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Scientia Horticulturae 172 (2014) 292–299

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Scientia Horticulturae

journa l h om epa ge: www.elsev ier .com/ locate /sc ihor t i

he accumulation of tannins during the development of ‘Giombo’ andFuyu’ persimmon fruits

agda Andréia Tessmer, Ricardo Alfredo Kluge, Beatriz Appezzato-da-Glória ∗

niversity of São Paulo/ESALQ, Department of Biological Sciences, C.P. 13418-900 Piracicaba, SP, Brazil

r t i c l e i n f o

rticle history:eceived 7 November 2013eceived in revised form 16 April 2014ccepted 18 April 2014

eywords:natomystringencyiospyros kaki L.annin cells

a b s t r a c t

Tannins are responsible for the astringency of persimmon fruits. The present study compared the develop-ment of ‘Giombo’ (PVA) and ‘Fuyu’ (PCNA) persimmon fruits until maturity to determine if the differencein astringency between the two cultivars is related to the early accumulation of tannins in the cells orto differences in the pattern of tannin accumulation during cell differentiation, cell density or the con-centrations of total and soluble tannins. Persimmon flowers and fruits were collected from a commercialorchard in Mogi das Cruzes during the 2010–2011 harvest season at predetermined stages until the fruitreached commercial maturity. Structural analyses were performed by light, scanning and transmissionelectron microscopy, and quantitative analyses of tannin cell density, tannin cell index and total andsoluble tannins were also performed. In both cultivars, the ovary already exhibited a small number oftannin cells. Throughout development, the ‘Giombo’ persimmon possessed higher tannin cell density andhigher 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 thetonoplast, as amorphous distributions occupying the entire vacuole, as homogeneous distributions withcircular, 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 degradedpectins, indicating fruit ripening and senescence.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

The persimmon (Diospyros kaki L.) belongs to the family Ebe-aceae. It is native to Asia, specifically China, which is the largestroducer of this fruit (FAOSTAT, 2009). Currently, Brazil is theourth largest producer of persimmons worldwide, with a produc-ion of 164,495 tonnes from a planted area of 8652 hectares in 2010IBGE, 2010).

In Brazil, the most cultivated persimmon cultivars includeRama Forte’ and ‘Giombo’, which belong to the pollination-variantnd astringent (PVA) group, and ‘Fuyu’, which belongs to theollination-constant non-astringent (PCNA) group. PVA cultivarsontain high levels of soluble tannins, which are responsible for

he astringency of these cultivars, and they require deastringencyreatment prior to consumption (Edagi and Kluge, 2009).

∗ Corresponding author. Tel.: +55 19 34294136x206; fax: +55 19 34348295.E-mail addresses: magdatessmer@yahoo.com.br (M.A. Tessmer), rakluge@usp.br

R.A. Kluge), bagloria@usp.br (B. Appezzato-da-Glória).

ttp://dx.doi.org/10.1016/j.scienta.2014.04.023304-4238/© 2014 Elsevier B.V. All rights reserved.

Persimmon accumulates soluble condensed tannins (CT) in fruit,which is responsible for its strong astringency trait, during theearly stages of fruit development (Akagi et al., 2010, 2011). Con-densed tannin, also called proanthocyanidin, is a phenolic oligomerresulting from the polymerisation of flavan-3-ol units, which con-sists of two types of subunits, extension and terminal units (Dixon,2005).

Analysis of method by thiolysis degradation, condensed tan-nin persimmon consist two types of flavan-3-ols: catechin (C)and gallocatechin (GC), and their gallate ester forms, C-3-O-gallate(CG) and GC-3-O-gallate (GCG) (Matsuo and Itoo, 1978). Thecis/trans configuration of flavan-3-ols form 2,3-cis-epicatechin-3-O-gallate (ECG) and 2,3-cis epigallocatechin-3-O-gallate (EGCG)(Tanaka et al., 1994). According to Akagi et al. (2011) and Novilloet al. (2014), the epigallocatechin (EGC) and epigallocatechin-3-O-gallate (EGCG) constitute the main subunit components of solubletannins (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, theonly study of structure and ultrastructure in the early appear-ance of tannin cells was performed in persimmon ovary and leaf

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rimordium, and thus, it was not possible to determine therganelle responsible for the biosynthesis of tannin (Yonemorit al., 1997). Quantitative studies of cells and soluble tannins dur-ng the development of astringent and non-astringent persimmonruits have been performed by Yonemori and Matsushima (1985,987a).

According to Yonemori and Matsushima (1985), the tanninells in PCNA fruits stop enlarging prematurely and are smallern size at the final stage of development, whereas tannin cells inariable-pollination non-astringent (PVNA) and astringent (PVA)nd pollination-constant astringent (PCA) fruits continue theirrowth. The number of tannin cells per unit area is little variable inhe fruits of four groups and cessation of cell growth in PCNA coinci-ent with reduction of soluble tannins and astringency (Yonemorind Matsushima, 1985, 1987a).

The above studies were performed in ‘Fuyu’ persimmons, andtudies of ‘Giombo’ development have been limited. The presenttudy aimed to compare the development of ‘Giombo’ (PVA) andFuyu’ (PCNA) fruits until maturity and to clarify whether the dif-erences in astringency of these fruits are related to the earlyccumulation of tannins in tannin cells, to differences in the pat-erns of tannin accumulation during cell differentiation, to tanninell density or to the concentrations of total and soluble tannins.

. Materials and methods

.1. Plant material

The flowers and fruits of the ‘Giombo’ (PVA) and ‘Fuyu’ (PCNA)ultivars were used and were obtained from Mogi das Cruzes, Sãoaulo (23◦31′ S, 46◦11′ W) at an altitude of 742 m from September010 to March 2011. For both cultivars, the following developmenttages were standardised visually (Fig. 1A–L) and characterisedased on measurements of the flowers and fruits:

Stage a Appearance of flower buds: buds approximately 0.5 cmn diameter and 1.0 cm in length for ‘Giombo’ and 0.8 cm in diam-ter and 1.5 cm in length for ‘Fuyu’. Stage b Opening of the calyx ofhe flower bud: bud approximately 0.8 cm in diameter and 1.4 cmn length for ‘Giombo’ and 1.0 cm in diameter and 1.5 cm in lengthor ‘Fuyu’. Stage c Elongation and change in the colour of the corollarom white to light yellow: bud approximately 0.8 cm in diameternd 1.6 cm in length for ‘Giombo’ and 1.1 cm in diameter and 1.7 cmn length for ‘Fuyu’. Stage d Opening of the corolla (anthesis): flowerpproximately 0.9 cm in diameter and 1.5 cm in length for ‘Giombo’nd 1.2 cm in diameter and 1.3 cm in length for ‘Fuyu’. Stage e Dry-ng and fall of the corolla: fruit approximately 1.0 cm in diameternd 0.9 cm in length for ‘Giombo’ and 1.0 cm in diameter and 0.9 cmn length for ‘Fuyu’.

From stage e (October) (Fig. 1F), monthly samples were taken.Fuyu’, which exhibits precocious maturation, was collected untilebruary, and ‘Giombo’, which matures later, was collected untilarch. Quantitative analyses were performed from stage e. Sam-

les of four fruits were used to determine the cell density and cellndex, and three replicates of three fruits were used to determineoluble and total tannins.

All stages of ‘Giombo’ and ‘Fuyu’ were subjected to analysis byight microscopy (LM). Stages a–d were analysed by transmissionlectron microscopy (TEM), and stage c ‘Giombo’ were analysed byryo-SEM.

.2. Light microscopy (LM)

Sections of the ovaries and of the equatorial regions of theruits were removed for light microscopy analysis. The sam-les were fixed in Karnovsky’s solution (Karnovsky, 1965), and a

ulturae 172 (2014) 292–299 293

vacuum pump was used to remove air from the intercellular spaces.The samples were then dehydrated in an ethanol series to 100%ethanol and embedded in hydroxyethyl methacrylate (Leica His-toresin) for block preparation (4 replicates of each stage). The blockswere then sectioned in a rotary microtome at a 5–6 �m thick-ness to yield cross- and longitudinal sections. The sections werestained with 0.05% toluidine blue in phosphate buffer and citricacid at pH 4–6 (Sakai, 1973) for conventional histological analyses,treated with ferric chloride to detect phenolic compounds (tannins)(Johansen, 1940) or treated with ruthenium red to detect pectins(Strasburger, 1924). The sections were mounted onto slides with“Entellan” synthetic resin, and the images were captured with aLeica DM LB trinocular microscope coupled to a Leica DC 300 Fvideo camera and processed on a computer to prepare the illustra-tions.

2.3. Transmission electron microscopy (TEM)

Two-millimetre-thick samples were removed from the ovaryand equatorial regions of the fruits and immediately fixedin modified Karnovsky’s solution (2.5% glutaraldehyde, 2.5%paraformaldehyde and 0.05 mM CaCl2 in 0.1 M sodium cacodylatebuffer, 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 for2 h. The samples were dehydrated in an ascending acetone series upto 100% and embedded in Spurr’s resin. The blocks were sectionedusing a Leica UC6 ultramicrotome, and the sections were counter-stained with 5% uranyl acetate and 2% lead citrate for 30 min at eachstep (Reynolds, 1963). Observations and photomicrographs weremade using a Zeiss EM-900 transmission electron microscope oper-ating at 50 kV with the electron micrograph scales printed directlyon the photomicrographs.

2.4. Cryo-scanning electron microscopy (Cryo-SEM)

One-millimetre-thick samples of the ovary were obtained withthe aid of a steel blade. The samples were placed into a metalsample holder, frozen in liquid nitrogen at −210 ◦C and immedi-ately transferred to the Cryo-Trans (CT 15000C, Oxford InstrumentsLtd., Oxford, England), which was coupled to a scanning elec-tron microscope (Jeol JSM 5410, Tokyo, Japan) and operated underfreezing (−130 ◦C) and vacuum conditions (10−2 bar pressure).In the Cryo-Trans, the sample was fractured at −180 ◦C, andthe sections were sublimated at −90 ◦C for 15 min to eliminatepotential excess surface water in the tissue. The samples werecoated with gold in the Cryo-Trans at 10−2 bar and 40 mA andthen observed under the microscope with a 10 kV voltage and aworking distance of 15 mm. The images were captured on a com-puter.

2.5. Tannin cell density and tannin cell index

Samples were prepared for LM, the cross-sections were stainedwith toluidine blue, and images were captured. Four 1 mm2 areaswere defined in these images to count the number of parenchymaand tannin cells using the Image Tool 3.0 software for Windows.The following formulas were used: Density = number of tannincells/mm2 and Index (%) = S/(S + E) × 100, where S is the number oftannin cells per unit area and E is the number of parenchyma cellsin the same area.

2.6. Levels of total and soluble tannin

Tannin concentrations were determined spectrophotometri-cally using the Folin–Ciocalteu reagent (50%) according to the

294 M.A. Tessmer et al. / Scientia Horticulturae 172 (2014) 292–299

Fig. 1. Development stages of flowers and fruits of ‘Giombo’ (A–K) and ‘Fuyu’ (L) persimmons. (A) Appearance of the flower buds (stage a). (B) Opening of the calyx of theflower bud (stage b). (C) Elongation and change in the colour of the corolla from white to light yellow (stage c). (D–E) Opening of the corolla (anthesis) (stage d). Note thea ) Fru( m thej e i). Fr

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trophied stamens (arrow). (F) Drying and fall of the corolla (October, stage e). (GDecember, stage g). (I) Fruits from the fifth harvest (January, stage h). (J) Fruits fro). (L) Fruits from the seventh and final harvest of ‘Fuyu’ persimmon (February, stag

ethod of Taira (1995). A 1g sample of pulp was ground, cen-rifuged with methanol 80% to soluble tannins and other sampleith HCl 0.56 N and NaOH 2,5 N addition to the total tannins

nd subsequently diluted with distilled water to a final volume of00 ml. A 1 mL aliquot was removed, and Folin–Ciocalteu reagent50%), 1.0 mL of supersaturated sodium carbonate solution and.5 mL of distilled water were then added to the aliquot. Thebsorbance of the resulting solution was measured at 725 nmn a spectrophotometer (Biochrom, Libra S22 model). Gallic acidolution (0.1 g L−1) was used as a standard, and the results werexpressed in g 100 g−1 pulp.

.7. Experimental design

The experimental design was completely randomised in a 2 × 6actorial scheme, with two cultivars and six and five harvest periods

or ‘Giombo’ and ‘Fuyu’, respectively. The data were subjected tonalysis of variance (ANOVA) for each variable, and the sampleeans were compared with Tukey’s test (p = 0.01 and p = 0.05) using

he statistical package SASM-Agri (Canteri et al., 2001).

its from the third harvest (November, stage f). (H) Fruits from the fourth harvest sixth harvest (February, stage i). (K) Fruits from the seventh harvest (March, stage

= fruit; Ov = ovary; Pe = petals; Se = sepals.

3. Results

The ‘Giombo’ and ‘Fuyu’ cultivars bear flowers with highlyornate, tetramerous, gamosepalous and gamopetalous verticils,with the sepals quite evident due to their size since early flowerbud development (Fig. 1A–C). These cultivars are female diclinousplants with superior ovaries and bifid stigma that exhibit atro-phied and hairy stamens (Fig. 1D). The ‘Giombo’ (PVA) cultivarproduces fruits parthenocarpically; that is, the fruits form withoutpollination, and the ovules degenerate and disappear. By contrast,the ‘Fuyu’ persimmon contains pollinators and produces pollinatedflowers and fruits with seed formation. The cycle of fruit devel-opment in ‘Giombo’ and ‘Fuyu’ began with the appearance of thefloral buds (stage a) between August and September, and the fruitsreached maturity at 210–240 and 150–180 days, respectively.

During stage a, the early floral bud development of ‘Giombo’

(Fig. 2A) and ‘Fuyu’, the ovary expanded due to intense meriste-matic cell division (Fig. 2B). These meristematic cells were smalland isodiametric and contained evident nuclei. At this stage, theearly accumulation of tannins facilitated the differentiation of

M.A. Tessmer et al. / Scientia Horticulturae 172 (2014) 292–299 295

Fig. 2. Accumulation patterns of tannins in the ovaries and fruits of ‘Giombo’ (A, B, C, D, E, F, G, J, L, M, N, O, Q) and ‘Fuyu’ persimmons (H, I, K, P). LM photomicrographs:A, B, C, E, H, I, K, L, N, P. Cryo-SEM electron micrographs: D, G, J, O, Q. TEM electron micrographs: F, M. (A) Ovaries in stage a. Arrows indicate the appearance of the firsttannin cells. (B) Cell division (*). (C and D) Early accumulation of tannins near the cell wall (arrows). In C, note the reaction with ferric chloride. (E–G) Vesicular structurespresent in the early accumulation of tannins (arrows). (H) Vesicles with projections found in the tonoplast. (I and J) Amorphous tannins filling the vacuole (arrows). (K)D ith thet annint C = tan

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ruse-type crystals under polarised light. (L) Homogeneous tannin accumulation whe homogeneous, circular, unfilled areas of the previous figure (arrows). (N and O) Tannin accumulation. (Q) Pores in the tonoplast (arrows). MC = meristematic cell; T

annin cells that were dispersed or grouped in the central region ofhe ovary.

In the beginning and concurrent with the accumulation that

ccurred near the cell wall (Fig. 2C and D), severe vesiculation wasbserved in the cytoplasm of the tannin cells (Fig. 2E–G). Such vesi-les formed projections that were connected to the tonoplast fromne side to the other side of the cell wall in ‘Giombo’ and ‘Fuyu’

formation of circular, unfilled areas (arrow). (M) Vacuolar spaces corresponding to cell with vacuole filled with dense, homogeneous tannins. (P) Different patterns ofnin cell; Dr = druse; Ov = ovary; CW = cell wall; Pe = petal; Tn = tannin; Vc = vacuole.

(Fig. 2H). During stage b, the vacuole was gradually occupied by theamorphous tannin (Fig. 2I and J). Idioblasts with polyhedral druse-like crystals dispersed throughout the ovary were also observed for

‘Giombo’ and ‘Fuyu’ (Fig. 2K).

Apart from the aforementioned patterns of accumulation, fromstage c of fruit development in both cultivars, tannin cells withhomogeneous tannin accumulation with circular, unfilled areas

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ere also observed (Fig. 2L). Ultrastructural analysis demonstratedhat the circular, unfilled areas observed by light microscopy werereas that were apparently not filled with the tannins in the vacuoleFig. 2M). Consequently, in these areas, the content of the vacuoleas filled homogeneously (Fig. 2N and O), and cells with differentatterns of accumulation were observed in stage c (Fig. 2P). Poresere also detected in the tonoplast (Fig. 2Q).

In stage c, the ovary was covered by a uniseriate epidermis,nd the subepidermal layer exhibited an accumulation of phenolicompounds that characterised the hypodermis (Fig. 3A and B). InFuyu’, sclereids were observed (Fig. 3A), while in ‘Giombo’ (Fig. 3B),he sclereids were absent in the first layer, and the parenchyma5–6 layers) cells were smaller in size and contained dense cyto-lasm.

During stage d, the anthesis period, the same features describedbove for the epidermis and the hypodermis were also observed.eginning at this stage, sclereids were also observed in the periph-ral layers of the pericarp of ‘Giombo’ (Fig. 3C). The cell expansionrocess continued in the other parenchymal layers, and more elon-ated tannin cells were observed in both cultivars, with some of theannin cells accompanying vascular bundles (Fig. 3D). Different pat-erns of tannin accumulation were observed in the tannin cells inhe parenchyma, and the vacuoles occupied most of the cell volumen the cells in which accumulation was pronounced (Fig. 3E).

In stage e, the post-anthesis period, the pistil dried out, and theorolla fell off in ‘Giombo’ and ‘Fuyu’. The number of cells in the fruitontinued to increase, which was confirmed by the high density ofannin cells obtained in October and the maintenance of this densityn subsequent months (Fig. 4A). In both cultivars, the most frequentype of tannin accumulation was the homogeneous type, and theruses that were previously dispersed throughout the parenchymaere now concentrated in the outer layers, between the sclereids

nd the epidermis.During the remaining stages of development (stages f–j) of

Giombo’ and ‘Fuyu’, after cessation of cell division and differen-iation, there is a separation of the epidermal cells in ‘Giombo’Fig. 3F), while these cells exhibit high anticlinal walls due to celllongation in the periclinal direction in ‘Fuyu’ (Fig. 3G). The cuticlelso became thicker and began filling the intercellular spaces thatppeared in the epidermis (Fig. 3F). The sclereids were present untilhe end of fruit maturation, imparting hardness and resistance tohe pericarp (Fig. 3G). The size of the space between the vacuolend the cell wall increased in the tannin cells (Fig. 3H). The inter-ellular spaces in the parenchyma were enlarged (Fig. 3F and G) andecame filled by polysaccharides, particularly pectin (Fig. 3I), due tohe parietal degradation that characterises fruit ripening and senes-ence. Druses were no longer observed in the peripheral layers ofhe pericarp of either cultivar beginning at stage f of development.

The tannin cell density was highest in October for both the ‘Fuyu’nd ‘Giombo’ cultivars (Fig. 4A). The tannin cell density of ‘Giombo’as higher than that of ‘Fuyu’ throughout development. The den-

ity values remained stable over time for the two cultivars, whichndicated that cell division ceased in November.

The tannin cell index, which takes into account the number ofannin cells in relation to the number of parenchyma cells, varieduring development in both cultivars (Fig. 4B). ‘Giombo’ exhibitedigher index values than ‘Fuyu’ each month, except for October andecember.

The total and soluble tannins decreased throughout devel-pment in ‘Fuyu’. By contrast, despite fluctuations, the levelsemained high throughout development in ‘Giombo’ (Fig. 4C and). At the end of development and maturation, the levels of

oluble tannin, which are the compounds responsible for astrin-ency, remained higher in ‘Giombo’ than in ‘Fuyu’, reaching valuesf 0.785 g 100 g−1 and 0.141 g 100 g−1 in ‘Giombo’ and ‘Fuyu’,espectively (Fig. 4D).

ulturae 172 (2014) 292–299

4. Discussion

In the astringent cultivar ‘Giombo’ and the non-astringent culti-var ‘Fuyu’, early differentiation of tannin cells occurred at the stageof flower bud appearance. At this stage, the meristematic tissueis undergoing intense cell division, and differentiated cells can beidentified only by early accumulation of tannin in different pat-terns. Yonemori et al. (1997) also reported the occurrence of tannincells before anthesis in ‘Fuyu’ and ‘Hiratenenashi’.

Different patterns of tannin accumulation emerged in the twocultivars; some of the events within these patterns occurred duringdifferent stages of fruit development, while others occurred simul-taneously. Although several patterns have been reported (Martiniet al., 2008; Franceschi et al., 1998; Yonemori et al., 1997; Chafe andDurzan, 1973), the patterns of events observed in the present studyhave not been previously reported. The presence of polyphenolsnear the cell wall, which characterises the first stage of accumu-lation observed in the present study, was previously describedby Yonemori et al. (1997) in the ovaries of ‘Fuyu’ and ‘Hiratene-nashi’ persimmons and by Franceschi et al. (1998) in the secondaryphloem of Picea abies (Norway spruce).

The development of many vesicular structures during the earlystages of tannin accumulation observed in the present study wasalso described in the cotyledons of Theobroma species (Sterculi-aceae) (Martini et al., 2008). The vesicles increased in size andcoalesced to form a new vacuole (Chafe and Durzan, 1973) or fuseddirectly to the central vacuole (Yonemori et al., 1997). A similarphenomenon should occur in persimmons because these vesiclesare not found in more advanced stages of accumulation.

The pattern of the homogeneous distribution of tannins with cir-cular, unfilled areas observed in both cultivars was also observed inTheobroma species (Martini et al., 2008), in Picea abies (Franceschiet al., 1998) and in Picea glauca (Pinaceae) (Chafe and Durzan, 1973).Franceschi et al. (1998) described that electron-dense material cor-responded to condensed tannins, while the homogeneous tanninswith circular, unfilled areas most likely corresponded to the solu-ble phase. The ultrastructural analysis in persimmons not shownthat these areas correspond to soluble tannins and are filled withcontent in sequence.

Yonemori and Matsushima (1987a, 1987b) described the pres-ence of pores in the tannin cell wall in ‘Fuyu’; the appearance ofthese pores closely coincided with the period of tannin accumula-tion and cell growth period that, according to the authors, precedestannin condensation. The presence of pores was visualised in thetonoplast but not in the cell wall as reported by Yonemori andMatsushima (1987a, 1987b). The final stage of accumulation, whichwas homogeneous and dense, has also been observed in other stud-ies of tannin cells in persimmons development (Yonemori et al.,1997) and in the final stages of maturation the inside of the cell isalmost totally taken up by a large vacuole, which is full of solublematerial (Salvador et al., 2007).

The different patterns of accumulation described in the two cul-tivars did not result from artefacts caused by the fixation method,as proposed by Martini et al. (2008), Durzan et al. (1973) and Curgy(1968). According to Martini et al. (2008), the polyphenolic cellsexhibit a complex cytoarchitecture, and after fixation in glutaralde-hyde, the phenolic secretion displays only one type of content orfragments into several spherical droplets. However, the patternsrepeat themselves in frozen samples prepared without fixation, asverified by other authors (Cole and Aldrich, 1971).

The possible organelles involved in the synthesis of tannins arethe plastid (Mueller and Beckman, 1974), dictyosome (Ginsburg,

1967) and endoplasmic reticulum (Chafe and Durzan, 1973; Martiniet al., 2008). Recently, Brillouet et al. (2013) proposed a new modelof tannin biosynthesis in which the tannins are polymerised in anew chloroplast-derived organelle called the tannosome. Spheres

M.A. Tessmer et al. / Scientia Horticulturae 172 (2014) 292–299 297

Fig. 3. Cross-sections (A–C; E–I) and longitudinal section (D) of the developmental stages of the ovaries and fruits of the ‘Giombo’ (B, C, D, F, H, I) and ‘Fuyu’ (A, E, G) persimmoncultivars. (A) Sclereids, druses (arrowheads) and tannin cells with early accumulation of tannins in the outer layers of the parenchyma during stage c (arrow). (B) Sclereidsabsent during stage c. Note the cell layers with evident nuclei (*) and cells with different accumulation patterns (arrows). (C) Appearance of sclereids in the parenchymaduring stage d. (D) Elongated tannin cells at different stages of accumulation during stage d. (E) Tannin cells with homogeneous filling after reaction with ferric chlorideduring stage e. (F) Fruit under cell expansion (stage g). Note the spaces between the epidermal cells (arrows) and the accumulation of phenols in the hypodermis. (G-H-I) Fruitmaturation stage (stages i and j) showing the accumulation of pectins (*), stained with ruthenium red, in the intercellular spaces (G, I) and tannin cells with filled vacuoles.PC = phenolic cell; TC = tannin cell; Cu = cuticle; Ep = epidermis; Sc = sclereids; Pa = parenchyma.

298 M.A. Tessmer et al. / Scientia Horticulturae 172 (2014) 292–299

Fig. 4. Density (A), tannin cell index (B) values and levels of total (C) and soluble (D) tannins in ‘Giombo’ and ‘Fuyu’ persimmon fruits during development (six and fiveharvest periods, respectively). Uppercase letters compare the cultivars during each harvest period, and lowercase letters compare the effect of the harvest periods for eachc The v

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ultivar. Mean values with different letters are significant by Tukey’s test (p = 0.01).

re formed from thylakoids with tannins in their interior, and thepheres are encapsulated by a membrane formed from the dou-le membrane of the chloroplast, promoting transport through theytosol to the vacuole, where they are invaginated by the tono-last, aggregated and stored in the vacuole. It was not possible to

dentify the origin of the vesicular structures that proliferated dur-ng the early tannin accumulation in the cells in the ultrastructuralnalysis performed in the present study.

In addition to idioblasts that accumulated tannins, crystals werelso present, and sclereid differentiation was observed during thearly stages of development. Crystals have been associated withissue calcium regulation, increased rigidity of plant tissues, pro-ection against herbivory and metal detoxification (Franceschi andakata, 2005; Nakata, 2003). During the final stages of fruit devel-pment and maturation, the druses were no longer observed, mostikely due to calcium reallocation into the outer layer of cells, whichre more resistant in the pericarp of persimmon fruit.

The patterns of tannin accumulation in the non-astringent cul-ivar ‘Fuyu’ (PCNA) and in the astringent cultivar ‘Giombo’ (PVA)ere similar; however, the two cultivars could be differentiated

ased on the number of tannin cells and the levels of total andoluble tannins throughout their development. Throughout theirevelopment, ‘Giombo’ exhibited higher tannin cell density, solu-le tannin concentrations and total tannin concentrations than the

Fuyu’ cultivar. Similarly, lower and more stable tannin cell countsere reported for ‘Fuyu’ (PCNA) compared with ‘Hiratenenashi’

PVA) (Yonemori and Matsushima, 1987b).

According to Yonemori and Matsushima (1985, 1987a), the

umber of tannin cells per unit area varies little in the fruits of theour persimmon types, and the cessation of cell growth in PCNAultivars is coincident with the reduction of astringency.

ertical bars represent the standard error.

Only the ‘Fuyu’ cultivar exhibited amounts of soluble tanninsin mature fruits (0.141 g 100 g−1) acceptable for consumption; val-ues of approximately 0.1% are required to produce no perceptionof astringency, according to Antoniolli et al. (2000) and Salvadoret al. (2007). By contrast, the ‘Giombo’ cultivar requires treatmentto remove the astringency before marketing due to the higher levelsof soluble tannins.

4.1. Conclusions

The difference in the astringency of the fruits of the two analysedcultivars was related to the tannin cell density and the levels oftotal and soluble tannins, as there was no difference in the earlyaccumulation of tannins or in the patterns of accumulation duringcell differentiation.

Acknowledgements

The authors thank the São Paulo Research Foundation (Fundac ãode Amparo à Pesquisa do Estado de São Paulo – FAPESP) for finan-cial support: process 2010/16392-7. The authors also thank TheNational Council for Scientific and Technological Development(Conselho Nacional de Desenvolvimento Científico e Tecnológico– CNPq) for funding (302776/2010-9). The authors thank Ph.D.Alejandra Salvador Pérez (IVIA) and Isabel Pérez-Munuera (Uni-

versidad Politécnica de Valencia) for assistance with Cryo-SEManalysis. The authors thank the persimmon producers in Mogidas Cruzes, SP, Brazil, who kindly provided material for thisstudy.

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