'Fuyu' Persimmon Fruits

Scientia Horticulturae 172 (2014) 292–299 Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti The accumulation of tannins during the development of ‘Giombo’ and ‘Fuyu’ persimmon fruits ∗ Magda Andréia Tessmer, Ricardo Alfredo Kluge, Beatriz Appezzato-da-Glória University of São Paulo/ESALQ, Department of Biological Sciences, C.P. 13418-900 Piracicaba, SP, Brazil a r t i c l e i n f o a b s t r a c t Article history: Tannins are responsible for the astringency of persimmon fruits. The present study compared the develop- Received 7 November 2013 ment of ‘Giombo’ (PVA) and ‘Fuyu’ (PCNA) persimmon fruits until maturity to determine if the difference Received in revised form 16 April 2014 in astringency between the two cultivars is related to the early accumulation of tannins in the cells or Accepted 18 April 2014 to differences in the pattern of tannin accumulation during cell differentiation, cell density or the concentrations of total and soluble tannins. Persimmon flowers and fruits were collected from a commercial Keywords: orchard in Mogi das Cruzes during the 2010–2011 harvest season at predetermined stages until the fruit Anatomy reached commercial maturity. Structural analyses were performed by light, scanning and transmission Astringency electron microscopy, and quantitative analyses of tannin cell density, tannin cell index and total and Diospyros kaki L. soluble tannins were also performed. In both cultivars, the ovary already exhibited a small number of Tannin cells tannin cells. Throughout development, the ‘Giombo’ persimmon possessed higher tannin cell density and higher levels of total and soluble tannins compared to ‘Fuyu’. The accumulation of tannins in the cells was homogeneous and restricted to the vicinity of the cell wall, in spherical vesicles connected to the tonoplast, as amorphous distributions occupying the entire vacuole, as homogeneous distributions with circular, unfilled areas in vacuoles, and as homogeneous distributions that completely filled the vacuoles. At the end of fruit development, the parenchyma cells displayed large intercellular spaces and degraded pectins, indicating fruit ripening and senescence. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Persimmon accumulates soluble condensed tannins (CT) in fruit, which is responsible for its strong astringency trait, during the The persimmon (Diospyros kaki L.) belongs to the family Ebe- early stages of fruit development (Akagi et al., 2010, 2011). Con- naceae. It is native to Asia, specifically China, which is the largest densed tannin, also called proanthocyanidin, is a phenolic oligomer producer of this fruit (FAOSTAT, 2009). Currently, Brazil is the resulting from the polymerisation of flavan-3-ol units, which con- fourth largest producer of persimmons worldwide, with a produc- sists of two types of subunits, extension and terminal units (Dixon, tion of 164,495 tonnes from a planted area of 8652 hectares in 2010 2005). (IBGE, 2010). Analysis of method by thiolysis degradation, condensed tan- In Brazil, the most cultivated persimmon cultivars include nin persimmon consist two types of flavan-3-ols: catechin (C) ‘Rama Forte’ and ‘Giombo’, which belong to the pollination-variant and gallocatechin (GC), and their gallate ester forms, C-3-O-gallate and astringent (PVA) group, and ‘Fuyu’, which belongs to the (CG) and GC-3-O-gallate (GCG) (Matsuo and Itoo, 1978). The pollination-constant non-astringent (PCNA) group. PVA cultivars cis/trans configuration of flavan-3-ols form 2,3-cis-epicatechin-3- contain high levels of soluble tannins, which are responsible for O-gallate (ECG) and 2,3-cis epigallocatechin-3-O-gallate (EGCG) the astringency of these cultivars, and they require deastringency (Tanaka et al., 1994). According to Akagi et al. (2011) and Novillo treatment prior to consumption (Edagi and Kluge, 2009). et al. (2014), the epigallocatechin (EGC) and epigallocatechin-3-O- gallate (EGCG) constitute the main subunit components of soluble tannins (proanthocyanidin) in astringent-type fruit. Tannins are found in the cell vacuoles that differentiate dur- ∗ ing the development of the flower buds and fruits. However, the Corresponding author. Tel.: +55 19 34294136x206; fax: +55 19 34348295. only study of structure and ultrastructure in the early appear- E-mail addresses: [email protected] (M.A. Tessmer), [email protected] (R.A. Kluge), [email protected] (B. Appezzato-da-Glória). ance of tannin cells was performed in persimmon ovary and leaf http://dx.doi.org/10.1016/j.scienta.2014.04.023 0304-4238/© 2014 Elsevier B.V. All rights reserved. M.A. Tessmer et al. / Scientia Horticulturae 172 (2014) 292–299 293 primordium, and thus, it was not possible to determine the vacuum pump was used to remove air from the intercellular spaces. organelle responsible for the biosynthesis of tannin (Yonemori The samples were then dehydrated in an ethanol series to 100% et al., 1997). Quantitative studies of cells and soluble tannins dur- ethanol and embedded in hydroxyethyl methacrylate (Leica His- ing the development of astringent and non-astringent persimmon toresin) for block preparation (4 replicates of each stage). The blocks fruits have been performed by Yonemori and Matsushima (1985, were then sectioned in a rotary microtome at a 5–6 ␮m thick- 1987a). ness to yield cross- and longitudinal sections. The sections were According to Yonemori and Matsushima (1985), the tannin stained with 0.05% toluidine blue in phosphate buffer and citric cells in PCNA fruits stop enlarging prematurely and are smaller acid at pH 4–6 (Sakai, 1973) for conventional histological analyses, in size at the final stage of development, whereas tannin cells in treated with ferric chloride to detect phenolic compounds (tannins) variable-pollination non-astringent (PVNA) and astringent (PVA) (Johansen, 1940) or treated with ruthenium red to detect pectins and pollination-constant astringent (PCA) fruits continue their (Strasburger, 1924). The sections were mounted onto slides with growth. The number of tannin cells per unit area is little variable in “Entellan” synthetic resin, and the images were captured with a the fruits of four groups and cessation of cell growth in PCNA coinci- Leica DM LB trinocular microscope coupled to a Leica DC 300 F dent with reduction of soluble tannins and astringency (Yonemori video camera and processed on a computer to prepare the illustra- and Matsushima, 1985, 1987a). tions. The above studies were performed in ‘Fuyu’ persimmons, and studies of ‘Giombo’ development have been limited. The present 2.3. Transmission electron microscopy (TEM) study aimed to compare the development of ‘Giombo’ (PVA) and ‘Fuyu’ (PCNA) fruits until maturity and to clarify whether the dif- Two-millimetre-thick samples were removed from the ovary ferences in astringency of these fruits are related to the early and equatorial regions of the fruits and immediately fixed accumulation of tannins in tannin cells, to differences in the pat- in modified Karnovsky’s solution (2.5% glutaraldehyde, 2.5% terns of tannin accumulation during cell differentiation, to tannin paraformaldehyde and 0.05 mM CaCl2 in 0.1 M sodium cacodylate cell density or to the concentrations of total and soluble tannins. buffer, pH 7.2) for 48 h. Air was removed from the samples for 5 min, and the samples were then post-fixed in 1% osmium tetroxide for 2 h. The samples were dehydrated in an ascending acetone series up 2. Materials and methods to 100% and embedded in Spurr’s resin. The blocks were sectioned using a Leica UC6 ultramicrotome, and the sections were counter- 2.1. Plant material stained with 5% uranyl acetate and 2% lead citrate for 30 min at each step (Reynolds, 1963). Observations and photomicrographs were The flowers and fruits of the ‘Giombo’ (PVA) and ‘Fuyu’ (PCNA) made using a Zeiss EM-900 transmission electron microscope oper- cultivars were used and were obtained from Mogi das Cruzes, São ◦ ◦ ating at 50 kV with the electron micrograph scales printed directly Paulo (23 31 S, 46 11 W) at an altitude of 742 m from September on the photomicrographs. 2010 to March 2011. For both cultivars, the following development stages were standardised visually (Fig. 1A–L) and characterised based on measurements of the flowers and fruits: 2.4. Cryo-scanning electron microscopy (Cryo-SEM) Stage a Appearance of flower buds: buds approximately 0.5 cm in diameter and 1.0 cm in length for ‘Giombo’ and 0.8 cm in diam- One-millimetre-thick samples of the ovary were obtained with eter and 1.5 cm in length for ‘Fuyu’. Stage b Opening of the calyx of the aid of a steel blade. The samples were placed into a metal ◦ − the flower bud: bud approximately 0.8 cm in diameter and 1.4 cm sample holder, frozen in liquid nitrogen at 210 C and immedi- in length for ‘Giombo’ and 1.0 cm in diameter and 1.5 cm in length ately transferred to the Cryo-Trans (CT 15000C, Oxford Instruments for ‘Fuyu’. Stage c Elongation and change in the colour of the corolla Ltd., Oxford, England), which was coupled to a scanning elec- from white to light yellow: bud approximately 0.8 cm in diameter tron microscope (Jeol JSM 5410, Tokyo, Japan) and operated under ◦ −2 − and 1.6 cm in length for ‘Giombo’ and 1.1 cm in diameter and 1.7 cm freezing ( 130 C) and vacuum conditions (10 bar pressure). ◦ − in length for ‘Fuyu’. Stage d Opening of the corolla (anthesis): flower In the Cryo-Trans, the sample was fractured at 180 C, and ◦ − approximately 0.9 cm in diameter and 1.5 cm in length for ‘Giombo’ the sections were sublimated at 90 C for 15 min to eliminate and 1.2 cm in diameter and 1.3 cm in length for ‘Fuyu’. Stage e Dry- potential excess surface water in the tissue. The samples were −2 ing and fall of the corolla: fruit approximately 1.0 cm in diameter coated with gold in the Cryo-Trans at 10 bar and 40 mA and and 0.9 cm in length for ‘Giombo’ and 1.0 cm in diameter and 0.9 cm then observed under the microscope with a 10 kV voltage and a in length for ‘Fuyu’.

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