Molecular Mechanism For The Regulation Of Fruit Development In Cucumis Sativus

Cucurbits, monoecious annual plant species, are widely grown in the world, and account for a large portion of agricultural production and consumption. However, abortion or abnormal growth of the ovaries occurs due to the unsuccessful pollination/fertilization, under environment factors such as low temperature, low light and high humidity in the green house, and thus, decrease the productivity and agricultural income. Therefore, it is important to study the physiological, cellular and molecular mechanisms of fruit development and the effects of phytohormones on it. In this study, we investigated the spatio-temporal changes of cell division, endoreduplication and cell expansion, as well as the expression patterns of the genes involved in these processes during cucumber fruit development. Meanwhile, the roles of brassinosteroids (BRs) in early fruit development were studied. Also, experiments were carried out to investigate the interactions between BRs, cytokinin and polyamine in cucumber fruit. The results are as follows:1. We investigated the temporal and spatial changes in cell division, endoreduplication and expression of cell cycle-related genes in developing cucumber fruits. Cell division was intense during the first 4 days after anthesis (DAA) and then decreased until to 8 DAA. Meanwhile, endoreduplication started at 4 DAA and increased gradually, accompanied with the increase in fruit weight. The appearance of endoredupliction associated with beginning of a rapid growth indicated that rapid cell expansion during fruit development started from 4 DAA. Cell division was mainly observed in the exocarp, while endoreduplication occurred mostly in the endocarp and pulp. Among the six cell cycle-related genes, the two mitotic cyclin genes (CycA and CycB) and CDKB showed their highest transcript levels within 2 DAA, while the transcripts of CycD3 and CDKA peaked at 4 DAA and 20 DAA, respectively. NAA, CPPU and EBR all induced parthenocarpic growth together with active cell division and enhanced transcripts of cell cycle-related genes. In comparison, GA₃ had little effect on the induction of parthenocarpy and the transcripts of cell cycle-related genes. 2. The expression patterns of expansin and XTH family genes in cucumber fruits and their responses to plant growth substances were studied. Most of the expansin family genes (CsExp3, 5, 6, 7, 8, 9 and 10) showed their highest levels during the rapid cell expansion phase (4-8 DAA) in fruit growth, suggesting the significant roles of these expansin genes in cell expansion in fruit. Similar roles of most of the expansin genes and XTH family genes were found in endocarp and/or pulp tissues of fruit, for their expression pattern are associated with the distribution of endoreduplication in different tissues of fruits. Each member of the expansin and XTH family genes has its unique expression pattern in response to different plant growth substances applied on the cucumber ovaries.3. The roles of BRs in early fruit development were studied. To address this, we manipulated BRs levels through application of exogenous BRs (24-epibrassinolide, EBR) or a BRs biosynthesis inhibitor (brassinazole, Brz) and examined their effects on early fruit development, cell division, expression of cyclin D3 genes in two cucumber cultivars that differ in parthenocarpic capacity. Application of EBR induced parthenocarpic growth accompanied with 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 application of EBR. cDNA sequences for CsCycD3;1, 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, BR6oxl and SMT transcripts, two genes involved in BRs synthesis exhibited feedback regulation. These results strongly suggest that BRs play an important role during early fruit development in cucumber.4. A cDNA library of genes which are preferentially expressed in the EBR-treated ovaries was constructed by suppression subtractive hybridization (SSH) to study the effects of BRs on the transcripts in cucumber fruit. We obtained 132 cDNAs including 118 unique genes and fall them into seven categories after analyzed by BLASTX and Go: phytosynthesis (8.7%), protein synthesis (19.1%), cell proliferation and differentiation (22.6%), cell enlargement (5.2%), metabolism(7.8%) and stress response (7.8%). This indicates that BRs may function in many biological processes when inducing parthenocarpic growth of cucumber fruit. The high occurrence of cell division related genes in the SSH cDNA library points to an important roles of BRs in cell division during fruit development. The appearance of auxin and gibberellin response factor genes in the library suggests that the interactions between BRs and other hormones may exist in regulating fruit growth. A strong expression pattern of gene encoding 14-3-3 protein was found in the EBR-treated ovaries, suggesting that the 14-3-3 protein pathway may be involved in the BRs regulation of cucumber fruit.5. Polyamine metabolism was studied in fruits treated by pollination, BRs and cytokinin. In the pollinated fruits, the contents of free polyamines and the expressions of four polyamine biosynthesis genes (ADC, SAMDC, SPDS and SPMS) were higher in early fruit development (with 4 DAA), and the free polyamines contents were highest in the exocarp among the three different fruit tissues. The contents of free polyamines were also increased in the EBR- and CPPU-treated ovaries with a greater extent in the ovaries treated by CPPU. The expression patterns of the four genes in the fruits treated by phytohormones were similar to those in the pollinated ones. These results suggest that polyamine biosynthesis is critical for the early fruit development of cucumber and BRs and cytokinin may interact with polyamines to regulate fruit development. Meanwhile, we found the free polyamine contents in the unpollinated ovaries were maintained at the levels as that in the ovaries at anthesis, and the expression levels of the four biosynthesis genes were quite high in the unpollinated ovaries. These suggest the polyamine biosynthesis plays an important role in delaying ovary senescence.