:(1'.- A... t. ‘..|“L~ -. .....,:_. I“ "l¢-:.‘..r m. .0. u.“ Iii. I:.,‘3~u Wu 4 . . --... 1..-. -... 325...: KKI‘W“: lumizi . n.6- \ 9- m. 1 . .~-.:. “.3... .1. .23. -4“. u..\i....m:‘.‘1. 1.1:”. -u 5.... WM~“\-~..u.~‘~~§ M ”-1.4 d. Lwlute d cell walls 27 Caa'as Related to Softening Enzymes The role of Cab'in fruit softening and its interaction with the enzyme polygalacturonase have been proposed by many researchers (Poovaiah, 1979; Buescher et al., 1981; Rigney 8 Wills, 1981: Buescher 8 Hobson, 1982: Tang 8 McFeeters, 1983: Davies 8 Dennis, 1983: Konno»et al., 1984: Brady et al., 1985; McFeeters et al., 1985). Poovaiah (1979) has reported.that an increase in polygalacturonase activity was suppressed by Ca”. Ca”’ starvation, particularly caused an increase of polygalacturonase activity which occurred accompanied by an absolute decrease in amount of pectic polysaccharides in the cell walls (Konno et al., 1984). According to Brady et a1. (1985) cell wall uronic acids of a firm and soft cultivar were equally susceptible to hydrolysis, suggesting that differences in the digestion of the walls by polygalacturonase were dependent on differences in Ca”’ content or distribution. It appears that Caa‘ associated with the cell wall-middle lamella and its removal regulate the rate and extent of degradation by polygalacturonase (Buescher 8 Hobson, 1982). Ca” chloride in brines did not alter the activity of other enzymes as catalase and lipoxygenase, but it stabilized peroxidase activity (Buescher et a1. , 1987) . It has been reported by Wills 8 Rigney (1979) that the presence of high concentrations of Ca2+ reduced the activity of both pectin methyl est: breakdown c The me pectins by methyl este An inc Ca” ions fr between adj wall extens bonds must acidificatj EXtrusion f from Catior this mechan solubiliZat PiCklj of tine in 28 methyl esterase and polygalacturonase reducing the rate of breakdown of cell walls. I The mechanism responsible for retarding demethylation of pectins by Cab'is probably associated with hindering pectin methyl esterase action (Hudson 8 Buescher, 1986). An increase in H? ions could result in displacement of Cab'ions from.the cross-linking of galacturonic acid residues between adjacent pectic polymers. This is the same process of wall extension during cell growth in which existing cohesive bonds must be broken and new bonds formed depending on acidification of the apoplast. Acidification due to H“ extrusion from the protoplast results in a displacement of ca?“ from cation exchange sites (Rushing 8 Huber, 1984). Through this mechanism which is non enzymic an increase in polyuronide solubilization can occur. storing and Holding Cucumbers Pickling cucumbers are normally stored for long periods of time in brine solution. If, however, fresh fruits need to be stored or held before being processed or placed in brine solution, the cucumbers should. be cooled as rapidly as possible to 7‘ to 10’C (Motes, 1977). Post harvest holding of cucumbers before brining is detrimental to final salt-stock pickle quality (Lee et al., 1982). The optimum conditions for holding cucumbers, accordi 10 ' C, 13' days. removal high te: in chil Ue Provide that 5 '4 been re] green-s authors Maximum Pickles 20' am h°1ding Cd” ‘3 Re Pickled picklin thsiCa prOceSS Propert 29 according to Motes (1977) are temperatures between 7° and 10'C, below 90 to 95% relative humidity for no longer than 10 days. The cucumbers should be processed immediately after removal from storage. Storage and holding of fresh fruit at high temperatures after being removed from storage can result in chilling injury. Uebersax et a1. (1986) reported that common cold storage provided improved quality stability of processed spears, and that 5'C temperature did.not increase "chilling injury" as has been reported in previous studies. These conditions allow for green-stock storage for up to 10 days. According to the same authors, temperatures within the range of 21°C to 1'C provided maximum quality of processed spears. However, texture of pickles significantly decreased with increased holding time at 20' and 30'C, even though no significant differences for holding time were detected at 5‘C (Lee et al., 1982). Caz’ as Related to Texture of Pickles Retention of firmness during processing and storage of pickled cucumbers is a major quality consideration in the pickling industry (McFeeters et al., 1980). In addition, the physical and chemical structures of the cell walls of processed fruits are the main determinants of textural properties (McFeeters 8 Lovdal, 1987). Ca2+ has been associated with the loss of tissue firmness during 130: 1978: find McFeeters 1985: Bue Caz‘ firmness Fleming c maximized brines co after des 1980: Bue HOWa asSociate in freSh- especiall fresh‘Pélc Peri and intac were Exp Fleming e 30 during post harvest processing of cucumbers (Fleming et al., 1978: Hudson 8 Buescher, 1980: Buescher et al., 1981: Tang 8 McFeeters, 1983: Buescher 8 Hudson, 1984: McFeeters et al., 1985: Buescher 8 Burgin, 1988: McFeeters 8 Fleming, 1989). Caa'has been shown to be beneficial in assuring cucumber firmness retention (Buescher 8 Hudson, 1984: Hudson, 1984: Fleming et al., 1987: Fleming et al., 1988), which was maximized in pickles processed from fermentation and storage brines containing Cab'chloride and treated with Caa'chloride after desalting (Buescher et al., 1979: Hudson 8 Buescher, 1980: Buescher et al., 1981: Buescher 8 Burgin, 1988). Howard 8 Buescher (1990) have reported that firmness was associated with the amount of bound Caa'and the supply of Ca2+ in fresh-pack cucumbers. Firmness was improved by blanching, especially in Ca”’ chloride solution in fermented and fresh-pack pickles (Sistrunk 8 Kozup, 1982). Pericarp and locular tissues of pickles were much firmer and intact after 1 and 4 months in storage when the pickles were exposed to Caz" chloride (Hudson 8 Buescher, 1980) . Fleming et a1. (1988) have shown that the addition of Caz+ acetate to the cover brine served as a source of Cab'to help ensure cucumber firmness retention. It has been reported by several researchers (Fleming et al., 1978: Buescher et al., 1979: Buescher 8 Hudson, 1986) that cucumber firmness is maintained even at lower concentration of NaCl in the brine Solution, if Cab'salts are also presen avoids exce: 1988) . ca” and 801 Excess cucumbers a by P°lygalz texture by Cab increa: Present, a: Pickles We 9t al‘l IE 2+ even thong] C°uld no t The I: Contml wa (Jar-(’18, 31 also present. The addition of Ca2+ to the brine solution avoids excessive shriveling of the cucumbers (Fleming et a1. , 1988). Caz’ and Softening of Cucumbers Excessive Softening is a common problem in fermented cucumbers and is caused by solubilization of pectic substances by polygalacturonase during storage. However, breakdown of texture by PG is inhibited by Ca2+ (Sistrunk 8 Kozup, 1982) . Ca” increased fruit firmness even when high levels of PG were present, and the resistance to softening persisted when the pickles were transferred to solutions without Ca2+ (Buescher et a1. , 1981) . Low endogenous Caz” concentrations (2-3 mM Ca”) slowed softening of blanched cucumber mesocarp tissue, even though the endogenous Ca2+ concentration in the cucumbers could not be controlled (McFeeters 8 Fleming, 1989). The percentage of Cay-bound is clearly substantial to control water-soluble uronic acid polymers in the cell walls (Jarvis, 1982) , and the amount of bound Ca2+ is dependent on the degree of pectin methylation (Howard 8 Buescher, 1990). Demethylation is believed to change the configuration of pectin macromolecules which contributes to loosening of middle lamella-cell wall components and softening (Hudson 8 Buescher, 1986) . Galacturonans are rapidly deesterified during cucumber fermentation (Tang 8 McFeeters, 1983: Hudson 8 Buescher, 1985). H0“ during stOJ Buescher, 2 Ca” c2 and Ca” 0 provided 5 softening 1 appears to 0f pectin Pectins (H1 Howey. Netins ta: 1933) - Th: or gelatio by the act ions Dre Se brine (Van Utili potential (BUESche r as c‘ '1':- fine The p frequently 1979) ' C; 32 1985). However, Caa'chloride added in the brine of cucumbers during storage can retard demethylation of pectins (Hudson 8 Buescher, 1986). Caa'chloride, Caa'nitrate, Caa’acetate, Cab'hydroxide, and Ca2+ oxide added in the brine of processed cucumbers provided similar increases in firmness and prevention of softening by polygalacturonase (Buescher et al., 1981). Ca2+ appears to protect against tissue softening by cross-linking of pectin macromolecules, and by reducing demethylation of pectins (Hudson 8 Buescher, 1986). However, when PME is activated and demethylation of pectins takes place gives a firmer product (Tang 8 McFeeters, 1983). ’This firming effect is attributed to the crosslinking or gelation of the pectic substances which were demethylated by the action of the enzyme pectin methyl esterase with Ca”’ ions present in the tissue when Caa'chloride is added to the brine (Van Buren et al., 1962; Van Buren, 1968). Utilization of Cab’in fermentation brines provides the potential for reducing losses caused by pectinolytic softening (Buescher et al., 1981). Caa'Transport in Plants The problems that arise with Cab'nutrition in plants are frequently related to redistribution and mobility (Ferguson, 1979). Ca2+ is a highly immobile macronutrient in plants (Biddulph e' Along its 1 subcellular sites Withi] as membrane macromolecu vacuole (Be 1979). Ca2+ up roots. Hox more Comple ion‘3011cent (1976) has important I The U] paSSiVe ( c restricteC1 (KirkaI 19 largely in ClarkSOn ' Gaze . thEir 10w catiOns may way (Ferguc 33 (Biddulph et al., 1959: Ringoet et al., 1968: Hanger, 1979). Along its pathway Caz+ can be fixed through absorption by subcellular organelles, adsorbed onto cation ion exchange sites within the cell walls, bound to cellular structures such as membranes and other surfaces, bound to proteins and other macromolecules, or precipitated as insoluble salts within the vacuole (Bell.8 Biddulph, 1963: Shear'8 Faust, 1970; Ferguson, 1979). Caa'uptake is very much associated with water uptake by roots. However, within the plant the distribution is much more complex and is also dependent on hormonal effects and ion-concentration gradient effects (Kirkby, 1979). Bangerth (1976) has concluded that auxins.produced.by the seeds play an important role in the accumulation of Cab'within the fruits. The uptake of C3” by roots is generally thought to be passive (Christiansen 8 Foy, 1979), and appears to be restricted. to a region immediately' behind. the root tip (Kirkby, 1979). Uptake and movement across the root cortex is largely in the free space pathway, the apoplast (Ferguson 8 Clarkson, 1976: Ferguson, 1979). The apparent reluctance of Cab'ions to move in the symplast of the root may be due to their low activity in the cytoplasm where most divalent cations may be electrostatically bound or sequestered in some way (Ferguson 8 Clarkson, 1976). Caa'movement in.plants is unidirectional, moving up from the roots and generally routed to meristematic zones and young tissue (Ha accumulati stele in 5 But, Caz’ plant and plants whl translocat becomes 51 constitute especially inhibiting Poor mltr; Cab uptake 1979: Kir] Both environmel °°mmon1y sometimes contribut resulting generatio and watEJ moistlire also res“ duration ' 34 tissue (Hanger, 1979). Chino (1979) reported that little accumulation of Ca2+ is found in the boundary region of the stele in soybean plant and within the stele in corn plant. But, Caz’ is highly concentrated at the endodermis in corn plant and at the outer cell layers of the root in soybean plants where Ca“M is easily translocated. However, Caz+ translocation to the shoot is greatly reduced when endodermis becomes suberized (Ferguson 8 Clarkson, 1976) , because it constitutes a barrier to apoplastic movement (Ferguson, 1979) , especially if growth stops (Kirkby, 1979) . Any root growth inhibiting factor as low temperature, inadequate aeration, poor nutrient status, or H‘ ion concentration will restrict Caz" uptake and. hence also impair Ca2+ translocation (Jacobsen, 1979; Kirkby, 1979). Both uptake and transport processes are subject to environmental influences (Wiersum, 1979). Caz’ deficiency is commonly associated with water deficiencies, however, sometimes excess water as well as water fluctuation have been contributing factors (Geraldson, 1979). Excess water resulting in rapid leaching of Ca2+ from the root zone and/or generation of anaerobic conditions inhibiting uptake of Ca2+ and water induces Caz+ deficiency. Conversely, low soil moisture resulting in poor water movement through the plant also results in Ca” deficiency in crops. Other environmental factors which decrease Ca2+ levels are high light intensity and duration, and adverse temperature (Millaway 8 Wiersholm, 1979) . During and nutrien accumulatic immobility Bollard, photosynth; be suffici needs of (Hanger, Cellular a be a back gradient A150 area to tramSpire Caz“ 35 During rapid growth, the translocation of photosynthates and nutrients through the phloem increases rapidly. But, Ca2+ accumulation occurs at a relatively slow rate due to the immobility of Ca2+ in phloem tissues (Biddulph et al., 1959: Bollard, 1970: Marschner, 1974) . May be when the photosynthates moved into the organ by mass flow, there would be sufficient water delivered by the phloem to satisfy the needs of the 'tissue without any import through the xylem (Hanger, 1979) . In addition, if phloem water exceeded the cellular and transpirational needs of the tissue, there could be a back flow of water in the xylem down a water potential gradient (Zimmermann, 1966). Also, rapid growth causes a marked fall in the surface area to volume ratio, therefore water loss through transpiration by the fruit becomes quite low (Hanger, 1979) . Caz‘ is notoriously immobile in phloem tissues (Biddulph et al., 1959: Biddulph et al., 1961: Zimmermann, 1966: Ringoet et al., 1968: Bollard, 1970: Ferguson 8 Clarkson, 1976: Raven, 1977: Ferguson, 1979: Hanger, 1979: Marschner, 1986) . For several reasons (high pH and high phosphate concentration) the concentration of free Ca” in the phloem sap has to be very low (Raven, 1977) . The high phosphate levels together with a high phloem-sap pH of 8.0-8.2 have important consequences because, if Caz+ is also in high concentrations, insoluble Ca?-+ phosphate precipitates are formed and probably could interfere with the normal functioning of the sieve tubes (Hanger, 1979) . re- to 19 fr wk Ca 01 36 So, there is general agreement that, in order for the requirements of growing tissue to be met, most of the Cab'has to be translocated via the xylem into the tissue (Marschner, 1986). This phenomenon is of particular importance to developing fruit (Bollard, 1970). Fruits and seeds are often the sites where the results of Cab'immobility are manifested. The low Caa'content of these tissues is thought to arise because most of the material moving into developing fruits is carried by the phloem, particularly in the later stages of development (Ferguson, 1979). Most of the difficulty of Caa'to move in symplastic pathways is supported by the relative immobility of Cab'in the phloem, its slow rates of diffusion across layers of cells such as in fruit cortical tissue, and lack of redistribution from aging leaves to the other parts of the plant (Ferguson, 1979). The ratio of K‘ to Ca” in cucumber fruits is considerably higher'than.those for leaves, suggesting that these fruits are relatively Cab'deficient (Bollard, 1970). The deficiency of Ca” in low-transpiring tissues has been very well documented (Bell 8 Biddulph, 1963; Gerald 8 Hipp, 1968: Shear 8 Faust, 1971: Tibbitts 8 Palzkill, 1979) . According to Shear (1975) subsequent enlargement of the fruit dilutes the Cab'in the fruit, and excessive size may reduce the Ca” concentration below that necessary for normal cell £111 8 1 ac pi de me cc CC 37 functions. This dilution effect has been reported by Widders 8 Price, 1984: Engelkes, 1987: and.Enge1kes et al., 1990 to be accentuated through ontogeny, in which Caa'concentrations in pickling cucumber fruit tissues declined during fruit development. Hanger (1979) and.Marschner (1986) also found a negative correlation between the growth rate and the Ca2+ content of growing fruits. It has been known that under conditions of high transpiration, no water enters the fruits directly from the xylem so under these conditions no Caa'is supplied to the fruits (Bollard, 1970). High transpiration rates direct the xylem volume flow to the high transpiring leaves decreasing the Cay'influx into low transpiring organs as in the case of fruits. Inhibition of transpiration (by high relative humidity or during the dark period) usually favors the direction of the xylem volume flow toward low-transpiring organs. The rate of xylem flow from the roots to the shoots under’ conditions of low 'transpiration is determined by the root pressure (Marschner, 1986). Root pressures are known to develop in plants when transpiration is slowed to a rate that is less than the rate of water influx into the roots. As root pressure builds up, a positive pressure develops in the xylem causing the flow of liquid through the xylem ducts resulting in guttation from hydathodes (Tibbitts 8 Palzkill, 1979). Thus, to prevent a Cab'deficiency caused by a lack of root pressure, conditions must be altered to reduce transpiration and increase Cab'and water uptai Stron‘ Ca”) move transpirat gradients (Bell 8 B rooting me for the l< or(Jams, be decreases or fmits 1986), The through ‘ fl°“’v but ale. 196: diVaIEnt (232+ 38 water uptake by the roots (Geraldson, 1979). Strontium (which is equally immobile in the phloem as Ca”) moved into potato tubers only when the foliar transpiration rates were lowered and the water potential gradients were more favorable for water flow to the tubers (Bell 8 Biddulph, 1963). Thus, water availability in the rooting medium particularly during the dark.period is crucial for the long-distance transport of Caa’into low-transpiring organs, because high osmotic potential of the soil solution decreases both root pressure and Cab'influx into young leaves or fruits and induces Ca?” deficiency symptoms (Marschner, 1986). Increasing Caa'Mobility The upward movement of Caa’in the transpiration stream through the xylem shows that Ca2* ions do not move by mass flow, but by a series of exchange reactions along negatively charged sites on the walls of the xylem vessels (Biddulph et al., 1961). So, movement can be promoted by the presence of divalent cations and by chelation of the Ca” ion (Hanger, 1979). Sprays of Caa’salts may supply sufficient added Ca2+ to control Ca” deficiencies in leafy vegetables. However, the Ca” from sprays must move into the fruits through their surface, and only very limited quantities can be supplied in this way (Shear, 1975). 39 Chelators of Ca” in the sap reduces the degree of adsorption of Caa’to negatively charged sites within the cell wall, potentially promoting its mobility in the xylem (Bradfield, 1976). So, when the ionic charge of the Cafi'ion is neutralized by chelation with either synthetic (e.g. ethylene diamine tetra acetic acid) or natural (citric and malic acids) Chelators, Cab'is moved more freely through the plant (Hanger, 1972). Chapter I THE 1 AC' influe develo increa Conway (Poova I cucunn 0“ pa: quali- the :f fnut inhih or Pc by ex in E (Eng been deve whet THE RELATIONSHIP OF BNDOGBNOUB Caz’ AND POLYGALACTURONASE ACTIVITY WITH TBXTURAL QUALITY OF PICKLING CUCUMBERS Introduction Cam' is thought to play a physiological role in influencing fruit quality, besides improved growth and development, by reducing respiration (Faust 8 Shear,1972) , increasing fruit firmness (Cooper 8 Bangerth,1976: Sams 8 Conway,1984), and, delaying fruit ripening and senescence (Poovaiah,1979: Leshem et al.,1982). Limited research, however, has been conducted on pickling cucumbers to determine the effects of a low fruit Cab'status on parameters affecting quality for processing. An important quality parameter in fresh-pack pickled cucumber products is the firmness and integrity of both the green and processed fruit stock tissue. The strength of the cell wall structure , and the inhibition of the breakdown of pectic polymers during growth or post harvest handling (Rigney 8 Wills, 1981) are affected by endogenous Ca”) However, the endogenous Caa'concentration in pickling cucumber fruit decline during fruit ontogeny (Engelkes,1987). Although plant environment and.genotype have been shown to significantly affect the Ca”’ status of developing fruit (Engelkes et al.,1990) it was not known whether the variability in various fruit quality parameters 40 such as fr different f Caz’ tissue Ca” h intermolecu cohesion (H between po] major struc with the 1. (Ferguson, cell wall insoluble et al. ’ 19 progressix al. , 1979) that the ; walls Ceme component: integrity pOm'aiah I 41 such as fruit firmness observed among cultivars or from different fields might be ultimately due to excessively low Cab'tissue levels. Ca2+ has been shown. to Ibe important serving as an intermolecular adhesive in plant cell walls maintaining cell cohesion (Hanson, 1984) by acting as a cross-linking component between polygalacturonide chains (Davis 8 Dennis, 1983). The major structural change associated with softening is concerned with the loosening of the cell wall by loss of cell cohesion (Ferguson, 1984: Wills 8 Rigney, 1979). These changes in the cell wall have been accompanied by a conversion of the insoluble pectic substance to soluble pectic substance (Lampi et al., 1958: Mohsenin, 1970) and have been related with the progressive dissolution of the middle lamella (Ben-arie et al., 1979). It.has been reported.by Poovaiah 8 Leopold (1973) that the loss of Cah'was followed by solubilization of cell walls cementing substances and by hydrolysis of the structural components of the wall while cell cohesiveness and structural integrity of the cell wall were preserved by Ca2+ (Glenn 8 Poovaiah, 1990). Loss in firmness, however, has been associated with the activity of the cell wall-degrading enzymes (Dilley, 1970). Polygalacturonase (Pilnik 8 Voragen, 1970: Pressey 8 Avants, 1975: Ben-Arie 8 Sonego, 1979: Buescher et al., 1981: Tucker 8 Grierson, 1982), pectin methyl esterase (Bell et al., 1951: McFeeters et al., 1985: Hudson 8 Buescher, 1986), cellulase (Pesis e! Lazan et 1982) , aC glucanase in cell we shown to degradati 1939). Ca2+ enzyme p0 Wills, 19; et a1 , ' 19 in Plant c been a1 SO 42 (Pesis et al., 1978), fi-galactosidase (Konno et al., 1984: Lazan et al., 1989), fi-1,3-glucanase (Buescher 8 Hobson, 1982), acid phosphatase, peroxidase, xylanase, and fi(1,4)-D- glucanase (Jarvis, 1984) have been reported to be implicated in cell wall degradation. However, polygalacturonase has been shown to be the primary determinant of cell wall polyuronide degradation (Buescher 8 Hobson, 1982; Giovannoni et a1. , 1989). Cak'has been reported to have an interaction with the enzyme polygalacturonase (Buescher et al., 1981: Rigney 8 Wills, 1981: Davies 8 Dennis, 1983: Konno et al., 1984: Brady et a1. , 1985: McFeeters et a1. , 1985) suppressing its activity in plant cell walls (Poovaiah, 1979). Polygalacturonase has been also thought to be influenced by the level of endogenous Ca” in mature fruit undergoing ripening (Rigney 8 Wills, 1981). It has been shown by Brady et a1 (1985) that cell wall uronic acids of a firm. and soft cultivar' were equally susceptible to hydrolysis, suggesting that differences in the digestion of the walls by polygalacturonase were dependent on differences in Cafl'content or distribution. It appears that Ca” associated with the cell wall-middle lamella and its removal regulate ‘the rate {and extent. of’ degradation. by polygalacturonase (Buescher 8 Hobson, 1982) . Both, the increase in polyuronide solubilization and the loosening of' wall matrix via Caz’ removal could reduce the physical res: pot: enz: to I mac: pot« sof' t0 1 tis cue the act pic bar 1301 Ex; Jul pla Uni dul- 43 resistance encountered by the enzyme as it migrates to potential sites of hydrolysis, increasing the efficiency of enzymic activity (Rushing 8 Huber, 1984) . In fact, Ca“2+ appears to protect against tissue softening by cross-linking of pectin macromolecules (Hudson 8 Buescher, 1986) providing the potential for reducing losses caused by pectinolytic softening. The objectives of the present study, therefore, were: a) to evaluate the effect of modified endogenous Ca2+ within fruit tissues on texture of immature freshly harvested pickling cucumber fruit and after post harvest storage, b) to determine the influence of low Ca2+ levels in fruit tissues on the activity of polygalacturonase in freshly harvested and stored pickling cucumber fruit, and c) to verify the effect of post harvest storage duration and temperature on fruit firmness and polygalacturonase activity. Materials and Methods Three experiments were conducted in this study. Experiments 1 and 2 were carried out during April through July, and July through October, 1985, respectively, in the Plant Science Greenhouses at Michigan State University. Experiment 3 was conducted in the Fitotecnia Greenhouse at Universidade Federal do Parana in Brazil, South America, during the months of March through June of 1989. Plant plants (c (Experimer (Experimer two expel supplied < network oi in Experin by hand. medified Containin. 0.05 KCl, 0.0005 Hz- di"3t"11Ylen FertiliZQ solution At cultm:e s ExPeT-‘imer Experimer (Table 1' 44 Plant Culture. Pickling cucumber (Cucumis sativus L.) plants (cv.Castlepik 2012) were grown in sand culture (Experiments 1 and 2) and. in pure silica quartz stone (Experiment 3) in 8 liter plastic containers. In the first two experiments, the nutrient treatment solutions were supplied directly to the plants from a reservoir tank via a network of tubing and a timer controlled pumping system.while in Experiment 3 the nutrient treatment solutions were supplied by hand. Plants were irrigated daily until anthesis with a modified Hoagland nutrient solution (Johnson et al., 1957) containing (mM): 3.0 KNO3, 1.0 Ca(NO3)2, 1.0 NaHZPO‘, 0.5 M9804, 0.05 KCl, 0.025 H3303, 0.002 MnSO‘, 0.002 211804, 0.0005 C1180“, 0.0005 HZMoO‘, and 0.15 Fe-chelate (DTPA - disodium ferric diethylene triamine penta-acetate from Miller Chemical 8 Fertilizer Corporation). The pH of the nutrient culture solution was 5.0. At flowering, the Ca” concentrations in the nutrient culture solutions were modified (macronutrients) to contain in Experiments 1 and 2, 0.01, 1.0 and 20.0 mM Ca2+, and in Experiment 3, 0.00, 0.01, 0.1, 1.0, 10.0 and 20.0 mM Ca2+ (Table 1). These treatments were continued throughout the reproductive period so as to modify predominately the Caz“ nutrition of the developing fruit. Female flowers were hand- pollinated daily as soon as the flowers opened, from 10 a.m. until noon (Experiments 1 and 2), and from 9 a.m. until noon (Experiment 3). Average greenhouse temperatures ranged from Table 1 " Experimeni arui 2 Saltsz NaHZPO‘ Ca (N03) 2. 4r KNO3 HgSO‘ CaCl2 H9(No3)2 Hume3 \ Experiment \- Salts \ Nafi ZPO‘ HgSo‘ - 71120 “(N09 2- 4H. CECIZ , 21.120 KNO3 K01 NHNO k X nut a1" 1957 .r' 45 Table 1 - Composition of Caa’treatment solutions applied to pickling cucumber plants beginning at anthesis. Experiment 1 Concentration (mM) and 2 Cab’treatments (mM) Salts‘ 20.0 1.0 0.01 NaI-IZPO,‘ 1.0 1.0 1.0 Ca(NO3)2.4HZO 1.0 0.5 0.01 K1903 3.0 3.0 3.0 Mgso, 1.0 0.5 0.5 Cac12 19.0 0.5 - M9(N03)2 - 0.5 0.5 mam, 1.0 1.0 1.5 Experiment 3 Concentration (mM) Caa’treatments (mM) Salts 20.0 10.0 1.0 0.1 0.01 0.00 NaH 2P0, 1.0 1.0 1.0 1.0 1.0 1.0 MgSO‘.7H20 0.5 0.5 0.5 0.5 0.5 0.5 Ca(N03)2.4H20 10.0 5.0 1.0 0.1 0.01 - CaC12.2HZO 10.0 5.0 - - - - K1103 - 3.0 3.0 2.8 3.0 3.0 KCl 3.0 - - 0.2 - - 1111,1103 - 3.5 7.5 8.5 8.5 8.5 ‘ Micronutrient salt concentrations as specified by Johnson et al. , 1957. 23-30 (Expe: 25'C provi Sanpl diam: 100 1. of e frui‘ frui deio sap Stet Par1 imm. mea Fe:- “31 die tis 46 23-30'C during the day and 15-20'C during the night (Experiments liand 2), and from.25-32'C during the day and 20- 25'C at night (Experiment 3). No supplimental lighting was provided. Sample Collection In the experiment 1, an average of three 4.5 cm diameter fruits were collected from each plant. At harvest, 100 pl of the pedicel exudate from the excised fruit and 30 ul of extracellular solution from the endocarp region of the fruit were collected. To obtain extracellular solution, fruits were fractured without the use of a knife, rinsed with deionized water, gently blotted dry and the subsequent exuding sap collected with micropipettes. All exudate samples were stored at -20’C in plastic vials for Cab'analysis. Two cross-sectional 0.5 cm thick slices from the middle part of each fruit were measured for fruit firmness immediately after harvest, and the average of the two measurements used in the statistical analysis. Firmness of pericarp, mesocarp, and endocarp tissues (Fig; 1) was measured using an Instron Universal Machine with a probe of 32 mm in diameter, 20 cm/min. From the remaining portion of the individual fruits, approximately 25 g samples of pericarp tissue, fruit wall tissue excluding the carpel, (Frost 8 Kretchman, 1989) and endocarp tissue were collected, and place were and q analy plant post fresh Prevj greet Tis$t Caz‘t E 47 placed into plastic trays, and weighed fresh.(g). The samples were dried in a forced-air oven at 60'C for 72 hr, weighed, and ground in a mortar and pestle prior for tissue Ca2+ analysis. In Experiment 2, five fruits were collected from each plant. Individual fruit were subjected to the five following post harvest storage treatments prior to textural analysis: - 0 days - 3 days at 5°C plus 24 hr at 25°C - 5 days at 5'C plus 24 hr at 25‘C - 3 days at 25‘C - 5 days at 25'C Textural firmness of pericarp and endocarp tissues for freshly harvested and stored fruits were measured as previously described in the Experiment 1 (see Figure 1 for green stock tissue identification for texture evaluation). Tissue samples were also collected from individual fruits for Caa'analysis. For assay of polygalacturonase (PG) activity, samples (50 9 fr wt) of both pericarp and endocarp tissues (Experiment 1) and of endocarp tissue only.(20 9 fr wt; Experiment 2) were excised from each fruit following the post-harvest treatments. The fresh fruit was then frozen at -70°C (Experiment 1) or -20°C (Experiment 2) for approximately 4 weeks prior to analyses. GREE 48 GREEN STOCK TISSUE IDENTIFICATION FOR TEXTURE EVALUATION PERICARP ENDOCARP MESOCARP FIGURE 1. Illustration of Cucumber Tissue (0.5 cm cross-sectional slices) for cylindrical probe punch evaluation. harvc wrap All 1 fruii whicl firm: was 1 Shima the c For I sampl 22'c. CASan weigh. the 8 Were 1 oxidi: boilir Plate. added digeSt subsam; 49 In Experiment 3, one fruit, 4.5 cm diameter, was harvested from each plant and wrapped immediately in a plastic wrap to minimize loss of moisture, and to reduce respiration. A11 fruits were then stored at 1'C for 20-24 days. Although fruits were generally in excellent condition, those fruits which had fruit rot after storage were discarded. Fruit firmness of pericarp and endocarp tissus of the stored fruit was measured by an Autograph Model Apparatus - AG 5,000 A Shimadzu. A single, 0.5 cm thick cucumber slice was cut from the center of each cucumber fruit for firmness measurements. For polygalacturonase (PG) analysis, fresh endocarp tissue samples (20 g) were obtained from each fruit and stored at - 22'C. Caaanalysis Aliquots of dried ground tissue, 0.2 g dry wt, were weighed and placed into 50 ml volumetric flasks. Following the addition of 10 ml concentrated nitric acid, the flasks were topped with a marble and the tissues were permitted to oxidize overnight at 25'C. Oxidation was completed by gently boiling and.evaporating off the nitric acid.on an electric hot plate. Just before achieving dryness, hydrogen peroxide was added a drop at a time until the color disappeared. Tissue digests were then partially diluted with deionized water and subsamples stored in sealed 20 ml plastic vials at room temperatui achieve a were deter by atomic In El lanthanum (1000 pg concentra' Pericarp a 2380 atom P°1Y981 no The hydrolyti acid spectmph Shown to k nanomole resulting that flue cyclizati 50 temperature. LaCl3 was added as an internal standard to achieve a concentration of 1000 ug/ml. Ca” concentrations were determined in fruit tissue extracts and in fruit exudates by atomic absorption spectrophotometry (IL Video 12). In Experiment 3, strontium chloride (SrClz) instead of lanthanum chloride (LaCls) was added to all diluted solutions (1000 ug/ml) to ‘avoid background interference. Ca2+ concentrations in the unknown samples of both fruit tissues, pericarp and endocarp were determined by a Perkin-Elmer Model 2380 atomic absorption spectrophotometer. Polygalacturonase Assay The assay of polygalacturonase activity is based on the hydrolytic release of reducing groups from polygalacturonic acid using 2-cyanoacetamide which was analyzed spectrophotometrically (Gross, 1982). 2-cyanoacetamide has shown to be effective for spectrophotometric quantification of nanomole amounts of reducing carbohydrate in solution resulting in the formation of ultraviolet-absorbing products that fluoresce intensely after their direct condensation and cyclization with reducing sugars (Honda et al., 1982). Enzy Gros cont beer extr cucr was The plac cem the 14,1 ...] con‘ Con: azi. ma 1 fOr car wasi inc. eXt; 51 Enzyme extraction The extractability of polygalacturonase described by Gross (1982) was modified for cucumber fruit, because in contrast to the general behavior of pectic enzymes, it has been shown that cucumber polygalacturonase is readily extracted.by water (Pressey'8 Avants, 1975). Partially thawed cucumber tissues were placed in a beaker, and to each sample was added sodium chloride. to achieve a concentration of 0.2 M. The tissue with NaCl was homogenized using a tissumizer, and placed into culture tubes in an ice bath" The tissue was then centrifuged at 17,000 x g for 10 min. An aliquot (1.0 ml) of the clear supernatant was taken, and placed into a 12,000 to 14,000 MW cut off membrane (spectrapor) and a number of five samples were dialyzed in each 1,000 ml erlenmeyer with continuous stirring for 12 hr at 4'C. The dialysis solution consisted of 36 mM maleic acid, 0.33 M NaCl, and 0.02% sodium azide to avoid microbial growth. The pH was adjusted to and maintained at 6.2 with 1 N NaOH. The desalted extract was used for polygalacturonase assay. All extraction procedures were carried out at 4’C. Reaction mixtures (0.2 ml total volume) containing 37.5 mM Na-acetate (pH 4.4), 0.2% polygalacturonic acid (PGA), washed with 80% ethanol, and 30 ul of enzyme extract were incubated at 30‘C for up to 3 hr. Different amounts of enzyme extract were previously tested, and the amount which showed the best reducing terminat Then, 0. final v0 amiimme the reac placed 5 determi: Tr Amount from a linear; nanomo; I absorb statis l were were treat cgu e“do. fres Sigr 52 the best linearity was chosen. For quantifying released reducing groups with 2-cyanoacetamide, reactions were terminated with 1.0 m1 of cold 100 mM borate buffer (pH 9.0). Then, 0.2 m1 of 1% 2-cyanoacetamide was added, yieldind a final volume of 1.4 ml in each tube. The samples were mixed and immersed in a boiling water bath for 10 min. to terminate the reactions. After equilibration to 25°C, the samples were placed into quartz cuvettes, and the absorbance at 276 nm was determined using a Beckman spectrophotometer (Gross, 1982). Triplicates analysis were made of each sample extract. Amount (nanomoles) of galacturonic acid was then estimated from a standard curve in which absorbance at 276 nm was linearly related to galacturonic acid concentration up to 60 nanomoles. In Experiment 3, a Van Potter Homogenizer was used and absorbance was measured by a DMS 80 spectrophotometer. Statistical Analysis Randomized complete block designs with 8, 12 and 4 blocks were used in Experiments 1, 2, and 3, respectively. Plants were sorted in blocks according to size with 1 plant per treatment. Analysis of variance was conducted on fruit tissue Ca.” concentration, on firmness of pericarp, mesocarp, and endocarp tissues, on fruit exudates, and on PG activity of freshly harvested 4.5 cm diameter fruit. The Least Significance Difference (LSD) test.was used to compare means. Correlation analysis were conducted where appropriate. gr01 rep: tis.- har eXp whi 082* con f ro Ca2* app res 0f Whi lea Gog aff Pla D01 53 Results Plants in all 3 experiments exhibited vigorous vegetative growth prior to flowering. The Caa'treatments applied during reproductive development to the cucumber plants modified the tissue Caa’concentrations (dry weight basis) in the fruit at harvest in all experiments (Table 2). In experiment 1, pericarp tissue Ca2+ concentrations expressed.on.a¢dry weight.basis ranged from 0.21% to 0.95% Cab' while.Ca”'levels in endocarp tissue ranged from 0.08% to 0.21% Ca”. In both experiments (2 and 3) pericarp tissue Ca” concentrations ranged from approximately 0.1% to 1.8% Ca”, and from 0.08% to 2.4% Ca”, respectively, while endocarp tissue Caa’ concentrations which were much lower ranged from approximately 0.05% to 0.3% Ca”, and from 0.05% to 0.5% Ca”, respectively. The symptoms of Caa'deficiency did not appear until 2 to 3 weeks after the onset of fruit development. The deficiency of Caa'was first observed.in.the youngest leaves of the plants which were poorly supplied with Ca” (0.01 mM Caz‘) . The leaves became curled, and at more advanced stages necrosis occurred at the leaf margins. However, Ca?” treatments did not affect the rate of fruit growth. The period of time from planting through harvest varied from 43-50 days, and from pollination until harvest from 10 to 11 days. The nutrient Table 2 -1 Ca” Treatments ls: 54 Table 2 - Effect of Ca” treatments in the nutrient culture cucumber respectively. 1, 2, and solution on Caa’concentrations of fruit tissues and in exudates from excised pedicels and from the seed cavity region of fractured 4.5 cm diameter pickling fruit in experiments 3: Experiment 1 Cab' Tissue Caa'(% dry wt) Ca”(mM) Treatments (mM) pericarp endocarp Seed Cavity Pedicel 0.01 0.21 0.08 3.34 28.81 1.0 0.37 0.13 4.63 30.12 20.0 0.95 0.21 4.26 42.30 LSD (0.05) 0.13 0.047 NS 9.76 Experiment 2 Ca*' Tissue Cab'(% dry wt) Treatment (mM) Pericarp Endocarp 0.01 0.13 0.05 1.0 0.48 0.18 20.0 1.83 0.38 LSD (0.01) 0.16 0.05 Experiment 3 0.00 0.11 0.05 0.01 0.08 0.05 0.1 0.16 0.15 1.0 0.31 0.24 10.0 1.38 0.54 20.0 2.47 0.50 LSD (0.05) 0.81 0.35 NS nonsignificant culture a not the cavity. fruit ra treatmen' (extrace from 3.3 were fou both ped from the PiIllness 55 culture solution Ca2+ treatments affected pedicel exudate, but not the extracellular sap Caz’ concentrations from the seed cavity. Pedicel Ca2+ concentration was much higher than within fruit ranging from 28 to 42 mM Caz’ depending on the Ca2+ treatment while Ca2+ concentration in the apoplastic (extracellular sap) solution of fruit endocarp tissue ranged from 3.3 to 4.6 mM Caz“ (Table 2) . No significant correlations were found between fruit firmness and Ca” concentrations of both pedicel exudate and extracellular sap Ca2+ concentration from the endocarp tissue . Firmness of Fresh and Stored Fruit Firmness of pericarp, mesocarp, and endocarp tissues in fresh fruit was not affected by nutrient solution Caz“ concentration in Experiment 1 (Table 1, Appendix) . However, Caz’ treatments in the nutrient solution significantly affected the firmness of the pericarp tissue, but-not of endocarp tissue after 3 and 5 days of storage in Experiment 2 (Table 3) . The low Ca2+ treatment (0.01 mM Ca”) fruit showed the lowest firmness at 5 days of storage (0.62 Kg), suggesting a greater amount of softening. Consistent with this observation, the rate of softening in the pericarp tissue between 3 and 5 days of storage was also significantly affected by the Ca” treatments (Table 3) . Softening was Table 3 _ 082* Cone . (M) 0.01 20.0 LSD(0.05 I’Signf l . (NS). f. 56 Table 3 - Effect of Caa'tieatment level on firmness and on the rate of softening between 3 and 5 days of storage of 4.5 cm diameter pickling cucumber fruit tissues (Experiment 2). Fruit Firmness (kg) Rate of Softening Caa’ (daysq) Conc. (mM) Storage Period (days) 3 5 3 - 5 Pericarp Endocarp Pericarp Endocarp Pericarp Endocarp 0.01 0.77 0.18 0.62 0.13 0.030 0.010 1.0 0.81 0.18 0.67 0.15 0.028 0.006 20.0 0.74 0.16 0.70 0.14 0.008 0.004 Lso(0.05)z 0.052 NS 0.054 NS 0.014 NS ‘ Significant LSD at P<0.05 or the F value was non-significant (NS). Table 4 -‘ Storage Temp . - ('C) 2 5 ° F test Sit \— Fruit Tis: PeriCa pG ac‘vtivi (unit-5X10 * . I nonsigfiit 57 Table 4 - Influence of storage temperature and duration on firmness and PG activity of pickling cucumber fruit tissues in Experiment 2. Fruit Firmness (Kg) PG activity Storage (unitsx103/g) Temp. ('C) Pericarp Endocarp Endocarp tissue 5‘ 0.72 0.17 1.66 25' 0.71 0.15 1.98 F test sign. NS * *** Fruit Tissue Storage Period (days) LSD (5%) 0 3 5 Pericarp 0.93 0.77 0.65 0.030 Endocarp 0.19 0.17 0.14 0.010 PG activity 1.82 1.71 1.93 0.180 (unitsx103/g) ***, *, NS significant at the 0.1% level, 5% level and nonsignificant. 58 Figure 2. Correlation between Ca2+ concentration in pericarp tissue and the rate of softening of pericarp tissue between 3 and 5 days of storage of pickling cucumber fruit. 60 significantly higher in the lower Ca” treatments (0.01 and 1.0 mM Caz“) than in the higher Cal" treatment (20.0 mM Ca”) as shown in Table 3. Although a similar relationship between Ca2+ concentration in nutrient solution and rate of softening was observed in endocarp tissue, the difference between the means was not significant (Table 3). Ca2+ concentration in pericarp tissue was found to be negatively correlated (r=-0.43) with the rate of softening between 3 and 5 days of storage (Fig. 2). Also, a positive correlation was observed between fruit Caz‘ concentration (% dry weight) and fruit firmness after 5 days at 5°C (Fig. 3). Fruit firmness of the endocarp tissue was influenced by the storage temperature (5' vs. 25°C) showing a significantly lower firmness at 25'C (Table 4). But, storage temperature did not affect pericarp and endocarp firmness or rate of softening during 3 and 5 days of storage, even though softening continued to occur during that period of time (data not presented). However, a significant effect of storage period (0-5 days) on fruit firmness of both pericarp and endocarp tissues was observed (Table 4). Fruits stored at 1'0 for 24 days showed a positive correlation between the fruit Caz’ concentrations and fruit firmness (r=0.68, Fig. 4) compared to the correlation coefficient found for the same parameters after 5 days storage at 5'C (r=0.61, Fig. 3). wk t1 be 61 It was observed during the period of storage, especially when fruit were stored for 24 days, that the fruits grown at the higher Ca2+ concentration came out of storage in much better condition, exhibiting good color and without any visible symptoms of rot. PG Activity in Fresh and Stored Fruit The levels of enzyme activity (PG) found in the fresh fruit (Experiment 1) ranged from 0.96 to 1.29 units x 107/9 (Table 1, Appendix) while in the stored fruit the levels were observed to be higher ranging from 1.39 to 2.71 units x 104/g (data not shown), and from 0.92 to 2.92 units x 104/g (Fig. 7) in Experiments 2 and 3, respectively. Polygalacturonase (PG) activity in the fresh fruit tissues was not affected by the Cab’treatments in Experiment 1 (Table 1, Appendix). However, PG activity was negatively correlated (r=-0.46) with Caa'concentrations in the pericarp tissue of fresh.pickling cucumber fruit (Fig. 5). Endocarp PG activity was significantly higher at 25’C than at 5’C and increased significantly after 5 days of storage (Table 4). Consistent with these findings, a negative correlation was found between the endocarp tissue firmness and PG activity in the endocarp tissue after 3 days of storage (Fig. 6). 62 Figure 3. Correlation between Ca2+ concentration in endocarp tissue and fruit firmness of endocarp tissue of pickling cucumber fruit after 5 days of storage at 5°C. 63 md m ouswwm Es Ea .5 .028 :8 nd to 3 No ..o 0.0 «b _ t lib Lb lib I» b «t x. (b Aufiu.nv on so 9% m .33 18.0 a a 3 12.0 «q \wxhlq <4 \«‘\\...\\ d film—HO < dd < < \\.\<\«\4\4.<< a a 4 ..\\\\.\\\\\ < ION.O 4 . 8a 5.0 fl .. fimNd x cud + 9.0 n > 0nd (5M) ssanau mas 64 Figure 4. Correlation between Ca?’ concentration in pericarp tissue and firmness of pericarp tissue in the 24 days at 1'C stored pickling cucumber fruit. 65 O ov AV mammt 950:0 on o. Q Cm oocoo DO vaDMwE .N .5 0.. 0.0 0.0 .0 no mm. . m 0 V IIIIIIIIIIIIIIIII ... . A... v n. v c v v a mum... 0N W M * mm. W.N S + no»... u» on W 5 ( 66 Figure 5. Correlation between Ca2+ concentration in the pericarp tissue and polygalacturonase activity of 5.0 cm diameter fresh pickling cucumber fruit. 68 Figure 6. Correlation between polygalacturonase activity and fruit firmness in the endocarp tissue of pickling cucumber fruit after 3 days at S'C. 69 o muswam A t o of x mcza E254 on, . . 0.... ....N. Go to Q—N. .VpN .O—N. . . . . — . . . 0.0 on so 98 n .36 . mm ....o m. M o oo o o H o nllloo.||.|| m.I.I|..n~u..l...w0l o M 8 0 mo ll 1N0 N o 3 o S 8 ..nd \I x and .. u t W x Rod .. «No ...... r ‘2 o 70 Figure 7. Correlation between Ca2+ concentration in endocarp tissue and.polygalacturonase activity in the endocarp tissue in pickling cucumber fruit stored at 1°C for 24 days. Asi found bet activity < In t in the Cu the nutri Obtained range 01 Pickling in field to 0.7% ‘ weight i Pericarp fr°m 2.4 ranged f A c (From: & diStal 72 .A significant negative correlation (r=-0.58) also was found between Ca2+ concentration in endocarp tissue and PG activity of fruits stored at 1'C for 24 days (Fig. 7). Discussion In the present study, the endogenous Caa'concentration in the cucumber fruit was modified by the Ca” concentration in the nutrient solution. The range of tissue Caa'concentrations obtained in the fruit were quite broad in comparison with the range of concentrations typically found in field-grown pickling cucumbers (Engelkes et a1. , 1990) . Ca” concentrations in field produced 4-4.5 cm diameter cucumbers vary from 1.0% to 0.7% dry weight in pericarp tissue and 0.65% to ~0.2% dry weight in endocarp tissue. The Cab'concentrations found in pericarp tissue in Experiments 1, 2, and 3 (Table 2) ranged from 2.4% to 0.08% of dry weight while in the endocarp tissue ranged from 0.5% to 0.05% dry weight. A Ca2+ gradient typically exists within cucumber fruit (Frost & Kretchman, 1989) extending from the proximal to the distal end. The significantly lower endocarp tissue Ca2+ concentrations in comparison with the pericarp tissue Ca?“ concentrations (Table 2) might be related to a similar causal factor. There is general agreement that, in order for the requirements of growing fruit to be met, most of the Cab'has to be translocated via the xylem into the tissue (Marschner, 73 1986). The low endocarp vs. pericarp tissue Ca2+ concentrations could be in part due to a slow rate of import of Caa'via the xylem.into the endocarp as it.has been reported by Engelkes et al.(1990). The same authors have pointed out that the imported Cay'passes first through the pericarp which in turn would deplete the Caa'from the apoplastic solution entering the endocarp, the seed cavity of the cucumber fruit. Also, endocarp tissue may contain fewer vascular tissues than the jpericarp 'tissue (Engelkes, 1987). Pericarp ‘would. be supplied mostly by the xylem.while endocarp by the phloem. As Cab’is not readily translocated in phloem tissues the rate of Caa'import within the endocarp would be lower. Xylem Caz‘ concentrations of 2-3 ml! Caz‘ (White et al., 1981), 4.7 mM Cab'(Harschner, 1986), and 4 mM Ca”’(Wilcox et al., 1977) in herbaceous plants, and 4.5 mM Caz+ in apples (Bradfield, 1976) have been reported. The xylem exudate has also been measured in cucumber plants, and Caa'concentrations of 4 mM Cab'have been found (Engelkes et al., 1990). These results are similar to what it was observed in the present study in the extracellular sap collected from the endocarp tissue of the cucumber fruit (Table 2), thus confirming the apoplastic (extracellular) origin of this sap. However, values in the xylem exudate as high as 5.7 mM Ca2+ (control plants) and ranging from approximately 7 to 14 mM Ca” in field-grown cucumber plants sprayed with foliar fertilizers have been observed (unpublished data). 4 Pec vasculal concentl concentJ Nicotiaz concentl exudate respect: total e1 Mchen 4 rePOJ’.‘te< extent fruit (y Hanger! Caz‘ in . 74 Pedicel exudate, believed to have originated from phloem vascular tissues, was found to be extremely high in Ca2+ concentration (Table 2) . Marschner (1986) showed a Ca2+ concentration around 2.08 mM Cal“ in the phloem exudate of Nicotiana plants while Van Die & Tammes (1975) found concentrations as low as 0.25 and 0.3 mM Ca2+ in the phloem exudate of Yucca flaccida and Arenga saccharifera, respectively. In cucumber (Cucumis sativus L.) plants the total endogenous Caz“ found in phloem exudate as reported by Mchen et a1. (1981) was 0.08 mM Ca”. However, it has been reported that an accumulation of Caz" occurs to a very large extent in the pedicels while only limited amounts reach the fruit (Wieneke, 1969; Wieneke, 1974; Terblanche et al., 1979; Hanger, 1979) . Terblanche et a1. (1979) found values of 140 mM Ca” in the pedicels of apple tissues. According to them, Ca2+ is accumulated in the phloem parenchyma of the pedicel in the form of rectangular crystals. This might be a contributing factor to low fruit Caz’. However, the results from the present study are consistent with what Hanger (1979) reported: during the period when the Ca"!+ concentrations in the pedicel rose sharply, proportionally more Ca2+ entered the fruit (Experiment 1, Table 2). 75 Ca” and Fruit Firmness The role of Ca” in fruit firmness has been widely reported (Cooper & Bangerth, 1976; Badawi et al., 1981; Buescher et al., 1981; Sams & Conway, 1984; McFeeters et al., 1985; Abbott et al., 1989). However, in fresh pickling cucumber fruit, Ca2+ treatments were observed not to significantly affect the firmness of the various fruit tissues in Experiment 1 (Table 1, Appendix). Supporting this result Ca”’ ion concentration. in 'the :mesocarp tissue showed no significant correlation with tissue firmness of fresh cucumber fruit as McFeeters & Lovdal (1987) reported. Caa'has been associated with the regulation of ripening and senescence processes of fruits during post-harvest storage. Membrane structure and function, and. cell wall structure are thought to be involved in the action of Ca2+ (Ferguson, 1984). The role for Ca” in maintaining fruit firmness during post-harvest processing of cucumbers (Hudson & Buescher, 1980; Tang & McFeeters, 1983) has also been reported. However, the physiological processes involving Caz” during storage and post-harvest processing do not take place in freshly harvested fruit in the immature stage of development. For this reason, endogenous Cab'may not have a major role in influencing texture of fresh pickling cucumber fruit. 76 Alternatively, Caa’concentrations as low as 0.21% and 0.08% of dry weight in pericarp and endocarp tissues, respectively might be sufficient for maintaining structural integrity in the cell wall of fruit tissues (Table 2). The length of storage affects the physical properties of the fruits (Bourne, 1983). A decrease in firmness has been observed from 0 to 5 days of storage for both pericarp and endocarp fruit tissues (Table 4). The significantly higher firmness and lower rate of softening found in the pericarp tissue of the high Cab'treatment (20.0 mM Ca”) after 5 days of storage (Table 3) in Experiment 2 suggests that Cab'might have an important role as a firmness maintaining agent during storage. Such a role for Ca2+ in maintaining firmness of fruit tissue during storage has been reported for other fruit crops (Cooper & Bangerth, 1976; Badawi et al., 1981: El-Hammady et al., 1987; Abbott et al., 1989; Tomala & Sadowski, 1989). Observing Table 3 in Experiment 2, it can be noted that there was a large amount of variability in the rate of softening at any level of Ca2+ in the tissue. The same variability was observed in Fig. 2 indicating that other factors besides Caa’are probably also influencing the rate of pericarp softening. Anatomical characteristics of the cucumber mesocarp tissue have been analysed by Goffinet (1977) and the average cell number (density) has been found to have a significant relationship to texture. Cell density in the cucumber tissues at harvest could.be a factor influencing the 77 pericarp firmness during storage. Also, differences in turgor are largely responsible for the variations encountered in relation to rigidity and crispness of the plant tissue, considering that water is a structural component of the matrix gel (Bourne,l983). Water'content.of the fruits at harvest time can.also account at least in part for the variability observed in the rate of pericarp softening in Fig. 2. Temperatures during the storage period (3 to 5 days) can not be discounted as a factor having a role in the variability observed in Fig. 2, since the fruits in this study were stored either at 5'C or 25°C. Fruit softening during storage has long been associated to changes in the amount and.character of the cell wall middle lamella polysaccharides (Pilnik 8 Voragen, 1970). Van Buren (1979) reported that fruit softening is accompanied by increased pectic substance solubility during storage. Several enzymes have been identified as being capable of hydrolyzing the glycosidic, linkages of cell. wall polymers including principally polygalacturonases, fi-galactosidases, and cellulases (Pilnik &‘Voragen,l970). Thus, cell density, water content of fruits at harvest, different temperatures during the storage period, as well as the activity of softening enzymes are factors which.might be influencing in addition to endogenous Cab'the rate of pericarp softening (Table 3 and Fig. 2). Bourne (1983) reported that fruits soften at a slower rate in cool storage than at ambient temperature. Bourne (1982) also showed that fruit firmness decreases with 78 increasing temperatures over the range 0-45'C. The results reported here indicate that fruit firmness in the endocarp tissue was significantly lower at 25'C than at 5°C storage temperature (Table 4) . However, no significant differences in fruit firmness were found in relation to storage temperature for pericarp tissue. Perhaps, if the storage period had been extended for a longer period of time, greater differences in fruit tissue firmness might have been manifested between the storage temperature treatments. The positive correlations between fruit Ca2+ concentration (% dry weight basis) and fruit firmness (r=0.61) after 5 days of storage (Fig. 3) and firmness of fruits stored at 1°C for 24 days (r=0.68, Fig. 4) add support for endogenous fruit Ca?" having a role in fruit softening during storage. Increasing the Ca” content of fruit leads to an increase in firmness retention of fruits. Enhanced Cab’content was also reported to minimize fruit decay, perishability, loss in fruit moisture content, and. ultimately' extend. the storability’ of fruit (Tomala.& Sadowski, 1989). ‘Van Buren (1979) reported that the addition. of Ca”' to fruits increased their' firmness and decreased softening during storage. Fruit firmness was correlated positively with tissue Caa'concentrations (Lau et al., 1973; Fallahi.et al., 1985) both.before and after storage at 0'C (Sams & Conway, 1984) . The same results have been found by Facteau (1982). In contrast, low Caz“ levels in fruits have been associated with poor keeping quality during 79 storage (Bramlage et a1. , 1974) . Thus, considerable evidence has been presented that Ca” influences firmness in the fruit tissues during storage. Ca” and PG Activity The interaction of Caa’with the enzyme.polygalacturonase has been very well documented (Buescher & Hobson, 1982; Tang & McFeeters, 1983; McFeeters et al., 1985). The mechanism by which Cab'ions has been shown to be effective in suppressing polygalacturonase activity (Poovaiah, 1979) seems to involve the crosslinking of pectin molecules by eletrostatic interactions between adjacent negatively charged. carboxyl groups of the pectin (Van Buren, 1979) conferring strength to the cell walls, and protecting them from degradation by polygalacturonase (Buescher & Hobson, 1982). The range of polygalacturonase (PG) activity levels found in the present study were much higher (Table 1, Appendix; Fig. 7) in comparison with the levels of polygalacturonase found by Pressey & Avants (1975) for the small,medium,large and.mature cucumberS‘which were 0.29, 0.63, 0.87, and 1.56 units>x log/g, respectively. In the present study, PG activity was not affected by the Caa'treatments in freshly harvested pickling cucumber fruit (Table 1,.Appendix). It‘was noted though that the PG activity levels in these fresh cucumber fruits were relatively low due 80 to the fact that 5 cm fruit are quite immature and still rapidly growing prior to harvest. According to McFeeters & Lovdal (1987) , a positive correlation was observed between the concentration of total cell wall sugars and mesocarp tissue firmness of fresh cucumber fruit accounting for 73% of the firmness variation. This suggests that a physical characteristic such as tissue firmness may be influenced as much by the concentration of cell wall polysaccharides in a tissue as the detailed molecular structures of those polysaccharides. In addition, a decrease in rhamnose, arabinose, and galactose has been observed in fresh cucumbers during fruit development (Wallner,l978; McFeeters & Lovdal, 1987). Since these sugars are known to be associated with polysaccharides other than the pectin substances (McFeeters & Lovdal,l987), other enzymes than polygalacturonase might be involved in the softening process of fresh pickling cucumber fruit. Wallner (1978) has supported this idea suggesting that the removal of neutral sugar polymers which serve as cross- links could weaken the complex network and then contribute directly to a loss of fruit firmness. In addition, the modification of neutral sugar side chains may affect the activity of PG on the polyuronide main chains. Another interesting finding supporting the results in Table 1 (Appendix) is that the degree of pectin methylation in the mesocarp tissue of fresh cucumber fruit increases during fruit 81 development (McFeeters,l986; McFeeters 8 Lovdal,l987) . Pectin methylation decreases the degree of Ca2+ bonding (Ferguson, 1984) . A negative correlation, however, was observed between PG activity and fruit Caz” concentration (Fig. 5). No significant correlation was found with endocarp Ca"3+ concentration. Table 4 shows that a significantly higher PG activity was present in the fruit stored for 5 days at 25°C. The action of softening enzymes in stored fruits can be controlled by low temperature. Fresh fruits soften as much in an hour at 32°C as in a day at 10°C, or in a week at 0°C (Mohsenin, 1970). Endocarp tissue firmness appeared to decline with an increase in PG activity after 3 days of storage (Fig. 6) . The correlation (r=-0 . 58) between endogenous Caz” concentrations and PG activity in the endocarp tissue of fruits stored at 1°C for 24 days (Fig. 7) also adds support for endogenous Ca2+ having an influence on PG activity. Caz” binding and release from the cell wall-middle lamella appears to regulate the rate and extent of degradation by polygalacturonase (Buescher 8 Hobson, 1982). This effect is apparent at chilling temperatures (0°or 5°C) by the increase in pectin demethylation (McFeeters, 1986) . Subsequently, more free carboxyl groups of pectin will be available for Ca2+ cross-linking (Hanson,l984). While PG is the most widely described enzyme to be implicated in the fruit softening process, the variability 82 observed in the correlation shown in Fig. 7 and the complexity of the cell wall suggest that other enzymes might be involved. Caz“ has been reported to limit the loss of galactose suggesting an involvement of fi—galactosidase (Huber,l983) . McFeeters et a1. (1985) reported that the effectiveness of Ca“ in maintaining firmness at high levels of methylation in acid conditions suggests that other types of polysaccharide/Ca‘?+ interactions may have an important role, and that the ocurrence of CaZ*/galactose interactions in plant cell walls should be considered. In conclusion, endogenous Ca2+ in fruit tissue appears to have a role in causing more rapid softening during post harvest storage. However, the results are not conclusive. It is difficult to affirm that much of the variability in firmness found in commercially produced cucumbers can be attributed to low Ca2+ levels, considering the great differences observed between the range of Ca“ concentrations found in the fruit tissues from all experiments and the range normally found in pickling cucumbers grown in the field. Other factors such as fruit growth, fruit anatomy and water content of fruits at harvest, and the activity of softening enzymes other than polygalacturonase, such as ,6- galactosidase, might be influencing texture of the fresh pickling cucumber fruit during storage. As Ca” is widely recognized to have a role in regulating numerous physiological processes it is possible that it might be involved in several 83 of these factors. Future research is needed to verify if these factors are really important in improving the texture of fresh cucumbers, and what are the interactions between Ca” with them, and how they might be controlled. 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McFeeters, R.F., Fleming, H.P., and Thompson, R.L. 1985. Pectinesterase activity, pectin methylation, and texture changes during storage of blanched cucumber slices. J. Food Sci. 50(1): 201—205, 219. McFeeters, R.F., and Lovdal, L.A. 1987. Sugar composition of cucumber cell walls during fruit development. J. Food. Sci. 52(4):996-1001. Mohsenin,, N.W. 1970. Physical properties of plant and animal materials. Gordon and Breach Sci. Publ. , New York. 742 p. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 89 Pesis, E., Fuchs, Y., and Zanberman, G. 1978. Cellulase activity and fruit softening in avocado. Plant Physiol. 61:416-419. Pilnik, W., and Voragen, A.G.J. 1970. Pectic substances and other uronides. In: Biochimestry of Fruits and Their Products. Hulme, A.C. Vol. I. 53-81. Poovaiah, B.W. 1979. Role of calcium in ripening and senescense. Commun. Soil Sci. Plant Anal., 10(182):83-88. Poovaiah, B.W., and Leopold, A.C. 1973. Inhibition of abscission by calcium. Plant Physiol. 51:848-851. Pressey, R., and Avants, J.R. 1975. Cucumber polygalacturonase. J. Food Sci. 40:937-939. Rigney, C.J., and Wills, R.B.H. 1981. Calcium movement, a regulation factor’ in ‘the initiation of 'tomato fruit ripening. HortScience. 16(4):550-551. Rushing, J.W. , and Huber, P.J. 1984. In Vitro characterization of tomato fruit softening - the use of enzymically active cell walls. Plant Physiol. 75:891-894. Sams, C.E., and Conway, W.S. 1984. Effect of calcium infiltration of ethylene production, respiration rate, soluble polyuronide content and quality of cultivar Golden-Delicious Apple Malus domestica fruit. J. Am. Soc. Hortic. Sci. 109 (1):53-57. Tang, H.L., and.McFeeters, R.F. 1983. Relationships among cell wall contituents, calcium and texture during cucumber fermentation and storage. J. Food Sci. 48:66-70. Terblanche, J.H., Wooldridge, L.G., Hesebeck, I., and Joubert, M.1979. The redistribution and immobilisation of calcium in apple trees with special reference to bitter pit. Commun. Soil Sci. Plt. Anal. 10 (182):195- 215. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 90 Tomala, R., and Sadowski, A. 1989. Some factors determining quality and storage ability of Spartan apples I. Variation of different characteristics due to the season. Fruit Sci. Rep. 16(2):59-66. Tucker, G.A., and Grierson, D. 1982. Synthesis of polygalacturonase during tomato fruit ripening. Planta, 155:64-67 . Van Buren, J.P. 1979. The chemistry of texture in fruits and vegetables. J. Texture Studies, 10:1-23. Van die, J., and Tammes, P.M.L., 1975. Phloem exudation from monocotyledonous axes. In: Encyclopedia of Plant Physiology - Vol I. Transport in plants I. Zimmermann, M.H., Milburn, J.A. Springer-Verlag Berlin, Heidelberg, New York. 196-222. Wallnery S.J. 1978. Postharvest: structural integrity. Journal of Food Biochemistry 2:229-233. White, M.C., Decker, A.M., and Chaney, R.L. 1981. Metal complexation in xylem fluid. I. Chemical composition of tomato and soybean stem exudate. Plant Physiol. 67:292- 300. Wieneke, J. 1969. Calcium Translokation und atiologie der stippigkeit bein apfel. Erwerbsobstbau. 11:225-231. Wieneke, J. 1974. Untersuchungen zur translokation van “Ca im apfelbaum. III Ca-Auswaschung im Verlaufe der Vegetationsperiode. Gartaenbauwissenschaft. 39:161-171. Wilcox, G.E. Mitchell, C.A., and Hoff, J.E. 1977. Influence of nitrogen form on exudation rate, and ammonium, and cation composition of xylem exudate in tomato. J. Amer. Soc Hort. Sci. 102:192-196. Wills, R.B.H., and Rigney, C.J. 1979. Effect of calcium on activity of mitochondria and pectic enzymes isolated from tomato fruit. J. Food Biochem. 3:103-110. Chapter II INFLUENCE OF BNDOGBNOUB Caa'ON TEXTURAL QUALITY OF FRESH-PACK PROCESSED CUCUMBBRB Introduction Firmness is a critical parameter of quality of pickled cucumbers. Efforts have focused on maintaining the firmness of cucumbers during and after fruit processing. Caz” has been reported to be an important factor in maintaining the tissue firmness during processing of cucumbers (Hudson 8 BueScher, 1980: Tang 8 McFeeters, 1983; Buescher 8 Hudson, 1984; Buescher 8 Burgin, 1988: McFeeters 8 Fleming, 1989), especially after fresh-pack processing (Fleming at al., 1978: Buescher et al., 1981: McFeeters et al., 1985). The cucumber firmness retention was maximized in pickles by processing and storing in brines containing Cab'chloride or treating with Ca” chloride after desalting (Buescher et al., 1979: Buescher et al., 1981). Howard 8 Buescher (1990) have reported that firmness was associated with the amount of bound Caz“ and the supply of Ca2+ in fresh-pack cucumber pickles. The role of Cab’in maintaining the strength of the cell wall structure is believed to be through binding to the pectic polysaccharide matrix. Thus, the percentage of Ca”- bound is clearly substantial to control water-soluble uronic acid polymers in the cell walls (Jarvis, 1982), considering that a decline in the content of bound Caa'is associated with fruit maturation (Marschner, 1986) and with an increase in polygalacturonase activity (Poovaiah, 1979). 91 92 Demethylation is believed to change the configuration of pectin macromolecules which contributes to loosening of middle lamella-cell wall components and softening (Hudson 8 Buescher, 1986) . Although galacturonans are rapidly deesterified during pickle processing (Tang 8 MCFeeters, 1983: Hudson 8 Buescher, 1985) , Ca” maintained firmness, regardless of degree of pectin methylation in the tissue (McFeeters et al., 1985). It has been reported by McFeeters 8 Fleming (1989) that high endogenous Caz’ concentrations in the cucumbers are involved in maintaining fruit firmness in pieces of mesocarp tissue after blanching. In McFeeters's study, however, fruit were obtained from diverse sources and thus potentially variable in other physiological parameters. In light of these observations, the objectives of this study were: a) to modify the Caa'content of cucumber fruit in a culturally controlled manner in order to evaluate the influence of endogenous fruit tissue Ca2+ concentrations on fruit firmness prior and subsequent to fresh-pack.processing; b) to determine the role of endogenous Caa'concentrations on post-processing softening of cucumber fruit tissues during accelerated aging at 46°C: c) to verify the effect of Ca2+ added to the brine solution on tissue firmness after fresh- pack processing of pickling cucumber fruit: d) to evaluate the effects of refrigeration at 4.4'C on firmness of fresh-pack processed cucumber spears and slices. 93 Materials and Hethods Plant culture. Pickling cucumber (Cucumis sativus L.) plants (CV. Castlepik 2012) were cultured hydroponically in sand within plastic containers during the months of July through October of 1990 in the Plant Science Greenhouses at Michigan State University. The vines were grown upright on string, and the plants were irrigated daily with a modified Hoagland's nutrient solution containing the following nutrient concentrations (mM) until anthesis: 3.0 KNO3, 1.0 Ca(N03)2, 1.0 NaHzPO“ 0.5 MgSO‘, and micronutrients according to Johnson et a1. (1957). The Cay'concentrations in the nutrient culture solution. were :modified from. anthesis 'through. harvest 'to contain 0.01, 0.1, 1.0, 10.0, and 20.0 mM Caz’ (Table 1). Pistillate flowers were hand-pollinated daily as soon the flowers opened, from 9 a.m. until noon. The average greenhouse temperature ranged from 25 to 32°C during the day and 18 to 23°C during the night while the relative humidity ranged from 65 to 75% during the day and 75 to 85% during the night. Fruit sampling Approximately 11 days after pollination, 9 fruits per treatment were harvested when they attained a diameter of 4.0- ' 94 4.5 cm. Three slices, 0.7 cm thick were cut from the center of each cucumber. One was used for fresh fruit firmness analysis, while the other two were fresh-pack processed. The remaining halves were cut into 8 spear segments, with 2 spear segments being separated into pericarp and endocarp tissue for Ca” concentration determination, and the remaining 6 spear segments of each fruit fresh-pack processed together with the 2 slices. Leaf samples (lamina and petiole) were also collected from the fifth node from the apex of each plant. One combined sample was collected from five plants per treatment per block, and analysed for Ca2+ concentration for both lamina and petiole. The two half spears were separated into pericarp and endocarp tissue for Ca2+ concentration determination, placed into pre-weighed trays, and the fresh weight (g) of each tissue determined. The samples were dried in an oven at 60°C for 72 hr, weighed again, and ground in a mortar and pestle. Leaf tissues were ground in a Wiley mill to pass a 20-mesh screen a 95 Table 1 - Composition of Caa’treatment solutions applied to pickling cucumber plants beginning at anthesis. Concentration (mM) Saltsz Caa'treatments (mM) 20.0 10.0 ,1.0 0.1 0.01 NaI-IZPO‘ 1.0 1.0 1.0 1.0 1.0 MgSO‘ 1.0 1.0 0.5 0.5 0.5 CaClz 12.0 2.0 - - - KNO3 - - 6.0 5.3 6.0 KCl 6.0 6.0 - 0.2 - NH,N03 - - 2.0 3.0 3.0 Nam3 - - 3.0 3.0 3.0 ’ Micronutrient salt concentrations as specified by Johnson et a1. , 1957. 96 Ca2+ analysis Tissue samples of 0.1 g previously dried at 60°C from pericarp and endocarp tissues, respectively were weighed and put in labeled digestion tubes. Then, 0.5 ml of 30% hydrogen peroxide (H202) was added to each digestion tube, and the tubes were swirled gently. Subsequently, 1 ml of perchloric acid (HClO,) was added to each tube and small funnels were placed on top of the digestion tubes to act as a condenser. The tubes were heated in a Tecator digestor for 5 min. at 300°C in a perchloric acid.hood, .After cooling for 10-15 min., 1 m1 of 30% hydrogen peroxide was added to each tube and heated again on digestor for 30 min. without the funnels. The volume of the solution was made to 50 ml with deionized water, the sample was mixed and stored in a 20 ml plastic vial at 5°C. Following the same procedures in relation to Caz“ standard solutions and dilutions for the unknown samples of pericarp and endocarp tissues were done as in Chapter 1. The Ca” concentrations in the digests were determined by atomic absorption spectrophotometry using an IL Video 12 Model. 97 Fresh-Pack Processing and Textural Evaluation Six spear segments and two slices from each fruit were fresh-pack processed. The following treatment CaCl2 concentrations; 0.0, 5.0, and 20.0 mM Ca”, were applied to the brine solution prior to fresh-pack processing. In addition, post-processing softening of the spear and slice tissues was evaluated by measuring firmness at three times, 0, 5, and 20 days after incubation at 46°Ctto accelerate aging and thus the softening processes. Fruit firmness measurements of pericarp and endocarp tissue were conducted of fresh fruit, immediately after fresh- pack processing, and following two incubation periods at 46°C. Firmness measurements were also taken from all jars before refrigeration, and after 3 and 6 weeks refrigeration at 4.4°C. Illustration of cucumber segments used for green stock analyses and fresh-pack processed spears are shown in Fig. 1. Textural firmness was determined using the Kramer Shear Press, Model TMS-90, Food Technology Corp. located in the Food Science laboratory. .A PTA-300 (136.1 Kg) transducer with a speed from 0.503 to 0.549 cm/sec, and a 0% to 2% graph scale ranging from 0. N to 20. N was used. The maximum penetration force expressed in newtons was the only parameter used for firmness evaluation. One measurement for each tissue, pericarp and endocarp, was taken from each of the 6 spear segments and from the 2 slices 98 FRESH-PACK SPEAR TEXTURE STEM END FIGURE 1. Illustration of Cucumber Segments Used For Green Stock Analysis and Fresh-Pack Processed Spears. 1) Green Stock Texture, 0.7 cm Cross- Sectional Slice Prepared For Probe Analysis. 2) Green Stock Calcium Analysis Segments A and a. 3) Fresh-Pack Spears Segments B and b + C and c + D and d. 99 from each jar. The average was used for statistical analysis for either spears or slices. The cucumber fruits were processed at the Food Science laboratory according to the following procedure: From each fruit 6 half spears were packed in 12-oz jars (350ml), and covered with brine solution. The jars were loosely closed, and passed through a steam (86°C) tunnel line for 5 minutes to blanch the fruit. After processing the jars were tightly closed.and.boiled.in water for'10 min., cooled in tap water, and held at 24°C for 4 days for equilibration. Occasionally, the jars were inverted to help assure equilibration of soluble components (McFeeters et al., 1989). The brine contained 1.5 M NaCl, 0.2 M acetic acid and 4 mM 802, added as sodium bisulfite. Ca” was added to the cover brines as Caz‘ chloride (CaClz) to achieve 5.0 and 20.0 mM Ca2+ brine treatments. Measurements of the pH of brines after equilibration with cucumber tissue were conducted using an Fisher Accumet Model 810 pH meter with a combination electrode. Following 4 days at 24°C either fruit firmness measurements were taken from the spear and slice tissues, or firmness measurements were taken after 5 and 20 days incubation at 46°C. The jars were equilibrated at room temperature before firmness measurement. 100 Statistical Analysis A randomized complete block design with 4 blocks was arranged by plant size, with 5 treatments per block, and 5 plants per treatment. 9 fruits were analysed per treatment, with 45 fruits per block, totaling 180 fruits for the entire experiment. For the analysis from the fresh-pack processed slices, 4 an° treatments (20.0, 10.0, 1.0, 0.1 mM Ca”), and 2 incubation periods at 46°C (5 and 20 days) were evaluated. Analysis of variance using a RCBD with 1 factor (Ca2+ concentration) were conducted on Caa’concentration of fruit and leaves, and firmness of fresh pickling cucumber fruit tissues. Analysis of variance was conducted to evaluate the potential treatment effects on texture and the rate of tissue softening of pickle fruit tissues following fresh-pack processing, during accelerated aging and refrigeration. The effect of Ca” concentration in brine solution on fruit firmness and rate of tissue softening of fresh-pack processed spears and slices during accelerated aging and refrigeration 'was also investigated. The means were compared using the LSD test. Correlation analysis were also investigated between the dependent variables; pericarp and endocarp endogenous Ca”, fruit tissue firmness, and rate of tissue softening, following fresh-pack 101 processing, during accelerated aging, and during refrigeration of processed spears and slices. The relationships between the dependent variables were investigated either by linear regression (tissue Caz’ x fruit Firmness) or by non-linear regression (tissue Ca2+ x tissue softening rates) analysis. Results Plant growth was vigorous with no stress symptoms observed on the foliage. The period of fruit growth, from pollination through harvest varied between 10 and 11 days with Ca2+ treatments having no effect. Ca2+ treatments in the nutrient culture solution applied to the plants at anthesis significantly modified the fruit and leaf endogenous Ca2+ concentrations (Table 2). Pericarp tissue Ca2+ concentrations expressed on a dry weight basis ranged from 0.46 to 1.15 % Caz“ while Ca2+ levels in endocarp tissue ranged from 0.15 to 0.26 % Ca”. Ca2+ concentrations in the petiole tissue ranged from 0.99 to 3.86 % dry weight while Caz" levels in the lamina tissue ranged from 2.14 to 5.14 % (Table 2). Firmness of both pericarp and endocarp tissues in freshly harvested cucumber fruit, however, were not affected by the Caz+ treatments (Table 2) . 102 Table 2 - Influence of Caa'concentration in nutrient culture solution on fruit and leaf tissue Ca2+ concentrations and on tissue firmness of fresh pickling cucumber fruit. Caa' Cab’concentration (% dry wt) Fruit Firmness Conc. Fruit Leaf (newtons) (mM) pericarp endocarp petiole lamina pericarp endocarp 0.01 0.48 0.15 0.99 2.14 16.9 4.4 0.1 0.46 0.16 1.02 1.87 17.2 4.2 1.0 0.56 0.18 1.59 2.81 17.9 4.6 10.0 0.95 0.25 2.99 5.24 17.5 4.4 20.0 1.15 0.26 3.86 5.14 17.3 4.5 LSD(0.01) 0.09 0.02 0.44 1.53 NS NS NS nonsignificant 103 Incubation of Fresh-Pack Processed Spears The brine pH after equilibration with the cucumber spears and slices was 2.5 in 0 mM Ca, 3.0 in 5 mM Ca, and 3.2 in 20 mM Cab'processing brine solution. In fresh-pack processed spears, the Ca2+ fertilization treatments had a significant effect on pericarp tissue firmness immediately following processing. This firming effect on pericarp tissue was still apparent even when averaged over all Cay'brine treatments (Table 3). The pericarp tissue firmness was significantly higher in plants fertilized with 20 mM Cab'as compared to the 0.01 mM Cab'treatment. Significantly higher firmness was also found in the pericarp tissue of plants fertilized with 20 and 10 mM Ca2+ in relation to the 0.1 and 0.01 mM Caz’ treatments following fresh-pack processing. Consistent with these results endogenous Cab'concentration in the pericarp tissue expressed as mM was positively correlated with pericarp tissue firmness following fresh-pack processing and (Fig. 2). No differences were found in endocarp tissue firmness as a result of the Caz+ treatment in the nutrient culture solution in freshly processed pickles. However, endogenous Ca2+ concentration in endocarp tissue, expressed as mM, showed a 104 Figure 2. Correlation between Ca2+ concentration (mM) in pericarp tissue and firmness of pericarp tissue following fresh-pack processing at 46°C of cucumber spears in O‘mM Caa'brine solution. 2105 N ouswflm 92.5 momm: am room (SN01M3N) ssanaL-J aux/0133a 106 Figure 3. Correlation between Cab'concentration (mM) in endocarp tissue and firmness of endocarp tissue after 5 days incubation at 46 ° C of cucumber spears in 0 mM Cab'brine solution. 107 0.1.5 momm: am woe (SNOlMEIN) ssanaL-i davoooNa 108 Table 3 - Effect of Ca” concentration in nutrient culture solution on firmness and on the rate of pericarp softening of fresh- ack processed cucumber spears averaged over all Ca+ brine treatments and in 0 mM Caa'brine during incubation at 46°C. Ca2+ Tissue Firmness (newtons) Rate of Softening (newtons/day) Conc. (mM) Pericarp Pericarp Incubation Time (days) 0-5 5-15 0 5 20 days days 0.01 10.26 4.69 2.93 1.11 0.12 0.1 10.90 6.10 2.88 0.96 0.21 1.0 12.85 6.38 2.95 1.29 0.23 10.0 12.68 5.90 3.31 1.35 0.17 20.0 13.86 5.55 3.33 1.66 0.14 LSD(0.05) 3.16 NS NS 0.35 NS 0 mM Caa'brine 0.01 7.07 2.45 2.68 0.92 -0.02 0.1 6.79 2.48 2.06 0.86 0.03 1.0 9.93 2.26 2.07 1.53 0.02 10.0 10.94 2.90 2.54 1.60 0.03 20.0 13.55 2.84 2.50 2.14 0.03 LSD(0.01) 4.60 NS NS 0.91 NS NS nonsignificant 109 positive correlation with endocarp tissue firmness after 5 days incubation at 46°C (Fig. 3). Cab'treatments did not affect the pericarp firmness after 5 and 20 days incubation (Table 3). Following accelerated aging at 46°C for 5 or 20 days, the Cab'firming effect was lost due to the large amount of softening of spears from all treatments (Table 3). The high levels (10 and 20 mM) of Ca2+ concentration in the nutrient solution showed the higher rates of pericarp softening after 5 days incubation at 46°C when averaged over all Ca2+ brine treatments (Table 3) . Higher rates of softening were also observed in the higher Caa'treatments after 5 days incubation in pericarp tissue in 0 mM Ca?” brine solution (Table 3). Consistent. with these results, the rate of' pericarp softening after 5 days incubation at 46°C appears to increase as the endogenous Caa'concentration increased in the pericarp tissue up to approximately' 8 mM (Fig. 4). At higher endogenous tissue Caa'levels, the rate of fruit softening is relatively constant (Fig. 4) . The Caz" treatments did not affect the rate of softening of pericarp tissue between 5 and 15 days incubation at 46°C (Table 3). No interaction was found between the effects of Ca” concentration in the nutrient solution and Caa’concentration within the brine solution on fruit tissue firmness or on the rate of softening either following fresh-pack processing or Figure 4 . 110 Correlation between endogenous Ca2+ concentration and the rate of softening of pericarp tissue in spears and slices of fresh- pack processed pickling cucumber in 0 mM Cab brine solution during 5 days incubation at 46'C. 111 3.5 :5 55.5.”. a ouswfim 0.0. 0:: 0.9 0.0— 0.0 0.0 0.... 0.N 0.0 . O-O p L . p . _ . _ p _ . h 0.0 . nu lllll Ill § NQd l " ... / . /m 80% 1o 0 o/ r / [No /o e cozobaofi mxoo 0 cute 10.0 2.0.0 + 3-x n00 n > . 0.0 . . .\ a. one u ._ \\ a I . . .\ mmfiam o _ U a \\ ‘4 .. IIIJPIIII.lllw.lllunlll.|||\.\\\\ d. d. ulny.mu d 4. u 4 532.305 mxoo m ..oto 10.0 «N I X050 + «xenodl n > (AVO/SNOlMElN) 0NlN3L-IOS .40 31w (AVG/SNOIMBN) ONINBlJOS 90 31w 112 accowuficmwmcoc mz mz mo.o mo.o mn.o om.o Hm.o m2 mn.o «H.H nm.a Amo.ocomq No.0 00.0 0v.0 mh.0 av.m mb.N vm.n ab.n «H.HH 00.vH 0m H0.0 NN.0 no.0 00.H mo.~ ¢H.~ m~.n m0.m 0¢.n mh.HH m «0.0 0H.0 No.0 H¢.H mb.d 0N.N 0H.m hm.N mm.~ 00.0 0 taco ammo name name om m a on m o maum mic maum mno finance mane coauaaoocH mucooocm muoofluom Quoooocm QuoOwuom A250 .ocoo Afm>sov ocficmuuom no ovum aazv mcou30cv unmcahwm mounds .8oO .O.ov us mcwmc omumuoaoooo vowuso museum monasoso ooumoooua_xoomicmouu mo mcficmuuom momma» mo cums on» so can unocsuwu so co«us~om downs 0:» ca cofiumuucoocoo.&mo mo uoouum I e dance 113 during 5 or 20 days of accelerated aging at 46°C in processed spears. The addition of Cay'to the processing brine solution of 5 and 20 mM Caa'enhanced the post-processing firmness of both pericarp and endocarp tissues regardless of whether the spears were incubated.at high temperature for'5 or 20¢days (Table 4). The 20 mM Caa'brine treatment showed a significantly higher pericarp firmness following fresh-pack processing, and a significantly higher pericarp and endocarp firmness during accelerated aging as compared to the 0 and 5 mM Caz“ brine treatment. Spears with 5 mM Cab’showed significantly higher pericarp firmness following fresh-pack and after 20 days incubation in pericarp and endocarp tissues than the 0 mM Caa' treatment. The Ca‘2+ brine treatments had no effect on fruit firmness of endocarp tissue following fresh-pack processing (Table 4). The firmness retention due to Cab'was much larger in pericarp than in endocarp tissue. The effect of Caa'addition to the brine solution on the rate of softening of fruit tissues aftem's days incubation and between 5 and 15 days incubation at 46°C is also shown in Table 4. Spears with 20 mM Caa'added to the brine solution showed significantly lower rates of pericarp softening after 5 days incubation at 46°C than the spears with.0 and 5 mM Ca2+ in the processing brine. In contrast, higher pericarp softening was observed in the spears with 20 mM Caa'added to Table 5 - Effect of Caa'concentration.in the nutrient culture solution.on.pericarp firmness of unincubated fresh- pack processed cucumber spears averaged over all Cah'brine treatments and in 0 mM Caa'brine during refrigeration at 4.4°C. 114 Ca”' Tissue Firmness (newtons) Conc. (mM) Pericarp Refrigeration Time (weeks) 0 3 6 0.01 10.26 11.25 9.64 0.1 10.90 11.61 10.61 1.0 12.85 13.85 11.38 10.0 12.68 12.63 10.77 20.0 13.86 13.93 12.47 LSD(0.05) 2.37 2.24 NS 0 mM Cab'brine 0.01 7.07 7.83 7.64 0.1 6.79 7.48 6.67 1.0 9.93 11.06 8.36 10.0 10.94 10.55 8.53 20.0 13.55 13.04 11.38 LSD(0.05) 3.28 4.05 NS NS nonsignificant Figure 5. 115 Correlation between Cab'concentration (mM) in pericarp tissue and firmness of pericarp tissue of ‘unincubated fresh-pack. processed cucumber spears in 0 mM Cab'brine after 3 and 6 weeks refrigeration at 4.4°C. PERICARP FIRMNESS (NEWWONS) PERICARP FIRMNESS (NEWTONS) I116 20",? Y = 5.21 + 0.50 x ° 7 15.0-1 r = 0.625 / ‘ / 12.0- o o/ / .. o . o oo //’6 o 3.0-'- ,/-°°’ °° ° ° : o . o 4.0: ° 2 3 weeks refrigeration 20.0 J . Y = 4.66 + 0.40 X 15.0: r = 0.51! A . A / 12.0- A A// A AA // a 0 ' A A// - " A / u ’/ A AA 2 AA AA 4.0- A A : 6 weeks refrigeration 0. 0 . r I r , 0. 0 5. 0 10. 0 15.0 20.0 Co2+ in Pericarp Tissue (mM) Figure 5 117 the brine solution in comparison with the spears in 0 or 5 mM Caa’between 5 and 15 days incubation at 46°C. No significant effect of Cab'concentration in the brine solution was found on the rate of endocarp softening during the same period of incubation (Table 4) . Refrigeration of Fresh-Pack Processed Spears Unincubated Fresh-Pack Processed Spears Ca2+ in the nutrient culture solution maintained the pericarp tissue texture during refrigeration of fresh-pack processed spears. The textural effect within the pericarp tissue was extended up to 3 weeks refrigeration at 4.4°C in both 0 mM Ca” brine and when averaged over all Caz“ brine treatments (Table 5). After 3 weeks refrigeration at 4.4°C, firmness was significantly higher in the 1 and 20 mM Ca2+ treatments as compared to the 0.01 and to the 0.1 and 0.01 mM Caz" treatments, respectively. No significant effect of Ca2+ treatments on pericarp tissue firmness was observed after 6 weeks refrigeration either averaged over all Cab" brine treatments or in.0 mM Cab'brine (Table 5). Cab'treatments had no effect on endocarp tissue texture during refrigeration. In 0 mM Cab' brine solution, the effects of Ca2+ fertilization treatments on pericarp texture at 0 and 3 weeks of refrigeration were more pronounced. Refrigeration maintained firmness of the 20 mM Cab'treatment in pericarp 118 tissue until 3 weeks as compared to the 0.1 and 0.01 mM Ca2+ treatments (Table 5). Consistent with these results, positive correlations were found between the pericarp Cab'concentration and pericarp firmness of fresh-pack processed spears when no Caa'was added to the brine solution after 3 weeks refrigeration at 4.4°C (Fig. 5). In addition, a positive correlation was observed between the endogenous pericarp Ca2+ concentration and pericarp tissue firmness of fresh-pack processed spears in 0 mM Ca2+ brine solution after 6 weeks refrigeration at 4.4°C (Fig. 5), even though no response in firmness was found to nutrient solution Cab'treatments. The Ca2+ fertilization treatments had no effect on the rate of tissue softening of unincubated fresh-pack processed spears either averaged over all Cab'brine treatments or in 0 mM Ca2+ brine solution during refrigeration at 4.4°C. 5 and 20 Days Incubated Spears Refrigeration had a significant effect on pericarp tissue texture in 5 days incubated processed spears after 3 weeks refrigeration when no Cay’was added to the brine solution (Table 6). No differences in tissue texture were found due to refrigeration for 20 days incubated processed spears. The 20 mM Ca2+ treatment showed a significantly higher firmness within the pericarp tissue than the 0.1 and 0.01 mM 119 Table 6 - Effect of Ca” concentration in nutrient culture solution on fruit firmness of 5 days incubated processed cucumber spears in 0 mM Caa' brine solution during refrigeration at 4.4°C. Tissue Firmness (newtons) Ca” Conc. Pericarp Endocarp (mM) 0 I Refrigeration T1me (weeks) 0 3 6 0 3 6 0.01 2.45 2.42 2.70 1.76 1.85 1.90 0.1 2.48 2.50 2.77 2.09 2.09 2.18 1.0 2.25 3.20 3.07 1.83 2.15 2.28 10.0 2.90 3.13 2.61 2.81 2.25 2.16 20.0 2.84 3.50 3.17 2.88 2.82 2.24 LSD(0.05) NS 0.81 NS 0.47 NS NS NS nonsignificant 120 Ca2+ treatments in 5 days incubated spears after 3 weeks refrigeration (Table 6). No effect of Ca2+ fertilization treatments on pericarp firmness was observed after 6 weeks refrigeration in 5 days incubated fresh-pack processed spears. The firming effect of 10 and 20 mM Caz" treatments as compared to the 1.0, 0.1, and 0.01 mM Ca” treatments on endocarp tissue texture before refrigeration in 5 days incubated processed spears was not extended after either 3 or 6 weeks refrigeration (Table 6). Ca2+ treatments in the nutrient solution had no significant effect on the rate of tissue softening of either 5 or 20 days incubated fresh-pack processed spears after 3 weeks refrigeration or between 3 and 6 weeks refrigeration. No interaction was found between the effects of Ca” concentration in the nutrient solution and Caa'concentration within the brine solution on fruit firmness or on the rate of softening of fresh-pack processed spears during refrigeration at 4.4°C. The effect of refrigeration on fruit firmness of 0, 5, and 20 days incubated fresh-pack processed spears when Ca2+ was added to the brine solution is shown in'Table 7. Regardless of storage time the Ca2+ concentrations in the brine solution affected firmness similarly. When 5 and 20 mM Cab'was added to the brine solution, a significantly higher pericarp firmness in the spears was obServed as compared to the spears 121 ucoofluficmfimcoc mz ea.e m2 ma.c m2 m2 mm.o oe.o mm.o Hm.o mm.c Ado.ocomq Hm.0 «0.0 HN.0 m0.0l N¢.H hm.m 0m.~ 0n.n em.m mb.n 0N NH.0 no.0 0m.0 ~H.0 m¢.H hm.d m0.~ HN.N Hm.m 0H.n m H0.0 00.0 no.0! NH.0 hm.H H0.H mh.a NH.~ N0.N hn.m 0 name on mH.0 mH.0 0a.0 0N.0 vn.0 mz Hm.0 0H-H mm.0 NH.H AH0.000mA H0.0I nH.0 m0.0l «H.0 0n.~ 0m.m me.m n0.HH eh.0a mH.HH 0N hH.0 00.0! -.0 hH.0I m0.H 0¢.~ «H.N NN.n om.m mn.n m no.0 H0.0 no.0 NH.0I mH.N mm.~ 0N.N 00.N mm.~ 0m.~ 0 when m 02 m~.0 02 02 nm.0 em.0 m2 mh.m ~n.~ mm.H Ado.ovomq no.0 50.0: o¢.0 mN.0I 0H.n ev.n ¢~.n ¢~.eH «0.0H 00.vH 0N no.0 em.0 «v.0 0H.0I mv.N vm.m 0N.m ha.0a nn.NH mn.aa m va.0 bN.0 0m.0 HH.0I om.H hm.~ ha.n mm.m mm.m m0.m 0 must 0 mln «I0 win nno 0 m 0 0 n 0 Amxmosv mafia coaunuomfinumm Anxomsv mafia sofiucuomfiumom Arse nuoooosm mucofiuom Quoooocm mucoHuom .ocoo :8 Aaoo\ncouzocv mcwcouuom mo oucm Amcouaocv unocaufim osmmwa .O.e.v um sowunuomfiummu ocauso museum nonesoso commoooum.xoomlnmmuu venomous“ m>np on can .m .0 ca 0ch0umom mammflu. HO GUM“ CO GEM mmmCEHHM UHDHM CO COMHflHUCGUCOU mCflHQ +~MU HO Homuwm l N. GHQMB 122 in 5 mM Caz‘ or without added Ca2+ (Table 7). This firming effect was maintained for 3 and 6 weeks by refrigeration when 20 mM Cab’was added. In addition, processed spears in 20 mM Caa'brine solution exhibited a significantly higher endocarp firmness as compared to the 0 and.5 mM Caa'brine solutions in unincubated spears (Table 7). Significantly lower rates of softening were observed in the endocarp tissue of unincubated spears when 20 mM Caa'was added to the processing brine. The same effect was observed for both pericarp and endocarp tissue in 5 days incubated as compared to 0 and 5 mM Ca” in the brine after 3 weeks and between 3 and 6 weeks refrigeration, respectively (Table 7). However, the decrease in the rate of softening caused by the Cab'ammendments to the brine solution was reversed in both 5 and 20 days incubated spears in pericarp and endocarp tissues after 3 weeks and between 3 and 6 weeks refrigeration, respectively. Cab'ammendments to the processing brine had no effect on the rate of softening of both.pericarp and endocarp tissues of unincubated spears refrigerated for more than 3 weeks or on the rate of pericarp softening refrigerated up to 3 weeks. Also, no effect was found of Caz’ amendments to the brine solution on rate of softening of 20 days incubated spears refrigerated up to 3 weeks (Table 7). 123 Incubation of Fresh-Pack Processed Slices Fruit tissues of fresh-pack processed slices were much softer immediately following blanching as compared to fresh tissue. Ca2+ concentration in the nutrient culture solution had little or no effect on firmness and rate of softening of processed slices in 0 mM Ca2+ in the processing brine. The exception was the significantly higher firmness observed in the pericarp tissue of plants fertilized with 10 mM or higher Caa'concentration as compared to 0.1 mM Cab'after 20 days of accelerated aging (Table 8). Also, adding support for Ca2+ having a role on fruit softening, the rate of pericarp tissue softening during aging of slices, after 5 days incubation at 46°C, was negatively correlated with tissue Ca2+ concentrations (Fig. 4). The rates of tissue softening following blanching differed greatly between spears and slices. Fruit textural measurements in processed slices following fresh-pack processing averaged 3.3 N in pericarp tissue and 2.9 N in endocarp tissue while the average of the textural measurements of processed spears from the same fruits was 10.2 N in pericarp tissue and 3.2 N in endocarp tissue following fresh-pack processing. Thus, the magnitude in pericarp firmness in processed spears was much higher than in processed slices. Differences in textural firmness between processed spears and slices from the'same fruits were as high as 14.9 N. 124 Table 8 - Effect of Ca2+ concentration in nutrient culture solution on fruit firmness of fresh-pack processed cucumber slices in 0 mM Caa'brine solution during incubation at 46°C. Tissue Firmness (newtons) Ca2+ Conc. Pericarp Endocarp (mu) . Incubation Time (days) 5 20 5 20 0.1 2.55 1.76 2.16 1.96 1.0 2.75 2.16 2.35 1.57 10.0 2.94 2.36 2.94 1.77 20.0 2.75 2.36 2.55 1.77 LSD(0.05) NS 0.42 NS NS NS nonsignificant 125 Amendments to the processing brine solutions of 20 mM Ca” enhanced the post-processing firmness of both pericarp and endocarp tissues regardless of whether the slices were incubated for 5 or 20 days at 46°C (Table 9). Processed slices in 20 mM Caz‘ brine solution showed significantly higher pericarp firmness as compared.to processed slices in 0 or SINM Ca2+ brine solution after either 5 or 20 days at 46°C. However, the rate of pericarp tissue softening was higher in slices processed in 20 mM Caa'brine solution as compared to slices processed either in 5 or without added Ca” in the brine solution between 5 and 15 days incubation at 46°C (Table 9). Slices processed in 20 mM Caz‘ brine solution showed significantly higher endocarp firmness as compared to slices processed in either 5 mM Caz‘ brine solution after 5 days incubation or 0 and 5 mM Ca2+ brine solution after 20 days incubation at 46°C (Table 9). Addition of 5 mM Ca2+ to the processing brine solution, however, did not enhance significantly the post-processing firmness of both pericarp and.endocarp tissues.of fresh-pack processed slices (Table 9). Caa'added to the processing brine solution had no effect on the rate of endocarp tissue softening between 5 and 15 days incubation at 46°C. The Caa’concentration in nutrient solution and in brine affected tissue firmness and the rate of softening during accelerated aging similarly in fresh-pack processed slices. 126 Table 9 - Effect of Cab'brine concentration on fruit firmness and on rate of softening of fresh-pack processed cucumber slices during incubation at 46°C. Tissue Firmness Rate of Softening (newtons) (newtons/day) Caz’ Conc. Pericarp Endocarp Pericarp Endocarp (mM) Incubation Time (days) 5-15 5-15 5 20 5 20 days days 0 2.75 2.16 2.50 1.76 0.04 0.05 5 2.53 2.43 2.28 1.94 0.01 0.02 20 5.12 3.24 2.80 2.45 0.12 0.02 LSD(0.05) 0.82 0.37 0.35 0.34 0.06 NS NS nonsignificant 127 Refrigeration of Fresh-Pack Processed Slices Caa'concentration in the nutrient culture solution had no effect on firmness and rate of softening of processed slices during refrigeration. Ca2+ addition to the processing brine solution enhanced the post-processing firmness and maintained pericarp tissue texture for 6 weeks refrigeration in 5 days incubated slices and up to 3 weeks refrigeration in 20 days incubated slices (Table 10). The effect of Caz" addition to the brine solution on endocarp tissue firmness was only apparent in 20 days incubated slices and up to 3 weeks refrigeration. In both pericarp and endocarp tissues, slices processed in 20 mM Ca2+ brine solution showed higher firmness as compared to slices processed in 0 or 5 mM Caa'brine solution (Table 10). An opposite effect, however, in relation to the rate of softening was observed in the endocarp tissue in 5 days incubated slices refrigerated up to 3 weeks and in both pericarp and endocarp tissues in 20 days incubated slices refrigerated for more than 3 weeks (Table 10). Cab'ammendments to the processing brine had no effect on the rate of softening of 5 days incubated slices either refrigerated for 3 weeks in pericarp tissue or between 3 and 6 weeks refrigeration in both pericarp and endocarp tissues. The rate of pericarp and endocarp softening in 20 days Handwuwcmwmco: mz 128 ~m.o m2 ha.o mz mz mm.o hv.o mz oo.o Hm.o AHo.o.omq mn.o mo.ol hm.o oo.o o¢.H mm.~ mv.~ m~.N HH.m ¢~.n om ea.o mo.o 0H.o oo.o mn.H oh.~ em.H 00.H 0H.N ne.~ m mo.o oo.o oo.o mo.o ~n.H me.H hh.a oh.a mm.a oa.m o msmo ea mz ma.o m2 m2 mz m2 mn.o no.o Hm.o no.o Amo.ovomq NH.o ha.o mo.o ma.o N¢.H m~.~ om.m om.¢ nh.¢ NH.m 0N oH.o no.ol ma.o oo.o! em.a mn.~ om.m «H.~ Nb.~ nm.~ m mo.o oo.o oo.o oo.o H~.~ on.~ om.m me.~ m>.~ m>.~ o when m min mlo oln nlo o n o o n o Amx0030 mafia cowuouooauumm Anxooav mafia sawuoumowuuem as: mucooocm munowumm mumooocm Qumofiuom .ocou so ~ Aaco\mcousocv anacouuom no ovum Ancouamcv mmocauwm commas + .O.v.v an cowuoumowuumu onwuso noowam umnasoso commmooua xoomlnmonu ooucnsocw mace om 0cm m 00 ocficouuom momma» mo much so one mmocauww uwsuu so cofiucuucoocoo ocean.&so no voouum 1 ca manna 129 incubated slices was also not affected by the Caz“ brine treatments after 3 weeks refrigeration (Table 10). Discussion This study is thought to be relevant to production of pickling cucumbers since Cab' concentrations within commercially produced fruit are highly variable (Engelkes et al.,1990), thus potentially affecting textural quality. Engelkes et a1.(1990) reported that Caa’concentrations within 150 g fresh weight fruit (similar in size to 4.5 cm diameter fruit), produced under' diverse environmental conditions, varied from approximately 0.7 to 1.0 % of dry weight in pericarp tissue and from 0.2 to 0.6 % in endocarp tissue. The range of Caz‘ concentrations induced experimentally in this study (Table 2) exceeded those reported by Engelkes et a1. (1990) for pericarp tissue but less for endocarp tissue. Caa'concentrations in the leaf lamina tissue were higher than in the petiole tissue (Table 2). Engelkes (1987) found in field-grown pickling cucumbers CaZ’ concentrations ranging from 1.85 to 3.10 % and 1.45 to 1.84 % of dry weight in leaf lamina and petiole tissue, respectively, while in the cucumber cultivar Castlepik 2012 the Ca” concentrations found were 2.56 % in leaf lamina tissue and 1.75 % in leaf petiole tissue. Carolus (1975) also reported Caa’concentrations around 3.71 % in the foliage of cucumbers. Thus, the field leaf Ca” 130 concentrations were intermediate to what was induced experimentally in the present study (Table 2). McFeeters. 8 Lovdal (1987) reported. that firmness of cucumber fresh fruit showed no relationship with Ca?" concentration in.mesocarp tissue. This is consistent with the results from this study (Table 2). Consistent also with the findings of McFeeters 8 Fleming (1989) blanched cucumbers submitted to accelerated aging at high temperature softened rapidly (Table 3,8) . High endogenous Cab’concentrations within pickling cucumber fruit were found to enhance the textural firmness of pericarp (fruit wall) tissue following blanching (Table 3). Maximum firmness of fresh-pack processed spears was achieved at fresh pericarp tissue Ca2+ concentrations of approximately 0.95 % dry weight and higher while in endocarp tissue the maximum firmness was achieved at Ca” concentrations of 0.25 % dry weight and higher. In processed slices maximum firmness was achieved at similar tissue Caa’concentrations, 0.97 % dry'weight for pericarp and 0.28 % for endocarp tissue. The effect of endogenous Ca2+ on pericarp firmness of processed spears was apparent even when Cab'was added to the solution prior to blanching and storage (Table 3). However, after accelerated aging at 46°C for 5 or 20 days (Table 3), the textural differences attributed to endogenous tissue Ca2+ concentrations in pericarp tissue disappeared within the processed spears. Softening associated.with aging of blanched 131 spear tissue also contributed to a reduction in differences among treatments. The changes in the chemical nature of the pectic materials are the primary cause of changes that occur in the textural properties of horticultural products (Van Buren,1979) . The decrease in protopectin and the increase in pectin, and thus in water insoluble and water soluble pectin, respectively, are caused by pectin methyl esterase and polygalacturonase (Pilnik 8 Voragen,1970: Jarvis,l984: Ben-Arie 8 Sonego,1979) . However, according to Bourne (1983) heat can also promote the depolymerization of the protopectin and the pectin resulting in great amount of fruit softening. The middle lamella is frequently stronger than the cell wall, with the result that when stress is applied the tissue breaks across cells walls allowing the contents of the cell to escape. In raw plant material the rupture occurs across the cell walls while in heated plant material rupture occurs in the middle lamella between intact cells (Bourne,l976). This fact could be a possible explanation to the great amount of softening observed in processed spears and slices during incubation at high temperatures. The relationships between tissue Ca” and firmness of pericarp tissue following fresh-pack processing support the hypothesis that endogenous Caz’ has a role in maintaining tissue firmness of fresh-pack processed spears. 132 The effectiveness of Cab'in maintaining the firmness of cucumber tissue after fresh-pack processing and during storage of pickles has been widely reported (Buescher et al., 1981: Tang 8 McFeeters, 1983; McFeeters et al., 1985: Fleming et a1. , 1987) . However, the effect of endogenous tissue Caz“ concentrations previously modified in.a culturally controlled manner on fresh-pack pickle firmness after blanching, and after accelerated aging at high temperatures had not been yet demonstrated. The effect of both endogenous tissue Ca2+ and Cab'addition to the brine solution on pickle firmness and on the rate of tissue softening during refrigeration had also not been evaluated. McFeeters 8 Fleming (1989) reported that the rate of softening of pericarp tissue in slices during accelerated aging was negatively correlated with endogenous Ca2+ concentration. A similar relationship was observed in blanched slices after 5 days (Fig. 4), however, a much different correlation was obtained when spears sections were processed from the fruit. The rate of softening of pericarp tissue in the spears was positively correlated with endogenous tissue Caa’ concentration (Fig. 4). The cause for this difference between spears and slices is not readily apparent, but it might be related to the effect of fruit section geometry on the blanching process or on they physical measurement of firmness with the shear press. 133 On the other hand, the endogenous Ca2+ concentration in the cucumbers tissue obtained by Mcfeeters 8 Fleming (1989) from a variety of sources and climates varied from 1.8 to 8.2 mM. In their study, endogenous Caa'seemed to be more effective in slowing softening at concentrations varying from 1.8 to 3.8 mM, that is, at very low Caa’concentrations. Evaluating the range of Ca”' concentrations in the pericarp tissue as presented in Fig. 4 (4.5 to 14.0 mM), it can be observed that the endogenous tissue Ca” concentrations induced in the present study are much higher than in McFeeters 8 Fleming's study. Also, even in the processed slices, a high degree of variability in the correlation between pericarp Caz“ concentration and the rate of pericarp softening (Fig. 4) was observed. Thus, the endogenous Ca2+ effect in slowing softening of either blanched spears or slices seems to occur within a relatively narrow range of Ca” concentrations within the tissue. At high tissue concentrations, variations in tissue Cab’has no effect on textural firmness. The differences in texture observed between spears and slices following fresh-pack.processing could be attributed to the larger surface area of the internal fruit exposed to the processing brine in the slices as compared to spears. In contrast, the internal tissue integrity in processed spears might have been protected during blanching, due to fruit section geometry. Such an effect has been observed by McFeeters 8 Fleming (1989) when pieces of mesocarp tissue 134 showed much higher softening rates as compared to cucumber slices. In the present study, cucumber slices exhibited higher softening rates as compared to cucumber spears. The beneficial effect of amending the processing solution with 20 mM Cab'was apparent. This might be due to the Ca” ions saturated the binding sites that inhibited softening (McFeeters 8 Fleming, 1989). However, this effect. was reversed in the pericarp tissue of processed spears and slices with longer'period.of incubation, and in pericarp and endocarp tissue during certain periods of refrigeration. This decline in texture is difficult to explain, but it seems to be related with high tissue firmness prior 'to incubation or refrigeration. The effect of Ca”' in ‘maintaining tissue firmness of brined cucumbers as well as in. many other vegetables has been based in the cross-linking of Cab'ion to pectin molecules by eletrostatic interactions between two negatively charged carboxyl groups of pectin (Van Buren, 1979). However, blocks of at least 14 consecutive demethylated carboxyl groups on adjacent polygalacturonan molecules are required for Cab’ion cross-linking according to Kohn (1975) . Pectin methyl esterase was inactivated by blanching at temperatures above 80°C, thus, maintaining high levels of methylation in the blanched cucumber tissues (McFeeters et al., 1985). Also, the apparent pK's of carboxyl groups ranges from about 3.8-4.5 in dilute solutions of pectic acid as it is neutralized with NaOH (Cesaro et a1, 1982). 135 In the present study, the pH of the fresh-pack processed pickles varied from 2.5 to 3.2 depending on the Ca2+ concentration in the processing brine. The blanching temperature was 86°C. Under these conditions, probably high levels of methylation were maintained due to the inactivation of pectin methyl esterase, and also a high level of protonation due to the pH. Thus, less negatively charged carboxyl groups on adjacent molecules would be available for Ca2+ cross-linking with the combination of a high degree of methylation and a high level of protonation. But, the fact that Ca2+ is so effective in firmness retention under these conditions, regardless of the degree of pectin methylation in the tissue (Tang 8 McFeeters, 1983: McFeeters 8 Fleming, 1989) , and also at low concentrations, suggests that in addition to binding the carboxyl groups of pectin molecules, the textural effects of Ca” are also due to binding at specific texture modification sites other than pectin carboxyl groups (McFeeters 8 Fleming, 1989). 'Thus, other types of polysaccharides/Ca” interations may have an important role (McFeeters et al., 1985). Losses in galactose from mesocarp cell walls have been reported by Howard 8 Buescher (1990) in fresh-pack pickles. Tang 8 McFeeters (1983) reported that galactose constitutes about 50% of the total hydrolyzable neutral sugars present in the cucumber cell wall while Wallner (1978) suggested that the removal of neutral sugars that serve as crosslinks could weaken the cell wall structure and 136 contribute to firmness loss, increasing also the susceptibility of pectin to degradationtby polygalacturonase. In conclusion, culturally modified endogenous tissue Ca2+ affected texture of fresh-pack pickles after blanching and during refrigeration, even when Ca"M was added to the brine solution before blanching and storage. Caa'addition to the processing brine also improved the texture of fresh-pack processed spears and slices either after accelerated aging or during refrigeration. Bibliography Ben-Arie, R. and Sonego, L. 1979. Changes in pectic substances in ripening pears. J. Amer. Soc. Hort. Sci. 104(4):500-505 Bourne, M.D. 1976. Texture of fruits and.vegetables. In: Rheology and Texture in Food Quality (J.M. deMan, P.W. Voisey, V.F. Rasper, and D.W. Stanley, eds.), Avi Publ. Co., Westport, CT. 275-307. Bourne, M.C. 1983. Physical properties and structure of horticultural crops. In: Physical Properties of Foods. Peleg, M., Baley, E.B., eds. Avi Publ. Co., Inc. Westport, CT. 207-228. Buescher, R.W. and Burgin, C. 1988. Effect of calcium chloride and alum on fermentation, desalting and firmness retention of cucumber pickles. J. Food Sci. 53(1) :296-297. Buescher, R.W. and Hudson, M. 1984. Softening of cucumber pickles by Cx-cellulase and its inhibition by calcium. J. .Food Sci. 49(3): 954-955. Buescher, R.W., Hudson, J.M. and Adams, J.R. 1979. Inhibition of polygalacturonase softening of cucumber pickles by calcium chloride. J. Food Sci. 44:1786-1787. Buescher, R.W., Hudson, J.M. and Adams, J.R. 1981. Utilization of calcium to reduce pectinolytic softening of cucumber pickles in low salt conditions. Lebensm. Wiss.n. Technol. 14:65-69. Carolus, R. 1975. Calcium relationships in vegetable nutrition and quality. Comm. Soil Sci. Plant Anal. , 6:285- 298. Cesaro, A., Ciana, A., Delben, G., Manzini, G. and Paoletti, S. 1982.. Physicochemical properties of pectic acid. I. Thermodynamic evidence of a pH-induced conformational transition in aqueous solution . Biopolymers. 21:431-449. 137 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 138 Engelkes, C.A. 1987. Cultivar and environmental impact on ontogenetic changes in the fruit calcium. status of pickling cucumbers. M.S. Thesis. Michigan State University. 180 p. Engelkes, C.A., Widders, I. and Price, H. 1990. Ontogenetic changes in calcium concentration and content in pickling cucumber fruit as influenced by genotype and environment. J.Amer. Soc. Hort.Sci. 115(4):555-558. Fleming, H.P. , McFeeters, R.F. , Daeschel, M.A. , Humphries, E.G. and Thompson, R.L. 1988. Fermentation of cucumber in anaerobic tanks. J. Food Sci. 53(1):127-133. Fleming, H.P., Thompson, R.L., Bell, T.A. and Hontz, L.H. 1978. Controlled fermentation of sliced cucumbers. J. Food Sci. 43:888-891. Howard, L.R. and Buescher, R.W. 1990. Cell wall characteristics and firmness of freshpack cucumber pickles affected by pasteurization and calcium chloride. J. Food Bioch. (14):31-43. Hudson, J.M. and Buescher, R.W. 1980. Prevention of soft center development in large whole cucumber pickles by calcium. J. Food. Sci. 45:1450-1451. Hudson, J.M. and Buescher, R.W. 1985. Pectic substances and firmness of cucumber pickles as influenced by CaClZ, NaCl and brine storage. J. Food Biochem. 9:211-229. Hudson, .J.M. and. Buescher, R.W. 1986. Relationship between degree of pectin methylation and tissue firmness of cucumber pickles. J. Food Sci. 51(1):138-140/149 Jarvis, M.C. 1982. The proportion of calcium-bound pectin in plant cell walls. Planta. 154:344-346. Jarvis, M.C. 1984. Structure and properties of pectin gels in plant cell walls. Plant, Cell and Environment. 7:153- 164. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 139 Johnson, C.M5, Stout, P.R., Broyerq T.C. and.Carlton, E.B. 1957. Comparative chlorine requirements of different plant species. Plant and Soil. Vol. 8:337-353. Kohn, R. 1975. Ion binding on polyuronates-alginate and pectin. Pure Appl. Chem. 42:371-397. Marschner, H. 1986. Mineral nutrition in higher plants. Academic Press Inc. (London) LDT. 674p. McFeeters, R.F. and Fleming, H.P. 1989. Inhibition of cucumber tissue softening in acid brines by multivalent cations: Inadequacy of the pectin "Egg Box" model to explain textural effects. J. Agric. Food Chem. (37):1053- 1059. McFeeters, R.F. and Lodval, L.A. 1987. Sugar composition of cucumber cell walls during fruit development. J. Food. Sci. 52(4):996-1001. McFeeters, R.F., Senter, M.M., and Fleming, H.P. 1989. Softening effects of monovalent ions in acidified cucumber mesocarp tissue. J. Food Sci. 54:366-370. Pilnik, W. and Voragen, A.G.J. 1970. Pectic substances and other uronides. In: Biochimestry of Fruits and Their Products. Hulme, A.C. Vol. I. 53-81. Poovaiah, B.W. 1979. Role of calcium in ripening and senescense. Commun. Soil Sci. Plant Anal., 10(182):83-88. Tang, H.L. and McFeeters, R.F. 1983. Relationships among cell wall contituents, calcium and texture during cucumber fermentation and storage. J. Food Sci. 48:66-70. Van Buren, J.P. 1979. The chemistry of texture in fruits and vegetables. J. Texture Studies, 10:1-23 Wallner; S.J. 1978. Postharvest. structural integrity. Journal of Food Biochemistry 2:229-233. Chapter III EVALUATION OF CALCIUN EERTILIZATION STRATEGIES FOR ENHANCING TEXTURAL QUALITY OE PICNLING CUCUNEERS Introduction Caz‘ has been shown to be important for normal cucumber plant growth and for fruit development (Frost 8 Kretchman, 1989) and quality (Cooper 8 Bangerth,1976). Cucumbers grown under Cab-deficient nutrient solution were spindly and produced small and mishapen fruit or no fruit (Staub et al., 1988) . Caz“ stress severely reduced cucumber root (Matsumoto 8 Kawasaki, 1981: Konno et al., 1984). Pillowing disorder has also been associated with decreasing Cal" levels in the cucumber mesocarp tissue (Staub et al., 1988). Environmental conditions, such as mild drought stress and genotype, have been also demonstrated to influence the Ca2+ concentration in pickling cucumber fruit (Engelkes et al., 1990). Thus, low soil moisture resulting in poor water movement through the plant can result in localized Ca?-+ deficiency (Geraldson, 1979). Tang 8 McFeeters (1983) and Fernandes 8 Widders (1986) reported that endogenous Ca2+ concentrations in fresh cucumber fruit tissue were highly correlated with the rate of pericarp softening in brine following blanching or during post-harvest storage. The question is how to enhance Caz“ levels within the fruit tissue. Ca2+ is the most immobile macronutrient in the plants. Thus problems that arise with Ca” nutrition in plants are 140 141 frequently related to redistribution and mobility (Ferguson, 1979). Caa'is not readily remobilized from mature leaves into developing fruit such as during periods of low uptake by the plant (Shear, 1975; Hanger, 1979: Bengtsson 8 Jensen, 1983). Due to the immobility of Caa'in phloem tissues (Biddulph et al., 1961: Zimmermann, 1966: Ringoet et al., 1968: Ferguson 8 Clarkson, 1976), Ca2+ accumulation within developing fruit occurs at a relatively slow rate (Hanger, 1979). In order to meet the requirements of growing tissues, most of the Cab'is thought to be translocated via the xylem into the fruit tissue through the transpiration stream.(Marschner, 1986). Water loss by transpiration from fruit, however, is quite low and declines due to an ontogenetic decrease in the surface area to volume ratio of the fruit (Hanger, 1979). All of these factors contributes to the low tissue Cab' concentrations in fruits. Foliar applications of nutrient solutions provide a potential means for supplying mineral nutrients directly to specific organs of a plant at critical stages of plant development (Biddulph et al., 1959; Mason, 1979: Tibbitts 8 Palzkill, 1979; Richardson 8 Lombard, 1979). However, foliar application is not considered to be an effective means of supplying Cab'to plants (Hanger, 1979). Chelating compounds enhance the solubility of Ca2+ in solution, and thus the potential absorption of Cab'by leaf tissue following foliar application (Millikan 8 Hanger, 1965: Wiersum, 1979). 142 In cucumber plants, fruit and root growth compete for photosynthates. Consequently, during fruit set and net growth, the rate of root growth is significantly reduced (McCollum, 1934: DeStigter, 1969; Barret 8 Amling, 1978) . The proportion of plant dry weight in the roots of fruiting plants was reported to decline during plant ontogeny from 0.38 to 0.09 while in defruited plants the proportion of roots decreases gradually from 0.37 to 0.30 (Hall, 1977). Sink competition between fruits and roots is important for the uptake of water and mineral nutrients (Stigter, 1969: Marschner, 1986). Root activity and thus nutrient uptake by the roots, in particular Cab, declined with the onset of the reproductive stage (Marschner, 1986). Caa'uptake by the root system was lower in fruiting than in non-fruiting trees (Hansen, 1973). It is, therefore, hypothesized that Caa'concentrations in xylem sap of pickling cucumber plants would decline during plant ontogeny, specifically during reproductive development, as a result of reduced new root growth. Thus, Caa'supply to developing fruit might be limited. The objectives of the present study were: a) to evaluate alternative Ca” fertilization strategies for enhancing the Caz+ concentration and thus textural quality of pickling cucumber fruit under field conditions, and b) to understand better how fruit set and growth might affect Caa'supply via the xylem to the fruit. 143 Materials and Methods A field experiment was conducted in 1990 at the Michigan State University Horticulture Research Center, in which pickling cucumbers, cv Flurry (Asgrow Seed Company, Kalamazoo, MI) were precision planted with a Heath vaccum planter on 2 July 1990. The cucumbers were planted into 9 flat beds spaced 2.3 In between centers with 3 rows per bed. Between row spacing was 71 cm and plants within the row were thinned to 7.6 cm between plants at the cotyledonary stage. A randomized complete block design was used for this experiment. Each block consisted of a bed of three rows, 53.5 m long,with 5 treatment plots each 7.7 m long and separated by 2.1 m of guard bed. All blocks were separated from neighboring blocks by guard rows. Complete blocks were replicated four times. The Ca” fertilizer treatments applied were : (1) Nutri-Cal foliar applied: A 2.5% solution of Nutri-Cal (25 ml/l) was prepared to which was added a surfactant, L-77 (Union Carbide) (0.5 ml/l) . Only deionized water was used in preparation of these solutions. At each application time, 912 ml of this solution was sprayed unto the foliage of each plot (7.7 m of bed). This application rate is equivalent to 6 quarts Nutri-Cal/acre (1.49 Kg Ca/ha) per application. Two applications of this formulation of Nutri-Cal were made: 9 and 16 August 1990. At these times, the cucumber 144 plants were at the following developmental stages, fruit set and during a period of rapid expansive fruit growth, respectively. (2) Nutri-Cal feliar applied: A 1.25% solution of Nutri-Cal (12.5 ml/l) was prepared to which was added 0.5 ml/l of L-77 surfactant. At each application time, approximately 912 ml of this solution was sprayed unto the foliage of each plot (7.7 m of bed). This application rate was equivalent to 3 quarts Nutri-Cal/acre or 304 g Ca2*/acre (0.745 Kg Cay/ha) per application. Three applications of this formulation of Nutri-Cal were made at 4 day intervals: 9, 13, and 16 August 1990. The intention was to make a total of 4 applications of the 1.25% Nutri-Cal solution so as to apply a total amount equivalent to treatment 1. The fruit however,reached harvestable maturity before the fourth application could be made. It must be noted, therefore, that the total cumulative application of Cab'was 912 g Cab/acre (2.24 Kg Cab/ha) for treatment 2 as compared to 1.2 Kg Cab/acre (2.979 Kg Cay/ha) for treatment 1. (3) CaCJ.2 foliar applied: A solution containing 9.744 g CaClZ/l plus 0.5 ml/l L-77 surfactant was prepared. At each application time, 912 ml of this solution was sprayed unto the foliage of each plot (7.7 m.of bed). This application rate is equivalent to 603 g Ca2*/acre (1.49 Kg Ca2*/ha) per application. In terms of net amount of Ca2+ applied, this treatment is identical to treatment 1, but without the.chelate 145 in the solution. Two applications of this solution were made during the fruiting period: 9 and 16 August 1990. (4) CaCllside dressed: At vine tip-over, 31 July 1990, 22.7 Kg Ca/acre (62.8 Kg/ha CaClz) were side-dress applied to the pickling cucumbers. A band of CaCl2 salt was placed into a furrow approximately 15.3 cm to the side of the row and 6.4 cm deep and covered up. (5) Control: To all control plants, a solution containing deionized water plus 0.5 ml/l of L-77 surfactant was applied foliarly. Applications were made on 9, 13, and 16 August 1990. Nutri-Cal (8% Ca2+ solution) is a commercial foliar fertilizer in which the Ca” is chelated by 2,3,4,5 trihydroxypentanedoic acid (a trihydroxygluterate). Fruit were once-over destructively harvested from 6.11 m of bed from each treatment plot on 20 August 1990. The fruit were then taken to a grading station, graded into commercially inportant size grades (#1a, 1b, 2a, 2b, and 3) including oversize fruit ( > 5 cm diameter) and misshapen fruit and then weighed. Fruit yield of fresh cucumbers was determined for each of the size grade fractions. A subsample of ten size #3 fruit (3.8 - 5.0 cm.diameter) were randomly collected and evaluated for fruit quality, including fruit L/D ratio (length/diameter), seed cavity diameter, % seed cavity, 146 relative seed size, internal quality defects including placental hollows and the separation of the carpels. Fruit texture was measured on an additional sample of eight size #3 fruit. A transverse slice, 7 mm thick, was cut from the middle section of each fruit. Texture (firmness) of the pericarp (fruit wall), and the endocarp (seed cavity) tissues were measured for each slice using a shear press (TMS- 90, Food Technology Corporation). A minimum of 2 replicate measurements were made for each tissue on each slice in order to obtain an accurate determination. Fruit tissue Ca2+ concentration was determined for the individual fruit evaluated for texture. Both pericarp and endocarp tissues were sampled and weighed. The fruit tissue samples were then dehydrated at 60°C in a ferce air drying oven for a minimum of 72 h and then reweighed. The tissue were ground in a Wiley mill to pass a 20 mesh screen and an 0.1 g subsample wet ashed in H202 and HClO‘ after the method of Adler and Wilcox (1985). Ca2+ concentration in diluted extracts was determined by atomic absorption spectrophotometry (Video 12 AA, Instrumentation Laboratory). Leaf samples (first fully expanded leaf approximately the 5th leaf from the vine apex) were collected from each treatment plot at 4 times during plant development for Ca2+ analysis. Times of collection were prior to foliar applications (8 August), mid-fruit development period (approximately 6-8 days after 1"t application) (15 August), harvest time (22 August), 147 and approximately a week following harvest (29 August, 1990). Leaves were separated into petiole and lamina tissue, rinsed with deionized water to remove potential surface contamination, dehydrated in a forced air oven at 60°C for 72 h and then ground in a Wiley mill. Following wet ashing as described by Adler 8 Wilcox (1985) , the extracts were analyzed for Cab’ by standard procedures for atomic absorption spectrophotometry. xylem Exudate Collection Xylem exudate collection was conducted during vegetative development (August 2), at anthesis (August 8), at mid-fruit development period (August 15) , at typical time. of first harvest (August 22), and a week following harvest (August 29) in 1990. Since a positive root pressure existed during the early morning hours, all collections were initiated at 8-8:30 a.m. Five vigorous healthy plants were sampled per replication. Two treatments were sampled, the control and the CaCl2 side-dress treatments with three replications. Sampling was taken at two times, at the 1“t and at the 2"“' hour after decapitation of the stem. Xylem Sap Collection Procedure Plants were decapitated immediatly above the cotyledonary node at approximately 8-8:30 a.m. The cut surface was rinsed with deionized water to remove any potential contamination, 148 and then gently blotted with filter paper. A section of acid rinsed rubber tubing (- 15 cm long) was gently slipped over the cut end of the stem and the open end of the tube was sealed with parafilm. After one hour, the exuded sap from the plant stump was collected from the tube using a transfer pipette and transferred to a 20 ml vial. After a second hour the collection was repeated. The vials were then frozen on dry ice and stored at -20°C for Cab'analysis. Defruiting Experiment In order to evaluate the effects of fruiting on the ability of the plants to take up Ca”, xylem exudate was collected from fruiting and defruited plants. Sample Collection In the center row of the guard bed, approximately 15 to 20 plants were completely deflowered on August 16, 1990. The plants had fruit up to approximately 2.5 cm in diameter at that time. On August 31, 1990 both xylem exudate, as previously described, and leaf samples were collected from 5 plants in each of 4 replications, from fruiting and defruited plants. Xylem exudate and leaf tissue samples were analyzed for Ca” concentration as described utilizing atomic absorption spectrophotometry. 149 Statistical Analysis All data. were tanalyzed statistically' by analysis of variance. The LSD test was used to compare treatment means if a significant F<.05 value was obtained. Correlations were investigated between tissue Ca” concentrations and fruit firmness. Results The 1990 growing season was ideal for pickling cucumber production. An excellent stand was obtained following precision seeding and the favorable environmental conditions during July and August resulted in good vine growth, fruit set and development. At no time was the crop exposed to a significant water stress as evidenced by the lack of foliar wilting. The Caa'treatments did not affect yield following a once over destructive harvest. The yield varied from 12.2 to 14.7 Kg/plot (13.97’ n3). The fresh weights of each of the individual size grade fractions of marketable fruit, #1a to #3, and of total yield were similar among all the treatments. Morphological quality parameters of #3 size grade pickling cucumber fruit were also not affected by either the foliar or the side-dress applications of Ca” as compared to the control. The firmness of fresh pericarp (fruit wall) tissue in pickling cucumber fruit was affected by the foliar application 150 on a 2.5% (v/v) solution of Nutri-Cal (Table 1). These fruit had significantly higher'pericarp tissue firmness as compared to fruit from non-Ca2+ fertilized plants (controls). The side- dress application of CaCl2 did not influence fruit texture. Endocarp (seed cavity) tissue firmness was much lower, approximately 5.5 newtons, than the firmness of pericarp tissue, 22.5 - 24.8 newtons (Table 1). The endocarp tissue was not affected by Caa'fertilization, regardless of whether foliar or side-dress applications were made. The concentration of Ca” within the pericarp tissue of size grade #3 fruit was enhanced by both the foliar application of Nutri-Cal (2.5%, v/v) solution and the side- dress application of CaCl2 (22.7 Kg Ca/acre) which showed significantly higher Ca” concentrations in pericarp tissue than the foliar applied with CaCl2 or non-Ca” fertilized plants (controls) (Table 1). The application of a more diluted Nutri-Cal solution (1.25%, v/v) also appeared to increase the tissue Cab'level above the control, however, the response was not statistically significant at the 5% level. None of the Cap'fertilization treatments influenced endocarp tissue Cab'concentration. It should be noted that Cafi'concentrations were also much lower in endocarp as compared to pericarp tissue. No correlation was found between tissue Caz” concentrations and fruit firmness (data not shown). 151 .muc0Hm Houuco0 HHO.ou u0fiau_00ucu 00aamn0 003 .thA .uc0u00uusm 0:0 mcwcfi0uco0.u0u03 .. .Aaxaa m.eo acoaucaom twosome nonaou Ham 00 00000 003 A0cwau00 cows: .uc0u00uusm 00002 SOOAHfimioc0ouo :00 beta .0000000050 4 u oz m.H mz mz m2 ao.o Amc.o u no can e.m a.mm n~.n ee.~ am.o em.o saonucoo e.m m.- on.n me.~ Hm.o oe.o 000000000 ~Hooo 4.0 e.n~ oa.n Ho.m on.o oo.o uaaaou ~Hooo n.m m.m~ ee.n mo.~ Hm.o no.c noaaou .»m~.Hc acouonuoz m.m a.e~ ea.n mo.~ Hm.o 50.6 unadoo ”Axm.~. Hoounuuoz 00000000 0000fiu0m 0cfia0q 0Hofiu0m 90000000 0000wn0m 50:0030cv 0cmmfiu M004 osmmwu.uashm 005008 #:0500000 000cauwm 0900“» A03 >00 *0 vasuu £0000 0000000000000.&00 soHu00HHQQ< .800 .0090“ 00030090 ocfiax0wm n* 00000 0000 no 00003000 :0 0:0 m0cmmfiu 000a 0:0 00:00 no 000w00uuc00c00 +~00 co ~H000 mo mcoHu0oflHQQ0 000000000 0:0 mcofiusHom ~0.000 90 Away 18:05.52 00 00030000300 00300 no 000000 I H 0.309 152 Leaf petiole Cab'concentrations ranged from 2.5 to 3.0% dry' ‘weight. ‘while lamina ‘tissue, concentrations ‘were comparatively higher, 3.15 to 3.4% dry weight (Table.1). Leaf tissue Caz’ concentrations were not affected by the various Cafi'fertilization treatments. Ca2+ concentration in the xylem sap was significantly higher in defruited plants than in fruiting (control) plants (Table 2) . Ca2+ concentrations (% dry weight) either in lamina or in petiole tissues in defruited plants were also higher than in the plants from the control, although statistically the differences were only significant at the 7% level (Table 2). The time of sampling highly influenced the Ca2+ concentration in the xylem exudate showing the second hour to be significantly higher in Cah'concentration than the first hour of sample collection (Table 2). Caa’concentrations within the xylem sap decreased during plant ontogeny (Fig. 1). Following the onset of flowering, the concentration of Ca2+ in the xylem sap decreased, and continued to decrease during reproductive development. A small increase was observed at 58 days, however. At this time the fruit, which had set, were approaching maturity (Fig. 1). Discussion Multiple foliar applications of Nutri-Cal (8% Ca) at a concentration of 2.5% (v/v) to pickling cucumber plants during 153 Figure 1. Changes in xylem exudate Ca” concentrations during vegetative and reproductive development of pickling cucumber plants (LSD<.01). 154 mm 02:25.1 mM.—LE. m><0 #4 Ru .0 H ouowfim 050026: 00 Lomco on o.o X tin .I 10.... ..WJ 0 V3 ..om 0.. O N .0 10.N— \.I w . W Ill\ 155 Table 2 - Influence of defruiting on Ca2+ concentrations in xylem exudate and leaf tissues and of time of sampling on xylem exudate Ca2+ concentrations in pickling cucumber plants. Treatments Xylem [Ca] (mM) Total Ca”’(% dry wt) Lamina Petiole Control 5.72 2.66 3.05 Defruited 6.74 3.95 3.68 F test sign. ** 7% 7% Time of sampling Ca”’concentration.in xylem exudate (mM) First hour 4.64 Second hour 7.79 F test sign. *** ***, **5 significant at the 0.1% and 1% level, respectively. 156 fruit set and development increased the firmness of pericarp tissue in fresh #3 size grade fruit. It is not known if this effect would be apparent in the tissue following fresh-pack processing. The physiological mechanism by which fruit firmness was increased by the foliar application of Nutri-Cal is not fully clear; The relatively high.pericarp tissue Cab'concentration in fruit from plants treated with Nutri-Cal (2.5% solution) might indicate a relationship between Cafi'levels in the fruit and tissue texture. However, the side-dress application of CaCl2 which resulted in the highest pericarp tissue Ca2+ concentration, 0.70% dry weight, did.not lead to firmer fruit. In addition, Caa'concentration within individual fruits was not correlated with textural firmness (data not presented). This suggests that. Caa' might. be involved indirectly in altering tissue texture or that there are other unknown factors which might be producing the response. Differences in apoplastic Cay’concentrations could be a factor influencing fruit firmness. It should also be noted that although sidedress applications of CaClz appear to have increased the pericarp tissue Caz" concentration (Table 1), there clearly was no effect of this treatment on fruit tissue texture (Table 1). This result is consistent with the notion that enhanced fruit Caz’ is not a primary factor affecting textural characteristics of fresh cucumber fruit. Changes related to sugars in pectic 157 polymers during cucumber fruit development must be considered. The positive correlation (r=0.85) between the fresh weight concentration of cell wall sugars and cucumber firmness found by McFeeters 8 Lovdal (1987) suggests that firmness could be predicted by the total cell wall sugar content. Also, according to the same authors, since the galacturonic acid/sugar ratios increased during cucumber growth, this could indicate that the pectic substances with fruit development had fewer or shorter neutral sugar side chains, but it could also mean that the amounts of these sugars in the hemicellulose fraction of the wall declined. They found high positive correlations between the galacturonic acid/rhamnose and galacturonic acid/arabinose molar ratios in mesocarp cell walls and the development of the cucumber fruit (diameter) (r=0.95 and r=0.96, respectively). It is well established from experiments in which pickling cucumber plants were cultured in nutrient solutions modified in Ca”’ concentration that high fruit Cab' levels were associated with firmer fruit after fresh-pack processing (Fernandes et al., 1990). One potential hypothesis which should be considered in evaluating the results of this study is that the chelator being used in the Nutri-Cal formulation, 2,3,4,5 trihydroxypentanedoic acid, might be inducing a specific physiological response that impacts ultimately upon fruit texture. At this time one can only speculate how this might 158 occur. Recent research has shown that applications of certain chelating compounds such as oxalate can induce a systemic resistance to foliar pathogens within cucurbit species such as cucumbers and squash (Doubrava et al., 1988). An associated physiological response is increased lignification of cell walls in leaf and potentially fruit tissue (Dean.8 Kuc, 1987). If lignification does occur in fruit tissue, one would expect that it would alter significantly textural characteristics. Research is needed to verify if the chelator’present.in.Nutri- Cal might have such physiological activity. The low amount of Caa'applied to the cucumber plants by the Nutri-Cal solution also places in question any potential effect of Cah’on fruit texture. In this study, the amount of Caa’applied (1.49 Kg/ha) achieved an increase in both fruit tissue firmness and in Cab'concentrations (Table 1). However, this amount corresponds only about 37% of the total amount of Cab’which accumulates in the fruit to be harvested (4.0 Kg Cab/ha). This assumption was made considering that a typical yield gives approximately 17,000 Kg/ha. This amount in turn corresponds to 680 Kg dry matter/ha considering that the dry weight/fruit weight ratio is 0.04 (Fernandes 8 Widders, 1990) and the mean Ca2+ concentration in whole cucumber fruit is 0.006 g/g dry weight (Engelkes,1987) . The question which needs to be addressed is whether increasing the amount of Ca2+ supplied by the Cab’fertilizers would influence the response 159 of pickling cucumbers in relation to tissue Caz’ concentrations and fruit firmness. Caz’ Concentrations in Xylem Bxudate The decrease observed in Ca2+ concentration in xylem exudate during the reproductive stage (Fig. 1) coincides with the decrease in cucumber root growth during fruit development observed by several researchers (McCollum, 1934; De Stigter, 1969: Van der Vlugt, 1986: Van der Vlugt, 1987: Van der Vlugt, 1990). Furthermore, De Stigter (1969) observed that the decline in root growth of cucumber plants continued until harvest when a recovery in growth occurred after the fruit were removed from the plant. He pointed out that it is possible that roots might have grown slightly if the fruit had been left on the plant. These findings are consistent with the results of the present study, with respect to the ontogenetic decline in Ca” concentration in the xylem exudate and the small increase at fruit maturity (Fig. 1) . The question is to what degree non growing roots continue to carry on their functions of water and mineral absorption (De Stigter, 1969: Marschner, 1986). As it has been reported by Hansen (1973), Caz” uptake by roots is lower in bearing than in non-bearing trees. It is possible that Ca2+ uptake and subsequent transport through the xylem is impaired during fruit development, due to 160 the root-fruit competition for assimilates and the associated reduction in root.growth. This is supported by the observation that defruited plants had a significantly higher Ca2+ concentrations in xylem exudate than fruiting plants (Table 2) . If one assumes that Caz“ concentrations in xylem sap remains constant over time, thentthe level of Ca”’in.the fruit would be a function of the amount of time that the fruit requires to reach maturity. The longer the fruit requires to reach maturity, the higher the concentration of Ca2+ at harvest time. However, subsequent enlargement of the fruit dilutes the calcium in the fruit (Shear, 1975). This dilution effect has been reported by Widders 8 Price (1984) , Engelkes, (1987) , and Engelkes et al.(1990) to be accentuated through ontogeny, in which Ca” concentrations in pickling cucumber fruit tissues declined during fruit development. The competition between sinks can also be observed in relation to Cab'concentrations in lamina and petiole in leaf tissues (Table 2). McCollum (1934) and Hall (1977) discussed the differential sensitivity showed by the non-fruit organs, roots and leaves, to fruit growth. In fruiting plants (Hall, 1977), the leaves exhibited slower growth rates and reduced lamina length which were not reversed until fruit growth rate declined, however, net assimilation rates of fruiting plants exceeded those of defruited ones during most of the post- anthesis period, irrespective of the use of functional or total leaf area as a basis for the calculation of this index. 161 In the defruited plant, according to McCollum (1934), a marked increase in development of all vegetative parts is observed, including the roots, accompanied by an increase in absorption of nutrients. The root-fruit relationships as related to the decreasing levels of Cab'in the xylem exudate during the reproductive stage of cucumber plants might provide a partial explanation for the low Ca2+ concentrations in pickling cucumber fruit (Engelkes, 1987; Engelkes et al.,1990). In conclusion, these results seem to support the hypothesis that Cab'concentrations in xylem sap and thus Caa' uptake decline during plant ontogeny mainly during fruit development as a result of reduced new root growth. As a consequence, the availability of Caz+ through the xylem to developing fruit is limited, and may account in part for the relatively low Caa'content in fruits. Although foliar applications of Ca” fertilization treatments enhanced fruit pericarp tissue Ca3’more than 15%, these changes were not related with changes in fresh tissue textural firmness. BIBLIOGRAPHY Adler, P.R. and Wilcox, G.E. 1985. Rapid perchloric acid digest methods for analysis of major elements in plant tissue. Comm. Soil Sci. Plant Anal. 16:1153-1163. Barret, J.E. III and. Amling, H.J. 1978. Effects of developing fruits on production and translocation of C“- labelled assimilates in cucumber. HortScience. 13 : 545-547 . Bengtsson, B. and Jensen, P. 1983. Uptake and distribution of calcium, magnesium and potassium in cucumber of different age. Physiol. Plant. 57(4):428-434. Biddulph, O. , Cory, R. and Biddulph, S. 1959. Translocation of calcium in the bean plant. Plant Physiol. 34:512-519. Biddulph, 0., Nakayama, F.S. and Cory, R. 1961. Transpiration stream and ascension of calcium. Plant Physiol. 36:429-436. Cooper, T. and Bangerth, F. 1976. The effect of Ca and Mg treatments on the physiology, chemical composition and bitter-pit development of Cox's orange apples. Scientia Horticulturae, 5:49-57. 7. Dean, R.A. and Kuc, J. 1987. Rapid lignification and response to wounding and infection as a mechanism for induced systemic protection in cucumber. Physiological and Molecular Plant Path. Vol. 31:69-81. De Stigter , H . C . M. 1969 . Growth relationships between individual fruits and between fruits and roots in cucumber. Neth. J. Agr. Sci. 17:209-214. Doubrava, N.S., Dean, R.A. and Kuc, J. 1988. Induction of systemic resistance to anthracnose caused by Colletotrichum lagenarium in cucumber by oxalate and extracts from spinach and rhubarb leaves. Physiological and Molecular Plant Pathology. 33: 69-79. 162 10. 11. 12. 13. 14. 15. 16. 17. 18. 163 Engelkes, C.A. 1987. Cultivar and environmental impact on ontogenetic changes in the fruit calcium status of pickling cucumbers. M.S. Thesis. Michigan State University. 180 p. Engelkes, C.A., Widders, I. and Price, H. 1990. Ontogenetic changes in calcium concentration and content in pickling cucumber fruit as influenced by genotype and environment. J.Amer. Soc. Hort.Sci. 115(4):555-558. Ferguson, LB. and Clarkson, D.T. 1976. Simultaneous uptake and translocation of magnesium and calcium in barley (Hordeum vulgare L.) roots. Planta. 128:267-269. Fernandes, R.F. and.Widders, I.E. 1986. The physiological importance of Ca status on quality of pickling cucumber fruit. Hortscience (Abstracts of 83"'Annual Meeting Amer. Soc. Hort. Sci.) 21(3):176 (507). Fernandes, R., Widders, I.E. and Uebersax, M. 1990. Influence of calcium fertilizattion and nutrition on textural quality of pickling cucumbers. Proceedings 1990 Pickling cucumber Improvement Coommittee Meeting, 17-18 October 1990, East Lansing,MI. Frost, D.J. and Kretchman, D.W. 1989. Calcium deficiency reduces cucumber fruit and seed quality. J. Amer. Soc. Hort. Sci. 114(4):552-556. Geraldson, C.M. 1979. Minimal calcium stress using the gradient mulch production system. Commun. Soil Sci. Plant Anal. 10(182):l63-169. Hall, A.J. 1977. Assimilate source sink relationships in capsicum annuum L. I. The dynamics of growth in fruiting and deflorated plants. Austr. J. P1. Physiol. 4(49):623- 636. Hanger, B.C. 1979. The movement of calcium in plants. Commun. Soil Sci. Plant Anal. 10 (182):171-193. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 164 Hansen, P. 1973. The effect of cropping on the growth and uptake of nutrients by apple trees at different levels of nitrogen, potassium, magnesium, and phosphorus. Acta Agr. Scand. 23:87-92. Konno, H., Yamaya, T., Yamasaki, Y. and Matsumoto, H. 1984. Pectic polysaccharide breakdown of cell walls in cucumber roots grown with calcium starvation. Plant Physiol. 76:633-637. Marschner, H. 1986. Mineral nutrition in higher plants. Academic Press Inc. (London) LDT. 674p. Mason, J.L. 1979. Increasing calcium content of calcium- sensitive tissues. Commun. Soil Sci. Plant Anal. 10(182): 349-371. Matsumoto, H. and Kawasaki, T. 1981. Changes of membrane- associated Mga'activated ATPase of cucumber roots during calcium starvation. Physiol. Plant. 52:442-448. McCollum, J.P. 1934. Vegetative and reproductive responses associated with fruit development in the cucumber. Cornell Univ. Agr. Expt. Sta. Mem. 163. McFeeters, R.F. and Lodval, L.A. 1987. Sugar composition of cucumber cell walls during fruit development. J. Food. Sci. 52(4):996-1001. Millikan, C. R. and Hanger, B. C. 1965. Effects of chelation and of certain cations on the mobility of foliar applied “Ca in stock, broad bean, peas, and subterranean clover. Aust. J. Biol. Sci., 18: 211- 226. Redmont, W.J. 1975. Transport of calcium in apple trees and its penetration into the fruit. Commun. Soil Sci. Plant Anal. 6(3):261-272. Richardson, D.G. and Lombard, P.B. 1979. Cork spot of Anjou pear : control by calcium sprays. Commun. Soil Sci. Plt. Anal. 10(182):383-389. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 165 Ringoet, A., Sauer, G. and Gielink, A.J. 1968. Phloem transport of calcium in oat leaves. Planta. (Berl.) 80:15- 20. Shear, C.B. 1975. Calcium related disorders of fruits and vegetables. HortScience, Vol. 10(4):361-365. Staub, J.E., Rousos, P. and Struckmeyer,B.E. 1988. Anatomical characterization and possible role of calcium in "Pillowy", A fruit disorder in processing cucumber. J. Amer. SOC. Hort. SCi. 113:905-909. Tang, H.L. and McFeeters, R.F. 1983. Relationships among cell wall contituents, calcium and texture during cucumber fermentation and storage. J. Food Sci. 48:66-70. Tibbitts, T.W. and Palzkill, D.A. 1979. Requirement for root-pressure flow to provide adequate calcium to low- transpiring tissue. Commun. Soil Sci. Plant Anal. 10(18 2):251—257. Van der Vlugt, J.L.F. 1986. Root death in cucumber under different competitive conditions of the roots. Acta Horticulturae. 178:121-127. Van der Vlugt, J.L.F. 1987. The case: Roots vs. fruits in the cucumber. I. The effect of the nitrogen concentration in the recirculating nutrient culture solution on root death in cucumber. Plant and Soil. 98:299-301. Van der Vlugt, J.L.F. 1990. VII. The verdict: fruit are accessory to root.death. Exudates are the cause ?1Norwu J. Agr. Sci. 4(1): 91-94. Widders, I. and Price, H. 1984. The effects of plant density on the mineral nutrition of two pickling cucumber cultivars, Tamor and Castlepik, Michigan State University Research Report, 18p. Wiersum, L.K., 1979. Effects of environment and cultural practices on calcium nutrition. Commun. Soil Sci. Plt. Anal. 10(182):259-278. 166 39. Zimmermann, M. 1966. Translocation of nutrients. In: The Physiology of Plant Growth and Development. M.B. Wilkins ed. McGraw-Hill. New York. 381-417. SUMMARY The objectives of this study were to evaluate the effects of modified endogenous calcium (Cab) within fruit tissues on texture and the rate of softening of fresh, stored and processed pickling cucumber fruit, to determine the influence of low tissue Ca2+ levels on polygalacturonase (PG) activity in fresh and stored fruit, and to verify the effect of Caz“ addition to the brine solution on texture after fresh-pack processing. Alternative Caz’ fertilization strategies for enhancing fruit Caz‘ concentrations in field- grown pickling cucumbers, and how fruit set and growth might affect Ca?” supply via the xylem to the fruit were also investigated. Firmness of freshly harvested fruit was not affected by the Ca2+ fertilization treatments (Table 1, Appendix), but C212" might have an important role on firmness retention during storage, considering the higher firmness and lower rate of softening in the pericarp tissue of the high Caz“ treatment (20.0 mM Caz’) after 5 days of storage (Experiment 2, Table 3) . The positive correlations between fruit Ca2+ concentrations and fruit firmness after 5 and 24 days of storage also add support for endogenous fruit Caz’ having a role in fruit softening during storage. Physiological processes involving Cab during storage and post-harvest processing do not take place in freshly harvested fruit. Perhaps, because in the freshly 167 168 harvested fruit still at the immature developmental stage, the macromolecules structures within the protoplast are well stabilized at the micromolar levels of free calcium (10*‘M or less), thus maintaining the normal cell functions. Any variation in cytosolic Cab'levels in response to environment (Kretsinger, 1977) as in the case of stored fruit has physiological implications as a liquid-gel phase change in the membranes. Leshem et al. (1986) reported that the passage of external Cab'across the membrane into the cytosol, or entry into the cytosol of Caa'from.Ca”-containing'organelles sets a "cascade effect" into motion due to the combination of Ca” with cytosolic calmodulin (CM). The complex CA:CM activates phospholipase-A.z which causes the liquid-gel phase change, hastening the membrane deterioration process. The fatty acid tails inutheibilayer'become "frozen" and completely lose their motional freedom. As a result, the membrane becomes rigid and the embedded proteins are no longer able to move. Leaks are formed and overall function impaired. External high Cab' concentrations maintain membrane integrity and consequentely firmness retention while within the cytosol Caz’ exerts an opposite effect. In the present study, the culturally modified endogenous Ca2+ within the cucumber fruit ranged from 0.08% to 2.4% Cab'and from 0.05% to 0.5% Cab’in pericarp and endocarp tissues, respectively. It was observed in the field experiment, however, that the 169 foliar Caz‘ applied affected only slightly the Ca2+ concentrations within the fruit tissues as compared with the broad range of Cab’concentrations found in the fruit tissues from greenhouse grown cucumbers. Thus, the relationships between the concentrations of Caa'within compartments as the apoplast, and fruit firmness in field cultured cucumbers should be considered in a further study. PG activity of fresh fruit was not affected by the Ca2+ fertilization treatments (Table 1, Appendix). In fresh fruit the concentration of total cell wall polysaccharides influenced more texture than the molecular structures of those polysaccharides (McFeeters 8 Lovdal, 1987). Also, the decrease in rhamnose, arabinose, and galactose observed during fruit development of fresh cucumbers suggests that other enzymes than polygalacturonase might be involved in the softening process of fresh pickling cucumber fruit. This is also applicable to the processed fruit. Losses in galactose from mesocarp cell walls have been observed in fresh-pack pickles (Howard 8 Buescher, 1990) . Thus, other enzymes such as fi-galactosidase could be involved in the softening process of cucumbers. It is interesting to verify also how the mechanism of Cab'action in the cell wall might relate to this enzyme. Caa'enhanced the textural firmness of fresh-pack pickles following blanching and during refrigeration, even when Ca”’ was added to the brine solution before blanching and storage. 170 Ca2+ added to the brine solution also improved texture of fresh-pack pickles after accelerated aging and during refrigeration. However, higher pericarp softening was observed in the processed spears and slices with 20 mM Caa'added to the brine solution as compared to 0 or 5 mM Cab'brine treatment during incubation at 46°C. The rate of softening was also higher in the high Cay'brine treatment (20 mM Ca”) than in the 0 or 5 mM Caz“ brine treatments during refrigeration of processed slices. Changes in solubility of the pectic polymers could have occurred during incubation time or refrigeration period since changes in solubility characteristics can be caused by exposure to low pH and elevated salt environment of the pickling solution (Buescher 8 Hudson, 1986) . Acidification due to HP ions in the brine solution could cause displacement of Caa'ion from.the cross-linking of galacturonic acid residues. Howard 8 Buescher (1990) reported that firmness of fresh-pack pickles coincided more closely with the amount of Cab'bound to the cell walls than to other cell wall characteristics. In the light of these comments, changes in bound Cab'should be investigated during either the incubation time or the refrigeration period. Foliar applications of 2.5 % Nutri-Cal (v/v) enhanced significantly pericarp firmness of fresh #3 size grade pickling cucumber fruit. The relatively high pericarp tissue Caa'concentration in fruit from plants treated with Nutri-Cal 171 (2.5 % solution) might indicate a relationship between Ca2+ levels in the fruit and tissue texture. A potential hypothesis is that the chelator being used in the Nutri-Cal formulation, 2, 3, 4,5 trihydroxypentanedoic acid, might be inducing a specific physiological response that impacts upon fruit texture. An associated physiological response is increased lignification of cell walls in leaf and potentially fruit tissue which would alter significantly textural characteristics (Dean 8 Kuc, 1987). Thus, investigations should be conducted to evaluate if the chelator used in the present study induces some physiological response such as increased lignification of cell walls in leaf and fruit tissues which is an important factor influencing texture. In this study, Caa' concentrations in xylem exudate decreased during the reproductive stage with a small increase at fruit maturity. A decrease in cucumber root growth during fruit development.has been observed (McCollum, 1934; Stigter, 1969: Van der Vlugt, 1990) extending until harvest when a recovery in growth occurred after the fruit were removed from the plant or at fruit.maturity (Stigter, 1969). It.is possible that Caa’uptake and transport through the xylem is impaired during fruit development, due to the root-fruit competition for assimilates and the reduction in root growth. Defruited plants also showed significantly’higher'Caa'concentrations in xylem exudate than fruiting plants. 172 In conclusion, the results to support the hypothesis that Cah'concentrations in xylem sap and thus Cab'uptake decline during plant ontogeny. This factor could contribute in part for the relatively low Cab’content in fruits. Appendix 173 Table 1 - Effect of Caa'tmeatment level on firmness and PG activity of fresh 4.5 cm diameter pickling cucumber fruit tissues (Experiment 1). 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