Partner 7: W. Vriezen and M. Mariani Characterization of abiotic-stress and abscisic acid mediated male sterility in tomato. The importance of abscisic acid (ABA) in the stamen has been demonstrated previously. For instance, exogenous ABA suppresses anther development and causes pollen abortion (Chandra Sekhar and Sawney, 1991) in tomato. ABA insensitivity on the other hand, also causes male sterility in Arabidopsis plants, as it is the case in the abscisic insensitive8 mutant (Brocard-Gifford and Finkelstein, 2004). However, the function of ABA in anther development is unknown, but some reports indicate a relation between ABA levels and temperature stress. In Arabidopsis the ABA response is enhanced by temperature stresses (Xiong et al., 1999). In tomato and many other species, a major limiting factor for fruit-set is the extreme sensitivity of pollen development and pollination to moderately high or low temperatures and inadequate humidity (Picken, 1984). The tomato stamenless-2 (sl-2) mutant has increased ABA levels in the anthers and is male sterile. Fertility is restored in sl-2 flowers at low temperatures which correlated with a decrease of ABA to normal levels (Singh and Sawhney, 1998). Previous research at the lab of Plant Cell Biology (P7) (C. Mariani, Radboud University Nijmegen) has demonstrated the involvement of ethylene in pollination (de Martinis et al., 2002; Sanchez and Mariani, 2002; Weterings et al., 2002), and in particular that ethylene regulates anther dehiscence (Rieu et al., 2003), a process dependent on water transport through aquaporins (Bots et al, 2004). In Arabidopsis, members of the aquaporin gene family are regulated by abiotic stresses like drought, salt or cold stress through ABA-dependent pathways (Jang et al, 2004). Recently, we started a new research on the hormonal regulation of tomato fruit initiation. A cDNA-AFLP experiment was performed to compare the transcriptomes from ovaries during fruit-set after pollination, with that of ovaries in which fruit-set was induced by gibberellin. From this experiment we isolated a PP2C gene, homologous to the rice and Arabidopsis ABI2 (Abscisic Acid Insensitive 2) and HAB1 (homology to ABI1/ABI2) genes. In Arabidopsis ABA-signalling is negatively regulated by ABI1/2 (Leung et al., 1997) and HAB1 (Saez et al., 2004). Our results show that the tomato PP2C gene is also strongly expressed in the anthers after dehiscence. The project within ADONIS aims to elucidate the role of ABA in tomato anthers during cold, drought and heat stress. To identify genes that are modulated by abiotic stress and by ABA, cDNA-AFLP will be used to compare the transcriptomes of anthers from ABA insensitive and hypersensitive tomato transgenic plants and anthers of wild-type plants. These transgenic plants will be constructed by over-expressing and silencing (using RNAi) the tomato anther orthologs of the Arabidopsis ABI1/ABI2/HAB1 PP2C-gene family. Transgenic plants will be made available to all partners interested. As the PP2C proteins might have redundant functions, the expression of the whole gene family in anthers will be mapped by Family Transcript Profiling, a cDNAAFLP based technique developed in our lab for simultaneous quantitative expression analysis of gene families. ABA - (hyper) sensitive plants will be exposed to different stresses. Genes modulated in anthers of these plants but not in insensitive ones will be characterized by DNA sequencing. As a control it is possible to simultaneously analyze the expression of these genes in the ABA-deficient tomato mutant flacca, which is blocked in the final step of ABA biosynthesis (Taylor et al., 1988). The identified genes might point-out stress-induced and ABA-related processes, whose role in stress resistance can be investigated further in transgenic plants. ABI1-family PP2C proteins mediate the expression of the majority of ABA responsive genes and appear to interact with multiple cellular targets (reviewed by Himmelbach et al., 2003). PP2C proteins likely function by dephosphorylation of other ABA signalling proteins. It is reasonable to think that this type of regulation may occur also in anthers, when exposed to various stresses evoking the ABA response. By analyzing the phosphorylation status of the anther proteome from the transgenic tomato plants (with silenced or over-expressed PP2C genes) in cooperation with P1 and P8, it should be possible to identify the targets of such a regulatory mechanism. Isolated proteins can be identified by protein profiling or sequencing at the laboratory of P3. Interactions between identified proteins can further be analyzed in cooperation with P2. At the laboratory of P7, partners in the training program can gain experience in using molecular biological techniques like cDNA-AFLP, histological techniques like RNA in situ hybridisation, immuno-localization and microscopy using Cryo-SEM, TEM, CLSM and LM. In addition, we have a very good cooperation with the Centre for Molecular and Biomolecular Informatics (Prof. Dr. G. Vriend; http://www.cmbi.kun.nl/gv/vriend_group/), specialized in protein homology modelling technology. Bots, M., Feron, R. Uehlein, N., Weterings, K., Kaldenhoff, R. and Mariani, T. (2004) PIP1 and PIP2 aquaporins are differentially expressed during tobacco anther and stigma development. J. Exp. Bot. in press. Brocard-Gifford, I., Lynch, T.J., Garcia, M.E., Malhotra, B. and Finkelstein, R.R. (2004) The Arabidopsis thaliana ABSCISIC ACID-INSENSITIVE8 locus encodes a novel protein mediating abscisic acid and sugar responses essential for growth. Plant Cell, 16, 406–421. Chandra Sekhar, K.N. and Sawney, V.K. (1991) Role of ABA in stamen and pistel development in the normal and solanifolia mutant of tomato (Lycopersicom esculentum). Sexual Plant Reprod. 4, 279-283. Martinis De, D., Cotti, G., Te Lintel Hekker, Harren, F.J.M., Mariani, C. (2002) Ethylene response to pollen tube growth in Nicotiana tabacum flowers. Planta 214, 806-812. Himmelbach, A., Yang, Y. and Grill, E. (2003) Relay and control of abscisic acid signaling. Curr. Opin. Plant Biol., 6, 470–479. Jang, J.Y., Kim, D.G., Kim, Y.O., Kim, J.S. and Kang, H. (2004) An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana. Plant Mol. Biol. 54, 713-725. Leung, J., Merlot, S., and Giraudat, J. (1997). The Arabidopsis ABSCISIC ACID–INSENSITIVE 2 (ABI2) and ABI1 genes encode redundant protein phosphatases 2C involved in abscisic acid signal transduction. Plant Cell 9, 759–771. Picken, A.J.F. (1984) A review of pollination and fruit set in the tomato (Lycopersicon esculentum Mill.) J. Hort. Sci. 59:1–13. Rieu, I., Wolters-Arts, M., Derksen, J., Mariani, C. and Weterings K. (2003) Ethylene regulates the timing of anther dehiscence in tobacco. Planta 217, 131-137. Saez, A., Apostolova, N., Gonzalez-Guzman, M., Gonzalez- Garcia, M.P., Nicolas, C., Lorenzo, O. and Rodriguez, P.L. (2004) Gain-of-function and loss-of-function phenotypes of the protein phosphatase 2C HAB1 reveal its role as a negative regulator of abscisic signalling. Plant J. 37, 354-369. Sanchez, A.M., Mariani, C. (2002) Expression of the ACC synthase and ACC oxidase coding genes after selfpollination and incongruous pollination of tobacco pistils. Plant Mol. Biol. 48, 351-359. Singh, S. and Sawhney, V.K. (1998) Abscisic acid in a male sterile tomato mutant and its regulation by low temperature. J. Exp. Bot. 49, 199-203. Taylor, I.B., Linforth, R.S.T., Al-Naieb, R.J., Bowman, W.R., Marples, B.A. (1988) The wilty tomato mutants flacca and sitiens are impaired in the oxidation of ABA-aldehyde to ABA. Plant Cell Environ 11, 739– 745. Weterings, K., Pezzotti, M., Cornelissen, M., and Mariani, C. (2002) 1-aminocyclopropane-1-carboxylatesynthase and oxidase transcript accumulation patterns during pollen tube growth in tobacco styles. Plant Physiol 130, 1190-1200. Xiong, L., Ishitani, M., and Zhu, J-K. (1999) Interaction of osmotic stress, temperature, and abscisic acid in the regulation of gene expression in Arabidopsis. Plant Physiol. 119, 205-211.
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