BACKGROUND
apple  fruit growth is mediated by cell production and expansion.
after bud-break, rapid growth within the ovary and floral-tube tissues is facilitated by intensive cell production.
this phase is followed by a period of temporary cessation of growth around bloom associated with quiescence in cell production, a phenomenon which likely prevents fruit growth in the absence of pollination and fertilization  <cit> .
cell production is re-initiated in response to signals generated during pollination and/or fertilization resulting in fruit set.
early fruit development is associated with intensive cell production-mediated growth which occurs until several weeks after fruit set  <cit> .
final cell number attained by the end of this period contributes greatly to the sink strength and thereby the growth potential of the fruit.
subsequent fruit growth is associated with post-mitotic cell expansion, a process which continues until maturity and contributes to the majority of fruit growth and increase in fruit size  <cit> .
enhanced fruit growth and increase in fruit size are mediated by changes in cell production or expansion.
for example, increase in fruit growth under higher carbohydrate availability during early fruit development is primarily associated with an increase in cell production  <cit> .
also, variation in fruit growth potential and fruit size across genotypes is associated with differences in cell number and size  <cit> .
although it is apparent that cell production and expansion are important determinants of fruit growth, our understanding of the molecular mechanisms and genes that regulate these processes remains limited.
cell production during fruit growth is potentially regulated by genes controlling the progression of the cell cycle  <cit> .
previous research indicated coordinated changes in the expression of core cell cycle genes during different phases of fruit growth in apple  <cit> .
expression of  <dig> such genes including b-type cyclin dependent kinases , a- and b-type cyclins, a wee kinase , and an atypical e2f transcription factor  was positively associated with cell production during fruit growth and development.
these genes displayed high expression before bloom and during early fruit development, stages primarily associated with rapid growth mediated by cell production.
subsequently, these genes displayed a sharp reduction in expression coincident with exit from cell production during fruit development.
additionally, five cell cycle genes including the kip related proteins , mdkrp <dig> and mdkrp <dig>  were negatively associated with cell production during different phases of fruit growth and development.
it is likely that upstream regulatory genes may, either directly or indirectly, coordinate changes in the expression of these cell cycle genes as well as other genes associated with cell proliferation, thereby regulating cell production during fruit growth.
such upstream regulators of cell production during fruit growth have not yet been definitively identified in apple.
recently, an auxin response factor  expressed during cell division and expansion phases of apple fruit development was co-localized to a major fruit size qtl, suggesting its involvement in regulating fruit growth  <cit> .
in other fleshy fruit such as tomato , fw <dig> , a fruit size regulator, inhibits cell production potentially through its association with a cell cycle gene, and thereby regulates fruit growth  <cit> .
also, sun, a gene involved in the regulation of tomato fruit shape may affect the patterns and orientations of cell proliferation during early fruit growth  <cit> .
beyond the above examples, little information is available regarding upstream regulators of cell production during growth of fleshy fruit.
identification and characterization of such genes is essential to develop a better understanding of fleshy fruit growth.
genes controlling organ growth are potential candidates for the regulation of growth of fleshy fruit.
many genes that regulate organ growth have been identified in arabidopsis  and other plants  <cit> .
one such gene, aintegumenta  is a key regulator of organ growth in arabidopsis.
ant is involved in the regulation of ovule development, floral organ growth and development, and organ size in arabidopsis  <cit> .
arabidopsis ant mutants display pleiotropic effects including a reduction in the size of floral organs and leaves  <cit> .
over-expression of ant in arabidopsis results in an increase in the duration of cell proliferation and enhances organ size in leaves, floral organs and siliques  <cit> .
additionally, ant mediates the effects of other genes involved in regulating organ growth.
in arabidopsis, argos  promotes cell production and growth, and positively regulates final organ size in an auxin-dependent manner  <cit> .
over-expression of argos in arabidopsis increases ant expression, and the effects of argos on organ growth are attenuated in the ant mutant, suggesting that ant mediates argos-dependent effects of auxin on organ growth.
ant expression is also affected by auxin response factor <dig> , a negative regulator of cell production and organ size in arabidopsis  <cit> .
ant is a member of the apetala2/ethylene response factor  family of transcription factors and is grouped within the ap <dig> sub-family.
members of the ap <dig> sub-family are defined by the presence of two ap <dig> domains separated by a conserved linker region which together constitute the dna binding domain  <cit> .
genes within the ap <dig> sub-family, including several aintegumenta-like  genes, are involved in the regulation of a multitude of plant growth and developmental processes.
for example, apetala <dig>  is involved in determining floral organ identity, regulating flower development, maintaining the stem cell niche in the shoot apical meristem, and regulating seed size  <cit> .
ap <dig> negatively regulates replum growth and valve margin formation during arabidopsis fruit development  <cit> .
plethora  genes are ails which function as master regulators of root growth and development in arabidopsis partly through their effects on promoting cell proliferation  <cit> .
atbbm  promotes cell production, and regulates embryo development and root growth  <cit> .
additionally, ail <dig> and ail6/plt <dig> are positive regulators of cell production and organ growth in arabidopsis as their over-expression leads to enhanced floral organ growth  <cit> .
whether ant and/or the ail genes are involved in regulating the growth of fleshy fruit has not been investigated previously.
it was hypothesized that the apple ant homolog regulate cell production during fruit development and therefore contribute to regulation of fruit growth.
here, the isolation and characterization of two ant genes from apple is reported.
evidence supporting the role of these genes in regulating cell production during different stages of fruit growth, across genotypes differing in fruit growth potential, and in response to carbohydrate availability is presented.
data from this study implicate ants in the regulation of fleshy fruit growth.
RESULTS
isolation of the apple ant genes
eight expressed sequence tags  with homology to the arabidopsis ant were identified from publicly available apple est databases.
the est displaying the highest similarity with the atant was designated as mdant and selected for the isolation of the full-length gene.
the 3′ race analysis of mdant revealed the presence of two ant genes which were designated as mdant <dig> and mdant <dig>  full-length sequences of these genes were determined as described in the ‘methods’ section.
mdant <dig> and mdant <dig> shared 93% homology at the nucleotide level  and 90% identity at the amino acid level.
nucleotide sequence identity also extended into the 5′  and 3′  regions of the open reading frame.
both genes encode putative protein products with  <dig> amino acids.
phylogenetic analysis of different plant ant transcription factors, including mdant <dig> and mdant <dig>  using their predicted protein sequences is shown in additional file  <dig>  mdant <dig> and mdant <dig> displayed higher sequence similarity with atant than with the other ails from arabidopsis .
mdant <dig> and mdant <dig> shared >50% amino acid identity with the arabidopsis ant and >75% identity with the peach  ant.
mdant <dig> and mdant <dig> displayed high sequence similarity with other plant ants within a stretch of around  <dig> amino acids containing the ap2-domain repeats and the linker region .
mdant <dig> and mdant <dig> displayed greater than 88% identity with atant in this region.
mdant <dig> and mdant <dig> contained a basic motif  similar to the nuclear localization signal in atant .
nineteen amino acids within the two ap2-domain repeats and the linker region essential for the dna binding activity of atant were identified in arabidopsis  <cit> .
all of these residues were conserved within the two apple ants.
seven potential apple ails were identified from the est databases and subsequently five were confirmed following comparisons with the apple genome database .
all these genes contained the well conserved ap2-domain repeats and the linker region.
expression of mdant <dig> and mdant <dig> is associated with cell production during fruit growth
fruit diameter in ‘gala’ increased by over 4-fold between  <dig> and  <dig> dafb  and continued to increase linearly during the rest of fruit development .
analysis of cell production within the fruit cortex indicated little change in cell number between  <dig> dafb and  <dig> dafb .
a rapid increase in cell number  was observed between  <dig> dafb and  <dig> dafb.
this was also reflected in the high relative cell production rates  observed especially around  <dig> and  <dig> dafb .
while the cell number continued to increase between  <dig> and  <dig> dafb , this occurred at a slower rate than that between  <dig> and  <dig> dafb.
the rcpr declined rapidly during this period, and reached basal levels by around  <dig> dafb.
cell number did not change greatly after this period.
the cortex cell area displayed little change during early fruit growth but increased from around  <dig> dafb, coincident with the period of decline in cell production .
most of the increase in cell area occurred during the later stages of fruit development and was associated with the majority of increase in fruit size .
mdant <dig> and mdant <dig> displayed largely similar patterns of expression during fruit development .
expression of mdant <dig> and mdant <dig> was generally high from bloom until around  <dig> dafb , coincident with the period of rapid cell production.
a sharp decline in expression was noted between  <dig> dafb and  <dig> dafb by ~8-fold and ~3-fold in mdant <dig> and mdant <dig>  respectively, and was coincident with the initial decline in cell production.
the expression of these genes declined further between  <dig> and  <dig> dafb, coincident with exit from cell production, and remained low throughout the rest of fruit development.
the above data indicate that the expression of mdant1and mdant <dig> was closely associated with cell production during early fruit growth.
in addition to mdant <dig> and mdant <dig>  the expression of five ails was studied during fruit development in ‘gala’.
mdail <dig>  mdail <dig>  and mdail <dig> displayed highest expression primarily before full bloom .
their expression declined rapidly during early fruit development and remained low throughout the rest of fruit development .
mdail <dig> and mdail <dig> also displayed a similar pattern except that the expression of these genes transiently increased by ~ <dig> and ~12-fold, respectively, between  <dig> to  <dig> dafb and was followed by low levels of expression throughout the rest of fruit development .
expression of mdant <dig> and mdant <dig> is enhanced in response to increase in carbohydrate availability
in ‘golden delicious smoothee’ , manual thinning at  <dig> dafb led to enhanced fruit growth and an increase in fruit size .
a 16% increase in fruit diameter was observed in thinned fruit by around  <dig> dafb , indicating that thinning resulted in a rapid increase in early fruit growth.
at maturity, thinned fruit had higher fruit diameter  and fruit weight  than un-thinned fruit.
enhanced fruit growth during early fruit development in thinned fruit was primarily associated with an increase in cell production in the fruit cortex.
cell production was similar between thinned and un-thinned fruit until around  <dig> dafb.
the extent of cell production in the fruit cortex was higher in thinned fruit between  <dig> and  <dig> dafb, than that in un-thinned fruit.
cell number in un-thinned fruit was lower than that in thinned fruit by ~30%  at  <dig> dafb, and remained lower during the rest of fruit development .
the rcpr was 3-fold higher in thinned fruit at  <dig> dafb .
cell area within the fruit cortex was significantly higher in thinned fruit in comparison to that in un-thinned fruit at  <dig> dafb  and  <dig> dafb .
these data indicate that increase in carbohydrate availability due to thinning enhanced fruit growth primarily by increasing cell production during early fruit growth and cell expansion at later stages.
expression of mdant <dig> and mdant <dig> was not significantly different between thinned and un-thinned fruit until after  <dig> dafb .
in comparison to un-thinned fruit, expression of mdant <dig> was almost 2-fold higher , while that of mdant <dig> was around 5-fold higher in thinned fruit at  <dig> dafb .
expression of mdant <dig> in thinned fruit was also significantly higher at  <dig> dafb , while that of mdant <dig> was significantly higher at  <dig> dafb  and  <dig> dafb .
interestingly, thinning resulted in a transient up-regulation in the expression of mdant <dig>  at  <dig> dafb, expression of mdant <dig> was >3-fold and >2-fold higher than that at  <dig> and  <dig> dafb respectively, in thinned fruit.
together, the above data indicate that enhanced expression of mdant <dig> and mdant <dig> due to thinning was associated with an increase in cell production.
mdant <dig> and mdant <dig> are differentially expressed across different fruit size genotypes during early fruit growth
fruit growth and development were compared across ‘gala’, a medium fruit size genotype, and gs, a large fruit size genotype.
‘gala’ flowers were in full bloom ~7 days prior to that of gs.
hence, growing degree days after bloom  were used to allow for comparison of fruit growth, cell production and gene expression parameters between ‘gala’ and gs.
both genotypes displayed a similar pattern of fruit growth, except that gs had a longer growing period of  <dig> gdd in comparison to  <dig> gdd in ‘gala’ .
the initial phase of fruit growth in ‘gala’ involved a rapid increase in fruit diameter which continued until around  <dig> gdd after which fruit diameter increased linearly until fruit maturity.
in gs, the initial phase of rapid fruit growth continued for a longer period  after which fruit diameter increased linearly until maturity.
final fruit diameter in gs was 23% higher than that in ‘gala’.
both genotypes displayed a similar number of cell layers within the floral-tube at full bloom .
‘gala’ and gs displayed differences in the pattern of progression in cell production within the fruit cortex.
in ‘gala’, cell number within the fruit cortex increased rapidly until around  <dig> gdd, continued to increase at a lower rate between  <dig> and  <dig> gdd, and remained largely unchanged thereafter.
in gs, increase in cell number within the fruit cortex was observed from around  <dig> gdd until around  <dig> gdd after which it remained largely unchanged .
cell number at maturity in gs was almost 54% higher than that in ‘gala’.
the rcpr maxima in ‘gala’ was around  <dig>  cell cell- <dig> gdd- <dig> while that in gs was around  <dig>  cell cell- <dig> gdd- <dig> .
however, the peak in rcpr in ‘gala’ was attained around  <dig> gdd while that in gs was attained at around  <dig> gdd .
potentially higher rcpr levels were maintained in gs than that in ‘gala’ from around  <dig> gdd until the end of the cell production period.
final area of the fruit cortex cells in gs was around 2-fold higher in gs than that in ‘gala’ .
mdant <dig> expression in ‘gala’ increased after bloom but was subsequently similar to that in gs until around  <dig> gdd .
after  <dig> gdd, transcript abundance of mdant <dig> declined rapidly in ‘gala’ but continued to remain high in gs until around  <dig> gdd.
around this period, mdant <dig> expression was substantially higher in gs  in comparison to that in ‘gala’.
expression of mdant <dig> appeared to be slightly higher in gs than that in ‘gala’ until around  <dig> gdd .
between  <dig> and  <dig> gdd, the expression of mdant <dig> declined in ‘gala’ by around 3-fold.
during a similar period  the expression of mdant <dig> in gs increased by >3-fold.
around  <dig> gdd, mdant <dig> expression was ~6-fold higher in gs in comparison to that in ‘gala’.
at later stages of fruit development, a period of post mitotic cell expansion-mediated growth, the ants displayed very low levels of expression in both the genotypes.
mdant <dig> and mdant <dig> are differentially expressed between the core and the cortex
apple is an accessory fruit  where the floral-tube tissue surrounding the ovary develops into the fleshy and edible region of the fruit  while the ovary develops into the core  <cit> .
laser capture microdissection  was used to isolate these tissues and the localization of mdant <dig> and mdant <dig> expression within these tissues during early fruit development was studied.
expression of two mads box genes, mdmads <dig> and mdmads <dig>  was also analyzed.
previous research indicated that mdmads <dig> was predominantly expressed in the cortex and the skin while mdmads <dig> was primarily expressed in the core tissue  <cit> .
in the present study, mdmads <dig> expression was clearly higher in the cortex tissue than in the core by  <dig> - to 11-fold during different stages of flower development and early fruit growth .
also, the expression of mdmads <dig> was consistently higher in the core tissue than in the cortex by 5- to 9-fold between - <dig> and  <dig> dafb .
these data are consistent with the previous report  <cit> , and demonstrate that the cortex and core tissues were effectively isolated using lcm.
mdant <dig> and mdant <dig> were expressed in the core as well as the cortex tissues during different stages of flower and early fruit development.
expression of these genes in both tissues was high before bloom and declined by up to 3-fold between - <dig> dafb and full bloom .
mdant <dig> expression was almost 2-fold higher in the ovary tissue than the floral-tube at - <dig> dafb.
between bloom and  <dig> dafb, mdant <dig> and mdant <dig> expression increased by 3-fold and 4-fold respectively in the cortex tissue, while it remained largely unaltered in the core.
at  <dig> dafb, expression of mdant <dig> within the cortex continued to be higher than that in the core, but the expression of mdant <dig> in the core reached levels similar to that in the cortex.
mdant <dig> and mdant <dig> expression is correlated with that of cell cycle genes
expression of several cell cycle genes was investigated in the core and cortex tissues isolated using lcm .
genes positively associated with cell production such as mdcyca2;3mdcycb1;1mdcdkb1;2mdcdkb2; <dig> and mddel <dig> <cit>  displayed expression patterns similar to that of mdant <dig> and mdant <dig> during early fruit growth and development.
at  <dig> dafb, expression of these genes was up to 3-fold higher in the cortex in comparison to that in the core.
mdant <dig> and mdant <dig> expression was significantly correlated with that of mdcyca2;3mdcycb1;1mdcdkb1;2mdcdkb2; <dig> and mddel <dig> .
mdkrp <dig>  a gene negatively associated with cell production, displayed a gradual increase in expression in the core tissue during early fruit growth while it declined steadily within the cortex.
at  <dig> dafb, mdkrp <dig> displayed ~10-fold higher expression in the core in comparison to that in the cortex.
mdkrp <dig> expression was not significantly correlated with either of the apple ant genes.
mdkrp <dig>  another gene negatively associated with cell production, displayed a minor increase in expression until  <dig> dafb in the core and the cortex.
at  <dig> dafb, mdkrp <dig> expression increased greatly  within the core but only slightly in the cortex, resulting in >6-fold difference in expression between these tissues.
mdant <dig> expression was weakly correlated with that of mdkrp <dig> .
discussion
several lines of evidence from this study suggest that mdant <dig> and mdant <dig> function as transcription factors in apple.
a motif of basic residues  is essential for the nuclear localization of ant, as replacement of two lysine residues within this motif resulted in a loss of nuclear localization in arabidopsis  <cit> .
in mdant <dig> and mdant <dig>  a major part of this element is conserved , strongly suggesting that these ants are targeted to the nucleus, consistent with their proposed roles as transcription factors.
the arabidopsis ant binds to the dna at a consensus site of  <dig> bases through two ap <dig> domains and a conserved linker region  <cit> .
mdant <dig> and mdant <dig> shared greater than 88% sequence identity with the arabidopsis ant within these regions.
all of the  <dig> residues identified as essential for the dna binding activity of the arabidopsis ant  <cit>  are conserved in the apple ants suggesting that they may bind to similar dna elements, further supporting their role as transcription factors.
domains within the amino-terminal region are also essential for the transcriptional activation properties of the arabidopsis ant  <cit> .
although the apple ants display limited conservation of residues with that of the arabidopsis ant in this region, it should be noted that other plant ants also display significant sequence divergence within this region, indicating that distinct, species-specific features may be required for the transcriptional activation properties of the ants.
mdant <dig> and mdant <dig> are expressed in regions associated with fruit growth and development .
mdant <dig> and mdant <dig> display high expression before bloom in the ovary as well as the floral-tube regions, strongly suggesting their association with cell production-mediated growth of the ovary and floral-tube tissues before bloom.
expression of mdant <dig> and mdant <dig> declines within these tissues during the period of temporary cessation of growth and quiescence in cell production .
subsequently, the expression of the ants increases sharply within the cortex tissue while little change in their expression is observed within the core tissue between bloom and  <dig> dafb, coincident with the resumption of growth and re-initiation of cell production in the cortex during early fruit development.
the sharp increase in expression at  <dig> dafb within the cortex is likely triggered by pollination and/or fertilization and may mediate fruit set.
mdant <dig> and mdant <dig> expression is high during the cell production-mediated phase of early fruit growth and subsequently declines greatly during exit from this phase.
this pattern of expression is conserved under conditions of different carbohydrate availability and across genotypes differing in their fruit growth potential.
together, the data presented here indicate that the expression of mdant <dig> and mdant <dig> is consistently and closely associated with cell production during fruit growth in apple.
therefore, it is proposed that ants are important components of a developmental program that controls the extent of cell production and thereby regulates fruit growth in apple.
cell production and fruit growth are limited by carbohydrate availability in many plant species  <cit> .
consistent with previous studies, increase in carbohydrate availability through manual thinning during early fruit development in gs enhanced fruit growth and final fruit size.
this was primarily achieved through sustained cell production in the fruit cortex during early fruit growth and a higher relative cell production rate, especially towards the later stages of the cell production phase.
these data indicate that carbohydrate limitation due to increased competition among sinks decreases the extent of cell production in the fruit cortex.
under conditions of higher carbohydrate availability, the expression of mdant <dig> and mdant <dig> was several-fold higher than that under carbohydrate limitation.
additionally, mdant <dig> was up-regulated  in response to an increase in carbohydrate availability in gs.
these data suggest that an increase in carbohydrate availability enhances the expression of the ant genes, especially mdant <dig>  thereby increasing the competence of the fruit cortex cells for cell production.
hence, it may be proposed that the ants, particularly mdant <dig>  mediate the effects of carbohydrate availability on cell production and fruit growth.
increase in competence for cell production may be achieved either through an increase in the proportion of fruit cortex cells undergoing proliferation or through an increase in the capacity of individual cortex cells for division.
increase in carbohydrate availability also led to a minor increase in cell area during later stages of fruit growth in gs, inconsistent with previously reported results in the apple cultivar, ‘empire’  <cit> , but consistent with results in tomato fruit  <cit> .
it is likely that an increase in sink strength as a result of higher fruit cortex cell number in thinned fruit may subsequently aid in increasing the extent of cell expansion.
comparison of apple genotypes differing in their growth potential further supports the proposed roles of the ants in regulating cell production.
although, it is possible that some of the differences observed between the two genotypes are due to environmental effects, the overall patterns of fruit growth and gene expression reported here were consistent with that observed in other studies during different years .
the initial cell number and the duration of the cell production phase were similar in ‘gala’ and gs , indicating that the higher final cell number within the fruit cortex of gs in comparison to that in ‘gala’ was due to differences in the pattern of progression in cell production during early fruit development.
gs fruit cortex cells displayed a more gradual increase in cell number after bloom in comparison to those of ‘gala’ which displayed a short-lived early burst in cell production.
in fact, the rcpr in gs reached the maxima around  <dig> gdd after that in ‘gala’.
subsequently, the rate of cell production in gs was higher than that in ‘gala’, especially between  <dig> and  <dig> gdd after bloom.
expression of mdant <dig> and mdant <dig> in the two genotypes matched their respective patterns of cell production.
expression of these genes in gs was sustained at higher levels for a longer duration while in ‘gala’, the expression of these genes displayed an initial rapid burst followed by a rapid decline.
the expression of both these genes was higher in gs than in ‘gala’ during the final stages of the cell production phase .
sustained competence for cell production as a result of this pattern of expression of the ants may allow for enhanced cell production and a higher final cell number in gs.
final cell number is often an important determinant of variation in fruit size across genotypes  <cit> .
differences in the pattern of expression of the ant genes during early fruit growth may affect the final cell number and thereby final fruit size across genotypes.
similarly, differences in the pattern of expression of fw <dig>  are thought to determine fruit size differences across tomato genotypes  <cit> .
hence, it is likely that mdant <dig> and mdant <dig> also function as regulators of fruit size in apple.
expression of the apple ant genes was correlated with that of several positive regulators of the cell cycle, including b-type cdks, a- and b-type cyclins, and mddel <dig> during different stages of flower and early fruit development.
during the period of exit from cell production , the expression of several cell cycle genes positively associated with cell production declined, while that of genes negatively associated with cell production increased  <cit> .
these changes in the expression of the cell cycle genes coincide with the decline in the expression of mdant <dig> and mdant <dig> observed in this study.
in fact, the expression patterns of the ant genes during fruit growth display high similarity with those of the core cell cycle genes involved primarily in the regulation of the g2-m phases of the cell cycle.
co-expression of these genes suggests coordinated regulation and their involvement in a common biological process  <cit> .
considering that the ant genes may function as transcription factors, it is possible that mdant <dig> and mdant <dig> regulate the expression of the core cell cycle genes and thereby coordinate cell production during fruit growth.
in arabidopsis, increased cell production as a result of the over-expression of ant was associated with the prolonged expression of d3-type cyclins  <cit> .
identification of the genes targeted for direct regulation by the ants is essential to test this hypothesis.
the general similarities in the expression patterns of mdant <dig> and mdant <dig> suggest overlapping roles for these genes in regulating flower and fruit development.
in arabidopsis, expression of four plt genes  in overlapping as well as specific regions of the root allows for plt concentration-dependent regulation of root growth and development  <cit> .
similarly in apple, the combined activity of mdant <dig> and mdant <dig> may have an additive effect on cell production and fruit growth.
however, certain key differences between mdant <dig> and mdant <dig> were also noted.
the expression of these genes in the core tissue differed slightly during early fruit development.
mdant <dig> and mdant <dig> also differed within the ap2-repeats and linker region in three residues .
if the dna binding characteristics are affected by the above residues, mdant <dig> and mdant <dig> may regulate different pools of downstream target genes.
together, the above data suggest that mdant <dig> and mdant <dig> may also have distinct roles in regulating fruit growth and development.
functional characterization of mdant <dig> and mdant <dig> and the identification of their downstream targets in vivo are essential to determine their specific roles in regulating fruit growth.
all of the ail genes studied here contained the characteristic ap2-repeats and the conserved linker region suggesting that they function as transcriptional regulators.
these genes displayed elevated expression during flower development and a sharp decline in expression during early fruit development, suggesting that they may be primarily involved in regulating flower growth and development in apple.
in arabidopsis, many of the ail genes are involved in regulating floral organ growth and development  <cit> .
mdail <dig> and mdail <dig> share significant amino acid identity with atail <dig> and atail <dig> respectively, genes which have been previously reported to regulate organ growth  <cit> .
further characterization of the tissue-specific patterns of expression and the functional characterization of the ail genes is essential to determine their specific roles in apple.
CONCLUSIONS
data presented here strongly suggest that mdant <dig> and mdant <dig> regulate cell production and fruit growth in apple by coordinating the expression of genes involved in cell proliferation.
mdant <dig> and mdant <dig> are a significant addition to the limited list of candidate upstream regulatory genes involved in the control of growth of fleshy fruit.
functional characterization of these genes and the identification of their downstream targets may greatly aid in unraveling the mechanisms involved in the regulation of fruit growth in apple and other fleshy fruit.

 apple fruit growth is mediated by cell production and expansion. after bud-break, rapid growth within the ovary and floral-tube tissues is facilitated by intensive cell production. this phase is followed by a period of temporary cessation of growth around bloom associated with quiescence in cell production, a phenomenon which likely prevents fruit growth in the absence of pollination and fertilization. cell production is re-initiated in response to signals generated during pollination and/or fertilization resulting in fruit set. early fruit development is associated with intensive cell production-mediated growth which occurs until several weeks after fruit set. final cell number attained by the end of this period contributes greatly to the sink strength and thereby the growth potential of the fruit. subsequent fruit growth is associated with post-mitotic cell expansion, a process which continues until maturity and contributes to the majority of fruit growth and increase in fruit size. enhanced fruit growth and increase in fruit size are mediated by changes in cell production or expansion. for example, increase in fruit growth under higher carbohydrate availability during early fruit development is primarily associated with an increase in cell production. also, variation in fruit growth potential and fruit size across genotypes is associated with differences in cell number and size. although it is apparent that cell production and expansion are important determinants of fruit growth, our understanding of the molecular mechanisms and genes that regulate these processes remains limited. cell production during fruit growth is potentially regulated by genes controlling the progression of the cell cycle. previous research indicated coordinated changes in the expression of core cell cycle genes during different phases of fruit growth in apple. expression of such genes including b-type cyclin dependent kinases, a- and b-type cyclins, a wee kinase, and an atypical e2f transcription factor was positively associated with cell production during fruit growth and development. these genes displayed high expression before bloom and during early fruit development, stages primarily associated with rapid growth mediated by cell production. subsequently, these genes displayed a sharp reduction in expression coincident with exit from cell production during fruit development. additionally, five cell cycle genes including the kip related proteins, mdkrp and mdkrp were negatively associated with cell production during different phases of fruit growth and development. it is likely that upstream regulatory genes may, either directly or indirectly, coordinate changes in the expression of these cell cycle genes as well as other genes associated with cell proliferation, thereby regulating cell production during fruit growth. such upstream regulators of cell production during fruit growth have not yet been definitively identified in apple. recently, an auxin response factor expressed during cell division and expansion phases of apple fruit development was co-localized to a major fruit size qtl, suggesting its involvement in regulating fruit growth. in other fleshy fruit such as tomato, fw, a fruit size regulator, inhibits cell production potentially through its association with a cell cycle gene, and thereby regulates fruit growth. also, sun, a gene involved in the regulation of tomato fruit shape may affect the patterns and orientations of cell proliferation during early fruit growth. beyond the above examples, little information is available regarding upstream regulators of cell production during growth of fleshy fruit. identification and characterization of such genes is essential to develop a better understanding of fleshy fruit growth. genes controlling organ growth are potential candidates for the regulation of growth of fleshy fruit. many genes that regulate organ growth have been identified in arabidopsis and other plants. one such gene, aintegumenta is a key regulator of organ growth in arabidopsis. ant is involved in the regulation of ovule development, floral organ growth and development, and organ size in arabidopsis. arabidopsis ant mutants display pleiotropic effects including a reduction in the size of floral organs and leaves. over-expression of ant in arabidopsis results in an increase in the duration of cell proliferation and enhances organ size in leaves, floral organs and siliques. additionally, ant mediates the effects of other genes involved in regulating organ growth. in arabidopsis, argos promotes cell production and growth, and positively regulates final organ size in an auxin-dependent manner. over-expression of argos in arabidopsis increases ant expression, and the effects of argos on organ growth are attenuated in the ant mutant, suggesting that ant mediates argos-dependent effects of auxin on organ growth. ant expression is also affected by auxin response factor, a negative regulator of cell production and organ size in arabidopsis. ant is a member of the apetala2/ethylene response factor family of transcription factors and is grouped within the ap sub-family. members of the ap sub-family are defined by the presence of two ap domains separated by a conserved linker region which together constitute the dna binding domain. genes within the ap sub-family, including several aintegumenta-like genes, are involved in the regulation of a multitude of plant growth and developmental processes. for example, apetala is involved in determining floral organ identity, regulating flower development, maintaining the stem cell niche in the shoot apical meristem, and regulating seed size. ap negatively regulates replum growth and valve margin formation during arabidopsis fruit development. plethora genes are ails which function as master regulators of root growth and development in arabidopsis partly through their effects on promoting cell proliferation. atbbm promotes cell production, and regulates embryo development and root growth. additionally, ail and ail6/plt are positive regulators of cell production and organ growth in arabidopsis as their over-expression leads to enhanced floral organ growth. whether ant and/or the ail genes are involved in regulating the growth of fleshy fruit has not been investigated previously. it was hypothesized that the apple ant homolog regulate cell production during fruit development and therefore contribute to regulation of fruit growth. here, the isolation and characterization of two ant genes from apple is reported. evidence supporting the role of these genes in regulating cell production during different stages of fruit growth, across genotypes differing in fruit growth potential, and in response to carbohydrate availability is presented. data from this study implicate ants in the regulation of fleshy fruit growth.  isolation of the apple ant genes eight expressed sequence tags with homology to the arabidopsis ant were identified from publicly available apple est databases. the est displaying the highest similarity with the atant was designated as mdant and selected for the isolation of the full-length gene. the 3′ race analysis of mdant revealed the presence of two ant genes which were designated as mdant and mdant full-length sequences of these genes were determined as described in the ‘methods’ section. mdant and mdant shared 93% homology at the nucleotide level and 90% identity at the amino acid level. nucleotide sequence identity also extended into the 5′ and 3′ regions of the open reading frame. both genes encode putative protein products with amino acids. phylogenetic analysis of different plant ant transcription factors, including mdant and mdant using their predicted protein sequences is shown in additional file mdant and mdant displayed higher sequence similarity with atant than with the other ails from arabidopsis. mdant and mdant shared >50% amino acid identity with the arabidopsis ant and >75% identity with the peach ant. mdant and mdant displayed high sequence similarity with other plant ants within a stretch of around amino acids containing the ap2-domain repeats and the linker region. mdant and mdant displayed greater than 88% identity with atant in this region. mdant and mdant contained a basic motif similar to the nuclear localization signal in atant. nineteen amino acids within the two ap2-domain repeats and the linker region essential for the dna binding activity of atant were identified in arabidopsis. all of these residues were conserved within the two apple ants. seven potential apple ails were identified from the est databases and subsequently five were confirmed following comparisons with the apple genome database. all these genes contained the well conserved ap2-domain repeats and the linker region. expression of mdant and mdant is associated with cell production during fruit growth fruit diameter in ‘gala’ increased by over 4-fold between and dafb and continued to increase linearly during the rest of fruit development. analysis of cell production within the fruit cortex indicated little change in cell number between dafb and dafb. a rapid increase in cell number was observed between dafb and dafb. this was also reflected in the high relative cell production rates observed especially around and dafb. while the cell number continued to increase between and dafb, this occurred at a slower rate than that between and dafb. the rcpr declined rapidly during this period, and reached basal levels by around dafb. cell number did not change greatly after this period. the cortex cell area displayed little change during early fruit growth but increased from around dafb, coincident with the period of decline in cell production. most of the increase in cell area occurred during the later stages of fruit development and was associated with the majority of increase in fruit size. mdant and mdant displayed largely similar patterns of expression during fruit development. expression of mdant and mdant was generally high from bloom until around dafb, coincident with the period of rapid cell production. a sharp decline in expression was noted between dafb and dafb by ~8-fold and ~3-fold in mdant and mdant respectively, and was coincident with the initial decline in cell production. the expression of these genes declined further between and dafb, coincident with exit from cell production, and remained low throughout the rest of fruit development. the above data indicate that the expression of mdant1and mdant was closely associated with cell production during early fruit growth. in addition to mdant and mdant the expression of five ails was studied during fruit development in ‘gala’. mdail mdail and mdail displayed highest expression primarily before full bloom. their expression declined rapidly during early fruit development and remained low throughout the rest of fruit development. mdail and mdail also displayed a similar pattern except that the expression of these genes transiently increased by ~ and ~12-fold, respectively, between to dafb and was followed by low levels of expression throughout the rest of fruit development. expression of mdant and mdant is enhanced in response to increase in carbohydrate availability in ‘golden delicious smoothee’, manual thinning at dafb led to enhanced fruit growth and an increase in fruit size. a 16% increase in fruit diameter was observed in thinned fruit by around dafb, indicating that thinning resulted in a rapid increase in early fruit growth. at maturity, thinned fruit had higher fruit diameter and fruit weight than un-thinned fruit. enhanced fruit growth during early fruit development in thinned fruit was primarily associated with an increase in cell production in the fruit cortex. cell production was similar between thinned and un-thinned fruit until around dafb. the extent of cell production in the fruit cortex was higher in thinned fruit between and dafb, than that in un-thinned fruit. cell number in un-thinned fruit was lower than that in thinned fruit by ~30% at dafb, and remained lower during the rest of fruit development. the rcpr was 3-fold higher in thinned fruit at dafb. cell area within the fruit cortex was significantly higher in thinned fruit in comparison to that in un-thinned fruit at dafb and dafb. these data indicate that increase in carbohydrate availability due to thinning enhanced fruit growth primarily by increasing cell production during early fruit growth and cell expansion at later stages. expression of mdant and mdant was not significantly different between thinned and un-thinned fruit until after dafb. in comparison to un-thinned fruit, expression of mdant was almost 2-fold higher, while that of mdant was around 5-fold higher in thinned fruit at dafb. expression of mdant in thinned fruit was also significantly higher at dafb, while that of mdant was significantly higher at dafb and dafb. interestingly, thinning resulted in a transient up-regulation in the expression of mdant at dafb, expression of mdant was >3-fold and >2-fold higher than that at and dafb respectively, in thinned fruit. together, the above data indicate that enhanced expression of mdant and mdant due to thinning was associated with an increase in cell production. mdant and mdant are differentially expressed across different fruit size genotypes during early fruit growth fruit growth and development were compared across ‘gala’, a medium fruit size genotype, and gs, a large fruit size genotype. ‘gala’ flowers were in full bloom ~7 days prior to that of gs. hence, growing degree days after bloom were used to allow for comparison of fruit growth, cell production and gene expression parameters between ‘gala’ and gs. both genotypes displayed a similar pattern of fruit growth, except that gs had a longer growing period of gdd in comparison to gdd in ‘gala’. the initial phase of fruit growth in ‘gala’ involved a rapid increase in fruit diameter which continued until around gdd after which fruit diameter increased linearly until fruit maturity. in gs, the initial phase of rapid fruit growth continued for a longer period after which fruit diameter increased linearly until maturity. final fruit diameter in gs was 23% higher than that in ‘gala’. both genotypes displayed a similar number of cell layers within the floral-tube at full bloom. ‘gala’ and gs displayed differences in the pattern of progression in cell production within the fruit cortex. in ‘gala’, cell number within the fruit cortex increased rapidly until around gdd, continued to increase at a lower rate between and gdd, and remained largely unchanged thereafter. in gs, increase in cell number within the fruit cortex was observed from around gdd until around gdd after which it remained largely unchanged. cell number at maturity in gs was almost 54% higher than that in ‘gala’. the rcpr maxima in ‘gala’ was around cell cell- gdd- while that in gs was around cell cell- gdd-. however, the peak in rcpr in ‘gala’ was attained around gdd while that in gs was attained at around gdd. potentially higher rcpr levels were maintained in gs than that in ‘gala’ from around gdd until the end of the cell production period. final area of the fruit cortex cells in gs was around 2-fold higher in gs than that in ‘gala’. mdant expression in ‘gala’ increased after bloom but was subsequently similar to that in gs until around gdd. after gdd, transcript abundance of mdant declined rapidly in ‘gala’ but continued to remain high in gs until around gdd. around this period, mdant expression was substantially higher in gs in comparison to that in ‘gala’. expression of mdant appeared to be slightly higher in gs than that in ‘gala’ until around gdd. between and gdd, the expression of mdant declined in ‘gala’ by around 3-fold. during a similar period the expression of mdant in gs increased by >3-fold. around gdd, mdant expression was ~6-fold higher in gs in comparison to that in ‘gala’. at later stages of fruit development, a period of post mitotic cell expansion-mediated growth, the ants displayed very low levels of expression in both the genotypes. mdant and mdant are differentially expressed between the core and the cortex apple is an accessory fruit where the floral-tube tissue surrounding the ovary develops into the fleshy and edible region of the fruit while the ovary develops into the core. laser capture microdissection was used to isolate these tissues and the localization of mdant and mdant expression within these tissues during early fruit development was studied. expression of two mads box genes, mdmads and mdmads was also analyzed. previous research indicated that mdmads was predominantly expressed in the cortex and the skin while mdmads was primarily expressed in the core tissue. in the present study, mdmads expression was clearly higher in the cortex tissue than in the core by - to 11-fold during different stages of flower development and early fruit growth. also, the expression of mdmads was consistently higher in the core tissue than in the cortex by 5- to 9-fold between - and dafb. these data are consistent with the previous report, and demonstrate that the cortex and core tissues were effectively isolated using lcm. mdant and mdant were expressed in the core as well as the cortex tissues during different stages of flower and early fruit development. expression of these genes in both tissues was high before bloom and declined by up to 3-fold between - dafb and full bloom. mdant expression was almost 2-fold higher in the ovary tissue than the floral-tube at - dafb. between bloom and dafb, mdant and mdant expression increased by 3-fold and 4-fold respectively in the cortex tissue, while it remained largely unaltered in the core. at dafb, expression of mdant within the cortex continued to be higher than that in the core, but the expression of mdant in the core reached levels similar to that in the cortex. mdant and mdant expression is correlated with that of cell cycle genes expression of several cell cycle genes was investigated in the core and cortex tissues isolated using lcm. genes positively associated with cell production such as mdcyca2;3mdcycb1;1mdcdkb1;2mdcdkb2; and mddel displayed expression patterns similar to that of mdant and mdant during early fruit growth and development. at dafb, expression of these genes was up to 3-fold higher in the cortex in comparison to that in the core. mdant and mdant expression was significantly correlated with that of mdcyca2;3mdcycb1;1mdcdkb1;2mdcdkb2; and mddel. mdkrp a gene negatively associated with cell production, displayed a gradual increase in expression in the core tissue during early fruit growth while it declined steadily within the cortex. at dafb, mdkrp displayed ~10-fold higher expression in the core in comparison to that in the cortex. mdkrp expression was not significantly correlated with either of the apple ant genes. mdkrp another gene negatively associated with cell production, displayed a minor increase in expression until dafb in the core and the cortex. at dafb, mdkrp expression increased greatly within the core but only slightly in the cortex, resulting in >6-fold difference in expression between these tissues. mdant expression was weakly correlated with that of mdkrp. discussion several lines of evidence from this study suggest that mdant and mdant function as transcription factors in apple. a motif of basic residues is essential for the nuclear localization of ant, as replacement of two lysine residues within this motif resulted in a loss of nuclear localization in arabidopsis. in mdant and mdant a major part of this element is conserved, strongly suggesting that these ants are targeted to the nucleus, consistent with their proposed roles as transcription factors. the arabidopsis ant binds to the dna at a consensus site of bases through two ap domains and a conserved linker region. mdant and mdant shared greater than 88% sequence identity with the arabidopsis ant within these regions. all of the residues identified as essential for the dna binding activity of the arabidopsis ant are conserved in the apple ants suggesting that they may bind to similar dna elements, further supporting their role as transcription factors. domains within the amino-terminal region are also essential for the transcriptional activation properties of the arabidopsis ant. although the apple ants display limited conservation of residues with that of the arabidopsis ant in this region, it should be noted that other plant ants also display significant sequence divergence within this region, indicating that distinct, species-specific features may be required for the transcriptional activation properties of the ants. mdant and mdant are expressed in regions associated with fruit growth and development. mdant and mdant display high expression before bloom in the ovary as well as the floral-tube regions, strongly suggesting their association with cell production-mediated growth of the ovary and floral-tube tissues before bloom. expression of mdant and mdant declines within these tissues during the period of temporary cessation of growth and quiescence in cell production. subsequently, the expression of the ants increases sharply within the cortex tissue while little change in their expression is observed within the core tissue between bloom and dafb, coincident with the resumption of growth and re-initiation of cell production in the cortex during early fruit development. the sharp increase in expression at dafb within the cortex is likely triggered by pollination and/or fertilization and may mediate fruit set. mdant and mdant expression is high during the cell production-mediated phase of early fruit growth and subsequently declines greatly during exit from this phase. this pattern of expression is conserved under conditions of different carbohydrate availability and across genotypes differing in their fruit growth potential. together, the data presented here indicate that the expression of mdant and mdant is consistently and closely associated with cell production during fruit growth in apple. therefore, it is proposed that ants are important components of a developmental program that controls the extent of cell production and thereby regulates fruit growth in apple. cell production and fruit growth are limited by carbohydrate availability in many plant species. consistent with previous studies, increase in carbohydrate availability through manual thinning during early fruit development in gs enhanced fruit growth and final fruit size. this was primarily achieved through sustained cell production in the fruit cortex during early fruit growth and a higher relative cell production rate, especially towards the later stages of the cell production phase. these data indicate that carbohydrate limitation due to increased competition among sinks decreases the extent of cell production in the fruit cortex. under conditions of higher carbohydrate availability, the expression of mdant and mdant was several-fold higher than that under carbohydrate limitation. additionally, mdant was up-regulated in response to an increase in carbohydrate availability in gs. these data suggest that an increase in carbohydrate availability enhances the expression of the ant genes, especially mdant thereby increasing the competence of the fruit cortex cells for cell production. hence, it may be proposed that the ants, particularly mdant mediate the effects of carbohydrate availability on cell production and fruit growth. increase in competence for cell production may be achieved either through an increase in the proportion of fruit cortex cells undergoing proliferation or through an increase in the capacity of individual cortex cells for division. increase in carbohydrate availability also led to a minor increase in cell area during later stages of fruit growth in gs, inconsistent with previously reported results in the apple cultivar, ‘empire’, but consistent with results in tomato fruit. it is likely that an increase in sink strength as a result of higher fruit cortex cell number in thinned fruit may subsequently aid in increasing the extent of cell expansion. comparison of apple genotypes differing in their growth potential further supports the proposed roles of the ants in regulating cell production. although, it is possible that some of the differences observed between the two genotypes are due to environmental effects, the overall patterns of fruit growth and gene expression reported here were consistent with that observed in other studies during different years. the initial cell number and the duration of the cell production phase were similar in ‘gala’ and gs, indicating that the higher final cell number within the fruit cortex of gs in comparison to that in ‘gala’ was due to differences in the pattern of progression in cell production during early fruit development. gs fruit cortex cells displayed a more gradual increase in cell number after bloom in comparison to those of ‘gala’ which displayed a short-lived early burst in cell production. in fact, the rcpr in gs reached the maxima around gdd after that in ‘gala’. subsequently, the rate of cell production in gs was higher than that in ‘gala’, especially between and gdd after bloom. expression of mdant and mdant in the two genotypes matched their respective patterns of cell production. expression of these genes in gs was sustained at higher levels for a longer duration while in ‘gala’, the expression of these genes displayed an initial rapid burst followed by a rapid decline. the expression of both these genes was higher in gs than in ‘gala’ during the final stages of the cell production phase. sustained competence for cell production as a result of this pattern of expression of the ants may allow for enhanced cell production and a higher final cell number in gs. final cell number is often an important determinant of variation in fruit size across genotypes. differences in the pattern of expression of the ant genes during early fruit growth may affect the final cell number and thereby final fruit size across genotypes. similarly, differences in the pattern of expression of fw are thought to determine fruit size differences across tomato genotypes. hence, it is likely that mdant and mdant also function as regulators of fruit size in apple. expression of the apple ant genes was correlated with that of several positive regulators of the cell cycle, including b-type cdks, a- and b-type cyclins, and mddel during different stages of flower and early fruit development. during the period of exit from cell production, the expression of several cell cycle genes positively associated with cell production declined, while that of genes negatively associated with cell production increased. these changes in the expression of the cell cycle genes coincide with the decline in the expression of mdant and mdant observed in this study. in fact, the expression patterns of the ant genes during fruit growth display high similarity with those of the core cell cycle genes involved primarily in the regulation of the g2-m phases of the cell cycle. co-expression of these genes suggests coordinated regulation and their involvement in a common biological process. considering that the ant genes may function as transcription factors, it is possible that mdant and mdant regulate the expression of the core cell cycle genes and thereby coordinate cell production during fruit growth. in arabidopsis, increased cell production as a result of the over-expression of ant was associated with the prolonged expression of d3-type cyclins. identification of the genes targeted for direct regulation by the ants is essential to test this hypothesis. the general similarities in the expression patterns of mdant and mdant suggest overlapping roles for these genes in regulating flower and fruit development. in arabidopsis, expression of four plt genes in overlapping as well as specific regions of the root allows for plt concentration-dependent regulation of root growth and development. similarly in apple, the combined activity of mdant and mdant may have an additive effect on cell production and fruit growth. however, certain key differences between mdant and mdant were also noted. the expression of these genes in the core tissue differed slightly during early fruit development. mdant and mdant also differed within the ap2-repeats and linker region in three residues. if the dna binding characteristics are affected by the above residues, mdant and mdant may regulate different pools of downstream target genes. together, the above data suggest that mdant and mdant may also have distinct roles in regulating fruit growth and development. functional characterization of mdant and mdant and the identification of their downstream targets in vivo are essential to determine their specific roles in regulating fruit growth. all of the ail genes studied here contained the characteristic ap2-repeats and the conserved linker region suggesting that they function as transcriptional regulators. these genes displayed elevated expression during flower development and a sharp decline in expression during early fruit development, suggesting that they may be primarily involved in regulating flower growth and development in apple. in arabidopsis, many of the ail genes are involved in regulating floral organ growth and development. mdail and mdail share significant amino acid identity with atail and atail respectively, genes which have been previously reported to regulate organ growth. further characterization of the tissue-specific patterns of expression and the functional characterization of the ail genes is essential to determine their specific roles in apple.  data presented here strongly suggest that mdant and mdant regulate cell production and fruit growth in apple by coordinating the expression of genes involved in cell proliferation. mdant and mdant are a significant addition to the limited list of candidate upstream regulatory genes involved in the control of growth of fleshy fruit. functional characterization of these genes and the identification of their downstream targets may greatly aid in unraveling the mechanisms involved in the regulation of fruit growth in apple and other fleshy fruit. 
two ant genes, mdant and mdant were isolated from apple and their expression was studied during multiple stages of fruit development. mdant and mdant expression was high during early fruit growth coincident with the period of cell production, rapidly declined during exit from cell production, and remained low during the rest of fruit development. cell divisioncell expansionfruit developmentfruit sizeorgan growth expression of the ants and cell cycle genes in the fruit core and cortex tissues isolated using laser capture microdissection was studied. during early fruit growth, expression of the mdants was higher within the cortex, the tissue that constitutes the majority of the fruit. fruit growth in apple is mediated by cell production and expansion. genes involved in regulating these processes and thereby fruit growth, are not well characterized. we hypothesized that the apple homolog of aintegumenta, an apetala2–repeat containing transcription factor, regulates cell production during fruit growth in apple. the effects of increase in carbohydrate availability during fruit growth were characterized. increase in carbohydrate availability enhanced fruit growth largely through an increase in cell production. expression of the ant genes was compared across two apple genotypes, ‘gala’ and ‘golden delicious smoothee’, which differ in the extent of fruit growth, largely due to differences in cell production. multiple lines of evidence from this study suggest that mdant and mdant regulate cell production during fruit growth in apple. ants may coordinate the expression of cell proliferation genes and thereby affect the competence of cells for cell production during fruit growth. together, data from this study implicate mdant and mdant in the regulation of fruit growth in apple. additionally, mdant and mdant expression was positively correlated with that of a- and b-type cyclins, b-type cyclin-dependent-kinases and mddel expression of mdant and mdant increased sharply by up to around 5-fold in response to an increase in carbohydrate availability.
 fruit growth in apple is mediated by cell production and expansion. genes involved in regulating these processes and thereby fruit growth, are not well characterized. we hypothesized that the apple homolog of aintegumenta, an apetala2–repeat containing transcription factor, regulates cell production during fruit growth in apple.  two ant genes, mdant and mdant were isolated from apple and their expression was studied during multiple stages of fruit development. mdant and mdant expression was high during early fruit growth coincident with the period of cell production, rapidly declined during exit from cell production, and remained low during the rest of fruit development. the effects of increase in carbohydrate availability during fruit growth were characterized. increase in carbohydrate availability enhanced fruit growth largely through an increase in cell production. expression of mdant and mdant increased sharply by up to around 5-fold in response to an increase in carbohydrate availability. expression of the ant genes was compared across two apple genotypes, ‘gala’ and ‘golden delicious smoothee’, which differ in the extent of fruit growth, largely due to differences in cell production. in comparison to ‘gala’, the larger fruit-size genotype, gs, displayed higher levels and a longer duration of mdant and mdant expression. expression of the ants and cell cycle genes in the fruit core and cortex tissues isolated using laser capture microdissection was studied. during early fruit growth, expression of the mdants was higher within the cortex, the tissue that constitutes the majority of the fruit. additionally, mdant and mdant expression was positively correlated with that of a- and b-type cyclins, b-type cyclin-dependent-kinases and mddel  multiple lines of evidence from this study suggest that mdant and mdant regulate cell production during fruit growth in apple. ants may coordinate the expression of cell proliferation genes and thereby affect the competence of cells for cell production during fruit growth. together, data from this study implicate mdant and mdant in the regulation of fruit growth in apple. cell divisioncell expansionfruit developmentfruit sizeorgan growth 