The long term goal of this research is to understand the sexual determination (SD) process in the model genetic system Zea mays. For unisexual plants like maize, SD is defined as the process that determines whether a flower will be staminate (the male tassel) or pistillate (the female ear). SD in maize involves the initial formation of bisexual flowers, followed by the developmentally programmed abortion of gynoecial primordia in the tassel (T1,T2), one of the two gynoecial primordia in the paired spikelets of the ear (E2) and the arrest of stamen initials in the ear. SD can be altered both genetically and physiologically [eg. by hormones such as gibberellic acid (GA) or by light]. Mutants in the GA pathway that cannot produce endogenous GA3 fail to arrest stamen in the ear leading to bisexual and staminate flowers. Elucidation of the mechanism(s) underlying SD in maize may also provide insight into the processes of cell death, cell protection and cell arrest. Molecular characterization of three genes which specifically affect sexuality, tasselseed1(TS1), tasselseed2(TS2) and silkless1(SK1), form the basis of the proposed experiments. ts1 and ts2 plants display virtually an identical phenotype - there is no abortion of T1, T2 or E2. Stamen are also arrested in the tassel leading to its complete feminization. Using a refined Ac tagging protocol, Dellaporta succeeded in cloning TS2 and showed that (i) it encodes a member of a short chain dehydrogenase family with greatest similarity to steroid dehydrogenase and is hypothesized to perform a catalytic role in the SD pathway, (ii) ts2 mRNA is found in all gynoecial cells - the three destined to die and the one that develops in the ear, (iii) ts2 expression in the tassel is responsible for TS2's SD role as judged by genetic mosaics created by Ac excision from the ts2-m1 allele, (iv) a deletion in the ts2 homolog of Tripsacum, a maize relative, is shown to be the cause of a mutation that feminizes the normally mixed staminate and pistillate inflorescence, and (iv) ts2 mRNA is not detected in a mutant ts1 background, suggesting that TS1 encodes a transcriptional activator of TS2. TS1 has been successfully tagged with a Mutator element and its isolation is said to be imminent. Finally, gynoecial programmed cell death has been shown to be associated with nuclear degeneration, whether this reflects necrosis or apoptosis will be addressed. In sk1 mutants, both E1 and E2 undergo cell death. In contrast, both E1 and E2 fail to abort in the sk1/sk1 ts2/ts2 double mutant. It is hypothesized that SK1 normally protects E1 from TS1,TS2 -mediated cell death either by a direct or indirect interaction between the SK1 and TS2 proteins.
Four specific aims are proposed. The first is to characterize the process of Ts2 induced cell death and to determine whether or not Ts2 expression is sufficient to signal a cell death response. The subcellular location of TS2 (and SK1, when available) will be determined using colloidal gold labelling with immunoelectron microscopy in collaboration with Joel Rosenbaum's lab. Nuclear localization will be assayed by transient transfection of GFP::TS2, N-terminal and C-terminal fusion constructs into onion epidermal cells. DNA degradation patterns in dying E2 cells will be assayed to determine whether apoptosis or necrosis is involved. Whether ectopic expression of Ts2 is sufficient to induce cell death will be addressed by assaying the ability of inducible Ts2 constructs to mediate cell death in maize BMS suspension cells The second aim is to clone and characterize Ts1 and to test the hypothesis that Ts1 activates Ts2 transcription. Ts1, which is reported to be tagged with the Mutator element will be cloned, characterized, its mRNA localized to see if it is concordant with Ts2 mRNA and its cDNA searched for previously identified sequence motifs. Nuclear localization will be tested as for Ts2, and a transient assay system will be developed to test the ability of constitutively expressed Ts1 to activate reporter genes fused to Ts2 promoter sequences.
The third aim i s to investigate whether (and how) Sk1 protects cells from TS-mediated cell death. Strategies to tag Sk1 with either Ac or Mutator are described. Alternatively, Sk1 may fall out of a two-hybrid screen for proteins that interact with Ts2. When available, sk1 will be characterized like Ts1, above. Of particular interest is the prediction that sk1 should be asymmetrically distributed in E1 and E2 if its role is to protect E2 from Ts2-mediated cell death. In he event that Sk1 cannot be isolated, it is proposed that Ts2 protein be assayed for post-translational modifications and subcellular loculation in wild type and sk1 mutant backgrounds.
The fourth aim i s to identify proteins that interact with Ts2 and Sk1 using both two-hybrid screen in yeast and enhancer and suppressor genetic screens. The fifth aim is to investigate the ancestral role of SD genes in the dicot Arabidopsis by gene cloning and knock outs. Homologs of Ts1, Ts2 and Sk1 will be sought among available Arabidopsis ESTs and, if found, attempts will be made to isolate T-DNA knock outs from a huge collection of T-DNA inserts in the Arabidopsis genome generated by the Dellaporta lab. This section follows from the interesting observation that Ts2 homologs in rice and Arabidopsis are expressed in vegetative cells that undergo programmed cell death including provascular and tapetal cells. It is hypothesized that SD homologs mediate a general program of cell death in plants and that the SD pathway may have been recruited from such an ancestral pathway.
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