A role of brassinosteroids in early fruit development in cucumber

Abstract

Brassinosteroids (BRs) are essential for many biological processes in plants, however, little is known about their roles in early fruit development. To address this, BR levels were manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz) and their effects on early fruit development, cell division, and expression of cyclin and cyclin-dependent kinases (CDKs) genes were examined in two cucumber cultivars that differ in parthenocarpic capacity. The application of EBR induced parthenocarpic growth accompanied by active cell division in Jinchun No. 4, a cultivar without parthenocarpic capacity, whereas Brz treatment inhibited fruit set and, subsequently, fruit growth in Jinchun No. 2, a cultivar with natural parthenocarpic capacity, and this inhibitory effect could be rescued by the application of EBR. RT-PCR analysis showed both pollination and EBR induced expression of cell cycle-related genes (CycA, CycB, CycD3;1, CycD3;2, and CDKB) after anthesis. cDNA sequences for CsCycD3;1 and CsCycD3;2 were isolated through PCR amplification. Both CsCycD3;1 and CsCycD3;2 transcripts were up-regulated by EBR treatment and pollination but strongly repressed by Brz treatment. Meanwhile, BR6ox1 and SMT transcripts, two genes involved in BR synthesis, exhibited feedback regulation. These results strongly suggest that BRs play an important role during early fruit development in cucumber.

Fig. 1.

Fruit set percentage of Jinchun No. 4 (A) and Jinchun No. 2 (B) after different treatments. (A) N, no-pollination; P, pollination; E, EBR treatment on unpollinated ovaries. (B) NP, natural parthenocarpy; B, Brz-treatment; BE, both Brz and EBR treatments. The numbers indicated the concentrations of the applied EBR and Brz (μM), respectively. Results are expressed as average ±SD, n=4.

Fig. 2.

Cross-section of a cucumber ovary of Jinchun No. 4 (A) and Jinchun No. 2 (B) after different treatments, and changes of ovary length in Jinchun No. 4 (C) and Jinchun No. 2 (D). (A, C), N, no pollination (open circles); P, pollination (filled squares); E, EBR treatment on an unpollinated ovary (open triangles). (B, D), NP, natural parthenocarpy (open squares); B, Brz-treatment (filled circles); BE, both Brz and EBR treatment (filled triangles). Brz and EBR were applied at concentrations of 1 μM and 0.2 μM, respectively. The numbers indicated the corresponding days after anthesis. Results are expressed as average ±SD, n=4.

Fig. 3.

Expression of cell cycle-related genes in response to pollination and EBR treatment in Jinchun No. 4 fruits at 1 DAA (white bars) and 4 DAA (grey bars). N, no pollination; P, pollination; E, EBR treatment on unpollinated ovaries. The data were obtained from three independent experiments. Each value in the graph shows mean with SD of three experiments. Expression levels produced by RT-PCR are expressed as a ratio to N at 1 DAA and 4 DAA, which was set at 1.

Fig. 4.

Alignment of the predicted amino acid sequences of the C. sativus CycD3;1 cyclin, MsCycD3 from M. sativa (Dahl et al., 1995). NtcycD3-1 From N. tabacum (Sorrell et al., 1999), At CycD3-1 from A. thaliana (Soni et al., 1995). Conserved residues are shaded. Dots indicate gaps required for maximum alignment of sequences. The cyclin box is boxed, the N-terminal pRB-binding motif is in bold and underlined with a dashed line, and the putative PEST-destruction sequences are underlined.

Fig. 5.

Phylogenetic tree derived from analysis of 12 full-length plant CycD3 genes using the CLUSTAL program. The other source Cyclin D3 were from NCBI database: Am, Antirrhinum majus; At, A. thaliana; Nt, N. tabacum; Ps, P. sativum; Sl, Solanum lycopersicum; Ls, Lagenaria siceraria. The accession numbers/gene identifiers of the sequences are shown.

Fig. 6.

Effects of pollination, Brz and EBR applications on the expression of CycD3 during fruit development in cucumber. (A) CycD3 expression in Jinchun No. 4 fruits. A, anthesis; N, no pollination; P, pollination; E, EBR treatment. (B) CycD3 expression in Jinchun No. 2 ovaries. NP, natural parthenocarpy; B, Brz treatment; BE, both Brz and EBR treatments. The numbers indicated days after anthesis.

Fig. 7.

Expression of Br6ox1 and SMT in response to EBR and BRZ treatments in Jinchun No. 4 and Jinchun No. 2 fruits. (A, C) N, no pollination for Jinchun No. 4 (open circles); P, pollination for Jinchun No. 4 (filled squares); E, EBR treatment on unpollinated ovaries for Jinchun No. 4 (open triangles); NP, natural parthenocarpy for Jinchun No. 2 (open squares). (B, D) NP, natural parthenocarpy for Jinchun No. 2 (open squares); B, Brz treatment for Jinchun No. 2 (filled circles); BE, both Brz and EBR treatments for Jinchun No. 2 (filled triangles). The data were obtained from three independent experiments. Each value in the graph shows mean with SD of three experiments. (A, C) Expression levels produced by RT-PCR are expressed as a ratio to N, which was set at 1; (B, D) expression levels are expressed as a ratio to NP, which was set at 1.