Expression profiling of cell cycle genes reveals key facilitators of cell production during carpel development, fruit set, and fruit growth in apple (Malusxdomestica Borkh.)

Abstract

Cell production is an essential facilitator of fruit growth and development. Cell production during carpel/floral-tube growth, fruit set, and fruit growth, and its regulation by cell cycle genes were investigated in apple (Malus×domestica Borkh.). Cell production was inhibited during late carpel/floral-tube development, resulting in growth arrest before bloom. Fruit set re-activated cell production between 8 d and 11 d after full bloom (DAFB) and triggered fruit growth. The early phase of fruit growth involved rapid cell production followed by exit from cell proliferation at ∼24 DAFB. Seventy-one cell cycle genes were identified, and expression of 59 genes was investigated using quantitative RT-PCR. Changes in expression of 19 genes were consistently associated with transitions in cell production during carpel/floral-tube growth, fruit set, and fruit growth. Fourteen genes, including B-type cyclin-dependent kinases (CDKs) and A2-, B1-, and B2-type cyclins, were positively associated with cell production, suggesting that availability of G2/M phase regulators of the cell cycle is limiting for cell proliferation. Enhanced expression of five genes including that of the putative CDK inhibitors, MdKRP4 and MdKRP5, was associated with reduced cell production. Exit from cell proliferation at G0/G1 during fruit growth was facilitated by multiple mechanisms including down-regulation of putative regulators of G1/S and G2/M phase progression and up-regulation of KRP genes. Interestingly, two CDKA genes and several CDK-activating factors were up-regulated during this period, suggesting functions for these genes in mediating exit from cell proliferation at G0/G1. Together, the data indicate that cell cycle genes are important facilitators of cell production during apple fruit development.

Fig. 1.

Carpel/floral-tube growth, cell production, and cell expansion in ‘Gala’ apple. Carpel/floral-tube diameter in king flowers was measured at 17, 7, and 0 d before full bloom. The number of cell layers within the floral tube and the cell area were determined using light microscopy at 26, 17, 7, and 0 d before full bloom. Error bars represent the standard error of the means (n=4).

Fig. 2.

Expression profiles of cell cycle genes during carpel/floral-tube growth in ‘Gala’ apple. (A) Hierarchical clustering was used to group cell cycle genes with similar expression patterns. Only genes exhibiting >2-fold alteration in expression were included in the cluster analysis (41 genes). Log₂-transformed data were used for the cluster analysis (n=4). Expression data for a given gene are shown relative to its expression at 26 d before full bloom. Numbers assigned to major clusters are indicated on the dendogram. The inset shows the colour legend used in the cluster representation (log₂ ratios). MdGAPDH and MdACTIN genes were used for normalization of expression data. (B) Fold change in expression of representative genes from each of the major clusters in ‘A’ is presented (clusters 1–4 from top to bottom). Data shown are fold changes in expression relative to expression at 26 d before full bloom. Error bars represent the standard error of the means (n=4).

Fig. 3.

Carpel/floral-tube growth, cell production, and cell expansion in ‘Pollinated’ and ‘Unpollinated’ flowers. Carpel/floral-tube diameter or fruit diameter was measured in ‘Pollinated’ and ‘Unpollinated’ flowers/fruits. The number of cell layers and the area of cells within the floral tubes/fruit cortex of ‘Pollinated’ and ‘Unpollinated’ flowers/fruits were determined using light microscopy. ‘Unpollinated’ flowers abscised during later stages of the fruit set period. Open and filled circles represent ‘Pollinated’ and ‘Unpollinated’ flowers, respectively. Error bars represent the standard error of the means (n=4).

Fig. 4.

Expression of cell cycle genes during the period of fruit set in ‘Pollinated’ and ‘Unpollinated’ flowers. (A) Genes exhibiting higher expression (>2-fold) in ‘Pollinated’ flowers in comparison with ‘Unpollinated’ flowers during the fruit set period are shown. MdCYCD3;4 was the only gene that displayed a >2-fold increase in expression in ‘Pollinated’ flowers at 0d after full bloom. (B) Genes exhibiting higher expression (>2-fold) in ‘Unpollinated’ flowers in comparison with ‘Pollinated’ flowers during the fruit set period are shown. Gene expression was determined by quantitative RT-PCR using MdGAPDH for normalization. Fold change in expression of a gene relative to its expression in ‘Pollinated’ flowers at 0d after full bloom is presented. White bars represent ‘Pollinated’ flowers and black bars represent ‘Unpollinated’ flowers. Error bars indicate the standard error of the means (n=4).

Fig. 5.

Fruit growth, cell production kinematics, and cell expansion during fruit development in ‘Gala’ apple. (A) Fruit diameter was measured from before full bloom until maturity. (B) The number of cell layers in the fruit cortex was determined at different stages of fruit development. (C) The relative cell production rate (RCPR) was determined during the period of fruit development. (D) The cell area of fruit cortex cells was measured during fruit development using light microscopy. Error bars represent the standard error of the means (n=5).

Fig. 6.

Expression of cell cycle genes during fruit development in ‘Gala’ apple. (A) Hierarchical cluster analysis was performed to group genes with similar expression patterns. Expression of 59 cell cycle genes was determined using quantitative RT-PCR. MdGAPDH and MdACTIN were used for normalization. Expression data for a given gene are shown relative to its expression at 0 days after full bloom (DAFB). Cluster analysis was performed using log₂-transformed data. Numbers assigned to major clusters are indicated on the dendogram. The inset shows a colour legend for the cluster representation (log₂ ratios). MdCYCB2;1 was not included in the cluster analysis as no expression was detected at 123 DAFB. (B) Expression of representative genes from each major cluster identified in (A) is shown (clusters 1–6 from top to bottom). Fold change in expression of a gene relative to its expression at 0 DAFB is presented. Error bars represent the standard error of the means (n=5).