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JXB Advance Access published online on April 5, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern049
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© The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

Shaping the shoot: the relative contribution of cell number and cell shape to variations in internode length between parent and hybrid apple trees

V. Ripetti1, J. Escoute2, J. L. Verdeil2 and E. Costes1,*

1INRA, UMR DAP (Développement et Amélioration des Plantes), CIRAD-INRA-SupAgro, Team ‘Architecture et Fonctionnement des Espèces Fruitières’, 2, Place Viala, F-34060 Montpellier, France
2CIRAD, UMR DAP (Développement et Amélioration des Plantes), CIRAD-INRA-SupAgro, PHIV (Plateau d'Histo-cytologie et Imagerie cellulaire Végétale), TA A96/03 Avenue Agropolis, F-34298 Montpellier, France

* To whom correspondence should be addressed. E-mail: costes{at}supagro.inra.fr

Received 2 January 2008; Revised 22 January 2008 Accepted 29 January 2008


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
Genetic control of plant size and shape is a promising perspective, particularly in fruit trees, in order to select desirable genotypes. A recent study on architectural traits in an apple progeny showed that internode length was a highly heritable character. However, few studies have been devoted to internode cellular patterning in dicotyledonous stems, and the interplay between the two elementary cell processes that contribute to their length, i.e. cell division and elongation, is not fully understood. The present study aimed at unravelling their contributions in the genetic variation of internode length in a selection of F1 and parent genotypes of apple tree, by exploring the number of cells and cell shape within mature internodes belonging to the main axes. The results highlighted that both the variables were homogeneous in samples collected either along a sagital line or along the pith width, and suggest that cell lengthening was homogeneous during internode development. They allowed the total number of cells to be estimated on the internode scale and opened up new perspectives for simplifying tissue sampling procedures for further investigations. Differences in internode length were observed between the genotypes, in particular between the parents, and partly resulted from a compensation between cell number and cell length. However, genetic variations in internode length primarily involved the number of cells, while cell length was more secondary. These results argue for an interplay between cellular and organismal control of internode shape that may involve the rib meristem.

Key words: Elongation, growth, histogenesis, Malusxdomestica Borkh, pith


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
The growing interest in organ cell patterns in plants stems from a number of studies that have shown how tissues are made up of a highly organized arrangement of cells (Coen et al., 2004; Dupuy et al., 2007). To account for the patterns observed, two theories, i.e. cellular versus organismal theory, are presently under debate (Inzé and De VeyIder, 2006). The cellular theory is based on the assumption that the cell-cycle process determines organ shape. It is supported by high correlations that have been found between the final individual organ size and the number of cells. Such correlations have been found in leaves of several annual species (Granier et al., 2000), and the preponderance of cell number on final internode length has been demonstrated in five unrelated trees (Brown and Sommer, 1992). However, the existence of a compensation phenomenon between cell number and cell size supports the organismal theory that assumes a control of number of cells and cell size directly from genetic information, at the organ level itself (Tsukaya, 2003). Such a compensation has been observed in several annual plants for both leaf (Cookson et al., 2005; Aguirrezabal et al., 2006) and internode development (Daykin et al., 1997).

Among the different plant organs, more attention has been paid to flower or leaf patterning (Coen et al., 2004; Aguizerbal et al., 2006; Cookson et al., 2007) than to axial organs such as shoot segments. However, shoot growth and development is, together with branching, a major feature defining plant architecture. Its genetic variation throughout higher plants has been widely investigated after the early study by Hallé et al. (1978). Within-species genetic variation has also been explored, especially in breeding programmes that aim at controlling plant size and shape. In grain crops, this led to the selection of dwarf genotypes with notably improved yields (Khush, 2001; Salamini, 2003). In fruit trees, tree size and shape are usually controlled by agronomic practices such as the use of dwarfing rootstocks, but their genetic variations could also be used to select trees that are easy to manage in the orchard (Laurens et al., 2000; Costes et al., 2006).

As argued by Tsukaya (2006), shoots usually have an indeterminate growth. However, the internodes which constitute the stems are determinate organs. Like all above-ground organs in higher plants, internodes originate from meristematic cells primarily located at the shoot apical meristem (SAM; Lyndon, 1998). While, in monocotyledonous plants, some meristematic cells remain inserted in mature tissues and form intercalary meristems that generate cells involved in internode growth and elongation, in dicotyledonous plants the internode tissues originate from meristematic cells that are located just below the apical dome and constitute the rib meristem (Esau, 1953; Howell, 1998). Anticlinal divisions of cells in the rib meristem give rise to parallel longitudinal files of cells that are involved in the differentiation of cylindrical parts of the plants (cortex and pith of the stem) (Esau, 1953). Moreover, histogenesis has been described as qualitatively similar in different dicotyledonous species (Brown and Sommer, 1992): cell division shifts progressively upwards while elongation and maturation of pith cells increases acropetally in developing internodes. The final cellular pattern of internodes thus appears as the result of an interplay between cell division and elongation that progress in inverse directions. Despite this global scheme demonstrated in different tree species, its application to the within-species genetic variations in internode length, and the relative contribution of cell number versus cell size in the final internode length are still poorly understood.

The aim of the study presented here was to investigate the cellular pattern of internodes in a selection of genotypes of a perennial crop, the apple tree, exhibiting contrasting internode lengths. The heritability of shoot and internode length was demonstrated by quantitative genetic approaches that have recently been initiated to elucidate the genetic basis of architectural variations in apple tree (De Wit et al., 2004; Segura et al., 2006; Kenis and Keulemans, 2007). Segura et al. (2006) studied a broad range of variables accounting for tree architecture, and showed that internode length was one of the most heritable characters among the numerous variables correlated to whole tree size. Furthermore, a strong QTL was detected for this character on linkage group 3 (Segura et al., 2007), distinguishing individuals belonging to different allelic classes for this QTL. However, further investigations into the molecular determinisms of internode length would need to dissect this integrated trait into elementary processes. In the present study, two genotypes with contrasting internode lengths and four F1 offspring derived from their cross and belonging to the two most extreme allelic classes for the QTL previously identified on LG3 were selected. On the basis of the histogenesis scheme presented above, it is assumed that the final cellular pattern of internodes may reveal which process (i.e. cell division versus elongation) or/and which part of their interplay is likely to be involved in the observed genetic variation. The sagital and transversal variations in the cell numbers and cell shape (i.e. length and width) over entire mature internodes sampled on 1-year-old main axes were investigated. In addition, because histological studies are time consuming, a further aim was to propose a simplified protocol for tissue sampling. The analysis of the spatial variation in cell number and shape led (i) to demonstrate that the variations in internode length among allelic classes and genotypes were correlated to the number of cells but not to the cell shape and (ii) to validate a method for estimating the cell number along the internodes.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary data
 References
 
Plant material
Initially, the two parents were chosen for their contrasted architecture: the ‘Starkrimson’ maternal parent displayed an erect growth habit with many short shoots and a tendency to irregular fruit bearing (Type II according to Lespinasse, 1992) while the ‘Granny Smith’ pollen parent displayed a weeping habit with long shoots and fruit-bearing regularity (Type IV). The F1 hybrid plants belonged to progeny derived from a cross between these two parents ‘Starkrimson’x‘Granny Smith’. Among the progeny, hybrids were selected within the two allelic classes displaying the strongest genotypic effect on internode length for the QTL mapped on linkage group 3 (Segura et al., 2007). Two hybrids were selected within each allelic class with an extreme phenotype (respectively, ad and bc classes obtained from an ‘abxcd’ cross).

In the winter of 2004, graft wood samples were taken from adult trees of the two parents and four F1 hybrids, all grown at the Melgueil INRA Montpellier experimental station. Two grafts were carried out for each of these six genotypes onto the ‘Pajam 1’ rootstock producing two replicates per genotype. The ‘Pajam 1’ rootstock is a clonal selection of M9 which confers low vigour, a short juvenile period, and substantial, regular productivity. Grafted plants were then grown in 4.0 l pots in a greenhouse. A single axis was permitted on each plant and two plant repetitions were considered for each genotype. Finally, 12 plants (or axes) were considered in total (two parents, four hybrids, two repetitions each; Table 1). All plants were irrigated with 0.5 l d–1 to maintain non-limiting growing conditions. Pests and diseases were controlled by conventional means in line with professional practices throughout the experiment.


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  		Table 1. Length of mature internodes sampled on the distal part of 1-year-old main axis of six apple genotypes corresponding to four allelic classes

 
In September 2005, internodes was sampled along the main axes before they stopped growing. Along these axes, internode length increases at the beginning of the growing season and decreases at the end of the growing season (see Supplementary Fig. S1 at JXB online). In terms of internode ranks, these variations roughly cover the first 20% of the final length at the bottom and the last 10% at the top of the main axes. By contrast, internode length varies with a homogeneous amplitude over the median ranks. Sampling was thus performed before the main axes entered into the last 10% of their final length, so it can be assumed that the selected internodes are representative of their genotype. Internode length was measured on the four mature internodes located distally at the time of sampling, i.e. below the cluster of unfolding leaves at the top of the shoot and starting from the most distal internode that ceased elongation (Fig. 1). These four mature internodes were then collected and immediately immersed in Carnoy fixative solution composed of 60% ethanol, 30% chloroform, and 10% acetic acid. After 12 h, the samples were transferred to 70% ethanol solution.


Figure 1
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  		Fig. 1. Schematic representation of internode samples from 1-year-old apple trees (left part). The organization of the photographs taken on a sagital section along the pith of distal mature internodes is illustrated in the right part (a): on each slide, three series of photographs covered pith width in the bottom, median, and top regions (bottom, median, and top rows, respectively) and three other series were taken along a median line in the same three regions (bottom, median, and top columns). Cells in an additional internode located two ranks lower were counted along a single median line along the slide (right part, b).

 
Histological observations
Samples corresponding to the most distal internode of each plant were prepared, excluding nodal tissues, and fixed with glue to the plate of an HM 650 V vibratome (Microm International, D-69190 Walldorf). Fresh sagital histological sections 20 µm thick were obtained. When less than 20 mm long, the entire internode was placed on a slide. Otherwise, the sample was cut transversally into two or three pieces. The corresponding histological sections were placed on different slides.

Histological sections were observed under a microscope (DM 6000, Leica) with differential interferential contrast (DIC). Two magnifications were used: x20 (HCI Plan Apo, Na 0.7) for measuring cell length and x1.25 (HCX PL Fluotar; Na 0.04) for measuring pith width. Photographs were taken on each slide at both magnifications using a Micropublisher camera (3.3 RTV; Q Imaging) coupled to the microscope and driven by Openlab software (Improvision, UK).

Photographs were taken of the histological sections (Fig. 1a). Three series were taken to cover transversally the pith width at the bottom, median, and top of each histological section. Each series contained a variable number of photographs depending on pith width and are called ‘row’ below: bottom, median, and top rows. The same histological section was used to take three other series of photographs along the median sagital axis in the pith, in the same three positions (Fig. 1a). Each series contained three photographs called ‘columns’: basal, median, and top columns. On each photograph, either on a row or a column, the length of all visible cells was measured by drawing a line between the cell walls on the computer screen (Fig. 2). The corresponding length was given in pixels, by the Openlab Improvision software. Measurements in micrometres (µm) were obtained by multiplying pixels values by the magnification, i.e. 0.17 and 2.74 for the x20 and x1.25 lenses used. In addition, for each photography in a row, the number of cell files was counted (Fig. 2).


Figure 2
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  		Fig. 2. Example of cell length measurement on the computer screen, and counting of cell files. The arrow was drawn manually between the cell walls using the computer mouse and the corresponding length was given, in pixels, by the Openlab Improvision software. This example also illustrates a portion of a photograph that contains three cell files.

 
The cell populations were characterized by three variables: (i) the mean cell length and width which both characterize the cell shape, (ii) the number of cells per surface unit (in µm2) or cell density. For photographs on a row, the mean cell width was calculated in two different ways: (i) by dividing the number of cell files in the row by the pith width, and (ii) by dividing the number of cell files per photograph by the photograph width. This allowed us to explore the homogeneity of mean cell width over the internode by comparing the mean values between bottom, median, and top rows, and over the pith width by comparing the mean cell width per photograph depending on the photograph relative rank within the row. In parallel, the homogeneity of cell length was explored along the internode by comparing the mean values between basal, median, and top columns and rows. Cell density was estimated by dividing the total number of cells counted by the measurement area. In rows, this surface corresponded to the product of the number of photographs and photograph area. In columns, it corresponded to 3-fold the photograph area. Cell density was compared between rows and columns. For the three variables studied, comparisons were also performed between genotypes and allelic classes.

The total number of cells contributing to the each sagital section was examined over its two main dimensions. In internode width, pith witdh was plotted against the total number of cell files in rows. In internode length, the total number of cells was estimated by dividing internode length measured along the entire internode by the mean cell length (in µm):

Formula (1)

To assess these last estimated values, additional measurements were performed, counting the total number of cells on an independent set of internodes. Six additional internodes were used for this, i.e. one internode from one plant per genotype. These internodes corresponded to those collected two nodes below the most distal mature internode. They were prepared as previously described for the most distal internode. Then, a series of photographs were taken along a line located along the sagital surface of the pith and covering the entire internode length (Fig. 1b). All cells in the internode were counted along this line, and this was done for all the photographs. Then, the number of cells counted in these six internodes was compared to the estimations performed on the most distal internodes.

Data analysis
Because of the small number of observed plants and histological samples per plant, comparisons were performed on median values using the non-parametric Kruskal–Wallis ANOVA. First, the length of the four most distal internodes on each plant were compared between the genotypes and allelic classes. Second, the mean cell width and length and the cell density were analysed. Comparisons of these variables were made for examining the within-pith homogeneity for each genotype separately before considering all genotypes together. In a last step, the effect of allelic class and genotype was estimated on the studied variables. When the ANOVA showed a significant effect, a Kruskal–Wallis multiple range comparison test was performed between the allelic classes and genotypes.

Linear regression and the corresponding Spearman correlation coefficients were estimated between the number of cell files and pith width on the one hand and between the total number of cells and internode length (from Goequation 1) on the other hand. Then the values obtained for six additional internodes were plotted on the second graph to determine whether they were included within the 95% confidence interval of the regression.

All the statistical analyses were performed by Stastistica® version 7.1 software.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary data
 References
 
Comparing internode length between genotypes and allelic classes
Contrasted internode length values were observed between genotypes and allelic classes (Table 1). Both these effects were significant according to the Kruskal–Wallis ANOVA. The two parents ranked in the expected order (i.e. ‘Starkrimson’ possessing relatively shorter internodes than ‘Granny Smith’), but the most extreme values were obtained with hybrids (1.20 cm in B023 and 3.20 mm in B013). As a consequence, more contrasted internode lengths were observed between recombinant allelic classes than between parental allelic classes.

Comparing the mean cell shape and cell density between samples at different positions within the pith
No obvious differences were detectable for these variables on the photographs examined on the screen. For internodes that were split into segments, a Kruskal–Wallis test was performed to check for a possible effect of segment position on the studied variables. Two internodes (belonging to ‘Granny Smith’ and B087 genotypes), that were longer than 20 mm were split into two segments, and one internode (belonging to B013 genotype) that was longer than 30 mm was split into three segments. Of these three internodes, that of the B087 genotype presented a slightly significant segment effect on the number of cells per surface unit and a highly significant effect on mean cell length (data not shown). By contrast, the segment had no significant effect on the studied variables in ‘Granny Smith’ and B013 genotypes.

The effect of pith sample position within the internode was then investigated. Row position had no significant effect on either the mean cell length or width in any genotype (data not shown). Two tests were performed successively for genotype B087 either including or not including the sample that presented a significant difference between segments. No significant effect was detected between row positions in either case. Similarly, no significant difference was found between rows when all the genotypes or allelic classes were considered together. The mean cell width was then analysed depending on the relative rank of the photographs along the rows, considering the three rows together (Fig. 3). This showed that the cells located at the pith periphery were slightly more narrow than those located over the central part. However, cell width was homogeneous in most photographs and the difference concerned only those located in the last 10% of the pith, on both sides.


Figure 3
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  		Fig. 3. Mean cell width per photograph, and associated standard deviation, depending on the relative rank of the photographs along the pith width. The effect of relative rank was estimated by the Kruskal–Wallis non-parametric test. The same letters on the top of the graph indicate a homogeneous group according to a multiple range comparison.

 
The homogeneity of mean cell length was then analysed by comparing the samples corresponding to basal, median, and top columns. Again, no significant difference was found for any genotype, including B087, or when all the genotypes, or allelic classes were considered together (data not shown). Similarly, no difference in cell density (i.e. mean number of cells µm–2) was found between rows and columns, for any genotype or when all genotypes were considered together. A slight difference in cell density between rows and columns was detected in one allelic class only, among the four classes (class ‘ad’; P=0.018; Table 2).


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  		Table 2. Cell density (i.e. number of cells per surface unit in µm) in pith tissues sampled along a row or a column in distal mature internodes of six apple genotypes

 
Comparing the mean cell shape, and the cell density between allelic classes and genotypes
The mean cell width and length were compared between the genotypes and allelic classes for the series of photographs taken over the three rows or the three columns, considered together. Cell density estimated from all the photographs was also compared between the genotypes and allelic classes.

Regarding cell width, no difference was detected either between the genotypes or allelic classes (data not shown). By contrast, the mean cell length and cell density were both significantly different depending on both these factors (Tables 3 and 4, respectively). Actually, the mean cell length and cell density showed inverse median values for each genotype and allelic class: the fewer the cells per surface unit, the longer the cells. In particular, the parents exhibited contrasting behaviour for both the variables, with ‘Starkrimson’ having fewer but longer cells than ‘Granny Smith’. Inverse values between cell density and cell length were also found for genotypes B023 and B122, belonging to the allelic class ‘ad’ while genotypes B013 and B087 exhibited a less contrasted behaviour.


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  		Table 3. Cell length (µm) in pith tissues sampled in distal mature internodes of six apple genotypes

 

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  		Table 4. Cell density (i.e. number of cells per surface unit in µm2) between pith tissues sampled in distal mature internodes of six apple genotypes

 
Regarding mean cell length (Table 3), both the genotypes and allelic classes had a significant effect. A pronounced difference was detected between the parents, and between the genotypes B023 and B122. Indeed, the mean cell length in B122 was relatively high (comparable to ‘Starkrimson’ parent) while in B023 the cells were quite short (comparable to that observed in ‘Granny Smith’). By contrast, the mean cell length was similar in both B087 and B122, and close to that observed in the ‘Starkrimson’ parent (even though B087 cannot be distinguished from ‘Granny Smith’). As a consequence, the mean cell length was higher in the allelic class ‘bc’ than in the class ‘ad’ and this allelic class was close to that observed in ‘Starkrimson parent’ while the allelic class ‘ad’ had a behaviour intermediate between the two parents.

Regarding cell density (Table 4), a highly significant difference was observed between the genotypes and allelic classes, with an inverse situation to that described for the mean cell length. The hybrid genotypes showed an intermediate behaviour compared with their parents and, in both allelic classes, the cell density was not significantly different between the genotypes. In the allelic class ‘ad’, B122 showed a low cell density, similar to that observed in the ‘Starkrimson’ parent, while B023 showed a higher cell density which cannot be distinguished from the two parents. In the allelic class ‘bc’, the cell density was homogeneous between the two genotypes but was slightly closer to the ‘Granny Smith’ parent than the ‘Starkrimson’ parent. As a consequence, when the mean values were calculated per allelic class, this factor had a highly significant effect on cell density: on average, the allelic class ‘ad’ behaved as the ‘Starkrimson’ parent while the allelic class ‘bc’ behaved as ‘Granny Smith’.

Estimating the number of cells in each internode and relative contribution of cell shape and cell number to genetic variations in internode length
The total of cell files in a row was plotted against the pith width (Fig. 4). A linear regression was estimated, and the corresponding correlation coefficient was significant (r2=0.63). In internode length, the total cell numbers were estimated from the median cell length value and the mean internode length for each observed internode, using Goequation 1. Despite highly significant differences between the genotypes (Table 3), cell length variations could not account for the range of variations seen for internode length (about 3 cm). In particular, the two genotypes with the lowest cell length values (B023 and ‘Granny Smith’; Table 3) also presented the shortest and longest internode, respectively (Table 1). Similarly, the two genotypes with the highest cell length value (B122 and ‘Starkrimson’) were relatively similar for internode length (1.45 and 1.80, respectively). This shows that cell length variations cannot explain the internode length variations observed between the genotypes. By contrast, the estimated number of cells was linearly related to internode length in all the genotypes (Fig. 5). The Spearman correlation coefficient for this linear regression was highly significant (r2=0.94). Moreover, when the total number of cells observed along a single line located in the middle of the pith for six additional internodes was plotted on the same graph (Fig. 5), the resulting points were all located within the 95% confidence interval of the linear correlation. This shows that genetic variations in internode length primarily involve the number of cells and demonstrates that cell length, as observed in internode samples, can be used to predict the total number of cells on the entire internode scale.


Figure 4
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  		Fig. 4. Relationship between pith width and number of cells estimated in mature internodes on six different apple genotypes. Cell numbers were calculated by summing the number of cells measured on a series of photographs along the pith width.

 

Figure 5
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  		Fig. 5. Relationship between total internode length (IN_length) and number of cells estimated in mature internodes on six different apple genotypes. Cell numbers were estimated by dividing internode length by mean cell length, as measured on 12 distal internodes (two per genotype). These 12 paired values were then used to estimate the parameters and correlation coefficient of the linear regression. Six additional value pairs, obtained from direct observation of the total number of cells per internode, were plotted on the same graph and were all located within the 95% confidence interval of the linear regression.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary data
 References
 
In the study presented here, the choice was made to focus the histological examinations on stem pith tissue rather than on the cortex or epidermis. This choice was based on previous studies on cell division and extension dynamics in different tissues as carried out on L. styraciflua L. (Brown et al., 1995a, b). These authors have established that the pith plays a predominant role over other tissues due to the pronounced increase in both cell number and cell length seen in this tissue during internode growth. This is also supported by the results of a study by Priestley (1929) where it was shown that cell vacuolization occurs first in the pith, before the cortex, and then in the epidermal layers. Since few studies have been devoted to histological examinations at the internode scale, the results presented here contribute to a comprehensive picture of cellular patterning on the organ scale. Our results highlight that the cell shape was similar in pith samples collected along a sagital line and along pith width, except at the pith borders where narrower cells were observed. These thinner cells may be due to different mechanical constraints and turgor pressure in the different tissues that constitute the stem, especially between the pith and the adjacent vascular tissues (Schopfer, 2006). Cell density was also found to be homogeneous when the samples were compared between the pith median part and the width, except in one allelic class. However, the fact that no significant difference was noted between the samples for cell length suggests that differences in cell density could result from thinner cells found at the pith periphery. Therefore, it is not very likely that this could have impacted on internode length. Cell shape homogeneity within the pith is consistent with previous studies describing histogenesis of trees (Brown and Sommer, 1992). These authors have shown that, after a period of cell division and elongation, the length of pith cells becomes relatively uniform throughout the internode. Because in dicotyledonous stems, and contrary to monocotyledonous stems, cell division and lengthening are separated both in space and time (Priestley, 1929, for dicotyledons; Kaufman et al., 1965; Hitch and Sharman, 1971, for monocotyledons), the cell length homogeneity demonstrated here suggests that cell lengthening was homogeneous during internode development. Moreover, these results provide a methodological basis for simplifying tissue-sampling procedures in future investigations on internode development. More particularly, if samples are carefully collected in the median part of the pith, avoiding the pith borders, basal, median, and top samples are unnecessary. This simplification will be particularly useful to enlarge the genetic bases of the material studied.

As expected, the genotypes were different in terms of internode length, particularly the parents. More pronounced differences in internode length were found in the F1 hybrids than between their parents. This may be interpreted as resulting from the fact that they were intentionally selected for the two most extreme classes of this trait within the progeny (Segura et al., 2007). This also shows that these F1 hybrids display heterosis (hybrid vigour) for this character, as previously found in poplar hybrid population (Pearce et al., 2004). While cell width did not significantly differ between the genotypes or the allelic classes, cell length was very different depending on these two factors. The parents showed significant differences for both cell density and cell length while the hybrid genotypes were not fully separated by the Kruskal–Wallis test. Moreover, the ‘Starkrimson’ parent showed fewer cells per surface unit and longer cells than the ‘Granny Smith’ parent. This shows the existence of a counterbalancing effect between cell number and cell length during internode development in apple tree stems. Since such a compensation phenomenon has previously been described in pea internodes (Daykin et al., 1997) and Arabidopsis thaliana leaves (Cookson et al., 2005, 2007; Aguirrezabal et al., 2006), it appears to be involved in the shaping of most aerial determinate organ, including the internodes of perennial plants.

Despite significant differences in cell length between the genotypes and allelic classes, the magnitude of the variations found means that it cannot explain the phenotypic variation observed in internode length. By contrast, the number of cells either estimated from samples or counted along a single line correlated closely with internode length variations. Similarly the number of cell files was significantly correlated to the pith width. These results are consistent with those reported by Brown and Sommer (1992) who stated that, in several woody species, the difference in internode length was due to cell number rather than cell length. The present histological investigation suggests that, in the absence of environmental constraints, cell division acts at the first order in both dimensions of organ shape. Referring to the global scheme of stem histogenesis previously introduced, the number of cells recruited in a given internode results from mother cell divisions in the rib meristem (Howell, 1998). However, as underlined by this author, relatively little attention has been paid so far to these cells. Our results suggest that more in-depth investigations on the mother cells of axial tissues would certainly improve the understanding of shoot shape determination.

From a methodological point of view, the linear relationship noted between the number of cells and internode length, and the corresponding high r2 value, can be used to predict with acceptable precision the number of cells per internode from cell length observations. This result, combined with the homogeneity of cell shape values in the different pith samples, will contribute to simplifying the sampling procedure used in future investigations. From a genetic point of view, the present results open up new perspectives for investigating the molecular determinisms of internode length in apple trees. In particular, the genetic variations in internode length highlighted for the studied genotypes could involve cell cycle regulation and its cross-talk with hormones (Vandenbussche and Van Der Straeten, 2004; Kepinski, 2006). In addition, despite the existence of a compensation between cell length and cell density, in particular in the parent genotypes, the most extreme internode lengths were obtained through the combination of a high number of cells and long cells (in B013 with the longest internodes) or a low number of cells and short cells (in B023 with the shortest internodes). This suggests that the underlying traits (cell numbers and cell length) could be genetically recombined. If confirmed on a larger genetic background, this result could open new possibilities for apple breeding programmes. Finally, the present study, based on the comparison of genotypes with contrasted internode length, but performed in the absence of abiotic stresses, provides arguments for an interplay between cellular and organismal control of plant morphogenesis (Inzé and De VeyIder, 2006; Tsukaya, 2006). Indeed, both the cell cycle and a compensation between the number of cells and the cell length were observed. However, a hierarchy existed between the two processes since the number of cells appeared to be the main factor involved in the genetic variations in internode length while the variations in cell length were more secondary. More fluctuating results have been obtained in the past by various authors when different species were compared or when variations in internode length were induced by environmental changes (Ross and Reid, 1992; Sommer et al., 1999; Ross et al., 2005). In particular, far-red light has been shown to stimulate internode length in bean, through an increase in cell division, elongation, and gibberellin levels (Beall et al., 1996), while blue light has been shown to decrease internode length by inhibiting cell division in soybean stems (Dougher and Bugbee, 2004). Similarly, GA3 applied during internode development promoted an increase in both cell number and cell size in dwarf pea (Daykin et al., 1997) while GA-insensitive alleles caused a reduction in cell length but had no effect on the number of cells in dwarf mutants of wheat (Miralles et al., 1998). These previous results, like others regarding leaf development (Cookson et al., 2005, 2007) suggest that (i) the main contribution made by cell number to genetic variations in internode length, as highlighted in the present study, is likely to result from the similar environmental conditions, especially light conditions, used to grow the plants; and (ii) further investigation, including fluctuating environmental conditions, could complement the present study in terms of the relative contribution made by cell number and cell shape to final internode length in apple trees.


   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary data are available at JXB online in Fig. S1 showing changes in internode length depending on the relative rank of the internode along the main axis.


   Acknowledgements
 
We thank V Segura and JL Régnard for constructive discussions on allelic class definitions and sampling procedures, M Lartaud for his help with Openlab software, S Feral and G Garcia for greenhouse maintenance, and M Jones for improving the English. We also thank the two anonymous referees for their helpful comments.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
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