Caracterización de los agregados; para caracterizar los agregados tanto la

TMl O overexpression in tobacco hetero logous system resulted in various alterations i n plant morphology. TMl O expression promoted early flowering, reduced apical dominance and poor seed development in transgenic t obacco l i nes. Though the mechanisms by which TMl O affected these traits i n tobacco are unknown, these suggest that Tj\;!J 0 _{may be involved in simil ar pathways in tomato ( Chung e t al., 1 994; Kang e t}

al., 1 995).

The early flowering o f tobacco transgenic l ines suggests that 7Ml O product cou l d h av e induced t h e expression o f genes i nvol ved in floral i nduction. I n A rabidopsis, t h e transition to flowering is controlled in at l east four pathways (Levy a n d D ean, 1 99 8 ;

Pineiro and Coupland, 1 998). Fl owering i n tobacco may b e controlled b y similar set o f genes a s i n Arabidopsis (Kempin et al., 1 993 ; Kell y e t a l . , 1 995). S imil arly, TMl O may have an effect on the pathways controlling apical dominance. Apical dominance i n tobacco can b e associated with specific l evels o f endogenous hormones. Elevated l evels o f cytokinins or reduced auxi n levels resu l t in increased axillary bud growth (Romano et al . , 1 99 1 ; S ano et al., 1 994; McKenzie et al . , 1 99 8 ; Eklof et al., 2000). C onverse ly, low levels of cytokinins or high auxi n levels promote apical dominance (Harrison and Kaufmann, 1 9 84; Romano et al . , 1 99 1 ) . The poor seed development in the transgenic plants as a result of steri le ovules indicates TM1 0 effect on ovule development. The TM 1 0 product may h ave resulted in the negative regulation o f factors required for proper seed development in tobacco. The steri le transgenic ovules may be due to embryo sac degeneration, abnormal ovule integument development or aberrant d i fferentiation of the megagametophyte (Ray et al. , 1 99 4 ; Western and Haughn, 1 999).

TMl () RNA or product may have disrupted the genetic pathway for normal ovule development.

TMl () is a tomato MADS-box gene with high homology to the A GL l 2 of A raoidopsis.

Tl\;f} 0, however, has a d i fferent expression pattern to what is known for A GL l

suggesting these t\I/O genes have d i fferent functions. The expression analysis and t he phenotypes o f the transgenic plants generated with IJ,f} () indicate it m ay regulate events in flower and fruit development.

CHAPTE R S General Discussion

Over the last decade, plant MADS-box genes have been studied extensively to reveal their control of floral development and by virtue of these roles in flower development, i ncluding the carpel and ovule, they are strong c andidates for regulating fruit development. This research was aimed at identifying the role of MADS-box genes in flower and fruit development. The approach used in this study was to identify new members of the tomato MADS-box family, which are expressed i n flower and fruit, to characteri se these genes using molecular techniques and to examine their functions using genetic methods. Tomato was chosen as a model crop, for this proj ect, because i t produces a berry frui t and has a good system t o study fruit development, unlike the sil ique produced by Arabidopsis, which is a good model of the Brassicaceae .

Two previously uncharacterised tomato MADS-box genes were obtained. Tlvf29 was isolated fro m a young fruit cD A l ibrary by screening with homologous MADS-box fragments and degenerate PCR was used to identify TM1 0, which was subsequentl y isolated using 3'-RACE P C R . For functional annotation of genes, sequences were fi rst analysed. This is based on the general assumption that genes with same sequence structures may have simi lar functional properties. Sequence homo logy searches, phy logenetic analysis, protein composition and structure identi fied homologous genes whose functions were known. In addition, northern hybridisation, reverse transcriptase PC R and in situ hybridisation techniques were used to define the temporal and spatial gene expression to give i ndications of where the genes function.

Despite the valuable i n formation that accrues from analysing gene sequence and expression patterns, it is only through functional analyses that the role of a gene can be estab lished with certainty. The pri mary strategy for studying gene function has been forward genetics, which begins with a mutant phenotype and screens for the loss-or­ function mutations (Martin, 1 99 8 ; Krysan el al. , 1 999). However, this strategy rarely

identify genes that act redundantly or whose loss of function do not result in remarkabl e phenotype. Reverse genetics on the other hand, begins with a gene sequence and determines its loss-of-function phenotype. The recent availability of genome sequences has created opportunities for reverse genetic tools, such as activation tagging which randomly activate the expression of g enes (Weigel et al. , 2000) and insertional mutagenesis, which disrupt gene expression through i nserting T -DNA or transposons (Krysan et al., 1 999) . Alternatively, transgenic techniques allow plant genes to be overexpressed by using cDNA fragments l inked to strong promoters or silenced through antisense and cossupression phenomena (Napoli et al. , 1 990; van der Krol et al., 1 990) . Though, the effectiveness of the transgenic techniques are variable and not controllable, they are particularly versatile and have been widely used to study gene functions ( Mizukami and Ma, 1 99 2 ; Pneuli et al. , 1 994ab; Angenent et al. , 1 995). In this proj ect, the transgenic methods were used to overexpress or to reduce the level of gene transcripts in tomato. In addition, tobacco was employed as a heterologous host to overexpress the MADS-box genes. The use of heterologous host provides i ndications of gene functions, which may be silent in the original host due to functional redundancy ( Kang et al. , 1 995).

Overall, the functional implications from the transgenic p henotypes generated from these transformations ,vere consistent with the inferences drawn from their sequence and expression patterns.

S u m m a ry of

fin d ings