2005tomato Fruit Growth Response to Temperature and Fruit Load in Relation to Cell Division Expansion 169

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Analysis of the Tomato Fruit Growth Response to Temperature andPlant Fruit Load in Relation to Cell Division, Cell Expansion and

DNA EndoreduplicationN. BERTIN*

Unite Plantes et Syste `mes de culture Horticoles, INRA, Site Agroparc, F-84914 Avignon Cedex 9, France

Received: 8 July 2004 Returned for revision: 20 September 2004 Accepted: 15 October 2004 Published electronically: 6 December 2004

Background and Aims To better understand the regulation of fruit growth in response to environmental factors, theeffects of temperature and plant fruit load on cell number, cell size and DNA endoreduplication were analysed.

Methods Plants were grown at 20/20 C, 25/25 C and 25/20 C day/night temperatures, and inorescences werepruned to two (‘2F’) or ve (‘5F’) owers.

Key Results andConclusions Despite a lower fruit growth rate at 20/20 C, temperature did not affect nal fruit sizebecause of the compensation between cell number and size. The higher cell number at 20/20 C (9 0 · 10 6 against7 9 · 10 6 at 25/25 Cand7 7 · 10 6 at 25/20 C) resulted from an extended period of cell division, and the smaller cellsize was due to a shorter period of expansion rather than a lower expansion rate. By contrast, the lower fruit growthrate and size of 5F fruits compared with 2Ffruits resulted from the slow down of cell expansion, whereas the numberof cells was hardly affected in the proximal fruit. However, within the inorescence the decreasing gradient of fruitsize from proximal to distal fruits was due to a decrease in cell number with similar cell size. Fruit size variationswithin each treatment were always positively correlated to variations in cell number, but not in cell size. Negativecorrelations between cell size and cell number suggested that cells of tomato pericarp can be seen as a population of competing sinks. Mean ploidy was slightly delayed and reduced in 5F fruits compared with 2F fruits. It was highestat 25/25 C and lowest at 25/20 C. Treatments did not affect ploidy and cell size in similar ways, but within eachtreatment, positive correlations existed between mean ploidy and cell size, though signicant only in the 2F-25/20treatment. ª 2004 Annals of Botany Company

Key words: Cell division, cell size, endoreduplication, ploidy, temperature, competition, tomato, Lycopersiconesculentum Mill.

INTRODUCTIONMuch attention has been paid to the environmental inuenceon fruit growth in greenhouse tomato crops, and optimumtemperature and light regime have been dened for fruitproduction (Pearce et al ., 1993; Adams et al ., 2001; Adamsand Valde´s, 2002). Since different processes are succes-sively involved in the control of growth during fruit ageing,the sensitivity to environmental variations is expected touctuate during fruit development, as shown for tempera-ture (De Koning, 1994; Adams et al ., 2001). Moreover,compensation between the numerous components of growthmay lead to underestimation of the fruit response. Forinstance, an increase of temperature effectively increases

the maximum tomato growth rate, but it is compensated forby a shorter period of growth, so that fruit weight may not besignicantly affected (Ho, 1996; Adams et al ., 2001). Todeepen our understanding and control of fruit growth inresponse to environmental uctuations, it is necessary todescribe better the individual processes involved in thisresponse during fruit development.

Tomato is a eshy fruit composed of different tissues: theepidermis, the pericarp (esh) and the placenta, and loculartissue including seeds (pulp). Increase in fruit volumeresults from biophysical limitation by epidermal extensibil-ity (Thompson, 2001) and from the development of pericarp

tissue which generally accounts for more than two-thirds of the total fruit weight (Ho and Hewitt, 1986). Both divisionand expansion activity in pericarp tissue are determinant fortomato growth. Whereas epidermal cells divide throughoutfruit development, cell division in the pericarp is limited to ashort period of fruit development and is located in theexternal tissue around the vascular bundles and in the hypo-dermis. Once cell division ends, cell expansion becomes thedominant way to increase fruit size. In tomato, large endo-reduplicated cells are located in the mesocarp (Bu ¨nger-Kibler and Bangerth, 1983).

Endoreduplication is an incomplete cell cycle that leadsto the increase of nuclear DNA content (D’Amato, 1964;Galbraith et al ., 1991), which in fruit pericarp reaches levelsup to 256C (C is the haploid nuclei DNA content) in cherrytomatoes as well as in large-size fruit cultivars (Bergervoetet al ., 1996; Joube`s et al ., 1999). Endoreduplication may beinvolved in the control of fruit growth since it was suggestedto set the size limit of a cell (Traas et al ., 1998). In Arabidopsis a positive relationship between endoreduplica-tion and cell size was reported in epidermis cells(Melaragno etal ., 1993).Amongpeaseed genotypes, a linearrelationship was reported between endoreduplication incotyledon cells and seed dry weight or mean cell volume(Lemontey etal ., 2000).In tomato, there is littleexperimentalevidence of any direct relationship between endoreduplica-tion and fruit size (Bu ¨nger-Kiblerand Bangerth,1983;Bertin

Annals of Botany 95/3 ª Annals of Botany Company 2004; all rights reserved

* For correspondence. E-mail [email protected]

Annals of Botany 95 : 439–447, 2005doi:10.1093/aob/mci042, available online at www.aob.oupjournals.org



et al ., 2003). However, it cannot be ruled out that endo-reduplication is involved in the control of cell growth inresponse to environmental variations, since it has beenrarely investigated.

Studies of fruit growth in response to environmentalvariations have been mainly focused on fruit expansion

processes (Ehret and Ho, 1986; Pearce et al ., 1993; Adamsetal ., 2001), though the very precocious control of nal fruitsize by cell division prior to anthesis has been well docu-mented at the fruit level (Bohner and Bangerth, 1988;Ho, 1996) and also at the gene level (Frary et al ., 2000).A decrease of the source : sink ratio on the plant diminishesnal fruit size by reducing both cell number and cell size(Bohner andBangerth, 1988; Bertin etal ., 2003).Many stud-ies have described the effects of temperature on cell cycleduration, cell division rate and cell expansion rate in root orshoot meristems and in leaves (Brown and Rickless, 1949;Lopez-Saez et al ., 1966; Francis and Barlow, 1998; Granieret al ., 2000; Tardieu and Granier, 2000), but more rarely infruit, though temperature is the primary climatic factoraffecting tomato fruit growth rate (Walker and Ho, 1977;Pearce etal ., 1993; Peet et al ., 1997; Willits and Peet, 1998).

In this study, the response of tomato fruit growth totemperature and plant fruit load was analysed, in relationto the cell number, cell size and nuclei DNA endoredupli-cation in fruit pericarp.

MATERIALS AND METHODS

Plant material and cultural conditions

The experiment was carried out in an 8 75-m 2 (21-m 3 )growth climatic chamber under controlled conditions.Seeds of tomato ‘Raı ¨ssa’ were sown in sand, and 12 homo-genous plants were pricked out at a developmental stage of about four or ve visible leaves, in 10-dm 3 pots lled with abalanced oxygenated nutrient solution, whose compositionwas checked every week and readjusted when necessary.Sowing took place in the growth climatic chamber itself under climatic conditions similar to those monitored afterplanting. Metal halide lamps were used to provide articiallighting. A 12-h photoperiod was applied with a photon uxof about 500 mmol m 2 s 1 PAR above the canopy. Airrelative humidity was maintained around 70 %. Fromanthesis of the rst truss, air was enriched to 800 ml CO 2 l 1

during the light period. Flowers were pollinated as theyopened with an electrical shaker, and all side shoots were

removed as they appeared.

Experimental treatments

Three successive experiments were conducted under thesame controlled conditions except the day/night air tem-perature regime which was successively set to 20/20 C,25/25 C and 25/20 C 6 0 5 C. Temperature of the nutri-tive solution was monitored to 22 C in all experiments. Ineach experiment, inorescences were pruned to ve owers(‘5F’ treatments) on six plants and to two owers on theother six (‘2F’ treatments). Pruning took place at 50 %anthesis of the truss. Plants were topped two leavesabove the tenth truss.

Observations and measurements

The developmental stage of each individual ower budwas recorded twice a week from appearance to fruit set(about 5 mm diameter). Anthesis time was considered asfull ower opening. As fruits developed, the equatorial dia-meter of the rst and second fruits (F1 and F2) on plantspruned to two owers per truss, and the diameter of the rst,third and fth fruits (F1, F3 and F5) on plants pruned to veowers, were recorded once a week with a caliper square.

Fruits F1 and F2 in the 2F treatments, and F1, F3 and F5in the 5F treatments were sampled as the rst truss ripened.The pericarp cell number was measured on all these fruits(except F2 in the 2F-25/20 C treatment) on the rst seventrusses. At this time the cell division activity had beencompleted in the rst four trusses. The ploidy level of peri-carp cells was measured on ovaries and fruits of differentages sampled on the rst eight trusses at the rst (F1) andfth (F5) positions. The mean cell volume was estimated bydividing the pericarp volume (measured by water displace-

ment) by the total number of pericarp cells. Previous meas-urements of cell area made in situ on pericarp slices (usingthe method described in Bertin et al ., 2003) conrmed thatthis ratio is a good indicator of cell size, considering that thetotal intercellular space of tomato pericarp is relativelyreduced (N. Bertin, unpubl. res.), and likely not to beaffected by treatments.

The number of pericarp cells was measured after tissuedissociation according to a method adapted from that of Bunger-Kibler and Bangerth (1983). Details of the appliedmethod are given in Bertin et al . (2002). The ploidy levelwas measured by means of a PARTEC ow cytometer(PARTEC Ploidy Analyser PA, GmbH, Germany),equipped with an HBO lamp for UV excitation (Bertinet al ., 2003). Three replicate measurements were made inmature fruit, but only one measurement in ower buds andyoung ovaries because of the small quantity of material.Mean endoreduplication level was calculated as:

mean C-value = Xn

i¼1

C i N i N tot

where n is the number of peaks of DNA content (max = 8) inthe sample, C i is the C value in the nuclei of peak ni (C 1 = 2,C 2 = 4 C 8 = 256), N i is the number of nuclei in thepeak n i and N tot is the number of nuclei in all peaks of thesample.

Statistical analysis

The effects of temperature or plant fruit load on cellnumber were analysed in interaction with the truss position(rst four trusses) by two-way ANOVA (Jandel ScienticSigmastat), and F -tests were used to determine the statisticalsignicance. When signicant effects were detected, aTukey test was applied for all pairwise comparisons of mean responses.

Fruit growth and mean ploidy curves were tted to three-parameter Gompertz and sigmoid functions, respectively.Parameter estimation was carried out using least squaresmethod (Jandel Scientic SigmaPlot). The difference

440 Bertin — Cell Population in Tomato Fruit



between two (or more) treatments was tested by comparingthe sum of the residual sums of squares for the two (or more)individual ttings ( SS i) with the residual sum of squaresfor the common tting to pooled treatments (SS c ) consider-ing that the statistic:

F = jSSc Pn

i¼1SS ij= n 1ð Þk ½

Pn

i¼1SS i= N data k ð Þ

follows Fisher’s law with ( n 1)k and ( N data k ) degrees of freedom. N data is the total number of data, n is the number of individual regression and k is the number of tted para-meters for each regression (three for Gompertz and sigmoidfunctions).

Correlations between fruit weight, cell number, cell sizeand mean ploidy, were analysed on fruits older than 30d afteranthesis (daa), by running the Pearson product Moment test(Jandel Scientic Sigmastat). Correlation coefcients arereported in the legends to Figs 2, 4 and 6 and statisticalsignicance of these correlations are given in the text.

RESULTS

Maximum plant fruit load was achieved on the 2F treat-ments at each of the three temperatures. On the contraryfor 5F treatments, numerous fruit abortions occurred in the25/25 C treatment and to a lesser extent in the 20/20 Ctreatment, where four to ve fruits actually set on each truss.To avoid confusion due to different source–sink balancesamong treatments, the fruit load effect was analysed only onthe 25/20 C treatment, whereas the temperature effect wasanalysed only among the 2F treatments.

Effect of temperature on fruit growth in relation to cellnumber and cell size when carbon supply is non-limiting

At 20/20 C, fruit growth (F1 + F2) was signicantly dec-reased ( P = 0 01) compared with 25/25 C and 25/20 Cwhich were similar (Fig. 1A). Differences in the growthpattern did not consist of a large difference in nal fruitsize, but rather in the duration of growth and in the max-imum growth rate.

The cell volume estimates on fruits of different ages andthe total number of pericarp cellsin the rst four trusses of 2Fplants are shown in Fig. 1B and Table 1 for the three tem-perature regimes. Theonset of cell expansion wasdelayed byabout 5 d at 20/20 C compared with 25/25 C and 25/20 C(Fig. 1B), and similar rates of increase in cell volume(slopes of the linear part of the curves) at all temperaturesled to lower nal cell size at 20/20 C. Since nal fruit sizeswere similar at the three temperature regimes (Fig. 1A), thelower cell size at 20/20 C was compensated for by a highernumber of cells. Indeed in the rst four trusses the numberof pericarp cells was higher at 20/20 C (P = 0 09) withoutany signicant interaction with the truss position (Table 1).On average on the rst four trusses, the number of cellswas 7 9 · 106 and 7 7 · 106 at 25/25 C and 25/20 C,respectively, against 9 0 · 106 at 20/20 C.

To analyse the correlation between fruit size, cell numberand cell size, fruits older than 30 daa, which were close totheir nal size were examined. A negative correlationbetween cell size and cell number conrmed the compensa-tion between cell size and cell number among temperaturetreatments (Fig. 2A). The negative correlation also heldwithin the 25/25 C (P < 0 01) and 25/20 C (P = 0 053)treatments, indicating that this compensation was not a spe-cic response to temperature. Similarly the variations of fruit weight within each temperature treatment were posi-tively correlated with the variations in cell number ( P <

0 001 at the three temperature regimes), but not with thosein cell size (Fig. 2B and C), except at 20/20 C (P = 0 02)where the correlation was signicant only due to oneextreme point

Effects of plant fruit load on fruit growth in relation to cellnumber and cell size

Plant fruit load effects were analysed on F1 in the 25/20 Ctreatment. Growth of F1 globally measured on the rstfour trusses was signicantly ( P < 0 05) reduced in the 5Ftreatment compared with the 2F treatment, due to the lowergrowth rate (Fig. 3A). In parallel a clear difference was

0

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F r u i t d

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)

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E s t i m a t e d c e l l v o

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F I G . 1. (A) Fruit size and fruit growth rate measured on 2F plants grown at20/20 C (dashed line), 25/25 C (dotted line) or 25/20 C (continuous line)day/night temperature. Three-parameter Gompertz functions were tted onfruit size measurements made on therst andsecondfruits (F1and F2)of therst four trusses. Adjustmentwas made on pooled datafrom six plants ( R2 >

0 95) and vertical bars indicate the standard error calculated on adjustmentsmade on individual plants. Daily fruit growth rates were obtained byderivative functions. (B) Change in cell volume during fruit ageing,estimated by dividing the pericarp volume by the total number of pericarp cells in 2F treatments. Each point is an individual fruit sampledat the rst or second positions (F1 and F2) on the rst eight trusses of fourplants grown at 20/20 C (open circles), 25/25 C (grey circles) or 25/20 C

(black circles) day/night temperature.

Bertin — Cell Population in Tomato Fruit 441



observed between the two treatments in the increase of cellvolume during fruit ageing (Fig. 3B). In contrast to the tem-peratureeffect (Fig.1B),thelowercellvolumein 5Ffruits wasmore likely due to the decline of the cell expansion rate fromabout 25 daa.

The number of cells in the pericarp of F1 was notsignicantly different between the 2F and 5F treatments(Table 1), but it signicantly varied among trusses

T A B L E 1. Number of pericarp cells ( · 10 6 ) measured in the rst (F1), and third and fth fruits (5F plants) of the rst four trussesand mean cell number in these four trusses

Truss 1 Truss 2 Truss 3 Truss 4 Mean of four trusses

2F-20/20 C F1 9.65 6 2.79 9.74 6 1.30 9 .53 6 2.79 7.09 6 1.58 9.00 6 1.252F-25/25 C F1 6.91 6 1.85 7.73 6 1.98 8 .87 6 0.52 7.91 6 1.25 7.86 6 0.792F-25/20 C F1 6.43 6 2.08 8.65 6 1.24 7 .78 6 1.16 7.94 6 0.61 7.70 6 0.915F-25/20 C F1 5.28 6 1.34 10 .05 6 1.40 9 .02 6 1.75 9.75 6 1.49 8.52 6 2.495F-25/20 C F3 6.18 6 2.84 5.15 6 1.78 5 .34 6 2.14 5.96 6 2.47 5.66 6 0.505F-25/20 C F5 4.69 6 2.03 3.45 6 1.26 5 .25 6 0.94 5.02 6 0.15 4.60 6 0.91

Data are means of four plants 6 95 % condence intervals.

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3 7 11 15

Cell number (10 6)

F r u i t

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t ( g )

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4 8 12 16 20Cell volume (nL)

F r u i t

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3 7 11 15

Cell number (10 6)

E s t i m a t e d c e l l v o l u m e

( n L ) A

F I G . 2. Correlations between (A) cell size and cell number, (B) fruit freshweight and cell number and (C) fruit fresh weight and cell size. Linesrepresent linear adjustments. Each point is an individual fruit older than30 daa sampled at the rst or second position (F1 and F2) in the rst fourtrusses of plants grown at 20/20 C [open circles and dotted line R = 0 17(A), 0 73 (B), 0 53 (C)], 25/25 C [grey circles and dashed line R = 0 66(A), 0 87 (B), 0 25 (C)] or 25/20 C [black circles and continuous line

R = 0 60 (A), 0 86 (B), 0 12 (C)] day/night temperature.

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F r u i t

d i a m e t e r ( m m

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t h r a t e ( mm d −

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Fruit age (days after anthesis)


( n L )

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F I G . 3. (A) Fruit size and fruit growth rate of the rst fruit (F1) of 2F(continuous line) and 5F (broken line) plants grown at 25/20 C. Three-parameter Gompertz functions were tted on fruit size measurements madeon therst four trusses. Adjustmentwas madeon pooled data fromsix plants( R2 > 0 85) and vertical bars indicate the standard error calculated on

adjustments made on individual plants. Daily fruit growth rates wereobtained by derivative functions. (B) Evolution of cell volume duringfruit ageing. Each point is an individual fruit sampled at the rst position(F1) on the rst eight trusses of four 2F (circles) and 5F (triangles) plants

grown at 25/20 C.




(P < 0 01), the rst truss containing fewer cells than thefollowing ones (signicant only in the 5F treatment). Onaverage on the rst four trusses the number of pericarp cellswas similar in the two treatments (8 5 · 10 6 and 7 7 · 10 6

cells for 5F and 2F, respectively).Analysis of fruits older than 30 daa showed that the

negative correlation between cell volume and cell numberwas still present within each treatment ( P = 0 053 and 0 059for 2F and 5F, respectively), but no compensation occurredacross the treatments (Fig. 4A), as was observed for

temperature (Fig. 2A). Similarly a signicant positive cor-relation ( P < 0 001) between fruit weight and cell numberexisted within each treatment (Fig. 4B), but no signicantcorrelation between fruit weight and cell volume wasobserved, either within each treatment or across the treat-ments (Fig. 4C).

Whereas the number of cells in the pericarp of F1 was notsignicantly affected by the plant fruit load, the numbers of

cells in the pericarp of F3 and F5 were signicantly reduced(Table 1). A two-way ANOVA was performed to assess thetruss and fruit effects on cell number of the rst four trussesin the 5F-25/20 C treatment. Except in the rst truss asignicant gradient of cell numbers existed, F1 containingmore cells than other fruits of the truss. Differences amongtrusses were not signicant and on the rst four trusses themean numbers of cells were 8 5 · 10 6 , 5 7 · 10 6 and 4 6 ·

10 6 in F1, F3 and F5, respectively. Compared with F1, thenal fruit size was reduced by 12 % and 19 % in F3 and F5,respectively, and the fruit growth curves signicantly ( P <

0 05) decreased from F1 to F5, due to a decreasing gradientin fruit growth rate. This could not be related to differencesin estimated cell size which did not vary clearly amongfruits of the same inorescence.

Considering fruits older than 30 daa, a positive correla-tion existed between fruit weight and pericarp cell number,but the compensation relationship between cell number andcell size which was observed in F1 (Fig. 4A), did not holdfor the third and fth fruits of the truss.

Effect of temperature and plant fruit load on the ploidy levelof pericarp cells and relationship with other fruit traits

The dynamic of mean ploidy during fruit ageing meas-ured on the rst eight trusses is presented in Fig. 5 for fruit

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F I G . 4. Correlations between (A) cell size and cell number, (B) fruit freshweight and cell number and (C) fruit fresh weight and cell size. Linesrepresent linear adjustments. Each point is an individual fruit older than30 daasampled at the rst position (F1) in the rst four trusses of 2F [circlesandfull line R = 0 60(A),0 86 (B), 0 12 (C)] and5F [triangles anddotted

line R=

0 56(A), 0 87 (B), 0 12 (C)] plants grownat 25/20 C day/nighttemperature regime.

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0 10 20 30 40 50Fruit age (days after anthesis)

M e a n

C - v a l u e o f p e r i c a r p c e l l s

N um

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F I G . 5. Mean C-value of pericarp cells measured in F1 and F2 for 2Ftreatments (circles) and in fruits F1 and F5 for 5F treatment (triangles).Each point is the mean of three measurements performed on an individualfruit sampled on the rst eight trusses of plants grown at 20/20 C (opencircles), 25/25 C (grey circles) or 25/20 C (blackcircles andtriangles) day/ night temperature. Lines represent three-parameter sigmoid functions ttedon data from each treatment ( R2 > 0 84). On the right axis, are indicated the

corresponding numbers of incomplete cell cycles.




load and temperature treatments. Ploidy of fruit pericarpwas similar in F1 and F5 fruits of the 5F treatment (notshown) and these data were pooled. Mean ploidy was initi-ally delayed in the 5F-25/20 C treatment compared withthe 2F-25/20 C treatment, but nal values were close to oneanother. Fitted curves were signicantly different at the 5 %error level, but not at 1 %.

Temperature signicantly changed the dynamic of pericarp cell ploidy ( P < 1 %). Mean ploidy was maximumat 25/25 C, but surprisingly it was higher at 20/20 C thanat25/20 C. Other measurements made in the 5F treatmentsat 20/20 C and 25/25 C agreed with these patterns (notshown). Differences in DNA amount between 25/20 Cand 25/25 C corresponded to less than one incompletecycle.

Nuclear DNA content was measured on some of the fruitsused for cell counting, so that correlative analysis betweenploidy and other fruit traits could be performed on fruitsolder than 30 daa which had almost reached their nal size(Fig. 6). In none of the treatments, was fruit fresh weightsignicantly correlated with the mean ploidy of pericarpcells and through the temperature treatments a negativecorrelation was even observed, though not signicant

(Fig. 6A and B). Within each treatment, positivecorrelations between cell size and mean ploidy (signicantonly in the 2F-25/20 C treatment P = 0 04; Fig. 6C and D)and negative correlations between cell number and meanploidy (signicant only in the 2F-25/25 C treatmentP = 0 013; Fig. 6E and F) could be noted. These correlationsheld across treatments only comparing the 20/20 C and25/25 C treatments, which was the only case where cellsize and cell number really compensated for fruit weight. Inthat case only, the increase in cell size at 25/25 C could berelated to an increase in ploidy. The low ploidy level mea-sured at 25/20 C for both 2F and 5F treatments could not berelated to any fruit traits. At this temperature the decrease incell size in the 5F treatment was not associated with lowploidy levels.

DISCUSSION

In accordance with the literature (Pearce etal ., 1993; Adamset al ., 2001) the maximum fruit growth rate was achieved at25/25 C (Fig. 1A). Actual fruit temperature was likely to behigher than 25 C, since air temperature was monitoredinstead of fruit temperature (Adams and Valde `s, 2002).

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C20

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F I G . 6. Correlationsbetween mean ploidyof pericarp cells andfruitfreshweight(A andB), cell volume(C andD) andcell number (E andF). Lines representlinear adjustments. Each point is an individual fruit older than 30 daa sampled on the rst four trusses at the F1 or F2 positions for the 2F treatments (circles)and at the F1 [open triangles and dotted line R = 0 29 (A), 0 22 (C), 0 29 (E)] or F5 [black triangles and dashed line R = 0 64 (A), 0 69 (C), 0 59 (E)]

positions for the 5F treatment. Plants were grown at 20/20

C [open circles and dotted line R=

0 25 (B), 0 007 (D), 0 33 (F)], 25/25

C [grey circles anddashedline R = 0 58(B),0 54 (D), 0 72 (F)] or 25/20 C [blackcircles andfullline R = 0 11(B),0 66(D), 0 43 (F) and triangles] day/night temperature.




High temperature may affect fruit growth through indirecteffects on plant development, maintenance respiration andassimilate availability. These indirect effects were avoidedhere by considering only the 2F plants in the analysis of theresponse to temperature.

In the range of temperature investigated in this study,

many authors reported no or negative effects of increasingtemperature on nal tomato size and attributed this to com-pensating effects on the rates of fruit growth and fruit devel-opment (De Koning, 1994; Ho, 1996; Adams et al ., 2001).The present investigation further indicated that this com-pensation is also due to inverse and compensating effects of temperature on cell number and cell size (Fig. 2A). Between20 and 25 C, an increase of temperature promoted cell size,but slightly decreased the number of cells, so that nal fruitsize was hardly affected (Fig. 1A). The increase in cellnumber at 20/20 C resulted from the extended period of cell division (Fig. 1B). Interestingly the different patterns of fruit growth at 20/20 C (slow) and 25/25 C (accelerated)resulted from differences in the duration of the cell divisionperiod and onset of cell expansion which mainly shortenedthe period of cell expansion at 20/20 C, but did not stronglyreduce the rate of cell expansion. This is in accordance withAdams et al . (2001) who hastened ower opening by heat-ing ower buds at 25 C. The shortening of the cell divisionperiod together with a reduction in the nal number of cellsat 25/25 C, suggested that the proportion of cycling cells inthe pericarp and its evolution during the division periodwere not affected by temperature between 20 and 25 C.Indeed, considering that the time required for a cell to dividedecreases in response to an increase of temperature with aminimum duration around 30 C for many species (Francisand Barlow, 1988), a compensation between the cell cycle

duration and the proportion of cycling cells did not occur,since fruits nally contained less cells at 25/25 C than at20/20 C.

Fruits produced at 25/20 C were close to those obtainedat 25/25 C with respect to fruit growth, and cell number andsize. Tomato plants are known to integrate day/night tem-peratures in terms of fruit yield (Hurd and Graves, 1984;Peet et al ., 1997) and thus the 25/20 C treatment can beconsidered as a constant 22 5 C temperature regime. How-ever, cell number and cell size were similar in the 25/25 Cand 25/20 C treatments, which is inconsistent with theabsence of compensation between cell cycle duration andproportion of cycling cells, except if the cell cycle durationis already minimum at 22 C in the tomato pericarp.

In contrast to what was observed in leaves of sunower,tobacco and pea (Granier et al ., 2000) cell division andtissue expansion did not have a common response to tem-perature in tomato, so that nal fruit size and cell numberwere not correlated throughout the temperature treatments(Fig. 2B). But within each treatment, the variations in cellnumber were always tightly and positively correlated withthe variations in fruit size, whereas the variations in cell sizewere not. Thus considering a given level of carbon andwater supply for fruit growth, cell division plays a dominantrole in the determination of intra-treatment variations of fruit size, which largely exceeded the inter-treatmentvariations.

Reduction of plant fruit load (2F against 5F) promoted thefruit growth rate and nal fruit size of F1, by increasingcell expansion without any inverse compensating effectson cell number. However, the absence of effects of fruitload on cell number may be due to the fact that the compar-ison of 2F and 5F treatments concerned only F1. Indeed F3

and F5 contained far fewer cells than F1 (Table 1) and aremuch more sensitive to the competition for assimilates thanF1 (Bangerth and Ho, 1984; Bohner and Bangerth, 1988;Bertin et al ., 2003). For instance, plant defoliation induces adecrease in both cell number and cell size more important indistal than in proximal fruits (Bohner and Bangerth, 1988).As observed by these authors on control trusses with sixfruits, the cell size did not vary among fruits within the sametruss in the 5F treatment, so that the gradient in fruit sizecould be totally attributed to the gradient in cell number.This indicated that the competition for assimilates in a trusswith six fruits, as currently conducted in a large-size tomato,is not high enough to affect cell size in distal fruits. It issuggested that the numerous cells in F1 were in competitionfor assimilates, whereas in F3 and F5 the low number of cells could be sufciently supplied to grow as much as thenumerous cells in F1. In that case, a negative correlationbetween cell number and cell size could be expected, whichwas true for F1 only. In F3 and F5, cells of the pericarp werelikely not to be in competition because of their low number.It is interesting to note that for F1 and F2, the negativecorrelation between cell size and cell number held withineach treatment (less at 20/20 C). Thus cells of the sametissue can be seen as a population of sinks in competitionfor the supplied assimilates. Any treatment that affectsthe number of cells without limiting the global supply of assimilates, is expected to have inverse effects on cell

number and cell size, and thus no effect on fruit size.This is what was observed among the temperature treat-ments. On the contrary, any treatment that affects the supplyof assimilates to fruits, as does truss pruning, with or with-out any effects on cell number should also affect fruit size.

Many studies describe the endoreduplication dynamicduring development of various species and plant organs,but it is still unclear if endoreduplication is involved inthe control of cell growth (Sugimoto-Shirasu and Roberts,2003). A link between cell size and average C-value wasfound in leaf epidermis (Melaragno et al ., 1993), in seed(Lemontey et al ., 2000) and in owers (Kudo and Kimura,2002; Lee et al ., 2004). In yeast cells, the ploidy-regulationof the cell cycle progression could explain the control of endoreduplication on cell size (Galitski et al ., 1999). Thehypothesis that endoreduplication determines cell growth isfurther supported by the fact that endoreduplication pre-cedes cell expansion (Traas et al ., 1998). In tomato fruitendoreduplication starts very early during the most inten-sive period of cell division and stops more or less with thecessation of cell expansion (Fig. 5). Nevertheless, relation-ships observed in yeast or in leaf epidermis, may not exist atthe level of fruit tissue such as tomato pericarp with millionsof cells at different states, some still dividing, other highlyendoreduplicated. A common point of all hypotheses raisedand whatever the controlling factor, is that the earlier theend of mitotic activity, the higher the ploidy level, since




endoreduplication starts as mitosis is blocked. In that casethe increase of endoreduplication at 25/25 C comparedwith 20/20 C would result only from the earlier cessationof mitotic activity at 25/25 C, which agrees with thedelayed onset of cell expansion at 20/20 C (Fig. 1). Asignicant effect of temperature on maize endosperm endo-

reduplication was also reported by Schweizer etal . (1995) toexplain inter-seasonal variations. Why was endoreduplica-tion minimum at 25/20 C (Fig. 5)? If the cell divisionperiod was as long at 25/20 C as at 25/25 C, as suggestedbefore, then endoreduplication was expected to be the samein both treatments. This may be an experimental artefact,but data were conrmed comparing 5F treatments at thethree temperature regimes (not shown). An effect of day/ night temperature uctuations on endoreduplication mayalso be assumed, but it has never been reported. Tempera-ture may also affect the rate of progression of nuclei fromlower C-value to higher C-value and contribute to increas-ing mean ploidy of the tissue, but such control has neverbeen investigated, although the rate of progression amongC-values is not constant (Schweizer et al ., 1995).

Absence of an effect of the competition among fruitswithin a truss or among trusses on endoreduplication hasbeen reported already, even when cell size is affected (Bu ¨n-ger-Kinbler and Bangerth, 1983; Bertin et al ., 2003). Traaset al . (1998) proposed that the increase in nuclear DNAcontent provides a given amount of DNA to support agiven future increase in mass, but the exact nal size israther dened by the fruit environment, so that endore-duplication is expected to be more or less loosely correlatedwith a range of cell size, as observed in Fig. 6 in the presentstudy. Thus rather than being involved in the control of cellgrowth, endoreduplication may initially determine the

potential size of the cell by controlling the switch fromcomplete to incomplete cell cycle, whereas actual cellsize would mainly depend on carbon supply to individualcells. Figure 6C and D suggests that the potential cell sizedetermined by DNA endoreduplication was not reacheddespite low competition (2F treatments) because cell sizewas the same at 25/25 C and 25/20 with high and lowploidy levels, respectively. Thus, endoreduplication seemsto be a poor indicator of actual cell size in tomato pericarp,since endoreduplication and cell size do not respond insimilar ways to the fruit environment.

ACKNOWLEDGEMENTS

This work was made possible by the technical assistance of B. Brunel and J. C. L’H ootel.

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