tomato fruit continues growing while ripening, affecting cuticle properties and cracking

Physiologia Plantarum 2012 Copyright © Physiologia Plantarum 2012, ISSN 0031-9317

Tomato fruit continues growing while ripening, affectingcuticle properties and crackingEva Domıngueza,† , Marıa Dolores Fernandezb,†, Juan Carlos Lopez Hernandezb, Jeronimo PerezParrab, Laura Espanaa, Antonio Herediaa and Jesus Cuarteroa,∗

aIHSM ‘‘La Mayora’’ (CSIC-Universidad de Malaga), Algarrobo-Costa, E-29750 Malaga, SpainbEstacion Experimental ‘‘Las Palmerillas’’, Fundacion Cajamar, E-04080 Almerıa, Spain

Correspondence*Corresponding author,e-mail: [email protected]

Received 20 February 2012; revised 12March 2012

doi:10.1111/j.1399-3054.2012.01647.x

Fruit cuticle composition and their mechanical performance have a specialrole during ripening because internal pressure is no longer sustained by thedegraded cell walls of the pericarp but is directly transmitted to epidermis andcuticle which could eventually crack. We have studied fruit growth, cuticlemodifications and its biomechanics, and fruit cracking in tomato; tomato hasbeen considered a model system for studying fleshy fruit growth and ripening.Tomato fruit cracking is a major disorder that causes severe economic lossesand, in cherry tomato, crack appearance is limited to the ripening process.As environmental conditions play a crucial role in fruit growing, ripening andcracking, we grow two cherry tomato cultivars in four conditions of radiationand relative humidity (RH). High RH and low radiation decreased the amountof cuticle and cuticle components accumulated. No effect of RH in cuticlebiomechanics was detected. However, cracked fruits had a significantlyless deformable (lower maximum strain) cuticle than non-cracked fruits. Asignificant and continuous fruit growth from mature green to overripe hasbeen detected with special displacement sensors. This growth rate variedamong genotypes, with cracking-sensitive genotypes showing higher growthrates than cracking-resistant ones. Environmental conditions modified thisgrowth rate during ripening, with higher growing rates under high RH andradiation. These conditions corresponded to those that favored fruit cracking.Fruit growth rate during ripening, probably sustained by an internal turgorpressure, is a key parameter in fruit cracking, because fruits that ripeneddetached from the vine did not crack.

Introduction

Tomato (Solanum lycopersicum) has been considereda model system for studying fleshy fruit growth andripening (Giovannoni 2001). Fruit ripening involves acomplex series of interrelated processes that includemodifications in texture, cell wall ultrastructure, compo-sition and others (Brady 1987). These alterations enable

Abbreviations – RH, relative humidity.

†These authors equally contributed to this work.

the fruit to accomplish its biological function of seeddispersal and have many horticultural implications: theyrender the fruit edible while at the same time limit shelflife and affect appearance, harvesting and handling tech-niques. Mechanical performance of the tomato fruit skinduring ripening is important because internal pressureis no longer sustained by the degraded cell walls of the

Physiol. Plant. 2012

pericarp but is directly transmitted to the outer cuticle.Almeida and Huber (1999, 2001) reported an increasein locular pressure during ripening because of theliquefaction of locular tissue (Cheng and Huber 1996).This liquid could infiltrate the pericarp apoplast andaffect fruit cracking susceptibility (Almeida and Huber2001). Thus, cuticle biomechanical behavior at ripeningcould affect fruit appearance, cracking resistance(Emmons and Scott 1997) as well as handling and furtherstorage (Chu and Thompson 1972).

The plant cuticle is an outer membrane that cov-ers aerial parts of the plant. It is an efficient barrier towater loss, attenuates ultraviolet light absorption andprotects from biotic and abiotic environmental stresses(Domınguez et al. 2011b). It is composed of a frameworkof cross-linked fatty acids, namely cutin, with waxesdeposited on the surface and inside the cutin matrix.The inner side of the cuticle contains a variable amountof polysaccharide material derived from the epidermalcell wall, which links the cuticle to the cell wall (Jeffree2006). A small fraction of phenolics, mostly flavonoidsin the case of tomato fruit cuticle (Hunt and Baker 1980),is also present in the cutin framework either trapped orchemically bonded (Domınguez et al. 2009a). In recentyears, the role of each cuticle fraction to the overallmechanical behavior of the tomato fruit cuticle has beenstudied (for a review, see Domınguez et al. 2011a). Thus,waxes and flavonoids have been shown to act as fillersincreasing the mechanical strength and stiffness of thecuticle (Petracek and Bukovac 1995, Domınguez et al.2009b). On the other hand, polysaccharides contributeto the linear elastic behavior of the cuticle, whereasthe cutin matrix is responsible for the high strain andviscoelastic behavior (Lopez-Casado et al. 2007).

Fruit cracking is an important disorder that has a neg-ative economic impact in fruit marketability especiallybecause cracks downgrade the quality of fruits as theycause poor appearance, reduce shelf life and even renderthe fruit unmarketable because of fungal infection (Peet1992). Cracking can occur during fruit growth and/orripening and affects several fruits such as tomato, pep-per, cherries and many others (Sekse 1995, Aloni et al.1998, Dorais et al. 2004, Huang et al. 2006). Cracksare assumed to occur when internal pressure exceedsthe breaking stress of the epidermis, mainly the cuti-cle (Ohta et al. 1997). In cherry tomato, cracking onlyappears during ripening (Bakker 1988, Ehret et al. 1993)and has been shown to have a genetic as well as anenvironmental component (Peet 1992). Genetic differ-ences in cracking susceptibility between genotypes havebeen associated with fruit shape and size, fruit growthrate, sugar content and fruit cuticle (Hankinson and Rao1979, Ehret et al. 1993, Emmons and Scott 1997, 1998).

Environmental conditions affect cuticle biomechan-ical properties as well as fruit cracking. The role ofenvironmental conditions, mainly relative humidity (RH)and temperature, on the mechanical performance of thetomato cuticle has been analyzed in vitro (Wiedemannand Neinhuis 1998, Matas et al. 2005), and both condi-tions were shown to decrease the elastic modulus andincrease the cuticle deformability, that is, they rendereda cuticle more easily deformable and less resistant tostress. Environmental variables were imposed during themechanical tests; however, in the present work, we haveinvestigated the mechanical properties of cuticles grownin different environmental conditions. There is a com-plex and well-documented relationship among tomatofruit cracking, cultivars, greenhouse environment andcultural practices (Dorais et al. 2004). Increases in irra-diance (Ehret et al. 1993), temperature (Aloni et al. 1998)and RH (Bertin et al. 2000, Leonardi et al. 2000) havebeen shown to have a positive effect on fruit cracking.Environmental temperature could have an indirect effectin fruit cracking by increasing photoassimilate supply tothe fruit (Walker and Ho 1977) and fruit growth rate(Pearce et al. 1993a, 1993b). RH can indirectly affectcracking through its influence on plant transpiration andfruit water status. High RH decreases leaf transpiration,which might result in increased fruit water supply andturgor pressure. Fruit cracking was observed more fre-quently at low vapor pressure deficit conditions (Bertinet al. 2000).

In tomato, variations of fruit growth rate during the day(Pearce et al. 1993a, 1993b, Guichard et al. 2001) havebeen suggested to significantly contribute to cracking.Several studies have analyzed the mechanical propertiesof the tomato fruit skin and isolated cuticle (Matas et al.2004a, Bargel and Neinhuis 2005, Domınguez et al.2011a). Hence, it has been proposed that the tomatofruit cuticle is of increasing importance for the structuralintegrity of the fruit during ripening and, as such, playsa prominent role in fruit cracking. Alteration of RH,water stress and irrigation or crop loading could favorthe occurrence of cracks through the intensification ofthese stresses (Milad and Shackel 1992, McFayden et al.1996, Børve and Sekse 2000).

In the present work, we have taken into account anumber of variables affecting fruit and cuticle growthduring ripening in order to reach a comprehensive pic-ture of this special period of fruit development. The roleof environmental conditions, mainly radiation and RH,on fruit cracking and cuticle development, composi-tion and biomechanics has been investigated in cherrytomato. To avoid differences due to fruit shape andsize, two cultivars with these same characteristics havebeen elected (Lopez-Casado 2006). At the same time,


changes in fruit diameter during ripening, most probablyattributed to variations in fruit internal pressure, havebeen recorded with the aid of special displacement sen-sors in genotypes with differential cracking sensitivity.A significant positive growth during fruit ripening andoverripening has been detected and discussed here forthe first time. This growth rate is genotype dependentand is affected by environmental conditions.

Materials and methods

Plant material

Solanum lycopersicum plants cv. Gardener’s Delight andCascada were used in this study. Gardener’s Delight wasselected because of its high incidence of fruit cracking ingreenhouse cultivation, whereas Cascada was selectedfor its low fruit cracking. Both cultivars show similar fruitgrowth pattern and have similar fruit size at ripening(Lopez-Casado 2006). Six hundred plants of Gardener’sDelight and Cascada, divided into four replicates of150 plants, were grown in two polyethylene multitunnelgreenhouses (630 m2 area per greenhouse) during twogrowing seasons, spring and winter. Experiments wereconducted at the Estacion Experimental Las Palmerillas,Fundacion Cajamar (36◦48′N; 2◦30′W and 151 ma.s.l), located in El Ejido, Almerıa, southeast of Spain.Two conditions of RH, high and low, were held inboth seasons, with each RH treatment in a differentgreenhouse. A combination of misting (1 l m−2 h−1)

during the day hours and passive ventilation was usedin spring to maintain a minimum RH of around 65%while the low RH treatment was determined by theexternal environmental conditions (minimum RH around50%); maximum RH was similar in both greenhouses,around 90%. Misting was used from March 1 (plantswith open flowers in the second to third truss) till the

end of experiment (end of June). In winter, heating(120 000 kcal h−1) during the night hours and passiveventilation was used to decrease the RH by modifyingthe temperature, obtaining values of 50–60% during theday and around 90% during the night. Environmentalconditions on the outside determined the high RH in thegreenhouse around 60–70% and 90% RH during dayand night, respectively. Heating system worked from lateOctober (plants with flowers in the second to third truss)until the end of experiment in March.

Tomato seedlings were grown in an insect-proofglasshouse, and plants of each tomato genotype weretransplanted to perlite bags (40 l, granulometry 1–5 mmØ) at a density of three plants per square meter at thefour-true-leaf growth stage. Six plants were transplantedper bag, fertirrigated with a standard nutrient solutioncommonly employed for tomato growth (Canovas 1995),supported by strings and pruned to a single stem. Fourreplicates per genotype with three lines of 50 plants/line/replicate were randomly distributed in each greenhouse.

The harvesting period lasted from March until theend of June in spring and from December until themiddle of March in winter. Temperature and RH ineach greenhouse were measured every 5 min duringthe growing period. Maximum, minimum and meantemperature, RH and radiation during the growing periodfor both experiments are shown in Table 1.

The following fruit stages of ripeness were considered(adapted from US Standards, cited by Grierson and Kader1986): immature green (fruit detached from the plant isunable to ripen), mature green (fruit entirely light greento dark green, but able to ripen detached from the plant),breaker (first appearance of external pink, red or tannishyellow color; not more than 10%), pink (over 10% butnot more than 30% red, pink or tannish yellow), lightorange (over 30% but not more than 60% pinkish or

Table 1. Maximum and minimum RH (%), temperature (Temp, ◦C) and radiation (MJ m−2 day−1) averages in spring and winter during the plantgrowth period in the greenhouses with high (+) and low (−) RH conditions.

Greenhouse RH+ Greenhouse RH− Radiation

Max RH Min RH Max Temp Min Temp MaxRH Min RH Max Temp Min Temp Max Min

SpringMarch 88.6 64.7 25.2 9.1 84.2 41.3 27.6 9.1 22.6 7.8April 91.9 70.7 25.2 11.4 90.1 53.2 27.5 11.6 27.4 7.2May 92.8 67.4 27.0 13.3 92.0 53.0 28.5 13.7 21.0 7.4June 92.8 64.3 30.9 16.1 92.1 50.8 32.0 16.5 29.8 12.3

WinterNovember 90.2 60.1 24.4 10.9 86.3 54.3 25.4 13.2 14.9 5.8December 91.8 68.2 21.2 9.6 85.7 57.2 21.7 11.0 10.8 3.2January 92.3 68.9 22.3 8.3 88.6 53.5 22.9 10.5 12.6 2.5February 92.6 67.1 23.4 10.3 86.6 59.5 24.1 11.5 17.1 1.8March 92.5 57.1 26.4 9.2 87.5 41.0 27.2 11.1 24.0 5.6


Fig. 1. Photographs of a dendrometer placed in the tomato fruit withthe aid of the custom-made holder designed for the purpose.

red), light red (over 60% but not more than 90% red),red (over 90% red) and overripe (over 90% red and somesoftening).

Dendrometers

Fruit diameter was recorded every 3 min with alinear variable displacement transducer (LVDT sensorsmodel 2.5 DF; Solartron Metrology, Bognor Regis,UK), also known as dendrometer, with a measurerange of 15–40 mm. Data were averaged every 15 minand stored in a datalogger (model CR800; CampbellScientific Inc., Logan, Utah, USA) connected to amultiplexor (model AM16/32; Campbell Scientific Inc.,Logan, Utah, USA).

Dendrometers were placed on aluminum and invar(iron/nickel alloy with a low expansion/dilation coeffi-cient) custom-made holders (see Fig. 1). Five dendrom-eters per genotype and RH were placed in fruits locatedin the middle of the truss. Fruit diameter was measured

before placing the dendrometers. Changes in fruit diam-eter were measured from breaker until fruits cracked oroverripened in spring and from mature green to ripe orcracked fruits in winter. Fruit growth was estimated fromthe differences in daily fruit diameter.

Cracking percentage

Ten plants per genotype, greenhouse and replicationwere selected to study the effect of RH and seasonon fruit cracking. Red ripe fruits were collected every4–7 days in spring and weekly in winter, and crackedand non-cracked fruits were counted and weighed.

Post-harvest cracking was measured in winter. Fortyfruits of Gardener’s Delight per replicate and RH werecollected with peduncle, at breaker stage, and allowedto ripe in well-aired boxes inside the greenhouse. At thesame time, a similar number of Gardener’s Delight fruitsper RH and replicate were labeled at the breaker stageand allowed to ripe in the plant. The number of crackedand not cracked fruit was counted once fruits reachedthe red stage.

Cuticle thickness and surface area

Three fruits per genotype and RH were harvested,and small pericarp pieces of each fruit were fixedin a formaldehyde, acetic acid and ethanol solution(1:1:18), dehydrated in an ethanol series (70–95%)and embedded in a commercial resin (Leica His-toresin Embedding Kit; Leica Microsystems, Heidelberg,Germany). Samples were cross-sectioned into 4-μmthick slices using a Leica microtome (RM2125, LeicaMicrosystems GmbH, Wetzlar, Germany). Sudan IV wasemployed to differentially stain the cuticle using the pro-tocol described by Jensen (1962), and a minimum of fiveslices per sample were inspected under a light micro-scope (Nikon, Eclipse E800, Nikon Instruments Europe,B.V., Amstelseen, Netherlands.).

Cuticle thickness was estimated from a minimum of30–50 measurements using an image capture analysisprogram (Visilog-Noesis 6.3). Central region betweenpegs of the cuticle covering epidermal cells was usedto estimate cuticle thickness because this area remainsalmost constant throughout a fruit or leaf and is notaffected by cuticle invaginations. Cuticular area wascalculated using the same program and a minimum of20 measures per genotype and stage.

Cuticle isolation

Cuticles were enzymatically isolated from tomato redripe fruits grown at different RH following the protocol


of Orgell (1955) as modified by Yamada et al. (1964), seePetracek and Bukovac (1995), using an aqueous solutionof a mixture of fungal cellulase (0.2% w/v; Sigma, St.Louis, MO) and pectinase (2.0% w/v; Sigma), and 1 mMNaN3 to prevent microbial growth, in sodium citratebuffer (50 mM, pH 3.7). Vacuum was used to facilitateenzyme penetration, and fruit samples were incubatedwith continuous agitation at 35◦C for at least 14 days.The cuticle was then separated from the epidermis,rinsed in distilled water and stored under dry conditions.

Cuticle components

Total cuticle waxes were removed by heating theisolated cuticles at 50◦C in chloroform:methanol (2:1v/v) for 2 h. Cutin isolates were obtained by refluxingdewaxed cuticles in a 6 M HCl solution for 12 h. Thisprocedure removed polar hydrolyzable components,mainly polysaccharides (Matas et al. 2004b). Cuticularcomponents (waxes, cutin and polysaccharides) wereestimated from 10 samples per genotype.

Mechanical tests

The cuticle mechanical properties of fruits, whosegrowth history was recorded by the dendrometers,were measured following the previous work of Mataset al. (2005). Rectangular uniform segments (3 × 9 mm)of isolated cuticles were removed, microscopicallyinspected to confirm the absence of small cracks andthen fixed to two hollow stainless-steel needles, by asmall amount of fast-drying super glue. The system wasenclosed in an environment-controlled chamber thatallowed control of temperature and RH. Each cuticlesample was held inside the extensometer chamber for atleast 30 min to equilibrate the temperature and humiditywith the medium before beginning the extension test.

The cross-sectional area of the samples and the lengthof the exposed surface of the sample between the twosupports were measured before mechanical extensiontests. The mechanical tests were performed as a transientcreep test to determine the changes in length of a cuti-cle segment by maintaining samples in uniaxial tension,under a constant load, for 1200 s, during which time thelongitudinal extension of each sample was recorded bya computer system every 3 s. Each sample was testedrepeatedly using an ascending sequence of sustainedtensile forces (from 0.098 N to breaking point by 0.098N load increments) without recovery time (Matas et al.2005). To determine stresses, the tensile force exertedalong the sample was divided by the representative cross-sectional area of the sample. To obtain the correspondingstress–strain curve and elastic modulus, the applied

stress was plotted against the total change in lengthafter 20 min. Breaking stress and maximum strain at thebreaking stress were also determined for each sample.

Strain–time and the corresponding stress–straincurves were calculated for a set of five to seven samplesof isolated cuticles at 25◦C and 40% RH. Six fruits perRH treatment were studied; three corresponding to fruitthat cracked and another three to fruit that did not crack.

Statistics

Simple regression analysis was used to fit fruit growth tostraight lines with one or two slopes. Mean comparisonwas used to determine whether the measured character-istics of the cuticle varied significantly as a function ofthe genotype, stage of fruit ripening and/or RH. Analyseswere performed using the IBM SPSS software package(IBM SPSS 2010), and data presented as means ± SEwith a level of significance of 5% (P = 0.05).

Results

Environmental conditions and fruit cracking

Cultural techniques applied to modify RH resulted insmall differences between RH during spring and winter(Table 1). Daily fluctuations in minimum and maxi-mum RH lowered the average RH differences betweengreenhouses with high and low RH. However, daily dif-ferences in RH between greenhouses were significantlydifferent from zero with an average of 18.5 ± 6.2% and13.0 ± 6.2% in minimum RH and 6.7 ± 2.9% and 9.1± 4.0% in mean RH (spring and winter, respectively).No significant differences were observed in maximumRH between RH treatments. Misting and heating slightlymodified the maximum (approximately 1.5◦C) and min-imum (approximately 1.8◦C) temperature, respectively,but they were not significant (Table 1).

Fig. 2 shows the percentage of fruit cracking in springand winter under two RH conditions for the two geno-types studied herein. Significant differences betweengenotypes were observed. Cascada showed a very lowpercentage of fruit cracking, not different from zero inmost of the harvestings, regardless of the RH and sea-son, whereas Gardener’s Delight, on the other hand,showed a high percentage of fruit cracking with dif-ferences between RHs in both seasons. Although thedifferences in RH obtained in both seasons were not ashigh as desirable (see Table 1), Gardener’s Delight con-sistently displayed a higher cracking percentage underhigh RH in spring and winter (Fig. 2A, B). There were fruitcracking differences between harvesting dates in bothseasons; especially on June 15 and 19 and February 15,


Sampling dates

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Fig. 2. Percentage of fruit cracking in Gardener’s Delight and Cascadaduring the spring (A) and winter (B) experiments under two RHconditions. Data correspond to the mean ± SE of four replicates,with over 200 fruits sampled per replicate.

the percentage of fruit cracking was abnormally lower.A general trend to increase fruit cracking throughout theharvesting period was observed in spring, whereas inwinter the opposite trend was found.

Percentage of cracking in fruits that ripened attachedand detached from the vine was estimated in winter forGardener’s Delight, the only genotype that did crack.Thus, fruits that ripened detached from the vine did notcrack regardless of the RH, whereas those that ripenedon the vine showed a high percentage of fruit cracking:73.1 ± 4.2% (high RH) and 56.2 ± 4.1% (low RH).Again, differences in fruit cracking depending on the RHwere observed with higher cracking percentage in highRH conditions. As both sets of fruits ripened at the same

time and under similar environmental conditions, thehigher fruit cracking on the vine points to a water andsolutes influx from the plant, which could increase fruitinternal pressure and hence fruit diameter.

Fruit growth during ripening

The study of possible changes in fruit diameter because ofdifferences in fruit internal pressure was approached withthe aid of special sensors, dendrometers, which recordedchanges in fruit diameter every 3 min. As cracking incherry tomato occurs during ripening, dendrometerswere placed on mature green fruits, some 10–15 daysbefore breaker, depending on the genotype, and leftuntil fruits cracked or overripened. Fifty-one fruits weremonitored in Gardener’s Delight and 40 in Cascada.Surprisingly, fruit growth, estimated from daily increasesin diameter, did not come to an arrest at the maturegreen stage but continued during ripening (Fig. 3). Thiswas true for all the fruits studied of both genotypesregardless of RH and growing season. The rate offruit growth manifested a biphasic behavior in mostof the studied fruits (Fig. 3A, C). Each of these twophases could be fitted to a straight line with differentslopes. A fruit was considered to show two growingrates when the difference between both slopes wassignificant. Regression deviations were only 4 and 7%of the regression value for the cases of fruits consideredto show one and two slopes, respectively. The first slopedenoted a higher rate of fruit growth and correspondedto mature green–light orange period of ripening. Thesecond slope was less pronounced indicating a decreasein the rate of fruit growth that corresponds to the lightorange–overripe period. This second rate of growthcontinued as long as fruits were maintained in the plant(Fig. 3, Table 2). Only in seven cases out of 91 recordedfruits, fruit growth ceased and the second slope was notdifferent from zero. Fruit cracking could be detected bya sudden and significant drop in fruit diameter (Fig. 3B).Breaker stage was attained during the first growing rate,before change of slope, i.e. decrease in growing rate,occurred near the light orange–red stages.

Table 2 shows the mean values of Cascada and Gar-dener’s Delight fruit growth rates (slopes) in spring andwinter experiments at high and low RH. Significantdifferences in fruit growth were observed between geno-types, regardless of the season and RH, during the maturegreen–light orange period, which corresponded to thefirst slope. In this sense, Gardener’s Delight showed a sig-nificantly higher rate of fruit growth (0.155 mm day−1)

compared with Cascada (0.076 mm day−1). Differencesbetween the first and second growth rate, althoughobserved in both genotypes, were more pronounced


Days to and from breaker

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Fig. 3. Diameter increase per day in two Gardener’s Delight fruit one that did not crack (A) and one that did crack (B) together with two Cascadafruit one with two slopes (C) and one with only one slope (D). Both slopes can also be easily observed in (A) as well as the day the Gardener’s Delightfruit cracked (B).

and statistically significant in Gardener’s Delight. InCascada, differences between both growth slopes weresmall, and in some cases only one slope was recorded(see Fig. 3C, D). In both genotypes, fruit growth ratewas slower in winter than in spring during the maturegreen–light orange period. An effect of the RH on thefirst growing rate was observed for both genotypes inspring and winter, with fruits displaying higher growth

values under high RH conditions. No significant differ-ences in the second slope, which corresponded to thelight orange–overripe period, were observed betweengenotypes or RH.

The possible effect of fruit growth on fruit cracking wasanalyzed in Gardener’s Delight, the cracking-sensitivegenotype. Fruits that eventually cracked showed asignificantly higher growth rate during the light

Table 2. Mean ± SE of the increase rate in fruit diameter (measured as the slope of the regression line) for the genotypes Cascada and Gardener’sDelight at two RH conditions and in two experiments, spring and winter. In Gardener’s Delight, rates of cracked and non-cracked fruit are alsoincluded. The number of fruit is between brackets.

Spring

RH+ RH−Genotype Fruits First slope Second slope First slope Second slope

Cascada Total 0.103 ± 0.013 (10) 0.053 ± 0.011 (6) 0.072 ± 0.010 (9) 0.045 ± 0.005 (5)Gardener’s Delight Total 0.205 ± 0.011 (10) 0.052 ± 0.008 (7) 0.156 ± 0.023 (12) 0.045 ± 0.015 (8)

Cracked 0.207 ± 0.007 (3) 0.205 ± 0.012 (5) 0.101 (1)Non-cracked 0.204 ± 0.015 (7) 0.052 ± 0.008 (7) 0.121 ± 0.021 (7) 0.037 ± 0.015 (7)

Winter

RH+ RH−Genotype Fruits First slope Second slope First slope Second slope

Cascada Total 0.074 ± 0.007 (10) 0.046 ± 0.005 (5) 0.053 ± 0.006 (11) 0.023 ± 0.006 (3)Gardener’s Delight Total 0.126 ± 0.007 (14) 0.042 ± 0.011 (9) 0.113 ± 0.007 (15) 0.026 ± 0.008 (11)

Cracked 0.114 ± 0.009 (8) 0.067 ± 0.013 (3) 0.124 ± 0.005 (6) 0.053 ± 0.002 (2)Non-cracked 0.142 ± 0.007 (6) 0.029 ± 0.013 (6) 0.106 ± 0.010 (9) 0.019 ± 0.009 (9)


orange–overripe period than fruits that did not crack(Table 2). In low RH conditions, cracked fruits also had ahigher growth rate during the mature green–light orangeperiod. This behavior could not be observed under highRH. Thus, Gardener’s Delight fruits that did not slowtheir growth upon entering maturation or delayed itwere those eventually cracked.

Cuticle composition and biomechanical parametersof the cuticle

The role of environmental conditions, mainly RH andradiation, on the amount of cuticle deposited duringfruit growth was studied at three stages of development,immature green, breaker and red in the two genotypes,Gardener’s Delight and Cascada. Table 3 shows themicrograms per square centimeter of cuticle and itscomponents measured in Cascada and Gardener’sDelight at red in two seasons and under two RH.Similar differences in cuticle accumulation due toenvironmental conditions were observed in all thedevelopmental stages studied; hence, only data at red arepresented here. The amount of cuticle was significantlyhigher in spring compared with winter because ofan increase in all cuticle components except waxes(Table 3). Average environmental conditions in springand winter were quite similar for RH and temperature,but radiation and daily sun hours (13.4 and 10.7 hin spring and winter, respectively) were significantlyhigher in spring (Table 1). Significant differences in theamount of cuticle and cutin were observed in springbetween genotypes at the three stages of development(immature green, breaker and red), but these differencescould not be observed during winter. In spring, RH hada noticeable effect on the amount of cuticle, cutin,polysaccharides and phenolics, which significantlydecreased under high RH in both genotypes at anystage of development. The effect of RH on waxeswas less pronounced and only significant at the redstage. In winter, this trend toward lower amount ofcuticle components under high RH was maintained,although the differences observed between RHs werenot significant, except for waxes that were higher underlow RHs for both genotypes at red.

Comparison of mechanical properties of Gardener’sDelight fruits that cracked and did not crack grownunder two RH conditions was carried out in spring(Fig. 4). Cuticle biomechanics showed a typical biphasicbehavior with an elastic component at low stressesand a viscoelastic component at higher stresses (datanot shown). Non-cracked fruits had a significant highermaximum strain and viscoelastic phase compared withcracked ones, whereas the elastic modulus and breaking

stress were similar (Fig. 4). At high RH, breaking stress,maximum strain and viscoelastic phase were higher innon-cracked than in cracked fruits. This trend was alsoobserved under low RH conditions, but differences wereonly significant for % viscoelastic phase. No clear effectof RH on cuticle biomechanics was observed exceptfor a significant lower viscoelastic phase under high RHin cracked fruits. This difference between RHs couldnot be observed in non-cracked fruits. As the Young’smodulus and breaking stress were similar in cracked andnon-cracked fruits and under high and low RH, cuticlestiffness was not modified. The effect on the viscoelasticphase indicated a higher deformability in non-crackedfruits under both conditions.

Discussion

Fruit growth continues from maturegreen to overripe

Tomato fruit growth can be divided into three periods(Ho and Hewitt 1986). A first period of slow growthmainly corresponds to the cell division stage (Bertinet al. 2007) with a gain of fruit weight less than 10%final weight. A second period of rapid growth mainlyattributed to cell expansion, and finally, a last periodof slow growth with little gain in fruit weight butwhere intensive metabolic changes take place (Ho andHewitt 1986). In the present work, the final period offruit growth has been recorded, because it is in thisperiod when fruit cracking takes place in cherry tomato.This growth during fruit ripening could be accuratelymodeled by simple linear functions, which allowedeasier comparison of fruits from different genotypesor different environments than asymmetric sigmoid(Monselise et al. 1978), Gompertz (Grange and Andrews1993, Bertin 1995) or Richards (Heuvelink and Marcelis1989) functions proposed to model the complete fruitgrowth period.

Mature green has been traditionally accepted to bethe developmental stage in which fruits raise theirfinal size, even though the ripening process is stillnot visible (Grierson and Kader 1986). Cheniclet et al.(2005) detected a period of fruit growth arrest aroundmature green, prior to a final cell expansion periodat the transition between mature green and breakerin cherry tomato, but they could not associate thisfinal increase in cell area with a measurable increasein fruit diameter. In this work, we have detectedcontinuous increase in fruit diameter that correspondedto a growth rate of 0.1–0.2 mm day−1 (Table 2) duringthe ripening period. This fruit growth, although 5–10times slower than growth during immature green, and


Table 3. Micrograms per square centimeter (mean ± SE) of cuticle and its components cutin, waxes, polysaccharides and phenolics for two genotypesCascada and Gardener’s Delight at red ripe. Cuticle quantitative analysis was performed on plants grown under two RH conditions (+ high and −low) and in two experiments, spring and winter. For each genotype, statistically significant differences (P ≥ 0.95) are indicated with different letters:bold indicates differences between RH within the same season, and italics indicate differences between seasons for the same RH.

Genotype Component Spring WinterRH+ RH− RH+ RH−

Cascada Cuticle 1185 ± 36 b a 1506 ± 35 a a 935 ± 49 a b 989 ± 34 a bCutin 860 ± 26 b a 1096 ± 26 a a 657 ± 35 a b 649 ± 22 a bWaxes 24 ± 1 a b 26 ± 1 a b 31 ± 2 b a 37 ± 1 a a

Polysaccharides 301 ± 9 b a 383 ± 9 a a 247 ± 13 b b 303 ± 10 a bPhenolics 99 ± 6 b a 130 ± 12 aa 50 ± 1 a b 48 ± 1 a b

Gardener’s Delight Cuticle 1018 ± 17 b a 1114 ± 17 a a 954 ± 25 a b 1015 ± 31 a bCutin 674 ± 11 b a 730 ± 11 aa 650 ± 18 a a 684 ± 23 a bWaxes 25 ± 0 ba 23 ± 0 a b 25 ± 1 b a 38 ± 2 a a

Polysaccharides 319 ± 6 b a 362 ± 5 a a 278 ± 9 b b 293 ± 14 a bPhenolics 89 ± 4 b a 104 ± 2 a a 70 ± 3 a b 65 ± 2 a b

with a limited repercussion in the final fruit size, isnot negligible. Tomato fruit grew at a constant ratefrom at least 15 days before breaker to 4–5 days afterbreaker. This growth was slowed down around lightorange and remained until overripe. It should be studiedif this decrease in fruit growth can be associated toevents that could take place later in ripening. Thus,ripening did not seem to influence fruit growth becausefruits continued growing even till overripe. Chenicletet al. (2005) postulated that the cell wall modificationsdescribed by Giovannoni (2004) could contribute tothe mechanism underlying cell expansion from maturegreen to ripe. However, cell wall degradation alonecould not lead to a significant increase in fruit diameter,because most of the cell volume is occupied by water,which is highly incompressible. Fruits grow at a constantrate from 15 days before breaker to light orange, whichsuggest the same causes of fruit growth operate beforeand after maturation.

Fruit growth can be attributed to the movement ofwater and carbohydrates from the stem to the fruit. Inmost tomato genotypes, such as those studied herein,these influxes are limited or prevented during ripen-ing with the development of an abscission zone in thepedicel. Hence, this abscission zone has been consid-ered to contribute to the cease of fruit growth. Althoughcessation of assimilate import to tomato fruit has beendocumented to occur around 15 days after breaker, somereports have demonstrated a net fruit influx for longerperiods (McCollum and Skok 1960, Windt et al. 2009)that could be attributed to some pedicel vascular bun-dles that remain unaltered in the abscission zone (Honget al. 2000). An incomplete closure of the vascular bun-dles could explain the small increases in fruit diametermeasured in the present work between mature green andoverripe. To our knowledge, this is the first report that

shows in detail and quantifies, with the aid of appropriatesensors, the tomato fruit growth during ripening.

Environmental conditions and fruit growth ratecontribute to trigger fruit cracking

Cracking is a major disorder that affects severalspecies. In the case of cherry tomato, fruits crackwhile they are on the vine or during harvesting,always between breaker and overripe stages. Crackinghas been shown to have an environmental as wellas a genetic basis (Cuartero et al. 1981). Presentwork has shown that the genetic background playsa more significant role than environmental variablesin fruit cracking, because Cascada always showeda much lower cracking percentage than Gardener’sDelight in any environmental condition, and differencesbetween genotypes were always higher than betweenenvironmental conditions. Fruit cracking increasesduring fruit overripening. Harvestings were evenlydistributed in order to compare fruit cracking amongharvestings. However, on June 15 and 19, the periodbetween harvestings was decreased, and hence, thiscould partially explain the low cracking observed asthe number of overripe fruits would be lower. Inaddition, average temperatures were somewhat lowerin the previous days. Similarly, a decrease in RH and anincrease in vapor pressure deficit in the days previousto the February 15 harvesting could partially explain thelow cracking percentage recorded.

Several environmental parameters such as RH, tem-perature and radiation have been suggested to play arole in fruit cracking. The differences between high andlow RH conditions achieved in the present experimentsin both seasons, although limited, were different enoughto demonstrate a positive correlation between high fruit


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Fig. 4. Histograms of the biomechanical parameters measured in cracked and non-cracked Gardener’s Delight fruit at red in two RH conditions(solid bar high RH and open bar low RH). Statistically significant differences (P ≥ 0.95) are indicated with different letters: bold indicates differencesbetween RH, and italics indicate differences between cracked and non-cracked fruits for the same RH.

cracking and high RH conditions, regardless of the sea-son. Moreover, the techniques applied to modify RHare the same employed in commercial greenhouses, andthe differences in RH obtained represent the range ofvariation attainable for growers. Small increases in RH,similar to those recorded in the experiments describedhere, have been shown to induce greenhouse fruit crack-ing in countries with climates as different as Japan (Ohtaet al. 1991), Spain (Maroto et al. 1995) and Canada(Ehret et al. 2008).

In the present work, fruit growth rate during ripeninghas been identified as another parameter that influencesfruit cracking on the vine. Cracking was only manifestedin fruits that ripened on the vine, because detachedfruits allowed to mature in well-aired conditions reachedthe ripe stage at the same time but did not crack.This points out to a fruit internal pressure, sustainedby plant attachment, as responsible for fruit crackingand probably fruit growth during this period too. Therate of fruit growth during ripening was higher inspring than in winter cultivation. A positive correlationbetween temperature and fruit growth rate has beenreported (Hurd and Graves 1985, Adams et al. 2001,Genard et al. 2007). However, in the present work, onlysmall variations in the average temperature could beobserved between winter and spring. Hence, irradianceand sun hours are most probably the main responsiblefor the increase in fruit growth rate between winterand spring conditions. RH also influenced fruit growth

during ripening, because higher rates of fruit growthcan be observed under high RH. These results are inagreement with previous reports on the role of RHon fruit growth (Romero-Aranda et al. 2002, Kawabataet al. 2005, Genard et al. 2007). This effect of radiationand RH on fruit growth rate is mostly due to changesin the growth period between mature green and lightorange, because no significant changes in growthrate could be observed between light orange andoverripe.

Fruit cracking susceptibility could be related witha higher growth rate between mature green–lightorange, regardless of the RH and the season, becausethe cracking-sensitive genotype, Gardener’s Delight,showed a higher growth rate at this stage than Cas-cada, the resistant genotype. Comparison of cracked andnon-cracked fruits for each RH and season suggests thatcracked fruits correspond to those that showed a higherfruit growth rate at both phases but especially during thelight orange–overripe period. Fruit growth was measuredin four additional cherry tomato genotypes in winter andspring. In all the cases, cracked fruits showed steeperslopes than non-cracked ones (Table S1), corroborat-ing the findings in Gardener’s Delight. Also, a relationbetween the steepness of the slopes and cracking per-centage was observed, supporting the results obtainedin Cascada and Gardener’s Delight. To our knowledge,this is the first demonstration of the influence of fruitgrowth rate in fruit cracking during the developmental


stage in which cherry tomato fruit cracking occurs: frombreaker to overripe.

Environmental conditions during fruit growthaffect cuticle accumulationand mechanical properties

It has been established that environmental conditionsplay a role on cuticle accumulation, but little researchhas been done to understand the influence of eachenvironmental component. Thus, irradiance has beenpositively correlated with cuticle development (Hullet al. 1975), although the experimental design employeddid not allow the elimination of RH and temperaturefrom the equation. The experiments presented hereinhave been designed to study the role of RH within twoconditions of radiation, spring and winter, with minimalchanges on temperature. The higher amount of cuticleand its components (cutin, waxes, polysaccharides andphenolics) observed in spring indicates that irradiancehas a positive effect on cuticle accumulation, regardlessof the RH. Comparison of RH within a season alsoindicated a role of RH on cuticle synthesis, becausehigh RH decreased the amount of cuticle deposited.The effect of RH was also observed for all cuticlecomponents except for waxes and phenolics, whoseaccumulation seems to be mostly dependent on radiationbut not on RH. These differences, although present inboth seasons, were only significant in spring, suggestingthat low irradiance minimizes the effect of high RH.High solar radiation has been reported to increase leafcuticle thickness in some species (Storey and Price1999, Tattini et al. 2005, Oliveira et al. 2008) in orderto absorb higher amounts of ultraviolet radiation dueto a correlative increase in phenolics (Krauss et al.1997, Grammatikopoulos et al. 1998). No differencesin cuticle thickness and degree of invagination wereobserved between seasons or RH although the amountof phenolic compounds was almost double in spring.The high amount of cuticle and cuticle componentsaccumulated in spring could be explained by the highermetabolic rate that can be observed in spring comparedwith winter together with a putative light-promotingeffect, as it has been described for flavonoid synthesisand accumulation (Pelletier and Shirley 1996, Jenkinset al. 2001, Agati and Tattini 2010). Light mainly, andtemperature probably too, promote fruit growth rateand higher cuticle synthesis. However, the role of highRH on cuticle accumulation needs further study. Fromthe results presented here, it could be concluded thatthe amount of cuticle and its components seems to becontrolled by an interaction between radiation, RH andmost probably also temperature.

It has been traditionally assumed that during ripening,fruit internal pressure is transmitted from the pericarpto the epidermal tissue, which should be stiff enoughto support this increase in pressure without sizechange. However, a small increase in size has beendetected during fruit ripening, and hence, the epidermisshould be able to accommodate this size change. RHhas been shown to influence this final fruit growthperiod by increasing its rate and thus play a rolein cracking. In addition, RH has been shown tomodify mechanical properties of isolated cuticles underlaboratory conditions (Matas et al. 2005, Lopez-Casadoet al. 2007). Water condensation, more probably inhigh than in low RH conditions, could contribute toincrease fruit cracking under high RH conditions becauseof its plasticizing effect on the cutin matrix mainly(Lopez-Casado et al. 2007). Analysis of the mechanicalproperties of cuticles showed that non-cracked fruits hada significantly higher maximum strain and viscoelasticphase compared with cracked fruits. These differences,although present under both conditions of RH, were onlysignificant under high RH, the condition that promotedfruit cracking. Thus, cracked fruits are covered by a lessdeformable (lower maximum strain) cuticle than non-cracked fruits. A more viscoelastic cuticle could betteradapt to changes in fruit size that take place during fruitmaturation.

Conclusions

In conclusion, cherry tomato fruit growth was notarrested at the mature green stage but continued untiloverripe. This growth could be accurately modeledby a simple linear function. Fruit growth rate duringripening varied among genotypes and was significantlyaffected by environmental conditions such as radiationand RH. This growth rate has been identified asan important parameter that influences fruit crackingbecause cracking-sensitive cultivars had a higher ratethan cracking-resistant ones. Thus, fruit cracking washigher just at higher RH and springtime, which arethe environmental conditions that produced higherfruit growth rate. Environmental conditions during fruitgrowth and development altered the accumulation ofcuticle and its components. Significant differences in thecuticle mechanical performance within a genotype wereobserved between cracked and non-cracked fruits. Thus,cracked fruits are covered by a less deformable cuticlethan non-cracked ones.

Acknowledgements – The authors are grateful for financialsupport received through grants AGL2009-12134 andTRA2009-0375 from the Plan Nacional de I+D, Ministry of


Science and Innovation, Spain. Collaborations of FundacionCajamar and Rijk Zwaan Iberica (Almeria, Spain) are alsoacknowledged.

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Supporting Information

Additional Supporting Information may be found in theonline version of this article:

Table S1. Mean ± SE of the increase rate in fruit diameter(measured as the slope of the regression line) for fourgenotypes in winter and spring.

Please note: Wiley-Blackwell are not responsible forthe content or functionality of any supporting materialssupplied by the authors. Any queries (other than missingmaterial) should be directed to the corresponding authorfor the article.

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tomato fruit continues growing while ripening, affecting cuticle properties and cracking

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