Plant Physiol. (1991) 97, 814-816 Received for publication March 5, 1991 0032-0889/91/97/081 4/03/$01 .00/0 Accepted June 14, 1991

Communication Cell Turgor Changes Associated with Ripening in Tomato Pericarp

Kenneth A. Shackel*, Carl Greve, John M. Labavitch, and Hamid Ahmadi Department of Pomology, University of California, Davis, California 95616

ABSTRACT to physically compress or penetrate external tissues. This force The pressure microprobe was used to determine whether the may change during softening as a result of reduced integrity turgor pressure in tomato (Lycopersicon esculentum Mill., variety in components but could also be a consequence of "Castelmart") pericarp cells changed during fruit ripening. The changes in the hydrostatic pressure (turgor) within fruit cells. turgor pressure of cells located 200 to 500 micrometers below Since cell turgor requires maintenance ofmembrane integrity, the fruit was uniform within the same tissue (typically the impact of Ca2" on firmness may also be understood as a ± 0.02 megapascals), and the highest turgors observed (<0.2 turgor-mediated consequence of the known membrane sta- megapascals) were much less than expected, based on tissue bilizing properties of Ca2" (3, 13). To our knowledge, there osmotic potential (-0.6 to -0.7 megapascals). These low turgor are no previous reports of changes in cell turgor associated values may indicate the presence of apoplastic solutes. In both with the ripening process in any fruit. Steudle and Wieneke intact fruit and cultured discs of pericarp tissue, a small increase (17) reported that elastic and hydraulic properties of cells in in turgor preceded the onset of ripening, and a decrease in turgor occurred during ripening. Differences in the turgor of individual apple fruit changed during fruit development, but in their intact fruit occurred 2 to 4 days before parallel differences in their system turgor was artificially modified by manipulating the ripening behavior were apparent, indicating that changes in turgor osmotic potential of the bathing medium. Using the pressure may reflect physiological changes at the cell level that precede microprobe technique (1 1), this communication reports direct expression of ripening at the tissue level. measurements of changes in turgor in the pericarp cells of ripening intact tomato fruit, and in a recently described in vitro ripening system, the tomato pericarp disc (2). MATERIALS AND METHODS Postharvest decreases in fruit firmness (fruit softening) are an important component of the increase in palatability that Plant Material and Experimental Design accompanies fruit ripening. If softening is not effectively Field-grown tomatoes (Lycopersicon esculentum Mill.) var. controlled in the postharvest environment, however, fruit "Castelmart" were harvested from university experimental susceptibility to mechanical damage and pathogen attack is plots at Davis, CA, at the mature-green 3 to 4 stage of greatly increased (16). Consequently, there have been several maturity, and, in most cases, were stored at 10°C for 1 to 2 d investigations into the cellular processes that regulate soften- before use. Disc preparation has been described previously ing and into postharvest handling procedures that can slow (2). Briefly, cylinders of pericarp tissue (10-mm diameter, 5- or stop the process (1, 8, 18). Most of the work concerning 7 mm thick) were excised from surface-sterilized fruits (about the biochemical basis of fruit softening has emphasized the 10 discs/fruit) and placed epidermis-side-down in multi-well metabolism of the cell wall/middle lamella complex. It is plates (Falcon 3047). Both discs and intact fruits were incu- reasonable to presume that softening is a physical conse- bated (ripened) at 20°C in boxes flushed with 3 volumes of quence of cell wall breakdown, since cells in ripening fruits saturated air per hour to prevent desiccation. Prelimi- are more readily crushed and/or can be moved past each nary experiments were performed on freshly cut discs from other more easily than cells in nonripening fruit. A straight- mature-green and red-ripe fruit, and two experiments were forward relation between fruit softening and the breakdown performed to follow turgor and color changes during ripening of fruit cell wall pectins and, to a lesser extent, hemicelluloses of discs and whole fruits. In experiment 1, fruits were har- has been developed in the literature (9). This remains the vested on September 19, 1990, and a total of six intact fruits dominant model explaining softening, although recent work and an initial population of 240 individual discs were ripened (e.g. 6) has indicated that softening requires more than the in culture (2). Color (see below) was measured nondestruc- digestion of pectin. Application of calcium to unripe fruit has tively on all discs (one measurement per disc) and fruits (three been shown to substantially reduce fruit softening in storage random measurements per fruit) on a daily basis. Any discs and slow the rate of ripening (14). This too has been inter- showing signs of microbial contamination or tissue damage preted in terms of cell wall integrity-the idea being that Ca2" were discarded. Periodic measurements of turgor were made has induced a stiffening of cell wall pectin gels (7). on each fruit, and on four discs with skin color close to that Fruit firmness is quantified by measuring the force required of the disc population mean for the day of measurement. 814 Downloaded from on May 11, 2019 - Published by www.plantphysiol.org Copyright © 1991 American Society of Plant Biologists. All rights reserved. RIPENING AND TURGOR IN TOMATO 815

Table I. Turgor of the Subepidermal Cells in Pericarp Discs Cut (+) axis (10). Disc color was corrected for the effect of the from Mature-Green and Red-Ripe Maturity Stage Tomato Fruit plastic (2). The pressure microprobe (11) was used to measure the Maturity Stage Turgor turgor of cells located 200 to 500 tsm below the epidermis in MPaa both whole fruit and pericarp discs. Tissue penetration and Mature-green 0.14b meniscus behavior were observed at x200 through a vertically Red-ripe 0.03 illuminated microscope with a long distance objective (15). a Mean of 17 cells sampled from seven individual fruits. b LSD The methodology used to measure the turgor of subepidermal (P < 0.01) of 0.02 was obtained from a simple one-way ANOVA. cells was essentially the same as that described by Cosgrove and Cleland (5), in which the epidermal and first few subepi- dermal cells were sacrificed to establish an observable menis- Turgor measurements were obtained from two cells in the cus external to the tissue. As was found by Cosgrove and center of each disc and three cells at random positions on Cleland (5), after meniscus establishment the measured turgor each fruit at each time ofsampling. After turgor measurement, of sequentially penetrated subepidermal cells was quite uni- discs were discarded and fruits were returned to the ripening form (typically ± 0.02 MPa). Difficulties oftip breakage were conditions. In experiment 2, fruits were harvested on October experienced, such as those described by Steudle and Wieneke 4, 1990, and a total of five intact fruits and an initial popu- (17), in the initial attempts to penetrate the fruit epidermis. lation of 120 individual discs were ripened. Disc and fruit These were solved by producing very short microcapillary tips color were measured nondestructively as in experiment 1, but (pulled with a Koph Mod. 750 micropipette puller) that were fruit color was sampled from the same three positions on each widened and sharpened with a modified jet-stream micro- fruit for each time of measurement. The same protocol as in beveler (12). For turgor measurements, individual pericarp experiment 1 was used for turgor measurement, but after discs were removed from culture and placed, epidermal-side- measurement, discs were sealed in plastic vials and frozen for up, in a plastic block with a cylindrical cavity of about the later determination of osmotic potential. same depth and diameter as the disc. In this way the tissue could be easily positioned during turgor measurements, while minimizing the exposure ofdisc cut surface area. A few drops Measurement of Fruit Color and Water Relations of water were placed in the cavity to prevent tissue drying Fruit color was measured directly on the fruit surface, and during turgor measurements, which were performed under disc color through the clear plastic base ofthe multi-well plate, laboratory conditions (diffuse fluorescent light and 25-30°C with a reflectance colorimeter (Minolta CR-200). Colors re- air temperature) and were completed within 40 min of re- ported are for the a* component of the L*a*b* uniform color moving discs from culture. During this period of time, there space (CIELAB), which measures hue on a green (-) to red was no detectable change in the turgor of the subepidermal cells located in the center ofthe 10-mm-diameter disc. Whole fruit turgor measurements were performed under the same Disc Osmotic Potential (MPa, exp. #2) -0.63 -0.74 -0.66 laboratory conditions. Osmotic potentials of sap extracted 0.16 V vI from frozen-thawed pericarp discs were determined with a vapor pressure osmometer (5500; Wescor, Inc., Logan, UT). a. 0.12 2 RESULTS AND DISCUSSION 0.08 0 In preliminary experiments, the turgor in discs cut from a: 0.04 mature-green fruit was always found to be higher than that in HD Disc (cxp. #1) - - 0.00 40 Fruit (exp. #1). -, 0.12 Disc (exp. #2) cam 20 2 cc. 0.08 0 0- . - ''- 0 - ..j.,_. o 0.04 0 .0 2 4 6 * 1 cc 14 -20 H 0.00 (exp. #2) 40 0 2 4 6 8 10 12 14 16 Fruit A cc DAYS AT 20 C FruitBB ------20 - cc- Fruit C ...... 0 Figure 1. Changes in subepidermal cell turgor and in surface color 0 (a*, measuring hue on a green [-] to red [+] axis, [10]) of tomato 0 pericarp discs (experiments 1 and 2) and intact tomato fruit (experi- 1- -20 ment 1 only) during ripening at 200C. Each point shows the mean ± 0 2 4 6 8 10 12 14 16 2 SE, and errors smaller than the symbols are hidden. The number of DAYS AT 20 C measurements (n) in each mean is: experiment 1 disc color n = 200, disc turgor n = 8, fruit color n = 18, fruit turgor n = 8; experiment 2 Figure 2. Changes in subepidermal cell turgor and in surface color disc color n = 100 and disc turgor n = 14. Only the initial, minimum, of individual intact tomato fruit (experiment 2) ripening independently and final values (d 1, 4, and 13, respectively) of disc osmotic potential at 200C. Each point shows the mean ± 2 SE, and errors smaller than in experiment 2 (n = 2, pooled SE = 0.3 MPa) are indicated at the the symbols are hidden. The number of measurements in each turgor top. mean is 2 and in each color mean is 3.

Downloaded from on May 11, 2019 - Published by www.plantphysiol.org Copyright © 1991 American Society of Plant Biologists. All rights reserved. 816 SHACKEL ET AL. Plant Physiol. Vol. 97, 1991 discs cut from red-ripe fruit (Table I). These turgors, however, logical changes at the cell or cell membrane level that precede were substantially less than those expected based on the range the expression of ripening at the tissue level. ofosmotic potentials found in these tissues (-0.6--0.7 MPa), Campbell et al. (2) have shown that ripening in pericarp even when discs had been partly or fully submerged in water discs (as judged by color change, increased C02 evolution and for 4 h (data not shown). Under such conditions, it is expected ethylene production, and, especially pertinent to this report, that tissue hydration would be essentially complete and that decreased tissue firmness and altered cell wall chemistry) cell turgor and cell osmotic potentials would be equal and follows the same course as ripening in intact fruit. The data opposite. It is possible that damage to the cell wall during shown in Figures 1 and 2 suggest that decreases in turgor penetration could result in erroneously low measured turgors, follow skin reddening in much the same way as wall chemical but the turgor of sequentially penetrated cells was uniform changes and softening (2). It is thus possible that some aspects (as described by Cosgrove and Cleland [5]) and stable imme- of tomato softening are the result of turgor loss as well as (or diately after penetration (as described by Shackel et al. [15]) instead of) altered wall integrity. Figure 2 also suggests that for all reported turgor values. Hence, there was no evidence physiological changes in cell membranes or symplast/apoplast to support the hypothesis that these low turgors were the result osmotic relations may precede the turgor loss that is associated of cell wall damage. One physiological mechanism that could with ripening. Further study of these changes may shed ad- ditional on the basis of fruit account for low turgors is the presence of solutes in the light physiological softening and other metabolic of the apoplastic space ofthis tissue, as suggested for hypocotyl tissue important aspects ripening process. by Cosgrove and Cleland (4). Regardless of the mechanism, LITERATURE CITED however, these data indicate that the turgor of the pericarp 1. Brady CJ (1987) Fruit ripening. Annu Rev Plant Physiol 38: cells of mature green tomato fruit is relatively low and is 155-178 further reduced during ripening. 2. Campbell AD, Huysamer M, Stotz HU, Greve LC, Labavitch During ripening in both discs and intact fruits, a decline in JM (I1990) Comparison of ripening processes in intact tomato turgor occurred, although the rate of ripening (as measured fruit and excised pericarp discs. Plant Physiol 94: 1582-1589 3. Christiansen NM, Foy C (I1979) Fate and function ofcalcium in by skin color) and also the absolute level of turgor were tissue. Commun Soil Sci Plant Anal 10: 427-442 different in different experiments (Fig. 1). In all cases, how- 4. Cosgrove DJ, Cleland RE (1983) Solutes in the free space of ever, the maximum turgor occurred 3 to 4 d after the start of growing stem tissues. Plant Physiol 72: 326-331 incubation, followed by a decline in turgor as ripening pro- 5. Cosgrove DJ, Cleland RE (1983) Osmotic properties of pea internodes in relation to growth and auxin action. Plant Physiol gressed. The osmotic potentials of the discs in experiment 2 72: 332-338 exhibited an overall pattern of change that was opposite that 6. DellaPenna D, Lashbrook CC, Toenjes K, Giovannoni JJ, Fischer of their turgor, namely an initial decrease followed by a RL, Bennett AB (I1990) Polygalacturonase isozymes and pectin depolymerization in transgenic rin tomato fruit. Plant Physiol subsequent increase (only initial, minimum, and final values 94: 1882-1886 are indicated in Fig. 1). The point of minimum osmotic 7. Grant GT, Morris ER, Rees DA, Smith RJC, Thom D (1973) potential, however, did not correspond to the point of maxi- Biological interactions between polysaccharides and divalent mum turgor, nor did the overall decrease in turgor correspond cations: the egg-box model. FEBS Lett 32: 195-198 8. Grierson D, Kader AA (I1986) Fruit ripening and quality. In JG to an overall increase in osmotic potential. Hence, there was Atherton, J Rudich, eds, The Tomato Crop-A Scientific Basis no simple relation between tissue osmotic potential and cell for Improvement. Chapman and Hall, London and New York, turgor potential during fruit ripening. If apoplastic solutes are pp 241-280 9. Huber DJ (1983) The role of cell wall hydrolases in fruit soften- present in these tissues, however, then this lack of relation ing. Hort Rev 5: 169-219 may not be surprising, since a change in partitioning ofsolutes 10. Hunt RWG (1987) Measuring Color. John Wiley & Sons, New between symplastic and apoplastic compartments could alter York, p 221 11. cell turgor without any change occurring in whole tissue Husken D, Steudle E, Zimmerman U (1978) Pressure probe technique for measuring water relations of cells in higher osmotic potential. If the initial decrease in whole tissue os- plants. Plant Physiol 61: 158-163 motic potential reflected osmotic changes in the symplasm, 12. Ogden TE, Citron MC, Pierantoni R (1978) The jet stream then it was large enough to easily account for the initial microbeveler: an inexpensive way to bevel ultra fine glass micropipettes. Science 201: 469-470 increase in turgor observed in this study. 13. Poovaiah BW, Glenn GM, Reddy ASN (1988) Calcium and fruit In experiment 1, the rate ofcolor change (ripening) in intact softening: physiology and biochemistry. Hort Rev 10: 107-152 fruits roughly corresponded to the average rate ofcolor change 14. Poovaiah BW (1986) Role of calcium in prolonging storage life in the discs (Fig. 1). However, individual fruits ripened inde- of fruits and vegetables. Food Tech 40: 86-89 15. Shackel KA, Matthews MA, Morrison JC (1987) Dynamic pendently, so that on the same date, color variation between relation between expansion and cellular turgor in growing fruits was higher than color variation within fruits. Independ- grape ( Vitis vinifera L.) leaves. Plant Physiol 84: 1166-1171 ent fruit ripening was also exhibited in experiment 2, and for 16. Sommer NF (1982) Postharvest handling practices and posthar- the vest diseases of fruit. Plant Dis 66: 357-364 three individual fruits that ripened normally during this 17. Steudle E, Wieneke J (1985) Changes in water relations and experiment, the order of fruit ripening was identical to the elastic properties of apple fruit cells during growth and devel- order of occurrence in the maximum observed fruit turgor opment. J Am Soc Hort Sci 110: 824-829 (Fig. 2). Since the maximum turgor in these fruits was exhib- 18. Wills RHH, McGlasson WB, Graham D, Lee TH, Hall EG (1989) Postharvest: An Introduction to the Physiology and ited 2 to 4 d before any change was apparent in fruit color, Handling of Fruit and Vegetables. AVI Publishing Co, these data suggest that changes in turgor may reflect physio- Westport, CT

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