Site-directed mutagenesis of GH3-like auxres to test their role in the regulation of tomato plasma membrane H???-ATPase in response to auxin

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Thesis (M.S., Biological Sciences (Molecular and Cellular Biology))--California State University, Sacramento, 2015. ; Auxins play an important role in plant growth and development in a variety of ways. Auxin is proposed to regulate cellular growth by activating the plasma membrane H???-ATPases. According to the Acid Growth Hypothesis, pumping of hydrogen ions into the apoplast upon activation of the pump acidifies that space activating cell wall loosening enzymes. This, in turn, allows turgor driven expansion of the cell. Plasma membrane H???-ATPases have been thought to be responsible for this acidification and perhaps to be key in other auxin regulated responses such as lateral root initiation and vascular differentiation. Plasma membrane H+-ATPases are encoded by a family of 8-11 closely related genes in higher plants. In order to understand the control of growth and development of plants by auxin it is important to identify auxin responsive isoforms and to define how they are regulated by auxin. Thus, members of our lab fused the first 2.3 kb of the promoter (including the transcriptional start site, the 5 ???UTR and the first???amino acids) of the tomato plasma membrane H???-ATPase- isoform LHA2 to the reporter gene GUS. Examination of reporter gene expression under this promoter in both vi tomato and Arabidopsis demonstrated that the LHA2 promoter directs expression of GUS in a pattern that is very similar to that of the synthetic auxin responsive promoter, DR5 fused to GUS with expression observed in multiple auxin-responsive sites in the plant that coincide with DR5:GUS expression as well as in other auxin responsive sites. Since the level and pattern of LHA2:GUS expression changed in response to the natural auxin IAA and NAA and the synthetic auxin 2,4-D, and to the transport inhibitors TIBA and NPA, it was concluded that LHA2 is an auxin regulated isoform. DNA sequences have been identified in the promoters of auxin-responsive genes that confer auxin responsiveness. One such auxin response element (AuxRE), with the sequence TGTCTC is present in three sites in the promoter of LHA2. The presence of these elements in the LHA2 promoter suggests that the auxin responsiveness of LHA2 is under the control of these elements. However, it has not been elucidated if they confer auxin responsiveness to LHA2 and, if so, whether all of the elements contribute to this responsiveness to some extent, and also whether these are the only auxin responsive elements within the promoter of LHA2. The purpose of this research was to test the hypothesis that if TGTCTC auxin responsive elements confer auxin responsiveness to the promoter of LHA2, then inactivating all of them should eliminate the auxin responsiveness of this gene. Also, if each individual AuxRE contributes to auxin responsiveness in a summative fashion, then inactivation of each element individually should reduce auxin responsiveness incrementally. Furthermore, if each element confers auxin responsiveness in a cell or vii tissue-specific manner, then inactivation of individual elements may affect expression in a subset of auxin responsive sites. Thus, the TGTCTC AuxREs were altered within the promoter of LHA2 using site-directed mutagenesis and changes in expression were observed primarily by histochemical detection in comparison both to the wild-type LHA2:GUS construct and the auxin-responsive DR5:GUS construct. The level of GUS expression were also determined using GUS activity assays. The Specific changes were in the construct described above that consists of the first 2.3 kb of the LHA2 promoter and 5???UTR fused to the GUS reporter gene as follows: 1. The GAGACA sequence at position -1880 was changed to GATACA and this construct was designated ??FA2; 2. The TGTCTC sequence located at position -1667 was converted to TGTATC and this construct was designated ??FA1. 3. The GAGACA sequence at position -827 was converted to GATACA and this construct was designated ??FA3. 4. In the fourth construct all three GH3-like Auxin responsive elements were changed as above in a single construct and this was designated ??FA-All. The levels of LHA2:GUS expression decreased in the columella cells of primary roots in ??FA-All and ??FA1in comparison to the wild-type promoter LHA2:GUS (3-16) lines and DR5:GUS. These levels also decreased in the columella cells of lateral roots in ??FA1 and ??FA2. Also, LHA2:GUS expression decreased in both primary and lateral root viii vasculature in ??FA3 lines and in lateral root primordia in ??FA2 lines. These findings were also consistent with the hypothesis that the three TGTCTC AuxREs are directing auxin responsiveness in specific cells and tissues. Furthermore, these AuxREs appear to confer auxin responsiveness incrementally as knocking out each element decreased LHA2:GUS expression to some extent. In ??FA1, ??FA2, and ??FA3 lines expression was observed in sites in which LHA2:GUS is not expressed including the primary and lateral root cortex near the root apical meristem. Expression in these lines was also observed to be at higher levels than is observed for the wild-type LHA2 promoter in some sites including the leaf near the petiole-leaf junction. This suggests that the TGTCTC element causes repression in some tissues since its removal causes up-regulation in these sites. ; Biological Sciences (Molecular and Cellular Biology)