Tissue and Cellular Localization of Tannins in Tunisian Dates

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Tissue and Cellular Localization of Tannins in Tunisian Dates (Phoenix dactylifera L.) by Light and Transmission Electron Microscopy Hédi Hammouda,*,†,§ Camille Alvarado,∥ Brigitte Bouchet,∥ Jamila Kalthoum-Chérif,†,‡ Malika Trabelsi-Ayadi,† and Sylvain Guyot§ †

Laboratoire d’Application de la Chimie aux Ressources et Substances Naturelles et à l’Environnement (LACReSNE), , Faculté des Sciences de Bizerte, 7021 Zarzouna-Bizerte, Tunisie ‡ Institut Préparatoire aux Études d’Ingénieurs de Tunis (IPEIT), Monfleury 1008 Tunis, Tunisie § INRA, PRP Team, UR1268 BIA, F-35650 Le Rheu, France ∥ INRA, BiBS Platform, UR1268 BIA, F-44316 Nantes, France ABSTRACT: A histological approach including light microscopy and transmission electron microscopy (TEM) was used to provide accurate information on the localization of condensed tannins in the edible tissues and in the stone of date fruits (Phoenix dactylifera L.). Light microscopy was carried out on fresh tissues after staining by 4-dimethylaminocinnamaldehyde (DMACA) for a specific detection of condensed tannins. Thus, whether under light microscopy or transmission electron microscopy (TEM), results showed that tannins are not located in the epidermis but more deeply in the mesocarp in the vacuole of very large cells. Regarding the stones, tannins are found in a specific cell layer located at 50 μm from the sclereid cells of the testa. KEYWORDS: fruits, tannins, DMACA, proanthocyanidins, histochemistry

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phloroglucinol groups corresponding to “activated” aromatic rings. Thus, the DMACA reacts on the phloroglucinol A-rings of condensed tannins and catechins on one of the positions 6 or 8 activated by the presence of multiple OH groups (Figure 1). This reagent does not react with the ring B. It does not react with flavones or flavonols, having a “deactivating” carbonyl group (CO) in position 4. The DMACA reagent was used by Feucht et al. (2004)6 and Cadot et al., (2006)7 for tannins localization in the nuclei of several tree species. The results show the existence of vacuolar flavanols by the appearance of blue color in different cell area studied. In addition, the localization of procyanidins was performed in the seeds of Brassica napus L. Procyanidins deposition was restricted to the seed coat because the embryo remained unstained. Moreover, DMACA stained seed sections showed that procyanidins were localized into the innermost cell layer of the inner integument, namely, the endothelium.8 Localization of tannins in grape berry9 and apple skin10 has been studied using transmission electron microscopy. Three groups of tannins were determined in grape berry:9 two groups are related to tonoplast with, on the one hand, proteins walls and to cell walls by osidic bonds on the other hand. The third group situated in the vacuolar sap, is constituted by some granulated tannins basically situated in the deep layer of peels and by some condensed tannins in big mass essentially localized in the superficial layer. More recently, localization of condensed

INTRODUCTION Flavonoids are naturally produced in the leaves, flowers, fruits, seeds, barks, and roots of many plant species. The flavonoid pathway produces a diverse array of plant compounds with functions in UV protection, as antioxidants, pigments, auxin transport regulators, defensive compounds against pathogens, and during signaling in symbiosis. In fruits, flavonoids are present under different subclasses including flavanols, flavones, flavonols, and anthocyanins. They are greatly involved in the nutritional and organoleptic qualities of fruits. Flavanols are divided into monomers (i.e., catechins) and oligomers and polymers of catechins referred to as proanthocyanidins or condensed tannins. For instance, tannins are responsible for the bitterness and the astringency of unripe fruits. Catechins are substrates of polyphenoloxidase activity, leading to oxidative browning of many fruits. The polyphenol profile of Tunisian varieties of dates including flavanols, flavonols, flavones, and hydroxycinnamates was recently characterized.1 Procyanidin polymers based on (−)-epicatechin structure were by far the most concentrated polyphenols in ripe dates, accounting for 95% of total polyphenols with an average concentration of 14 g/kg in the fresh edible parts of the fruit.1 Various histochemical methods can be used to reveal flavonoids in fruits. Indeed, a red color is obtained with nitrous reagent, allowing localization of the tannins in cottonseed,2 and other reagents such as toluidine blue solution were also used to locate the tannins in the chalaza.3 Generally, a specific and highly sensitive localization of flavanols on fixed tissue sections is performed after tissue staining by reaction with vanillin4 or with 4-dimethylaminocinnamaldehyde (DMACA).5,6 Under acidic conditions, the DMACA is a protonated electrophilic reagent which is likely to react with resorcinol or © 2014 American Chemical Society

Received: Revised: Accepted: Published: 6650

March 20, 2014 June 16, 2014 June 19, 2014 July 2, 2014 dx.doi.org/10.1021/jf5013715 | J. Agric. Food Chem. 2014, 62, 6650−6654

Journal of Agricultural and Food Chemistry

Article

Figure 1. Putative reaction mechanism of DMACA with flavanols in an acidic medium (according Delcour and De Varebeke4 by analogy with the reaction of vanillin).

Figure 2. Light micrographs of Deglet Nour dates pericarp and stone cross sections: (A,B) pericarp, (C,D) stone, (A,C) binocular observation of thick sections, (B,D) semithin sections stained with toluidine blue. M1, external zone of the pericarp including the epicarp; M2, mesocarp zone 2; M3, mesocarp zone 3; F, fibrous zone; en, endosperm; te, testa.

specific staining and TEM, the purpose of this study is to give more accurate evidence of condensed tannin localization in edible tissues of dates. Their localization in the stones is also considered because these compounds were recently characterized and quantified in this part of the fruits.

tannins in chlorophyllous tissues of several tracheophyta species was investigated,11 showing that tannins are polymerized in a new chloroplast-derived organelle, named the tannosome. More specifically concerning date tissues, ultrathin sections of frozen pericarp were observed by transmission electron microscopy by Shomer et al. (1998).12 These authors suggested that tannins were specifically located in the cells of the epicarp. However, only TEM observation cannot clearly make the difference between tannins and other polyphenolic classes such as flavonols which are also present in date epidermis tissues.1 Indeed, both flavonols and tannins are molecules containing CC conjugated double-bond systems. Therefore, after the treatment of tissues with osmium tetroxide, both flavonols and tannins containing zones would appear as highly electron-dense on the TEM pictures. Thus, the localization of tannins in dates tissues is still badly known with accuracy. By coupling light microscopy after

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MATERIALS AND METHODS

Solvents, Reagents, and Phenolic Standards. Ethanol and hydrochloric acid were from Merck (Allemagne). 4-Dimethylamino cinnamaldehyde (DMACA) and uranyl acetate were purchased from Sigma-Aldrich (France), and glutaraldehyde and LRwhite resin were purchased from Agar Scientific (UK). Water was purified using a MilliQ system from Millipore (France). Plant Materials. Ripe Deglet Nour dates (Phoenix dactylifera L.) were harvested during the 2009 season in the region of Kebili, Tunisia. Microscopy. Sample Preparation. For light and electron microscopy observations, the fruit was thawed out and blocks of tissue (1 mm3) were cut from the pericarp (including the epicarp and 6651

dx.doi.org/10.1021/jf5013715 | J. Agric. Food Chem. 2014, 62, 6650−6654


Article

Figure 3. Tannins localization with DMACA staining. Light micrographs of Deglet Nour dates pericarp cross sections M1 + M2 (thick sections): (A) witness pericarp, (B) pericarp stained with DMACA, and (C) pericarp stained with DMACA at higher magnification. T, tannins; M1, external zone of the pericarp; M2, mesocarp zone. mesocarp) and the stone were fixed with 3% glutaraldehyde in a 0.1 M phosphate buffer at pH 7.4 for 24 h at 4 °C, rinsed several times with 0.1 M phosphate buffer at pH 7.4, and then postfixed with 1% osmium tetroxide (OsO4) in deionized water. After being postfixed, tissues were rinsed 5 times with deionized water, followed by dehydration through a graded aqueous ethanol series (30, 50, 70, 85, 95, and 100%). The samples were then progressively infiltrated with London Resin White acrylic (LRW) according to the following schedule: 20, 40, 60, and 80% LRW/ ethanol and overnight in pure LRW. The infiltrated samples were finally embedded by LRW resin and polymerized for 48 h at 55 °C. For light microscopy, semithin sections (1 μm) were obtained using an ultramicrotome (UC7, Leica), mounted on glass slides, and stained with a solution of toluidine blue 1% in Na2CO3 2.5% for histology. The observation was carried out using an inverted epifluorescence microscope (Axiovert 135M, Carl Zeiss). The images are taken with the objectives ×10 and ×40. The software acquisition used is “Archimed”. Observations were taken using a color camera (Retiga 2000R, Microvision). For transmission electron microscopy, ultrathin sections (80 nm) contrasted with uranyl acetate 2% in aqueous solution were examined with a JEM-1230 (Jeol) with an accelerating voltage of 80 kV. The images were obtained with a camera (ES500W, Gatan) associated with software acquisition “Digital Micrograph.” Tannins Localization by Light Microscopy in Combination with DMACA Staining. Thick sections of the pericarp (150 μm) were obtained with a vibratome (HM 650 V, Microm). The operation consists of placing a drop of reagent DMACA (0.3% w/v) in a mixture of DMACA methanol/6 N HCl (1/1: v/v) on sections and has been incubated for 20 min at 4 °C. Rinsing is carried out several times in deionized water. The observation was made directly by light microscopy in bright field.5

revealed a fibrous zone corresponding to unstructured cells which encompasses the stone. Obviously, the cellular architecture of the stone is very different from that of the pericarp. Two main different tissue zones are clearly observable: the testa, corresponding to the peripheral zone, is approximately 100 μm thickness which surrounds the endosperm that corresponds to the inner part (Figure 2D). The testa shows several well-defined layers of cells. The most external is composed of small cells showing rectangular form and whose cell wall is very thick and sclerotized as can be deduced from the intense coloration by toluidine blue (Figure 2D). Then the most inner part of the testa is composed of two internal and parallel layers of well-organized cells situated approximately at 80 μm from the surface of the stone (Figure 2D). The most external layer appears more deeply stained by toluidine blue than the most internal one, suggesting that these two types of cells do not have the same composition. It is interesting to see that the testa shows this very particular tissue organization showing cells with different cell wall thickness, sizes, and orientations. This may confer to the testa very specific physical properties acting as a protective barrier. Below this external layer, an unstructured and inhomogeneous zone of around 50 μm thickness containing large free cells with very variable forms is observable (Figure 2D). Concerning the endosperm zone (Figure 2D) which corresponds to storage tissues, it is composed of large-sized cells only weakly colored by toluidine blue and orientated perpendicularly to the cells of the testa. This tissue likely contains proteins and oils the latter taking the form of globules of darker color clearly visible in the cells (Figure 2D). Localization of Tannins in the Pericarp. Tannins Localization by Light Microscopy in Combination with DMACA Staining. In our recent study, condensed tannins were characterized in date tissues by phloroglucinolysis associated with HPLC-UV and mass spectrometry.1 We showed that these compounds were oligomers and polymers of (−)-epicatechin highly concentrated both in the edible parts and the stones. The DMACA staining of condensed tannins allowed us to localize these compounds in the edible tissues of the date (Figure 3). Interestingly, the staining was not distributed in the whole tissues but was very specifically located in the external part of the mesocarp approximately at 1500 μm inside the

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RESULTS AND DISCUSSION Cellular Organization of the Date Tissues. On the basis of images obtained with a binocular stereo zoom microscope and light microscope, Figure 2 describes the morphology of the soft tissues corresponding to the pericarp (edible part of the fruit) (Figure 2A,B) and the hard tissues corresponding to the stone (Figure 2C,D), respectively. Soft tissues were divided into four zones. Regarding the edible tissues, the most external zone (M1) included the epidermis (epicarp) and some subepidermal layers. The second zone (M2) corresponded to mesocarp tissues containing some large cells as observed on the lower part of Figure 2B. The M3 zone including the main part of the mesocarp and the most inner part of the soft tissues (F) 6652



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Figure 4. Transmission electron micrographs of the mesocarp in the transition zone between M1 and M2. (A) tannins free cell (on the left) and tannins containing cell (on the right). (B) Observation at higher magnification. T, tannins (T).

Figure 5. Light micrographs of cross sections (semithin sections) of the stone of Deglet Nour dates: (A) witness stone, (B) stained with DMACA, (C) stained with DMACA at higher magnification. en, endosperm; te, testa.

Figure 6. Transmission electron micrographs in the stone: (A) form of a continuous deposit extremely dense to the electrons, (B) form of granular units, observation at higher magnification. CW, cell wall; T, tannins.

pericarp and with a thickness close to 1000 μm (Figure 3B). Higher magnification (Figure 3C) revealed that the tannins are located in 3−5 layers of very large spherical cells reaching 200 μm diameter and having thin cell walls. The intense blue coloration seems to fill almost completely the intracellular area suggesting the massive presence of these flavanols in the vacuoles. Although DMACA staining does not permit to distinguish between flavanol monomers (i.e., catechins) and condensed tannins (i.e., proanthocyanidins), it is likely that those flavanols mainly correspond to highly polymerized procyanidins as it was shown in our previous study.1 Localization of Tannins by Transmission Electron Microscopy (TEM). Polyphenols and particularly those containing o-diphenol groups in their structure are highly osmiophilic agents.13 Therefore, when they are present in the

tissues, condensed tannins are responsible for high electron density creating contrasted zones in TEM images. In a previously published work conducted on dates using TEM observation, Shomer et al. (1998)12 proposed that tannins were probably located in the subepidermal zone (M1) of the epicarp on the basis of high density areas of this tissue zone. This hypothesis was invalidated by our observations in light microscopy after specific staining of flavanols structures with DMACA. Tannins are specifically located in the mesocarp (M2) in large-sized cells situated at 1500 μm from the epicarp cells. Electron-dense precipitates are visible in the vacuoles of the mesocarp cells after the treatment of samples by osmium tetroxide (Figure 4A). These precipitates are in the form of granules of different sizes. The size of the granules is up to 0.2 6653



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μm in diameter (Figure 4B). The precipitates sometimes form clusters fused to tonoplast. Tannins Localization in the Dates Stone. Tannins Localization by Light Microscopy. The DMACA staining of cross sections of the stone allowed us to observe the organization of tannins (Figure 5B,C). Tannins are specifically located in a single cell layer situated approximately at 50 μm from the surface of the testa (Figure 5B). In addition, close to this layer, some very large cells containing tannins are observed in the unstructured zone of the testa. Interestingly, knowing that tannins are antimicrobial and antifungal agents,14 this organization makes us think that they are located in a specific cell layer, forming a defensive barrier that likely protects the stone against pathogen attacks. The flavonoid accumulation pattern was examined in young seedlings and mature tissues of wild-type Arabidopsis. Flavonoid accumulation in mature plants was localized in cauline leaves, pollen, stigmata, floral primordia and in the stems of young actively growing inflorescences.15 Tannins Localization by TEM. The tannins of these cells are characterized by a continuous deposit extremely dense to the electrons. It consists of similar elements and expressing the same characters. This layer is formed by rectangular cells stacked to one another. The observations by transmission electron microscopy allow us to have some information on the dimensions of the cells tannins whose sizes range from 20 to 40 μm (Figure 6A). One also notices that these cells are characterized by the plasmic membranes surrounded by a thick rigid cell wall. These tannins are present in cells of different forms. They mainly occur in the form of a continuous deposit extremely dense to the electrons (Figure 6A) and sometimes in the form of granular units (Figure 6B). Generally speaking, we did not detect the tannins localized in the cell wall as in the case of the grape proved by Amrani Joutei et al. (1994).9 To conclude, the observation of pericarp tissues by light and transmission electron microscopy revealed that tannins are located in the vacuoles of very large mesocarp cells. These cells are in turn located at 1500 μm from the surface. The microscopic approach has allowed us, for the first time, to locate the tannins in the stone. Those tannins are located in a specific cell layer located at 50 μm from the surface. From this work, we can conclude on the high specificity of tannins organization in the pericarp and the stone of the date. This very specific organization may be related to the physiological role of tannins as active components able to protect the fruit and the seed against pathogen attacks. Interestingly, tannins in the stones may be also involved in the germination mechanisms.16 As perspectives to this work, we would like to observe different stages of ripening fruits in order to have a better understanding of the building of the tannin pool in the fruit (i.e., structure and location) during growing and maturation.

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Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED DMACA, 4-dimethylaminocinnamaldehyde REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

This work was supported by the Tunisian Ministry of Higher Education and Scientific Research and by the Doctoral School “Vie Agro Santé” in France. 6654


Tissue and Cellular Localization of Tannins in Tunisian Dates

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