Mediterranean climate . In addition, osmotic stress formed by high sucrose levels in the medium was suggested to influence polyphenol metabolism and tannins production in tissue cultures of several plants [49, 50, 51]. A relatively high sucrose content also led to a higher content of tannins in the calli of B. verbascifolia, Tamarix tetrandra and Quercus acutissima grown in the dark, and only residual HTs were detected in the light [49, 50, 51, 52], as is reported also in the current study. These findings further support our outcomes that GA is produced mainly in the dark.
In this study, light was found to be a critical stimulus regulating ATs in outer peels (Fig. 3) and in the calli (together with high sucrose content) (Fig. 4). On the other hand, light irradiation and sucrose treatment appears to inhibit HTs production in the calli, while at the same sucrose concentration in the dark, the levels of HTs significantly accumulated. Together, these studies suggest that a combination of osmotic stress under light or dark conditions can significantly affect the levels of ATs and HTs.
These abiotic conditions also affect the levels of other metabolites in the calli. The reduction of phenylalanine and tryptophan at higher sucrose levels in pomegranate calli suggest that they are used as a substrate for polyphenols that accumulate under these conditions (Fig. 4a). Indeed, the levels of GA, ellagic acid and punicalagins increased significantly when the levels of sucrose increased in the medium (Fig. 4). Comparable results were also reported in Arabidopsis culture exposed to salt. The levels of shikimate increased together with shikimate-related metabolites such as in coniferin [53, 54]. In addition, cell suspension of grape berry exposed to strong light and high temperature (40 °C) showed high elevations of metabolites downstream to the phenylpropanoid pathway as resveratrol and its derivatives piceid, epigallocatechin and coumaroylated forms of peonidin and cyanidin, dihydroflavonol glucosides and quercetin-3-O-glucoside .
Besides the more abundant HTs, the pomegranate calli show that the levels of proline, serine and alanine, as well as total amino acids, together with several sugars and sugar alcohols (sorbitol, mannitol and inositol) also increased under osmotic stress conditions. Accumulations of these metabolites are well known to occur under osmotic stress to protect plants [55, 56]. Also, the contents of intermediates of the TCA cycle also increased significantly. This finding is similar to those reported for Arabidopsis culture exposed to salt [53, 54]. Cell suspension of grape berry exposed to high temperature and light stress also showed high elevations of sugars, serine, alanine, proline, glutamate and metabolites of the TCA cycle . However, the levels of threonine, methionine and isoleucine decreased under osmotic stress in the pomegranate calli. These amino acids are known to degrade to metabolites that feed the TCA cycle under stress to gain more energy .
Taken together, the results of this study strongly suggest that the biosynthesis of HTs and ATs competes for the same substrate, 3-DHS, which is used by DQD/SDH to form GA or shikimate (Fig. 1). The relative activity of DQD/SDH for shimikate and GA is regulated not only by the ratio of NADPH and NADP+, but also by the expression of PgSDH3, PgSDH3a and PgSDH4. Light with high sucrose promotes the synthesis of ATs in calli, while dark at the same sucrose concentration promotes the synthesis of HTs. Both groups of metabolites are used to protect the calli from these abiotic stresses/conditions.
Our results provide deeper insight on the factors that regulate the levels of HTs and ATs in the outer peels of pomegranate fruits. The importance of the outer peels is not just attributed to the fact that the customer’s decision to consume the fruit is based on the first impression of the phenotype of the peels, mainly color and ATs content, but also to the nutritional value of pomegranate juice (that has high levels of HTs, which are usually extracted into the juice in the industries). In addition, high levels of HTs in the peels are related to less physiological defects such as husk scald . The knowledge obtained in this study could be utilized for the development of new genetic markers for breeding pomegranates having high contents of ATs and HTs in the same fruits.
Materials and methods
Thirty-three pomegranate accessions were chosen for this study. Twenty-two of these accessions were collected from a farm in the Neve Ya’ar Research Center, ARO [registered in the Israel Gene Bank for Agriculture Crops (IBG, http://igb.agri.gov.il)] , and 11 accessions were collected from the Havat Hamataim operated by Northern R&D, Israel. The accessions differed in their peel colors. The fruits of the different accessions were harvested between September and November when the fruits were fully matured according to commercial practice. The fruits were transported via a ventilated car to the laboratory, where their outer peels were removed by a peeler. The peels were frozen in liquid nitrogen and kept at − 80 °C for further analyses. For the peel-tissue culture, the immature pomegranate fruits (3–4 cm in diameter) from accession UG28 were collected in June from Havat Hamataim.
Determination of HTs and ATs
HTs and ATs were analyzed using a high-performance liquid chromatography-diode array detector (HPLC-DAD). HPLC was performed with an UltiMate 3000 system consisting of a solvent delivery module LG-980-02, a pump LPG-3400SD, an autosampler WP53000TSL, a column compartment TCC3000SD and a detector DAD3000 UV/vis. The column was a Hydro-RP-C18 Synergi column, 3 mm × 100 mm, and particle size = 2.5 μm (Phenomenex, UK). Ten μl of water extracts of calli or outer peels were injected into the column. Elution was performed at a flow rate of 0.8 mL/min using a gradient of water/formic acid (99:1 v/v) (A) and acetonitrile (B) (Sigma-Aldrich, Rehovot, Israel.): 100% A and 0% B at time 0, 96% A and 4% B at 3.6 min, 85% A and 15% B for 22 min, 50% A and 50% B at 23 min, 20% A and 80% B from 25 to 28 min, 100% A and 0% B at 32 min until 35 min. The metabolites were detected at 360 nm for punicalagins, 280 nm for other HTs, 320 nm for GA, and 520 nm for ATs. Identification and quantification of punicalagin isomers, GA and ellagic acid were achieved by comparing the retention time and standard curves of the authentic standards (Sigma-Aldrich, Rehovot, Israel).
For ATs determination, a peak assignment was performed by the software based on UV/vis absorbance spectra and the retention times of ATs standards. The standard library was constructed from cyanidin 3,5-diglucoside and cyanidin 3-glucoside (Sigma Aldrich, St. Louis, MO). Each of these standards (50–100 μg/mL methanol) was injected separately, and the data acquired by the photodiode array detector with the 3D feature were incorporated into the system’s ATs standard library. Relative standard deviation for the retention times in repetitive runs was in the range of 0.4–1.9%. Individual ATs were quantified from the corresponding chromatogram peak area calculated by the software. Calibration curves (linear, R2 = 0.99) were constructed with standards for each of the ATs at four concentrations (0.01, 0.10, 0.25 and 0.50 μg/mL).
Determination of TPC, total ATs and metabolic profiling
Outer peel tissue culture
Immature pomegranate fruits of accession UG28 were washed with soap and water, the colored fruit peel was removed using a peeler, and small peel segments (measuring 1.0 ± 0.2 cm2) were cut and surface-sterilized as previously described . The outer peel segments were placed in half-strength Murashige and Skoog (MS) medium containing 1% sucrose and 1 mg/L of 6-benzylaminopurine (BAP). The cultures were maintained in a growth room at 22 °C in the dark. The explants were transferred every two weeks to fresh medium for 11 weeks. After the initial development of the calli, the explants were removed; the calli continued to grow on the same medium and continued to be transferred every two weeks for an additional 10 weeks. The calli were then transferred to MS with 1, 2.5, 5% or 7.5% sucrose, and half of the plants at each sucrose concentration were transferred to light under a 16 h light/8 h dark photoperiod with light intensity of 50 μmol m− 2 s− 1 .
The cultures were weighed every two weeks after being transferred to a fresh new medium until the end of the experiment in order to monitor growth rates (Additional file 3: Fig. S2). At the end of the experiment, the fresh and dry weights were measured.
Peels and calli extracts
Peels and the calli induced from the pomegranate outer peels were harvested, frozen in liquid nitrogen, and kept at − 80 °C until being dried with a lyophilizer, after which they were ground with a mortar and pestle into a fine powder. For water extraction, 50 mg of the dried tissue was mixed with distilled water at a ratio of 1:19 (v:v). The mixture was shaken for 40 min at 50 °C and then centrifuged for 20 min at 10,000 x g to remove the tissue debris. The supernatant was filtered through a 0.22 μm polytetrafluoroethylene (PTFE) filter and transferred to new tubes.
mRNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis
RT-qPCR was used to determine the expression levels of the three PgSDH genes in the calli grown in the dark and the light. Fifty mg of lyophilized dry calli were extracted using the Spectrum plant total RNA kit (Sigma). cDNA synthesis and qPCR procedures were carried out according to a previous study . To normalize variance among samples, we used the constitutive-ribosomal Protein S (PgRPSII) gene as an endogenous control . The values presented are means of three biological replicates, each with three technical replicates. The primers used for analyzing the expression of PgSDHs and PgRPSII are shown in Additional file 2: Table S3.
A phylogenetic tree analysis was made of selected dehydroquinate dehydratase/shikimate dehydrogenases taken from dicots. This phylogenetic tree was constructed from the six PgSDHs sequenced in this study and the 12 sequences available on public databases (NCBI) using Clustal Omega Phylogenetic options.
Statistical analysis of the metabolite and gene expression data was performed using JMP version 8.0 (SAS Institute Inc., Cary, NC). Significant differences between treatments were calculated according to the Kramer HSD-Tukey test (p < 0.05). The Pearson test was used for the correlation analysis. Phylogenetic trees were created using the MAFFT version 7  with the WAG model and a 1000 bootstrap. The SDH protein sequences were aligned using Clustal Omega.
Irit Bar-Ya’akov for helping in collecting the pomegranate fruits; Rotem Harel for the PgSDH3a sequences; Janet Covaliu for English editing.
RH, YH, RD and IM conducted the study; DH, LT and RA initiated the study; DH, RD and RH carried out the bioinformatics analysis; DH and RH collected and prepared the plant materials; RA, YH, RH and LT wrote the manuscript. All authors have read and approved the manuscript.
This research was supported by research grant award no. IS-4822-15 R from BARD, the United States-Israel Binational Agricultural Research and Development Fund. The funding contributed to the experimental costs, the data interpretation, the writing, and the English language editing. MIGAL supported the publication fees.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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