Comparison of genes constantly affected in the tga14 mutant at 3, 6, 11 and 24 hpi and in mock-treated plants identified 7 down-regulated (Fig. 4.3.3.1) and 10 up-regulated (Fig.
4.3.3.2) genes.
A function of 3 up-regulated genes was predicted based on amino acid sequences and the role of two proteins was already described. DUR3 (Degradation of Urea 3) has a high affinity to urea and it serves as the major transporter for urea up-take in A. thaliana (Kojima et al., 2007). The atdur3-1 and atdur3-3 mutants grew less vigorously on media with urea as the main nitrogen source. Moreover, expression of DUR3 was stimulated by urea and repressed by ammonium and nitrate and an increased DUR3 promoter activity was detected under low
- 122 - nitrogen conditions in the rhizodermis. Therefore, DUR3 function is suggested to be an adaptation to low urea levels in unfertilized soils (Kojima et al., 2007). Due to one potential TGA binding site in the DUR3 promoter a direct regulation of the gene by clade I TGA TFs could be possible. Since my experiments were done on fertilized soil I assume that lower levels of DUR3 expression should not have an impact on avrRPS4-triggered defense response in the tga14 mutant.
The ABC transporter NAP3 was suggested to be involved in the detoxification of Al (Huang et al., 2010). It was shown that nap3 mutants (atstar1) were more sensitive to Al and an earlier flowering was observed. The increased sensitivity to Al was complemented after expression of a NAP3 rice ortholog (OSSTAR1) that was also implicated in Al tolerance (Huang et al., 2009). NAP3 expression occurs at outer cell layers of root tips and in developing leaves (Huang et al., 2010). A binding site for TGA TFs in the promoter is missing. Since my plants grew without Al stress I assume no impact of low NAP3 transcript levels to resistance mediated by TGA1 and TGA4.
The third down-regulated gene with a predicted function as a glutathione-dependent oxireductase was ROXY9, which is described later (Chapter 5.4).
The functions of 5 of the 10 genes that were consistently higher expressed in the tga14 mutant were already described.
MPL1 plays an important role in defense against the green peach aphid and is highly induced after attack (Louis et al., 2010). Interestingly, it is the only TGA1/TGA4-suppressed gene that was induced after Pst avrRPS4 in wildtype Col-0 (Fig. 4.4.1.3). The mpl1 mutant showed an increased number of green peach aphids in infection studies, whereas over-expression under the control of the 35S promoter led to a lower number of attackers. The MPL1 protein exhibits lipase activity and MPL1-dependent lipids were suggested to have a positive influence on resistance against green peach aphids (Louis et al., 2010). Whether clade I TGA TFs play a role in defense against aphids remains to be tested. In order to find a possible link between increased susceptibility of the tga14 mutant after infection with Pst avrRPS4 and an elevated MPL1 expression, infection of the mpl1 mutant and 35S:MPL1 plants with Pst avrRPS4 has to be performed. With respect to ETI triggered by avrRPS4 I have to propose that MPL1 interferes with this defense response. The MPL1 promoter exhibits 1 potential binding site for TGAs and might therefore be a direct target gene of TGA1 and TGA4. For MPL1, it has to be postulated that clade I TGA TFs can repress promoters of genes.
- 123 - The -amylase RAM1 was shown to account for the major part of -amylase activity in rosette leaves and inflorescences. The ram1 mutant has normal starch levels, indicating that only small activities of RAM1 homologs are sufficient for starch degradation (Laby et al., 2001). The promoter of RAM1 exhibits 3 possible binding sites for TGA TFs.
ASN1 encodes an asparagine synthase and overexpression of the gene under the control of the 35S promoter increased the nitrogen levels in seeds. It is suggested that increased asparagine levels lead to a higher source to sink flux of the amino acid resulting in high amounts of nitrogen in seeds (Lam et al., 2003). The promoter of ASN1 shows 4 possible TGA TF binding sites.
The NAI2 protein is localized to endoplasmic reticulum (ER) bodies and a lack of NAI2 resulted in abnormal formation and a low number of these bodies. Therefore, a crucial function of NAI2 in the formation of ER bodies was implicated (Yamada et al., 2009). The promoter of NAI2 exhibits one potential TGA binding site.
Transcripts of LTP4 (Lipid Transfer Protein 4) were found specifically in guard cells, whereas they were up-regulated in all tissues in seedlings after NaCl treatment (Chae et al., 2010). The plant LTP proteins exhibit four conserved disulfide bonds with eight cysteine residues (Douliez et al., 2000) and can interact with phospholipid molecules and fatty acids in vitro (Zachowski et al., 1998). Two possible TGA binding sites are present in the LTP4 promoter.
Transcription factor SPL4 (SPL3, SPL5) is important in regulating flowering (Wang et al., 2009). The expression of SPLs is increased in the center of the shoot during the transition from juvenile to adult plants (Schmid et al., 2003; Wu and Poething et al., 2006) and overexpression results in early flowering (Wu and Poething, 2006). An early flowering of the tga14 mutant was not observed. The SPL4 promoter exhibits three possible binding sites for TGA TFs.
Furthermore, 5 ROXYs arranged in a tandem on chromosome 4 were constitutively co-regulated to higher transcript levels in the tga14 mutant and are described later (Chapter 5.4).
Taken together, it cannot be decided whether this basal deregulation of different genes influences the interaction with Pst avrRPS4. Since an interaction of ROXYs and TGA TFs was shown (Ndamukong et al., 2007) and a redox modification of clade I TGA TFs was
- 124 - suggested (Depres et al., 2003), the reciprocal changes in ROXY gene expression were the most promising alterations with respect to defense mechanisms.
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