Severe Shading Reduces Early Fruit Growth in Apple by Decreasing Cell Production and Expansion

in Journal of the American Society for Horticultural Science
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  • 1 Department of Horticulture, University of Georgia, 1111 Miller Plant Sciences, Athens, GA 30602

Shading during early fruit development reduces fruit growth and initiates fruit abscission in apple (Malus ×domestica). The mechanisms mediating the decline in fruit growth in response to shading are not well understood. In this study, the effects of shading during early fruit development on cell production and expansion were investigated. Additionally, the effects of shading on the expression of genes associated with carbohydrate metabolism, fruit growth, and cell production and expansion were investigated to develop a better understanding of the molecular mechanisms and to identify genes that mediate the reduction in fruit growth. Shading of isolated branches or entire trees ≈15 to 18 days after full bloom resulted in a sharp decline in fruit growth by 3 days after treatment. Reduction in fruit growth was consistently mediated by a decline in cell production within 3 days after treatment. Reduced fruit growth was also associated with lower cell size by 3 to 7 days after shading in two different years. These data indicate that the reduction in fruit growth as a result of shading is mediated by a reduction in cell production and expansion. The expression of two sorbitol dehydrogenase (SDH) genes, MdSDH1 and MdSDH2, was higher in the shaded fruit by up to 10-fold, suggesting an increase in SDH activity to meet the immediate respiratory demands of the developing fruit. The auxin response factor (ARF), MdARF106, displayed ≈3-fold higher expression in the shaded fruit, suggesting its involvement in regulating mechanisms that mediate the reduction in fruit growth. Two A2-type cyclins, MdCYCA2;2 and MdCYCA2;3, which are positively associated with cell production, displayed lower expression in the shaded fruit by up to 4.6-fold. Conversely, MdKRP4 and MdKRP5, cell cycle genes negatively associated with cell production, displayed 3.9- and 5.3-fold higher expression in the shaded fruit, respectively. Additionally, two genes associated with cell expansion, MdCOB1 (cobra1) and MdEXPA10;1 (expansin), displayed lower expression in the shaded fruit. Together, these data indicate that shading results in coordinated changes in the expression of carbohydrate metabolism-related genes, key transcription factors related to fruit growth, and genes associated with cell production and expansion. These changes may subsequently decrease the progression of the primary processes that mediate fruit growth.

Abstract

Shading during early fruit development reduces fruit growth and initiates fruit abscission in apple (Malus ×domestica). The mechanisms mediating the decline in fruit growth in response to shading are not well understood. In this study, the effects of shading during early fruit development on cell production and expansion were investigated. Additionally, the effects of shading on the expression of genes associated with carbohydrate metabolism, fruit growth, and cell production and expansion were investigated to develop a better understanding of the molecular mechanisms and to identify genes that mediate the reduction in fruit growth. Shading of isolated branches or entire trees ≈15 to 18 days after full bloom resulted in a sharp decline in fruit growth by 3 days after treatment. Reduction in fruit growth was consistently mediated by a decline in cell production within 3 days after treatment. Reduced fruit growth was also associated with lower cell size by 3 to 7 days after shading in two different years. These data indicate that the reduction in fruit growth as a result of shading is mediated by a reduction in cell production and expansion. The expression of two sorbitol dehydrogenase (SDH) genes, MdSDH1 and MdSDH2, was higher in the shaded fruit by up to 10-fold, suggesting an increase in SDH activity to meet the immediate respiratory demands of the developing fruit. The auxin response factor (ARF), MdARF106, displayed ≈3-fold higher expression in the shaded fruit, suggesting its involvement in regulating mechanisms that mediate the reduction in fruit growth. Two A2-type cyclins, MdCYCA2;2 and MdCYCA2;3, which are positively associated with cell production, displayed lower expression in the shaded fruit by up to 4.6-fold. Conversely, MdKRP4 and MdKRP5, cell cycle genes negatively associated with cell production, displayed 3.9- and 5.3-fold higher expression in the shaded fruit, respectively. Additionally, two genes associated with cell expansion, MdCOB1 (cobra1) and MdEXPA10;1 (expansin), displayed lower expression in the shaded fruit. Together, these data indicate that shading results in coordinated changes in the expression of carbohydrate metabolism-related genes, key transcription factors related to fruit growth, and genes associated with cell production and expansion. These changes may subsequently decrease the progression of the primary processes that mediate fruit growth.

Shading during early apple fruit development decreases fruit growth and induces fruit abscission and has been used to understand processes that affect thinning (Byers et al., 1985, 1990a, 1990b, 1991). During early fruit development, active sinks such as the growing shoots and fruit compete for limited carbohydrate and nutrient resources (Lakso et al., 1999). Shading during this period rapidly reduces canopy photosynthesis resulting in the decreased availability of assimilates and further enhances the competition among these sinks (Byers et al., 1990b; Morandi et al., 2011; Polomski et al., 1988; Zibordi et al., 2009). In such a context, the available carbohydrate and nutrient resources are channeled primarily in favor of shoot growth, whereas fruit growth is reduced. For example, shading of isolated branches during early fruit development results in reduced fruit growth but allows for continued shoot growth (Bepete and Lakso, 1998). The reduction in fruit growth and a subsequent decrease in sink strength may lead to the induction of fruit abscission mechanisms (Botton et al., 2011; Zhou et al., 2008; Zhu et al., 2011). A decrease in fruit relative growth rate (RGR) was apparent within 2 d after shading (Morandi et al., 2011; Zibordi et al., 2009). Shade-induced fruit abscission occurred ≈5 to 10 d after shading and peaked ≈15 d (Kolaric et al., 2011; McArtney et al., 2004; Zhu et al., 2011). Hence, reduction in fruit growth is an earlier response to shading. Although progress has been made in understanding the mechanisms that mediate shade-induced fruit abscission (Zhou et al., 2008; Zhu et al., 2011), mechanisms regulating the shade-induced reduction in fruit growth are not well understood.

Reduction in fruit growth resulting from shading may be mediated by a decrease in the extent of cell production and/or cell expansion, the primary mechanisms that mediate fruit growth. The contribution of these processes to shade-induced reduction in fruit growth has not yet been determined. Shading during early fruit development, a period of intensive cell production, is particularly effective in reducing fruit growth and inducing abscission (Byers et al., 1990b; Polomski et al., 1988). Furthermore, cell production has a high requirement for resources and this phase of fruit development displays the highest amount of respiration on a per unit fruit weight basis (Bepete and Lakso, 1997). Hence, cell production may be particularly sensitive to lower assimilate availability as a result of shading. However, considerable cell expansion also occurs during early fruit growth (Malladi and Johnson, 2011). Hence, it is also likely that shading may reduce fruit growth by altering the extent of early cell expansion.

The molecular mechanisms leading to a reduction in fruit growth resulting from shading are not well understood. Shade-induced reduction in fruit growth may be mediated by genes regulating carbohydrate metabolism, transcription factors associated with fruit growth, and final effectors of cell production and expansion. Changes in the expression of genes associated with sorbitol metabolism such as sorbitol dehydrogenase in response to shading may enable the developing fruit to respond to the decreased availability of assimilates. For example, SDH expression in the fruit decreased in response to shading (Zhu et al., 2011). Transcription factors such as the auxin response factor, MdARF106, a gene putatively associated with the regulation of fruit growth (Devoghalaere et al., 2012), may coordinate changes in gene expression to facilitate a reduction in fruit growth in response to shading. Core cell cycle genes such as the B-type cyclin dependent kinases (CDKs) and A- and B-type cyclins (CYCs) are positively associated, whereas others such as the KIP related proteins, MdKRP4 and MdKRP5, are negatively associated with cell production during different stages of apple fruit growth (Malladi and Johnson, 2011). Expansins (EXPAs) and cobra (COB) genes are associated with cell growth and orientation of cell expansion (Cho and Cosgrove, 2000; Roudier et al., 2005; Schindelman et al., 2001). These genes may function as the downstream effectors of cell production and expansion and may aid in coordinating changes in these processes in response to shading. Analyses of the changes in the expression of these genes resulting from shading may allow for a better understanding of the molecular mechanisms that facilitate the reduction in fruit growth.

It was hypothesized that a decrease in cell production and expansion contributes to the reduction in fruit growth in response to shading and that genes associated with these processes mediate the shade-induced reduction in fruit growth. To address these hypotheses, the effects of shading on fruit size, cell number, and cell area were analyzed at different stages of early fruit growth and changes in the expression of key genes associated with the regulation of fruit growth and, particularly, cell production and expansion, were investigated.

Materials and Methods

Plant material.

Mature trees of ‘Golden Delicious Smoothee’ on M.7a rootstocks were used in this study. Trees were grown and maintained at the Mountain Research and Education Center, University of Georgia, Blairsville, GA. The trees were maintained according to commercial apple production practices. Chemical or hand-thinning was not performed in either of the 2 years of this study.

Shading treatment.

In 2009, eight uniform trees were selected and assigned randomly either to the “shaded” treatment or used as “control” trees (n = 4). One major scaffold branch per tree, on the west side, was selected for these treatments. At 15 d after full bloom [DAFB (≈11 mm fruit diameter)], the selected branch within the shaded treatment was covered with black polypropylene, 80% shade material using a wire support framework built around the branch. Previous studies have reported significant fruit growth reduction in response to similar levels of shading (Bepete and Lakso, 1998; Zhou et al., 2008). The branches were shaded throughout the duration of the experiment (15 to 25 DAFB). The base of the branch, close to the tree trunk, was girdled to isolate the branch from the rest of the tree (Bepete and Lakso, 1998). Girdling has been found to have little direct impact on fruit growth for at least 10 d (Bepete and Lakso, 1998). The branches on the control trees were also girdled but were left uncovered. Temperature within the canopy of the shaded and control branches was recorded using sensors placed inside radiation shields. The average daily temperature during the period of the experiment was 18.9 ± 0.6 °C and 18.6 ± 0.7 °C within the branches in the shaded and the control treatments, respectively. The light levels within the canopy of the shaded and control treatments were measured during early afternoon at 0, 3, 7, and 10 d after treatment using a 1-m line quantum sensor (LI-191; LI-COR, Lincoln, NE). During the experimental period, the light levels within the canopy of the control branches were ≈585.1 μmol·m−2·s−1, whereas the canopy within the shaded branches received ≈59 μmol·m−2·s−1, indicating ≈90% shading. Ten fruit on each experimental unit (branch) were tagged and fruit diameter was recorded over the duration of the experiment. Fruit RGR (mm·mm−1·d−1) was calculated from the fruit diameter data using the formula [ln(D2)-ln(D1)]/T2-T1, where D2 and D1 are fruit diameter at time points, T2 and T1. Five fruit from each replicate were sampled at 0, 3, 7, and 10 d after shading. All of the samples were collected during the afternoon period (≈1400 hr). The samples were fixed in CRAF III fixative (3% chromic acid, 20% acetic acid, and 10% formalin) for histology or immediately frozen in liquid N2 for gene expression analyses.

In 2010, four trees each were assigned randomly to either the shaded or the control treatments (n = 4). Entire trees within the shaded treatment were individually covered with black polypropylene, 80% shade material using a metal framework support constructed around the trees at 18 DAFB (≈11 mm fruit diameter). The trees were shaded throughout the duration of the experiment (18 to 28 DAFB). Temperature sensors housed within a radiation shield and light sensors (SQ100; Apogee Instruments, Logan, UT) were placed at 1 m above ground level close to the tree canopy. The average daily temperature during the duration of the experiment was 18.1 ± 1.3 °C and 17.6 ± 1.3 °C in the shaded and the control treatments, respectively. The average daily light integrals (DLIs) over the duration of the experiment (18 to 28 DAFB) were 25.9 ± 3.3 mol·m−2·d−1 (maximum and minimum of 38 and 13 mol·m−2·d−1, respectively) and 3.8 ± 1.7 mol·m−2·d−1 within the canopies of the control and shaded trees, respectively. Severe weather at 6 d after the initiation of the experiment resulted in partial opening of the shade material over three of the replicates but was fixed on the same day. Analysis of the DLI data did not indicate an increase in light levels within the shaded treatment during this period. The ambient light levels were low from 6 to 10 d after treatment. Twenty fruit per tree were tagged at 0 d after treatment and were used to determine fruit diameter. Fruit RGR (mm·mm−1·d−1) was calculated as described previously. Fruit were sampled from the trees at 0, 1, 2, 3, 6, and 10 d after treatment and fixed in CRAF III fixative for histology. All of the samples were collected during the afternoon period (≈1400 hr). One branch (average of 42 fruit per branch) on each tree was tagged at 1 d after treatment and the number of fruit on it was monitored during the duration of the experiment (18 to 28 DAFB).

Cell number and cell area measurement.

The number of cell layers in the cortex and the cortex cell area were measured as described previously in Malladi and Johnson (2011). Briefly, samples fixed in CRAF III were sectioned using a vibratome (Microcut H1200; Bio-Rad, Hercules, CA). The sections were stained in toluidine blue and images were captured using a microscope (BX51, DP70; Olympus, Center Valley, PA). The number of cell layers between the petal vascular trace and the epidermis was counted manually to obtain the cell number data. The number of cells was determined at three locations within the fruit cortex, between the petal vascular trace and the epidermis. The average cell area from the three locations was used to determine the cortex cell area.

RNA extraction and cDNA synthesis.

RNA was extracted from fruit collected at 0 and 3 d after the initiation of the treatment in 2009. The extraction was performed as reported previously in Malladi and Hirst (2010). One microgram of total RNA was used for cDNA synthesis using oligo dT primers and ImProm II reverse transcriptase (Promega, Madison, WI) after treatment with DNase (Promega). Synthesis of cDNA was performed in a total volume of 20 μL, which was subsequently diluted 6-fold and stored at –20 °C until further analysis.

Quantitative reverse transcriptase–polymerase chain reaction.

All quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analyses were performed using a Light Cycler 480 (Roche Applied Sciences, Indianapolis, IN). One microliter of the diluted cDNA was used in a final reaction volume of 12 μL. The Light Cycler 480 SYBR Green I Master mix (Roche Applied Sciences) was used in all gene expression analyses. The reaction conditions involved the following cycles: 95 °C for 10 min and 40 cycles of 95 °C for 30 s and 60 °C for 1 min. Melt curve analyses performed at the end of the PCR cycles indicated a distinct single peak for all of the amplicons analyzed. Controls without a template and without the RT were used. Rarely, some negative controls displayed low amplification but this occurred only during the late stages of PCR cycling. The genes analyzed and the gene-specific primer sequences used in this study are indicated in Table 1. The primer efficiency was determined using LinRegPCR (Ruijter et al., 2009) using converted fluorescence data from the Light Cycler 480 and ranged from 1.58 to 1.91. All gene expression was normalized to the expression of three reference genes, MdACTIN (actin), MdGAPDH (glyceraldehyde 3-phosphate dehydrogenase), and MdCACS (clathrin adaptor complexes medium subunit family protein). Gene expression was calculated using the Cq (cycle number where the fluorescence threshold was crossed) values with correction for amplification efficiency (Pfaffl, 2001). The relative quantities (1/ECq, where E is the amplification efficiency) were normalized using the geometric mean of the relative quantities of the reference genes. The expression of a given gene relative to its expression in the control fruit at 0 d after treatment is reported here. The se of the expression was calculated as described in Rieu and Powers (2009).

Table 1.

List of the apple genes and the sequence of primers used in quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analyses.

Table 1.

Statistical analyses.

All statistical analyses and graph preparation were performed with JMP software (Version 9; SAS Institute, Cary, NC) and Sigmaplot 11 (Systat Software, San Jose, CA). The main effects of “shading” and “time after treatment” and their interaction effects were tested using repeated measures. Wherever the interactions were significant between the main factors, the simple effects were further analyzed using test of effect slices. For the gene expression data, the normalized relative quantities were transformed (log2) before statistical analyses. For the gene expression analysis, the genes of interest were ones that displayed significant interaction effects between the main factors, because in these cases, the effect of shading depended on the time after treatment.

Results

Shading reduces fruit growth by decreasing cell production and expansion.

In 2009, fruit within the control treatment increased in size by 8.3 mm between 15 and 25 DAFB, but little change in fruit diameter (2.2 mm) was observed in the shaded fruit during this period (Fig. 1). Fruit diameter was significantly smaller in the shaded fruit from 3 d after treatment. Similar to data from the 2009 study, shaded fruit in the 2010 study displayed only a minor increase in size (2.4 mm), whereas the control fruit displayed an increase in fruit diameter by 8.7 mm between 18 and 28 DAFB (Fig. 1). A significantly lower diameter was evident in shaded fruit from 3 d after treatment. Therefore, in both years of the study, shading resulted in a reduction in fruit growth within 3 d after treatment. In 2009, the fruit RGR (mm·mm−1·d−1) was significantly different at 3 d after treatment [control fruit = 0.05 ± 0.01 mm·mm−1·d−1, shaded fruit = 0.02 ± 0.01 mm·mm−1·d−1 (P = 0.018)]. In 2010, fruit RGR was significantly different between control and shaded fruit at 2 d after treatment [control fruit = 0.06 ± 0.01 mm·mm−1·d−1, shaded fruit = 0.02 ± 0.01 mm·mm−1·d−1 (P = 0.005)]. During the duration of the experiment (18 to 28 DAFB), shading did not have a significant effect on the extent of fruit drop. At 10 d after treatment, ≈54% of the fruit in the shaded treatment abscised, whereas 38% of the fruit abscised in the control treatment (P = 0.06, Student’s t test).

Fig. 1.
Fig. 1.

Effects of shading on apple fruit growth, cell number, and cell size. Shading was performed on branches in 2009 and entire trees in 2010 using 80% shade material. Fruit diameter, number of cell layers in the fruit cortex, and cortex cell area were determined in fruit sampled from the “shaded” and “control” treatments in 2009 and 2010. Error bars indicate the se (n = 4). Significant interaction effects between the factors, “shading” and “time after treatment,” were observed for all the fruit growth-related parameters in both years of the study (P < 0.05). Simple effects were analyzed using the test of effect slices. Asterisk indicates significant difference between the shaded and control fruit within the indicated time after treatment, as determined using the test of effect slices. All the differences indicated by asterisks were significant at α = 0.01 except for fruit diameter at 3 d after treatment in 2010 (P = 0.012).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 137, 5; 10.21273/JASHS.137.5.275

In 2009, the number of cell layers within the cortex increased by 1.47-fold over the duration of the experiment in the control fruit but only by 1.32-fold in the shaded fruit (Fig. 1). The number of cell layers within the fruit cortex of shaded fruit was significantly lower than that in the control fruit from 3 d after treatment. By the end of the experiment, cell number in the shaded fruit was only 82% of that in the control fruit. A similar pattern of change in cell number was observed in the 2010 study and the number of cell layers was significantly lower in the shaded fruit from 3 d after treatment (Fig. 1). Over the duration of the experiment, the cortex cell area increased by 1.72-fold and 1.48-fold in the control fruit in 2009 and 2010, respectively. In the shaded fruit, cortex cell area increased only by 1.17-fold and 1.13-fold in 2009 and 2010, respectively. In 2009, significant differences in cell area between the control and shaded fruit were evident at 7 and 10 d after treatment (Fig. 1). In 2010, lower fruit cortex cell area in the shaded fruit was evident from 3 d after treatment. Together, these data clearly indicate that the shade-induced decrease in fruit growth was associated with a reduction in cell production and expansion in the fruit cortex.

Altered expression of carbohydrate metabolism- and fruit growth-related genes resulting from shading.

The expression of two sorbitol dehydrogenase genes, MdSDH1 and MdSDH2, was higher in the shaded fruit at 3 d after shading by ≈10-fold and 2-fold, respectively (Fig. 2). The expression of two transcription factors (ARFs) putatively associated with fruit growth was investigated. The interaction effect between the factors, shading and time after treatment, was not significant for MdARF6 (Fig. 3). MdARF106 displayed higher expression in the shaded fruit at 3 d after treatment by 2.9-fold (Fig. 3).

Fig. 2.
Fig. 2.

Effect of shading on the expression of two sorbitol dehydrogenase (SDH) genes in apple fruit. Shading was performed on branches in 2009 using 80% shade material. Open box represents “control” fruit and closed box represents “shaded” fruit. Expression was determined using quantitative reverse transcriptase–polymerase chain reaction. The expression of a gene in relation to its expression at 0 d after treatment in the control fruit is presented. Error bars indicate the se (n = 4). Both the genes displayed a significant interaction between the factors, “shading” and “time after treatment” (P < 0.05). Simple effects were analyzed using test of effect slices. Asterisk indicates significant difference between the shaded and control fruit at the indicated time after treatment as determined using the test of effect slices (P < 0.01).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 137, 5; 10.21273/JASHS.137.5.275

Fig. 3.
Fig. 3.

Effect of shading on the expression of two transcription factors putatively associated with fruit growth in apple. Individual branches were shaded in 2009 using 80% shade material. Open box indicates “control” fruit and closed box indicates “shaded” fruit. Expression was determined using quantitative reverse transcriptase–polymerase chain reaction. The expression of a gene in relation to its expression in the control fruit at 0 d after treatment is presented. Error bars indicate the se (n = 4). Only MdARF106 expression displayed a significant interaction effect between the factors, “shading” and “time after treatment” (P < 0.05). Simple effects were analyzed using test of effect slices to determine differences between shaded and control fruit at each time after treatment for MdARF106. Asterisk indicates significant difference between the shaded and control fruit within the indicated time after treatment as determined by this test for MdARF106 (P < 0.01).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 137, 5; 10.21273/JASHS.137.5.275

Altered expression of cell production- and expansion-related genes resulting from shading.

The expression of 10 genes positively associated with cell production and two genes negatively associated with cell production was investigated (Fig. 4). Many of the positive regulators of cell production including four B-type cyclin dependent kinase (MdCDKB) genes, MdCYCA2;1, and two B1-type cyclins (MdCYCB1;1 and MdCYCB1;2) were affected by the main factors, shading and time after treatment. Significant interaction effects of these factors on the expression of these genes were not observed. At 0 d after treatment, the expression of these genes in the shaded fruit was never lower than 1.9-fold of that in the control fruit and were not significantly different (pairwise comparisons using Tukey’s honestly significant difference) except for MdCYCB1;1. The expression of the B2-type cyclin, MdCYCB2;2, was affected only by the factor time after treatment. The expression of two A2-type cyclins, MdCYCA2;2 and MdCYCA2;3, and two KRP genes, MdKRP4 and MdKRP5, displayed significant interaction effects between shading and time after treatment. The expression of MdCYCA2;2 and MdCYCA2;3 was lower in the shaded fruit by 4.6-fold and 3.6-fold, respectively, at 3 d after treatment. The expression of MdKRP4 and MdKRP5 was higher in the shaded fruit by 3.9-fold and 5.3-fold, respectively, at 3 d after treatment, consistent with their proposed roles as negative regulators of cell production during apple fruit growth.

Fig. 4.
Fig. 4.

Effect of shading on the expression of core cell cycle genes associated with cell production in apple fruit. Shading was performed using 80% shade material on individual branches in 2009. Open box represents “control” fruit and closed box represents “shaded” fruit. Expression analysis was performed using quantitative reverse transcriptase–polymerase chain reaction. Expression of a gene relative to its expression in the control fruit at 0 d after treatment is presented. Error bars indicate the se (n = 4). The interaction effects between “shading” and “time after treatment” were significant (P < 0.05) for MdCYCA2;2, MdCYCA2;3, MdKRP4, and MdKRP5 only. Asterisk indicates significant difference between shaded and control fruit at the indicated time after treatment for the above genes as determined by the test of effect slices (P < 0.01). All the other cell cycle genes (except for MdCYCB2;2) displayed significant main effects of shading and time after treatment but non-significant interaction between these factors. For MdCYCB2;2, only the factor, time after treatment, was significant.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 137, 5; 10.21273/JASHS.137.5.275

The expression of two genes putatively associated with directional cell expansion, MdCOB1 and MdCOBL4 (Cobra1 and Cobra-Like4), and several α-type expansin (MdEXPA) genes putatively involved in the loosening of cell walls, was investigated (Fig. 5). MdCOB1 expression in the shaded fruit was slightly higher (1.2-fold) at 0 d after treatment and was 1.6-fold lower at 3 d after treatment than that in the control fruit. MdCOBL4 expression was 1.9-fold higher in the shaded fruit at 3 d after treatment. The expression of MdEXPA10;1 was lower in the shaded fruit by 4.6-fold at 3 d after treatment. The expression of the other MdEXPA genes analyzed here was not significantly affected by shading.

Fig. 5.
Fig. 5.

Effect of shading on the expression of genes associated with cell expansion in apple fruit. Shading was performed using 80% shade material on individual branches in 2009. Open box indicates “control” fruit and closed box indicates “shaded” fruit. Expression was measured using quantitative reverse transcriptase–polymerase chain reaction. Expression of a gene relative to its expression at 0 d after treatment in the control fruit is presented. Error bars indicate the se (n = 4). The interaction effects between “shading” and “time after treatment” were significant for MdCOB1, MdCOBL4, and MdEXPA10;1 only (P < 0.05). Asterisk indicates significant difference between shaded and control fruit at the indicated time after treatment for these three genes as determined by the test of effect slices. Expression of MdCOB1 at 0 d after treatment and MdCOBL4 at 3 d after treatment were significantly different between the shaded and control fruit at α = 0.05, whereas MdCOB1 at 3 d after treatment and MdEXPA10;1 at 3 d after treatment were significantly different between shaded and control fruit at α = 0.01. MdEXPA8;1 and MdEXPA8;2 were unaffected by shading.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 137, 5; 10.21273/JASHS.137.5.275

Discussion

Several studies have indicated a reduction in fruit growth before fruit abscission in response to shading during early fruit development (Morandi et al., 2011; Zhou et al., 2008; Zhu et al., 2011; Zibordi et al., 2009). In both years of the current study, fruit growth was significantly lower in shaded fruit from 3 d after treatment indicating that a reduction in fruit growth is an early response to shading. The fruit RGR declined by 2 to 3 d after shading. These data are consistent with previous studies in which shading reduced fruit RGR by ≈50% at 2 d and fruit growth by 58% at 3 d after treatment (Morandi et al., 2011; Zhou et al., 2008). A reduction in fruit growth may decrease sink strength and subsequently lead to the activation of abscission mechanisms. Although the effect of shading on fruit drop was not significant within the duration of the experiment, it is likely that fruit abscission under shading continued at later stages. The high levels of shading used in this study have previously been shown to induce extensive abscission (Polomski et al., 1988; Zhu et al., 2011).

The shading treatments were imposed during the phase of fruit development involving intensive cell production. In both years of this study, shading resulted in a rapid decline in cell production within 3 d after treatment, coincident with the decrease in fruit growth. These data indicate that the reduction in fruit growth was partly mediated by a decline in cell production. Although the majority of cell expansion typically occurs during the later stages of fruit development, a considerable increase in cell area occurs during early fruit development and may contribute to early fruit growth. In fact, cell area in the control fruit increased by ≈1.5- to 1.7-fold over the duration of the experiment. These data are consistent with previous studies in which considerable cell expansion was noted during early fruit growth (Malladi and Hirst, 2010; Malladi and Johnson, 2011). In the 2009 study, cell expansion was affected by shading at 7 d after treatment, and in the 2010 study, it declined by 3 d after shading. Therefore, the data indicate that a decline in cell expansion contributes to the reduction in fruit growth. Together, these data indicate that shade-induced reduction in fruit growth is mediated by a decline in cell production and expansion. Progression of these processes mediating growth is dependent on the availability of carbohydrates. A reduction in photosynthate availability and subsequent changes in carbon metabolism resulting from shading may rapidly decrease the rates of cell production and expansion, thereby reducing fruit growth.

Sorbitol is the main translocated carbohydrate in apple and is converted to fructose through the activity of SDH (Bieleski, 1969). Interestingly, the expression of MdSDH1 and MdSDH2, genes known to be expressed in the fruit cortex (Nosarzewski and Archbold, 2007; Nosarzewski et al., 2004), was higher in the shaded fruit, suggesting higher SDH activity. A decrease in light levels, and subsequently photosynthesis, potentially decreases the extent of sorbitol translocated into the developing fruit. It may be hypothesized that the fruit responds through a rapid (and potentially transient) increase in SDH activity, which allows for a higher rate of conversion of the available sorbitol into fructose, thereby allowing the fruit to meet its immediate and high respiratory demand (Lakso et al., 1999). Hence, the effects of shading on the carbohydrate status within the developing fruit, and its metabolism, warrant further investigation. The expression of another SDH, MdSDH5, reportedly decreased initially in the fruit cortex in response to shading (Zhu et al., 2011). This may reflect differences in the expression among different SDH genes.

The shade-induced reduction in fruit growth may be mediated by changes in the expression of key transcription factors. The transcription factors, MdARF6 and MdARF106, have been investigated in relation to their potential roles in the regulation of fruit size (Devoghalaere et al., 2012). MdARF106 was colocalized to a region on chromosome 15 containing a quantitative trait locus associated with fruit size. Also, MdARF106 was expressed during the cell production and expansion phases of fruit growth consistent with a proposed role in mediating the effects of auxin on fruit growth (Devoghalaere et al., 2012). In the current study, MdARF106 expression was higher by 2.9-fold in the shaded fruit. The increase in MdARF106 expression may, in turn, regulate mechanisms that mediate the shade-induced reduction in fruit growth.

The shade-induced decline in cell production was associated with coordinated changes in the expression of core cell cycle genes, which are key facilitators of cell production (Malladi and Johnson, 2011). The expression of MdCYCA2;2 and MdCYCA2;3, genes positively associated with cell production (Malladi and Johnson, 2011), was lower in the shaded fruit. A2-type cyclins mediate the progression of the G2/M phase of the cell cycle. Members of this class of cyclins associate with CDKBs to prevent exit from mitotic cell production (Boudolf et al., 2009). Lower expression of these genes in response to shading may therefore facilitate the exit from cell production within the developing fruit cortex. KRPs are key facilitators of exit from mitotic cell production and are also involved in promoting entry into endoreduplication in other plants (Verkest et al., 2005; Weinl et al., 2005). In apple, MdKRP4 and MdKRP5 display an increase in expression in unpollinated fruit and during later stages of fruit development, consistent with their proposed roles in mediating the exit from cell production (Malladi and Johnson, 2011). MdKRP4 and MdKRP5 displayed a sharp increase in expression by approximately 4- to 5-fold in response to the decrease in light levels, indicating that they may mediate the shade-induced exit from cell production. Together, these data indicate that shading during early fruit development results in the coordinated alteration of core cell cycle gene expression, which may subsequently mediate the reduction in cell production and fruit growth.

The expression of several genes associated with cell growth was reduced by shading and preceded the decline in cell expansion. COBRA encodes a glycosyl-phosphatidyl inositol-anchored protein, which may regulate cell growth by affecting cellulose biosynthesis and by determining the orientation of cellulose microfibrils within the cell wall (Roudier et al., 2005; Schindelman et al., 2001). MdCOB1 expression was lower in the shaded fruit at 3 d after treatment. Lower MdCOB1 expression in response to shading may impair cell wall extensibility and contribute to the subsequent decline in cell expansion. Interestingly, expression of the COBRA-LIKE gene, MdCOBL4, was higher in the shaded fruit. Members of the COBL4 subgroup of COB genes are thought to function in secondary cell wall synthesis and may primarily contribute toward maintaining the mechanical strength of tissues (Brown et al., 2005; Ching et al., 2006; Li et al., 2003). As cell production and expansion decline, mechanisms involved in secondary cell wall synthesis may be activated and the higher MdCOBL4 expression may potentially be part of such a mechanism in the shaded fruit. The expansin family consists of multiple genes, which encode extracellular proteins that facilitate the loosening of cell walls, thereby allowing for cell expansion (Cho and Cosgrove, 2000; Sampedro and Cosgrove, 2005). The expression of the α-Expansin, MdEXPA10;1, was lower by over 4-fold in shaded fruit. Such a reduction in MdEXPA10;1 expression may reduce cell wall extensibility and contribute to the shade-induced decline in cell expansion and fruit growth.

The high level of shading used in the current study is known to induce extensive fruit abscission (Polomski et al., 1988; Zhu et al., 2011). Hence, it is possible that some of the fruit sampled for growth and gene expression analyses were derived from a population of fruit, some of which were destined to abscise. It will be interesting to investigate in future studies whether the shade-induced changes in fruit growth-related parameters and gene expression reported here are applicable to fruit that display a reduction in growth but are not programmed for abscission.

Data from this study are consistent with our hypotheses and clearly demonstrate that shade-induced reduction in fruit growth is facilitated by a decrease in cell production and expansion. Furthermore, the data indicate that the decrease in the extent of cell production and expansion resulting from shading may be mediated by coordinated changes in the expression of carbohydrate metabolism-related genes, transcription factors associated with fruit growth, and key genes associated with cell production and expansion.

Literature Cited

  • Bepete, M. & Lakso, A.N. 1997 Apple fruit respiration in the field: Relationships to fruit growth rate, temperature and light exposure Acta Hort. 451 319 326

    • Search Google Scholar
    • Export Citation
  • Bepete, M. & Lakso, A.N. 1998 Differential effects of shade on early-season fruit and shoot growth rates in ‘Empire’ apple HortScience 33 823 825

    • Search Google Scholar
    • Export Citation
  • Bieleski, R.L. 1969 Accumulation and translocation of sorbitol in apple phloem Aust. J. Biol. Sci. 22 611 620

  • Botton, A., Eccher, G., Forcato, C., Ferrarini, A., Begheldo, M., Zermiani, M., Moscatello, S., Battistelli, A., Velasco, R., Ruperti, B. & Ramina, A. 2011 Signaling pathways mediating the induction of apple fruitlet abscission Plant Physiol. 155 185 208

    • Search Google Scholar
    • Export Citation
  • Boudolf, V., Lammens, T., Boruc, J., Van Leene, J., Van Den Daele, H., Maes, S., Van Isterdael, G., Russinova, E., Kondorosi, E., Witters, E., De Jaeger, G., Inze, D. & De Veylder, L. 2009 CDKB1;1 forms a functional complex with CYCA2;3 to suppress endocycle onset Plant Physiol. 150 1482 1493

    • Search Google Scholar
    • Export Citation
  • Brown, D.M., Zeef, L.A.H., Ellis, J., Goodacre, R. & Turner, S.R. 2005 Identification of novel genes in arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics Plant Cell 17 2281 2295

    • Search Google Scholar
    • Export Citation
  • Byers, R.E., Barden, J.A. & Carbaugh, D.H. 1990a Thinning of spur delicious apples by shade, terbacil, carbaryl, and ethephon J. Amer. Soc. Hort. Sci. 115 9 13

    • Search Google Scholar
    • Export Citation
  • Byers, R.E., Barden, J.A., Polomski, R.F., Young, R.W. & Carbaugh, D.H. 1990b Apple thinning by photosynthetic inhibition J. Amer. Soc. Hort. Sci. 115 14 19

    • Search Google Scholar
    • Export Citation
  • Byers, R.E., Carbaugh, D.H., Presley, C.N. & Wolf, T.K. 1991 The influence of low light on apple fruit abscission J. Hort. Sci. 66 7 17

  • Byers, R.E., Lyons, C.G., Yoder, K.S., Barden, J.A. & Young, R.W. 1985 Peach and apple thinning by shading and photosynthetic inhibition J. Hort. Sci. 60 465 472

    • Search Google Scholar
    • Export Citation
  • Ching, A., Dhugga, K.S., Appenzeller, L., Meeley, R., Bourett, T.M., Howard, R.J. & Rafalski, A. 2006 Brittle stalk2 encodes a putative glycosylphosphatidylinositol-anchored protein that affects mechanical strength of maize tissues by altering the composition and structure of secondary cell walls Planta 224 1174 1184

    • Search Google Scholar
    • Export Citation
  • Cho, H.T. & Cosgrove, D.J. 2000 Altered expression of expansin modulates leaf growth and pedicel abscission in Arabidopsis thaliana Proc. Natl. Acad. Sci. USA 97 9783 9788

    • Search Google Scholar
    • Export Citation
  • Devoghalaere, F., Doucen, T., Guitton, B., Keeling, J., Payne, W., Ling, T.J., Ross, J.J., Hallett, I.C., Gunaseelan, K., Dayatilake, G.A., Diak, R., Breen, K.C., Tustin, D.S., Costes, E., Chagne, D., Schaffer, R.J. & David, K.M. 2012 A genomics approach to understanding the role of auxin in apple (Malus × domestica) fruit size control BMC Plant Biol. 12 7

    • Search Google Scholar
    • Export Citation
  • Kolaric, J., Plesko, I.M., Tojnko, S. & Stopar, M. 2011 Apple fruitlet ethylene evolution and MdACO1, MdACS5A, and MdACS5B expression after application of naphthaleneacetic acid, 6-benzyladenine, ethephon, or shading HortScience 46 1381 1386

    • Search Google Scholar
    • Export Citation
  • Lakso, A.N., Wunsche, J.N., Palmer, J.W. & Grappadelli, L.C. 1999 Measurement and modeling of carbon balance of the apple tree HortScience 34 1040 1047

  • Li, Y.H., Qian, O., Zhou, Y.H., Yan, M.X., Sun, L., Zhang, M., Fu, Z.M., Wang, Y.H., Han, B., Pang, X.M., Chen, M. & Li, J. 2003 BRITTLE CULM1, which encodes a COBRA-like protein, affects the mechanical properties of rice plants Plant Cell 15 2020 2031

    • Search Google Scholar
    • Export Citation
  • Mach, F.E. 2007 Istituto Agrario Di San Michele All'Adige computational biology web resources apple genome. 3 May 2012. <http://genomics.research.iasma.it/>

  • Malladi, A. & Hirst, P.M. 2010 Increase in fruit size of a spontaneous mutant of ‘Gala’ apple (Malus × domestica Borkh.) is facilitated by altered cell production and enhanced cell size J. Expt. Bot. 61 3003 3013

    • Search Google Scholar
    • Export Citation
  • Malladi, A. & Johnson, L.K. 2011 Expression profiling of cell cycle genes reveals key facilitators of cell production during carpel development, fruit set, and fruit growth in apple (Malus × domestica Borkh.) J. Expt. Bot. 62 205 219

    • Search Google Scholar
    • Export Citation
  • McArtney, S., White, M., Latter, I. & Campbell, J. 2004 Individual and combined effects of shading and thinning chemicals on abscission and dry-matter accumulation of ‘Royal Gala’ apple fruit J. Hort. Sci. Biotechnol. 79 441 448

    • Search Google Scholar
    • Export Citation
  • Morandi, B., Zibordi, M., Losciale, P., Manfrini, L., Pierpaoli, E. & Grappadelli, L.C. 2011 Shading decreases the growth rate of young apple fruit by reducing their phloem import Sci. Hort. 127 347 352

    • Search Google Scholar
    • Export Citation
  • Nosarzewski, M. & Archbold, D.D. 2007 Tissue-specific expression of SORBITOL DEHYDROGENASE in apple fruit during early development J. Expt. Bot. 58 1863 1872

    • Search Google Scholar
    • Export Citation
  • Nosarzewski, M., Clements, A.M., Downie, A.B. & Archbold, D.D. 2004 Sorbitol dehydrogenase expression and activity during apple fruit set and early development Physiol. Plant. 121 391 398

    • Search Google Scholar
    • Export Citation
  • Pfaffl, M.W. 2001 A new mathematical model for relative quantification in real-time RT-PCR Nucleic Acids Res. 29 2002 2007

  • Polomski, R.F., Barden, J.A., Byers, R.E. & Wolf, D.D. 1988 Apple fruit nonstructural carbohydrates and abscission as influenced by shade and terbacil J. Amer. Soc. Hort. Sci. 113 506 511

    • Search Google Scholar
    • Export Citation
  • Rieu, I. & Powers, S.J. 2009 Real-time quantitative RT-PCR: Design, calculations, and statistics Plant Cell 21 1031 1033

  • Roudier, F., Fernandez, A.G., Fujita, M., Himmelspach, R., Borner, G.H.H., Schindelman, G., Song, S., Baskin, T.I., Dupree, P., Wasteneys, G.O. & Benfey, P.N. 2005 COBRA, an arabidopsis extracellular glycosyl-phosphatidyl inositol-anchored protein, specifically controls highly anisotropic expansion through its involvement in cellulose microfibril orientation Plant Cell 17 1749 1763

    • Search Google Scholar
    • Export Citation
  • Ruijter, J.M., Ramakers, C., Hoogaars, W.M.H., Karlen, Y., Bakker, O., van den Hoff, M.J.B. & Moorman, A.F.M. 2009 Amplification efficiency: Linking baseline and bias in the analysis of quantitative PCR data Nucleic Acids Res. 37 e45

    • Search Google Scholar
    • Export Citation
  • Sampedro, J. & Cosgrove, D.J. 2005 The expansin superfamily Genome Biol. 6 242

  • Schindelman, G., Morikami, A., Jung, J., Baskin, T.I., Carpita, N.C., Derbyshire, P., McCann, M.C. & Benfey, P.N. 2001 COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis Genes Dev. 15 1115 1127

    • Search Google Scholar
    • Export Citation
  • Verkest, A., Manes, C.L.D., Vercruysse, S., Maes, S., Van der Schueren, E., Beeckman, T., Genschik, P., Kuiper, M., Inze, D. & De Veylder, L. 2005 The cyclin-dependent kinase inhibitor KRP2 controls the onset of the endoreduplication cycle during arabidopsis leaf development through inhibition of mitotic CDKA;1 kinase complexes Plant Cell 17 1723 1736

    • Search Google Scholar
    • Export Citation
  • Weinl, C., Marquardt, S., Kuijt, S.J.H., Nowack, M.K., Jakoby, M.J., Hulskamp, M. & Schnittger, A. 2005 Novel functions of plant cyclin-dependent kinase inhibitors, ICK1/KRP1, can act non-cell-autonomously and inhibit entry into mitosis Plant Cell 17 1704 1722

    • Search Google Scholar
    • Export Citation
  • Zhou, C.J., Lakso, A.N., Robinson, T.L. & Gan, S.S. 2008 Isolation and characterization of genes associated with shade-induced apple abscission Mol. Genet. Genomics 280 83 92

    • Search Google Scholar
    • Export Citation
  • Zhu, H., Dardick, C.D., Beers, E.P., Callanhan, A.M., Xia, R. & Yuan, R.C. 2011 Transcriptomics of shading-induced and NAA-induced abscission in apple (Malus × domestica) reveals a shared pathway involving reduced photosynthesis, alterations in carbohydrate transport and signaling and hormone crosstalk BMC Plant Biol. 11 20

    • Search Google Scholar
    • Export Citation
  • Zibordi, M., Domingos, S. & Grappadelli, L.C. 2009 Thinning apples via shading: An appraisal under field conditions J. Hort. Sci. Biotechnol. 84 138 144

    • Search Google Scholar
    • Export Citation

Contributor Notes

We thank the staff at the Mountain Research and Education Center, Blairsville, GA, for tree maintenance and help with the experiments. We thank Dr. Phillip Brannen, University of Georgia, for the trees used in this study, and Dr. Marc van Iersel and Sue Dove, University of Georgia, for help with the data loggers.

These authors contributed equally to this work.

Corresponding author. E-mail: malladi@uga.edu.

  • View in gallery

    Effects of shading on apple fruit growth, cell number, and cell size. Shading was performed on branches in 2009 and entire trees in 2010 using 80% shade material. Fruit diameter, number of cell layers in the fruit cortex, and cortex cell area were determined in fruit sampled from the “shaded” and “control” treatments in 2009 and 2010. Error bars indicate the se (n = 4). Significant interaction effects between the factors, “shading” and “time after treatment,” were observed for all the fruit growth-related parameters in both years of the study (P < 0.05). Simple effects were analyzed using the test of effect slices. Asterisk indicates significant difference between the shaded and control fruit within the indicated time after treatment, as determined using the test of effect slices. All the differences indicated by asterisks were significant at α = 0.01 except for fruit diameter at 3 d after treatment in 2010 (P = 0.012).

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    Effect of shading on the expression of two sorbitol dehydrogenase (SDH) genes in apple fruit. Shading was performed on branches in 2009 using 80% shade material. Open box represents “control” fruit and closed box represents “shaded” fruit. Expression was determined using quantitative reverse transcriptase–polymerase chain reaction. The expression of a gene in relation to its expression at 0 d after treatment in the control fruit is presented. Error bars indicate the se (n = 4). Both the genes displayed a significant interaction between the factors, “shading” and “time after treatment” (P < 0.05). Simple effects were analyzed using test of effect slices. Asterisk indicates significant difference between the shaded and control fruit at the indicated time after treatment as determined using the test of effect slices (P < 0.01).

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    Effect of shading on the expression of two transcription factors putatively associated with fruit growth in apple. Individual branches were shaded in 2009 using 80% shade material. Open box indicates “control” fruit and closed box indicates “shaded” fruit. Expression was determined using quantitative reverse transcriptase–polymerase chain reaction. The expression of a gene in relation to its expression in the control fruit at 0 d after treatment is presented. Error bars indicate the se (n = 4). Only MdARF106 expression displayed a significant interaction effect between the factors, “shading” and “time after treatment” (P < 0.05). Simple effects were analyzed using test of effect slices to determine differences between shaded and control fruit at each time after treatment for MdARF106. Asterisk indicates significant difference between the shaded and control fruit within the indicated time after treatment as determined by this test for MdARF106 (P < 0.01).

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    Effect of shading on the expression of core cell cycle genes associated with cell production in apple fruit. Shading was performed using 80% shade material on individual branches in 2009. Open box represents “control” fruit and closed box represents “shaded” fruit. Expression analysis was performed using quantitative reverse transcriptase–polymerase chain reaction. Expression of a gene relative to its expression in the control fruit at 0 d after treatment is presented. Error bars indicate the se (n = 4). The interaction effects between “shading” and “time after treatment” were significant (P < 0.05) for MdCYCA2;2, MdCYCA2;3, MdKRP4, and MdKRP5 only. Asterisk indicates significant difference between shaded and control fruit at the indicated time after treatment for the above genes as determined by the test of effect slices (P < 0.01). All the other cell cycle genes (except for MdCYCB2;2) displayed significant main effects of shading and time after treatment but non-significant interaction between these factors. For MdCYCB2;2, only the factor, time after treatment, was significant.

  • View in gallery

    Effect of shading on the expression of genes associated with cell expansion in apple fruit. Shading was performed using 80% shade material on individual branches in 2009. Open box indicates “control” fruit and closed box indicates “shaded” fruit. Expression was measured using quantitative reverse transcriptase–polymerase chain reaction. Expression of a gene relative to its expression at 0 d after treatment in the control fruit is presented. Error bars indicate the se (n = 4). The interaction effects between “shading” and “time after treatment” were significant for MdCOB1, MdCOBL4, and MdEXPA10;1 only (P < 0.05). Asterisk indicates significant difference between shaded and control fruit at the indicated time after treatment for these three genes as determined by the test of effect slices. Expression of MdCOB1 at 0 d after treatment and MdCOBL4 at 3 d after treatment were significantly different between the shaded and control fruit at α = 0.05, whereas MdCOB1 at 3 d after treatment and MdEXPA10;1 at 3 d after treatment were significantly different between shaded and control fruit at α = 0.01. MdEXPA8;1 and MdEXPA8;2 were unaffected by shading.

  • Bepete, M. & Lakso, A.N. 1997 Apple fruit respiration in the field: Relationships to fruit growth rate, temperature and light exposure Acta Hort. 451 319 326

    • Search Google Scholar
    • Export Citation
  • Bepete, M. & Lakso, A.N. 1998 Differential effects of shade on early-season fruit and shoot growth rates in ‘Empire’ apple HortScience 33 823 825

    • Search Google Scholar
    • Export Citation
  • Bieleski, R.L. 1969 Accumulation and translocation of sorbitol in apple phloem Aust. J. Biol. Sci. 22 611 620

  • Botton, A., Eccher, G., Forcato, C., Ferrarini, A., Begheldo, M., Zermiani, M., Moscatello, S., Battistelli, A., Velasco, R., Ruperti, B. & Ramina, A. 2011 Signaling pathways mediating the induction of apple fruitlet abscission Plant Physiol. 155 185 208

    • Search Google Scholar
    • Export Citation
  • Boudolf, V., Lammens, T., Boruc, J., Van Leene, J., Van Den Daele, H., Maes, S., Van Isterdael, G., Russinova, E., Kondorosi, E., Witters, E., De Jaeger, G., Inze, D. & De Veylder, L. 2009 CDKB1;1 forms a functional complex with CYCA2;3 to suppress endocycle onset Plant Physiol. 150 1482 1493

    • Search Google Scholar
    • Export Citation
  • Brown, D.M., Zeef, L.A.H., Ellis, J., Goodacre, R. & Turner, S.R. 2005 Identification of novel genes in arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics Plant Cell 17 2281 2295

    • Search Google Scholar
    • Export Citation
  • Byers, R.E., Barden, J.A. & Carbaugh, D.H. 1990a Thinning of spur delicious apples by shade, terbacil, carbaryl, and ethephon J. Amer. Soc. Hort. Sci. 115 9 13

    • Search Google Scholar
    • Export Citation
  • Byers, R.E., Barden, J.A., Polomski, R.F., Young, R.W. & Carbaugh, D.H. 1990b Apple thinning by photosynthetic inhibition J. Amer. Soc. Hort. Sci. 115 14 19

    • Search Google Scholar
    • Export Citation
  • Byers, R.E., Carbaugh, D.H., Presley, C.N. & Wolf, T.K. 1991 The influence of low light on apple fruit abscission J. Hort. Sci. 66 7 17

  • Byers, R.E., Lyons, C.G., Yoder, K.S., Barden, J.A. & Young, R.W. 1985 Peach and apple thinning by shading and photosynthetic inhibition J. Hort. Sci. 60 465 472

    • Search Google Scholar
    • Export Citation
  • Ching, A., Dhugga, K.S., Appenzeller, L., Meeley, R., Bourett, T.M., Howard, R.J. & Rafalski, A. 2006 Brittle stalk2 encodes a putative glycosylphosphatidylinositol-anchored protein that affects mechanical strength of maize tissues by altering the composition and structure of secondary cell walls Planta 224 1174 1184

    • Search Google Scholar
    • Export Citation
  • Cho, H.T. & Cosgrove, D.J. 2000 Altered expression of expansin modulates leaf growth and pedicel abscission in Arabidopsis thaliana Proc. Natl. Acad. Sci. USA 97 9783 9788

    • Search Google Scholar
    • Export Citation
  • Devoghalaere, F., Doucen, T., Guitton, B., Keeling, J., Payne, W., Ling, T.J., Ross, J.J., Hallett, I.C., Gunaseelan, K., Dayatilake, G.A., Diak, R., Breen, K.C., Tustin, D.S., Costes, E., Chagne, D., Schaffer, R.J. & David, K.M. 2012 A genomics approach to understanding the role of auxin in apple (Malus × domestica) fruit size control BMC Plant Biol. 12 7

    • Search Google Scholar
    • Export Citation
  • Kolaric, J., Plesko, I.M., Tojnko, S. & Stopar, M. 2011 Apple fruitlet ethylene evolution and MdACO1, MdACS5A, and MdACS5B expression after application of naphthaleneacetic acid, 6-benzyladenine, ethephon, or shading HortScience 46 1381 1386

    • Search Google Scholar
    • Export Citation
  • Lakso, A.N., Wunsche, J.N., Palmer, J.W. & Grappadelli, L.C. 1999 Measurement and modeling of carbon balance of the apple tree HortScience 34 1040 1047

  • Li, Y.H., Qian, O., Zhou, Y.H., Yan, M.X., Sun, L., Zhang, M., Fu, Z.M., Wang, Y.H., Han, B., Pang, X.M., Chen, M. & Li, J. 2003 BRITTLE CULM1, which encodes a COBRA-like protein, affects the mechanical properties of rice plants Plant Cell 15 2020 2031

    • Search Google Scholar
    • Export Citation
  • Mach, F.E. 2007 Istituto Agrario Di San Michele All'Adige computational biology web resources apple genome. 3 May 2012. <http://genomics.research.iasma.it/>

  • Malladi, A. & Hirst, P.M. 2010 Increase in fruit size of a spontaneous mutant of ‘Gala’ apple (Malus × domestica Borkh.) is facilitated by altered cell production and enhanced cell size J. Expt. Bot. 61 3003 3013

    • Search Google Scholar
    • Export Citation
  • Malladi, A. & Johnson, L.K. 2011 Expression profiling of cell cycle genes reveals key facilitators of cell production during carpel development, fruit set, and fruit growth in apple (Malus × domestica Borkh.) J. Expt. Bot. 62 205 219

    • Search Google Scholar
    • Export Citation
  • McArtney, S., White, M., Latter, I. & Campbell, J. 2004 Individual and combined effects of shading and thinning chemicals on abscission and dry-matter accumulation of ‘Royal Gala’ apple fruit J. Hort. Sci. Biotechnol. 79 441 448

    • Search Google Scholar
    • Export Citation
  • Morandi, B., Zibordi, M., Losciale, P., Manfrini, L., Pierpaoli, E. & Grappadelli, L.C. 2011 Shading decreases the growth rate of young apple fruit by reducing their phloem import Sci. Hort. 127 347 352

    • Search Google Scholar
    • Export Citation
  • Nosarzewski, M. & Archbold, D.D. 2007 Tissue-specific expression of SORBITOL DEHYDROGENASE in apple fruit during early development J. Expt. Bot. 58 1863 1872

    • Search Google Scholar
    • Export Citation
  • Nosarzewski, M., Clements, A.M., Downie, A.B. & Archbold, D.D. 2004 Sorbitol dehydrogenase expression and activity during apple fruit set and early development Physiol. Plant. 121 391 398

    • Search Google Scholar
    • Export Citation
  • Pfaffl, M.W. 2001 A new mathematical model for relative quantification in real-time RT-PCR Nucleic Acids Res. 29 2002 2007

  • Polomski, R.F., Barden, J.A., Byers, R.E. & Wolf, D.D. 1988 Apple fruit nonstructural carbohydrates and abscission as influenced by shade and terbacil J. Amer. Soc. Hort. Sci. 113 506 511

    • Search Google Scholar
    • Export Citation
  • Rieu, I. & Powers, S.J. 2009 Real-time quantitative RT-PCR: Design, calculations, and statistics Plant Cell 21 1031 1033

  • Roudier, F., Fernandez, A.G., Fujita, M., Himmelspach, R., Borner, G.H.H., Schindelman, G., Song, S., Baskin, T.I., Dupree, P., Wasteneys, G.O. & Benfey, P.N. 2005 COBRA, an arabidopsis extracellular glycosyl-phosphatidyl inositol-anchored protein, specifically controls highly anisotropic expansion through its involvement in cellulose microfibril orientation Plant Cell 17 1749 1763

    • Search Google Scholar
    • Export Citation
  • Ruijter, J.M., Ramakers, C., Hoogaars, W.M.H., Karlen, Y., Bakker, O., van den Hoff, M.J.B. & Moorman, A.F.M. 2009 Amplification efficiency: Linking baseline and bias in the analysis of quantitative PCR data Nucleic Acids Res. 37 e45

    • Search Google Scholar
    • Export Citation
  • Sampedro, J. & Cosgrove, D.J. 2005 The expansin superfamily Genome Biol. 6 242

  • Schindelman, G., Morikami, A., Jung, J., Baskin, T.I., Carpita, N.C., Derbyshire, P., McCann, M.C. & Benfey, P.N. 2001 COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis Genes Dev. 15 1115 1127

    • Search Google Scholar
    • Export Citation
  • Verkest, A., Manes, C.L.D., Vercruysse, S., Maes, S., Van der Schueren, E., Beeckman, T., Genschik, P., Kuiper, M., Inze, D. & De Veylder, L. 2005 The cyclin-dependent kinase inhibitor KRP2 controls the onset of the endoreduplication cycle during arabidopsis leaf development through inhibition of mitotic CDKA;1 kinase complexes Plant Cell 17 1723 1736

    • Search Google Scholar
    • Export Citation
  • Weinl, C., Marquardt, S., Kuijt, S.J.H., Nowack, M.K., Jakoby, M.J., Hulskamp, M. & Schnittger, A. 2005 Novel functions of plant cyclin-dependent kinase inhibitors, ICK1/KRP1, can act non-cell-autonomously and inhibit entry into mitosis Plant Cell 17 1704 1722

    • Search Google Scholar
    • Export Citation
  • Zhou, C.J., Lakso, A.N., Robinson, T.L. & Gan, S.S. 2008 Isolation and characterization of genes associated with shade-induced apple abscission Mol. Genet. Genomics 280 83 92

    • Search Google Scholar
    • Export Citation
  • Zhu, H., Dardick, C.D., Beers, E.P., Callanhan, A.M., Xia, R. & Yuan, R.C. 2011 Transcriptomics of shading-induced and NAA-induced abscission in apple (Malus × domestica) reveals a shared pathway involving reduced photosynthesis, alterations in carbohydrate transport and signaling and hormone crosstalk BMC Plant Biol. 11 20

    • Search Google Scholar
    • Export Citation
  • Zibordi, M., Domingos, S. & Grappadelli, L.C. 2009 Thinning apples via shading: An appraisal under field conditions J. Hort. Sci. Biotechnol. 84 138 144

    • Search Google Scholar
    • Export Citation
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