Scientia Horticulturae 172 (2014) 292–299

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Scientia Horticulturae

journal homepage: www.elsevier.com/locate/scihorti

The accumulation of 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 accumulation during differentiation, cell density or the con-

centrations 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 , in spherical vesicles connected to the

tonoplast, as amorphous distributions occupying the entire , as homogeneous distributions with

circular, unfilled areas in , 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 , 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.

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’. working distance of 15 mm. The images were captured on a com-

From stage e (October) (Fig. 1F), monthly samples were taken. puter.

‘Fuyu’, which exhibits precocious maturation, was collected until

February, and ‘Giombo’, which matures later, was collected until

2.5. Tannin cell density and tannin cell index

March. Quantitative analyses were performed from stage e. Sam-

ples of four fruits were used to determine the cell density and cell

Samples were prepared for LM, the cross-sections were stained

index, and three replicates of three fruits were used to determine 2

with toluidine blue, and images were captured. Four 1 mm areas

soluble and total tannins.

were defined in these images to count the number of parenchyma

All stages of ‘Giombo’ and ‘Fuyu’ were subjected to analysis by

and tannin cells using the Image Tool 3.0 software for Windows.

light microscopy (LM). Stages a–d were analysed by transmission

The following formulas were used: Density = number of tannin

electron microscopy (TEM), and stage c ‘Giombo’ were analysed by 2

cells/mm and Index (%) = S/(S + E) × 100, where S is the number of

Cryo-SEM.

tannin cells per unit area and E is the number of parenchyma cells

in the same area.

2.2. Light microscopy (LM)

2.6. Levels of total and soluble tannin

Sections of the ovaries and of the equatorial regions of the

fruits were removed for light microscopy analysis. The sam- Tannin concentrations were determined spectrophotometri-

ples were fixed in Karnovsky’s solution (Karnovsky, 1965), and a 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 the

flower 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 the

atrophied stamens (arrow). (F) Drying and fall of the corolla (October, stage e). (G) Fruits from the third harvest (November, stage f). (H) Fruits from the fourth harvest

(December, stage g). (I) Fruits from the fifth harvest (January, stage h). (J) Fruits from the sixth harvest (February, stage i). (K) Fruits from the seventh harvest (March, stage

j). (L) Fruits from the seventh and final harvest of ‘Fuyu’ persimmon (February, stage i). Fr = fruit; Ov = ovary; Pe = petals; Se = sepals.

method of Taira (1995). A 1g sample of pulp was ground, cen- 3. Results

trifuged with methanol 80% to soluble tannins and other sample

The ‘Giombo’ and ‘Fuyu’ cultivars bear flowers with highly

with HCl 0.56 N and NaOH 2,5 N addition to the total tannins

ornate, tetramerous, gamosepalous and gamopetalous verticils,

and subsequently diluted with distilled water to a final volume of

with the sepals quite evident due to their size since early flower

100 ml. A 1 mL aliquot was removed, and Folin–Ciocalteu reagent

bud development (Fig. 1A–C). These cultivars are female diclinous

(50%), 1.0 mL of supersaturated sodium carbonate solution and

plants with superior ovaries and bifid stigma that exhibit atro-

7.5 mL of distilled water were then added to the aliquot. The

phied and hairy stamens (Fig. 1D). The ‘Giombo’ (PVA) cultivar

absorbance of the resulting solution was measured at 725 nm

produces fruits parthenocarpically; that is, the fruits form without

in a spectrophotometer (Biochrom, Libra S22 model). Gallic acid

−

1 pollination, and the ovules degenerate and disappear. By contrast,

solution (0.1 g L ) was used as a standard, and the results were

−

1 the ‘Fuyu’ persimmon contains pollinators and produces pollinated

expressed in g 100 g pulp.

flowers and fruits with seed formation. The cycle of fruit devel-

opment in ‘Giombo’ and ‘Fuyu’ began with the appearance of the

2.7. Experimental design

floral buds (stage a) between August and September, and the fruits

reached maturity at 210–240 and 150–180 days, respectively.

The experimental design was completely randomised in a 2 × 6

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

factorial scheme, with two cultivars and six and five harvest periods

(Fig. 2A) and ‘Fuyu’, the ovary expanded due to intense meriste-

for ‘Giombo’ and ‘Fuyu’, respectively. The data were subjected to

matic cell division (Fig. 2B). These meristematic cells were small

analysis of variance (ANOVA) for each variable, and the sample

and isodiametric and contained evident nuclei. At this stage, the

means were compared with Tukey’s test (p = 0.01 and p = 0.05) using

early accumulation of tannins facilitated the differentiation of

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

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 first

tannin 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 structures

present 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)

Druse-type crystals under polarised light. (L) Homogeneous tannin accumulation with the formation of circular, unfilled areas (arrow). (M) Vacuolar spaces corresponding to

the homogeneous, circular, unfilled areas of the previous figure (arrows). (N and O) Tannin cell with vacuole filled with dense, homogeneous tannins. (P) Different patterns of

tannin accumulation. (Q) Pores in the tonoplast (arrows). MC = meristematic cell; TC = tannin cell; Dr = druse; Ov = ovary; CW = cell wall; Pe = petal; Tn = tannin; Vc = vacuole.

tannin cells that were dispersed or grouped in the central region of (Fig. 2H). During stage b, the vacuole was gradually occupied by the

the ovary. amorphous tannin (Fig. 2I and J). Idioblasts with polyhedral druse-

In the beginning and concurrent with the accumulation that like crystals dispersed throughout the ovary were also observed for

occurred near the cell wall (Fig. 2C and D), severe vesiculation was ‘Giombo’ and ‘Fuyu’ (Fig. 2K).

observed in the of the tannin cells (Fig. 2E–G). Such vesi- Apart from the aforementioned patterns of accumulation, from

cles formed projections that were connected to the tonoplast from stage c of fruit development in both cultivars, tannin cells with

one side to the other side of the cell wall in ‘Giombo’ and ‘Fuyu’ homogeneous tannin accumulation with circular, unfilled areas

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

were also observed (Fig. 2L). Ultrastructural analysis demonstrated 4. Discussion

that the circular, unfilled areas observed by light microscopy were

In the astringent cultivar ‘Giombo’ and the non-astringent culti-

areas that were apparently not filled with the tannins in the vacuole

var ‘Fuyu’, early differentiation of tannin cells occurred at the stage

(Fig. 2M). Consequently, in these areas, the content of the vacuole

of flower bud appearance. At this stage, the meristematic tissue

was filled homogeneously (Fig. 2N and O), and cells with different

is undergoing intense cell division, and differentiated cells can be

patterns of accumulation were observed in stage c (Fig. 2P). Pores

identified only by early accumulation of tannin in different pat-

were also detected in the tonoplast (Fig. 2Q).

terns. Yonemori et al. (1997) also reported the occurrence of tannin

In stage c, the ovary was covered by a uniseriate epidermis,

cells before anthesis in ‘Fuyu’ and ‘Hiratenenashi’.

and the subepidermal layer exhibited an accumulation of phenolic

Different patterns of tannin accumulation emerged in the two

compounds that characterised the hypodermis (Fig. 3A and B). In

cultivars; some of the events within these patterns occurred during

‘Fuyu’, sclereids were observed (Fig. 3A), while in ‘Giombo’ (Fig. 3B),

different stages of fruit development, while others occurred simul-

the sclereids were absent in the first layer, and the parenchyma

taneously. Although several patterns have been reported (Martini

(5–6 layers) cells were smaller in size and contained dense cyto-

plasm. et al., 2008; Franceschi et al., 1998; Yonemori et al., 1997; Chafe and

Durzan, 1973), the patterns of events observed in the present study

During stage d, the anthesis period, the same features described

have not been previously reported. The presence of polyphenols

above for the epidermis and the hypodermis were also observed.

near the cell wall, which characterises the first stage of accumu-

Beginning at this stage, sclereids were also observed in the periph-

lation observed in the present study, was previously described

eral layers of the pericarp of ‘Giombo’ (Fig. 3C). The cell expansion

by Yonemori et al. (1997) in the ovaries of ‘Fuyu’ and ‘Hiratene-

process continued in the other parenchymal layers, and more elon-

nashi’ persimmons and by Franceschi et al. (1998) in the secondary

gated tannin cells were observed in both cultivars, with some of the

phloem of Picea abies (Norway spruce).

tannin cells accompanying vascular bundles (Fig. 3D). Different pat-

The development of many vesicular structures during the early

terns of tannin accumulation were observed in the tannin cells in

stages of tannin accumulation observed in the present study was

the parenchyma, and the vacuoles occupied most of the cell volume

also described in the cotyledons of Theobroma species (Sterculi-

in the cells in which accumulation was pronounced (Fig. 3E).

aceae) (Martini et al., 2008). The vesicles increased in size and

In stage e, the post-anthesis period, the pistil dried out, and the

coalesced to form a new vacuole (Chafe and Durzan, 1973) or fused

corolla fell off in ‘Giombo’ and ‘Fuyu’. The number of cells in the fruit

directly to the central vacuole (Yonemori et al., 1997). A similar

continued to increase, which was confirmed by the high density of

phenomenon should occur in persimmons because these vesicles

tannin cells obtained in October and the maintenance of this density

are not found in more advanced stages of accumulation.

in subsequent months (Fig. 4A). In both cultivars, the most frequent

The pattern of the homogeneous distribution of tannins with cir-

type of tannin accumulation was the homogeneous type, and the

cular, unfilled areas observed in both cultivars was also observed in

druses that were previously dispersed throughout the parenchyma

Theobroma species (Martini et al., 2008), in Picea abies (Franceschi

were now concentrated in the outer layers, between the sclereids

et al., 1998) and in Picea glauca (Pinaceae) (Chafe and Durzan, 1973).

and the epidermis.

Franceschi et al. (1998) described that electron-dense material cor-

During the remaining stages of development (stages f–j) of

responded to condensed tannins, while the homogeneous tannins

‘Giombo’ and ‘Fuyu’, after cessation of cell division and differen-

with circular, unfilled areas most likely corresponded to the solu-

tiation, there is a separation of the epidermal cells in ‘Giombo’

ble phase. The ultrastructural analysis in persimmons not shown

(Fig. 3F), while these cells exhibit high anticlinal walls due to cell

that these areas correspond to soluble tannins and are filled with

elongation in the periclinal direction in ‘Fuyu’ (Fig. 3G). The cuticle

content in sequence.

also became thicker and began filling the intercellular spaces that

Yonemori and Matsushima (1987a, 1987b) described the pres-

appeared in the epidermis (Fig. 3F). The sclereids were present until

ence of pores in the tannin cell wall in ‘Fuyu’; the appearance of

the end of fruit maturation, imparting hardness and resistance to

these pores closely coincided with the period of tannin accumula-

the pericarp (Fig. 3G). The size of the space between the vacuole

tion and cell growth period that, according to the authors, precedes

and the cell wall increased in the tannin cells (Fig. 3H). The inter-

tannin condensation. The presence of pores was visualised in the

cellular spaces in the parenchyma were enlarged (Fig. 3F and G) and

tonoplast but not in the cell wall as reported by Yonemori and

became filled by polysaccharides, particularly pectin (Fig. 3I), due to

Matsushima (1987a, 1987b). The final stage of accumulation, which

the parietal degradation that characterises fruit ripening and senes-

was homogeneous and dense, has also been observed in other stud-

cence. Druses were no longer observed in the peripheral layers of

ies of tannin cells in persimmons development (Yonemori et al.,

the pericarp of either cultivar beginning at stage f of development.

1997) and in the final stages of maturation the inside of the cell is

The tannin cell density was highest in October for both the ‘Fuyu’

almost totally taken up by a large vacuole, which is full of soluble

and ‘Giombo’ cultivars (Fig. 4A). The tannin cell density of ‘Giombo’

material (Salvador et al., 2007).

was higher than that of ‘Fuyu’ throughout development. The den-

The different patterns of accumulation described in the two cul-

sity values remained stable over time for the two cultivars, which

tivars did not result from artefacts caused by the fixation method,

indicated that cell division ceased in November.

as proposed by Martini et al. (2008), Durzan et al. (1973) and Curgy

The tannin cell index, which takes into account the number of

(1968). According to Martini et al. (2008), the polyphenolic cells

tannin cells in relation to the number of parenchyma cells, varied

exhibit a complex cytoarchitecture, and after fixation in glutaralde-

during development in both cultivars (Fig. 4B). ‘Giombo’ exhibited

hyde, the phenolic secretion displays only one type of content or

higher index values than ‘Fuyu’ each month, except for October and

December. fragments into several spherical droplets. However, the patterns

repeat themselves in frozen samples prepared without fixation, as

The total and soluble tannins decreased throughout devel-

verified by other authors (Cole and Aldrich, 1971).

opment in ‘Fuyu’. By contrast, despite fluctuations, the levels

The possible involved in the synthesis of tannins are

remained high throughout development in ‘Giombo’ (Fig. 4C and

the (Mueller and Beckman, 1974), dictyosome (Ginsburg,

D). At the end of development and maturation, the levels of

1967) and (Chafe and Durzan, 1973; Martini

soluble tannin, which are the compounds responsible for astrin-

et al., 2008). Recently, Brillouet et al. (2013) proposed a new model

gency, remained higher in ‘Giombo’ than in ‘Fuyu’, reaching values

− −

1 1 of tannin biosynthesis in which the tannins are polymerised in a

of 0.785 g 100 g and 0.141 g 100 g in ‘Giombo’ and ‘Fuyu’,

new -derived organelle called the tannosome. Spheres respectively (Fig. 4D).

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) persimmon

cultivars. (A) Sclereids, druses (arrowheads) and tannin cells with early accumulation of tannins in the outer layers of the parenchyma during stage c (arrow). (B) Sclereids

absent during stage c. Note the cell layers with evident nuclei (*) and cells with different accumulation patterns (arrows). (C) Appearance of sclereids in the parenchyma

during stage d. (D) Elongated tannin cells at different stages of accumulation during stage d. (E) Tannin cells with homogeneous filling after reaction with ferric chloride

during 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) Fruit

maturation 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 five

harvest periods, respectively). Uppercase letters compare the cultivars during each harvest period, and lowercase letters compare the effect of the harvest periods for each

cultivar. Mean values with different letters are significant by Tukey’s test (p = 0.01). The vertical bars represent the standard error.

are formed from thylakoids with tannins in their interior, and the Only the ‘Fuyu’ cultivar exhibited amounts of soluble tannins

−1

spheres are encapsulated by a membrane formed from the dou- in mature fruits (0.141 g 100 g ) acceptable for consumption; val-

ble membrane of the chloroplast, promoting transport through the ues of approximately 0.1% are required to produce no perception

to the vacuole, where they are invaginated by the tono- of astringency, according to Antoniolli et al. (2000) and Salvador

plast, aggregated and stored in the vacuole. It was not possible to et al. (2007). By contrast, the ‘Giombo’ cultivar requires treatment

identify the origin of the vesicular structures that proliferated dur- to remove the astringency before marketing due to the higher levels

ing the early tannin accumulation in the cells in the ultrastructural of soluble tannins.

analysis performed in the present study.

In addition to idioblasts that accumulated tannins, crystals were

also present, and sclereid differentiation was observed during the 4.1. Conclusions

early stages of development. Crystals have been associated with

tissue calcium regulation, increased rigidity of plant tissues, pro- The difference in the astringency of the fruits of the two analysed

tection against herbivory and metal detoxification (Franceschi and cultivars was related to the tannin cell density and the levels of

Nakata, 2005; Nakata, 2003). During the final stages of fruit devel- total and soluble tannins, as there was no difference in the early

opment and maturation, the druses were no longer observed, most accumulation of tannins or in the patterns of accumulation during

likely due to calcium reallocation into the outer layer of cells, which cell differentiation.

are more resistant in the pericarp of persimmon fruit.

The patterns of tannin accumulation in the non-astringent cul-

tivar ‘Fuyu’ (PCNA) and in the astringent cultivar ‘Giombo’ (PVA) Acknowledgements

were similar; however, the two cultivars could be differentiated

based on the number of tannin cells and the levels of total and The authors thank the São Paulo Research Foundation (Fundac¸ ão

soluble tannins throughout their development. Throughout their de Amparo à Pesquisa do Estado de São Paulo – FAPESP) for finan-

development, ‘Giombo’ exhibited higher tannin cell density, solu- cial support: process 2010/16392-7. The authors also thank The

ble tannin concentrations and total tannin concentrations than the National Council for Scientific and Technological Development

‘Fuyu’ cultivar. Similarly, lower and more stable tannin cell counts (Conselho Nacional de Desenvolvimento Científico e Tecnológico

were reported for ‘Fuyu’ (PCNA) compared with ‘Hiratenenashi’ – CNPq) for funding (302776/2010-9). The authors thank Ph.D.

(PVA) (Yonemori and Matsushima, 1987b). Alejandra Salvador Pérez (IVIA) and Isabel Pérez-Munuera (Uni-

According to Yonemori and Matsushima (1985, 1987a), the versidad Politécnica de Valencia) for assistance with Cryo-SEM

number of tannin cells per unit area varies little in the fruits of the analysis. The authors thank the persimmon producers in Mogi

four persimmon types, and the cessation of cell growth in PCNA das Cruzes, SP, Brazil, who kindly provided material for this

cultivars is coincident with the reduction of astringency. study.

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

References IBGE – Instituto Brasileiro de Geografia e Estatística, 2010. http://www.ibge.gov.

br/home/estatistica/indicadores/agropecuaria/lspa/

Johansen, D.A., 1940. Plant Microtechnique. McGraw-Hill Book, New York, pp. 523.

Akagi, T., Ayako Katayama-Ikegami, A., Yonemori, K., 2011. Proanthocyanidin

Karnovsky, M.J.A., 1965. Formaldehyde–glutaraldehyde fixative of high osmolality

biosynthesis of persimmon (Diospyros kaki Thunb.) fruit. Sci. Hortic. 130,

for use in electron microscopy. J. Cell Biol. 27, 137–138.

373–380.

Martini, M.H., Figueira, F., Lenci, C.G., Tavares, D.Q., 2008. Polyphenolic cells and their

Akagi, T., Suzuki, Y., Ikegami, A., Kamitakahara, H., Takano, T., Nakatsubo, F.,

interrelation with cotyledon cells in seven species of Theobroma (Sterculiaceae).

Yonemori, K., 2010. composition analysis in persimmon

Rev. Brasil. Bot. 31, 425–431.

(Diospyros kaki Thunb.) fruit by acid catalysis in the presence of excess phloroglu-

Matsuo, T., Itoo, S., 1978. The chemical structure of kaki-tannin from immature fruit

cinol. J. Jpn. Soc. Hortic. Sci. 79, 275–281.

of the persimmon (Diospyros kaki L.). Agric. Biol. Chem. 42, 1637–1643.

Antoniolli, L.R., Castro, P.R.C., Kluge, R.A., Scarpare Filho, J.A., 2000. Remoc¸ ão da

Mueller, W.C., Beckman, C.H., 1974. Ultra structure of the phenol storing cells in

adstringência de frutos de caqui ‘Giombo’ sob diferentes períodos de exposic¸ ão

roots of banana. Physiol. Plant. Pathol. 4, 187–190.

ao vapor de álcool etílico. Pesqui. Agropec. Bras. 35, 2083–2091.

Nakata, P.A., 2003. Advances in our understanding of calcium oxalate crystal forma-

Brillouet, J.M., Romieu, C., Schoefs, B., Solymosi, K., Cheynier, V., Fulcrand, H., Verdeil,

tion and function in plants. Plant Sci. 164, 901–909.

J.L., Conéjéro, G., 2013. The tannosome is an organelle forming condensed tan-

Novillo, P., Salvador, A., Llorca, E., Hernando, I., Besada, C., 2014. Effect of CO2

nins in the chlorophyllous organs of Tracheophyta. Ann. Bot. 112, 1–12.

deastringency treatment on flesh disorders induced by mechanical damage in

Canteri, M.G., Althaus, R.A., Virgens-Filho, J.S., Giglioti, E.A., Godoy, C.V., 2001. SASM-

persimmon. Biochemical and microstructural studies. Food Chem. 145, 454–463.

Agri: Sistema para análise e separac¸ão de médias em experimentos agrícolas

Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain

pelos métodos Scoft-Knott, Tukey e Duncan. Rev. Bras. Agroc. 1, 18–24.

for electron microscopy. J. Cell Biol. 17, 208–212.

Chafe, S.C., Durzan, D.J., 1973. Tannin inclusions in cell suspension cultures of white

Sakai, W.S., 1973. Simple method for differential staining of paraffin embedded plant

spruce. Planta 113, 251–262.

material using toluidine blue. Stain Technol. 48 (5), 247–249.

Cole, G.T., Aldrich, H.C., 1971. Demonstration of myelin figures in unfixed,

Salvador, A., Arnal, L., Besada, C., Larrea, V., Quiles, A., Pérez-Munuera, I., 2007. Phys-

freezeetched fungus spores. J. Cell Biol. 51, 873–874.

iological and structural changes during ripening and deastringency treatment

Curgy, J.J., 1968. Influence du mode de fixation sur la possibilité d’observer des

of persimmon cv. ‘Rojo Brillante’. Postharvest Biol. Technol. 46, 181–188.

structures myeliniques dans les hepatocytes d’embryons de poulet. J. Microsc.

Strasburger, E., 1924. Handbook of Practical Botany. The MacMillan Company, New

7, 63–80.

York, pp. 532.

Dixon, R.A., 2005. Engineering of plant natural product pathways. Curr. Opin. Plant

Taira, S., 1995. Astringency in persimmon. In: Linskens, H.F., Jackson, J.F. (Eds.), Fruit

Biol. 8, 329–336.

Analysis. Springer, Hannover, pp. 97–110.

Durzan, D.J., Chafe, S.C., Lopushanski, S.M., 1973. Effects of environmental changes

Tanaka, T., Takahashi, R., Kouno, I., Nonaka, G., 1994. Chemical evidence for de-

on sugars, tannins, and organized growth in cell suspension cultures of white

astringency (insolubilization of tannins) of persimmon fruit. J. Chem. Soc. Perkin

spruce. Planta 118, 241–249.

Trans. 1, 3013–3022.

Edagi, F.K., Kluge, R.A., 2009. Remoc¸ ão de adstringência de caqui: um enfoque bio-

Yonemori, K., Oshida, M., Sugiura, A., 1997. Fine structure of tannin cells in fruit and

químico, fisiológico e tecnológico. Cienc. Rural 39, 585–594.

callus tissues of persimmon. Acta Hortic. 436, 403–416.

FAOSTAT – Food Agricultural Organization Statistical, 2009. Agricultural Data.

Yonemori, K., Matsushima, J., 1987a. Changes in tannin cell morphology with growth

http://faostat.fao.org/faostat/collections

and development of Japanese persimmon fruit. J. Am. Soc. Hortic. Sci. 112,

Franceschi, V.R., Krekling, T., Berryman, A.A., Christiansen, E., 1998. Specialized

817–821.

phloem parenchyma cells in Norway spruce (Pinaceae) bark are an important

Yonemori, K., Matsushima, J., 1987b. Morphological characteristics of tannin cells in

site of defense reactions. Am. J. Bot. 85, 601–615.

Japanese persimmon fruit. J. Am. Soc. Hortic. Sci. 112, 812–817.

Franceschi, V.R., Nakata, P.A., 2005. Calcium oxalate in plants: formation and func-

Yonemori, K., Matsushima, J., 1985. Property of development of the tannin cells

tion. Annu. Rev. Plant Biol. 56, 41–71.

in non-astringent type fruits of Japanese persimmon (Diospyros kaki) and its

Ginsburg, C., 1967. The relation of tannins to the differentiation of the root tissues

relationship to natural deastringency. J. Jpn. Soc. Hortic. Sci. 54, 201–208.

in Reaumuria palestina. Bot. Gaz. 128, 1–10.