Effects of overexpression of PIP5K2 variants in tobacco pollen tubes are not correlated

In the last section the in vivo activity of PIP5K2 T500A/D and PIP5K T430A/D combinations was tested by heterologous expression in tobacco pollen tubes. Tobacco pollen tubes expressing PIP5K2 T430A-EYFP, PIP5K2 T430D-EYFP and PIP5K2 T500A-EYFP depicted apical tip swelling as has been described for PIP5K2-EYFP (Stenzel et al., 2008) and thus, were thought to be functional in vivo. In contrast, expression of PIP5K2 T500D-EYFP had no influence on tobacco pollen tube morphology suggesting that this PIP5K2 variant was catalytic inactive in vivo. To test whether higher expression levels account for the observed morphological changes in pollen tubes expressing PIP5K2 T430A-EYFP, PIP5K2 T430D-EYFP and PIP5K2 T500A-EYFP compared to PIP5K2 T500D-EYFP, relative fluorescence intensities of transformed pollen tubes were measured with ImageJ (http://rsbweb.nih.gov/ij/).

67 Figure 3.21 depicts the relative fluorescence intensity patterns for pollen tubes expressing the indicated PIP5K2-EYFP variants and shows that the amount of pollen tubes decreases continually towards high fluorescence intensities for all of the investigated constructs. About 40 to 50 % of the pollen tubes transformed with PIP5K2-YFP, PIP5K2 T430A-EYFP and PIP5K2 T430D-EYFP constructs display low fluorescence intensities (Fig. 3.21 1-20). Pollen tubes heterologously expressing PIP5K2 T500A-EYFP and PIP5K2 T500D-EYFP are equally distributed between low and medium fluorescence intensities (Fig. 3.21 1-60). Although the fluorescence intensity of pollen tubes expressing PIP5K2 T500A-EYFP and PIP5K2 T500D-EYFP was similar, their effect on tobacco pollen tube morphology was severely different (Fig. 3.20 B), suggesting that the observed pollen tube phenotypes were not a result of altered expression level but indeed depended on the in vivo catalytic activity of the investigated PIP5K2-EYFP variants. An increased proportion of pollen tubes expressing PIP5K2 T500A-EYFP at high intensity was observed for 20 % of the pollen subjected to analysis and may contribute to the wild type-like pattern illustrated for that variant in Figure 3.20. As the high intensity is only observed in 20%

of the pollen tubes tested, this effect is not likely to substantially impair interpretation of the data shown in Figure 3.20.

Fig. 3.21: Fluorescence intensities of pollen tubes expressing PIP5K2-EYFP and the corresponding variants. PIP5K2-EYFP and variants were transiently expressed in tobacco pollen tubes and the relative fluorescence was measured after 12 h of growth. Over 100 pollen tubes per construct were analysed with light exposure of 0.2 s.

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3.11 3.11 Mutant complementation

Although the heterologous expression in tobacco pollen tubes gave first hints towards the in vivo functionality of PIP5K2 T430 A/D and PIP5K2 T500A/D combinations, it was next tested whether the investigated variants can also function in their homologous system. PIP5K2 is ubiquitously expressed in Arabidopsis, and although it has been postulated that Arabidopsis pip5k2 mutants show reduced lateral root formation (Mei et al., 2012), this phenotype was not observed during experiments performed in this thesis (data not shown). The disruption of an additional PI4P 5-kinase, PIP5K1, results in an Arabidopsis pip5k1 pip5k2 double mutant that has a severe phenotype as already described in section 2.17. By expressing PIP5K2 variants in the pip5k1 pip5k2 double mutant background and investigating the resulting phenotype, qualitative conclusions about the in vivo catalytic activity can be made.

To test whether the PIP5K2 variants investigated so far were able to complement the pip5k1 pip5k2 double mutant phenotype, PIP5K2 cDNA was expressed in the heterozygous pip5k1 PIP5K1 x pip5k2 pip5k2 mutant line under the control of a 1500-bp PIP5K2 promoter fragment as a fusion to a C-terminal EYFP tag. After transformation, the offspring was screened for pip5k1 x pip5k2 PIP5K2-EYFP mutants by amplifying the T-DNA tagged pip5k1 and pip5k2 alleles and the corresponding wild type alleles (Fig. 3.22 E). As can be seen in Figure 3.22 E, amplification of PIP5K1 and PIP5K2 wild type alleles results in a band with a size of 1200 bp.

PIP5K2 wild type alleles were also amplified in pip5k1 x pip5k2 PIP5K2 T500A-EYFP and pip5k1 x pip5k2 PIP5K2 T500D-EYFP mutant plants but were only about 750 bp in size. The local amplification of the genomic PIP5K2 sequence contains two introns that are absent from PIP5K2-EYFP cDNA used for transformation experiments (Fig. 3.22 F). The observed size difference after amplification of the PIP5K2 wild type allele in pip5k1 1 x pip5k2 PIP5K2 T500A-EYFP and pip5k1 x pip5k2 PIP5K2 T500D-EYFP mutant plants therefore is connected to the general absence of introns in cDNA and confirms that the transformation was successful.

pip5k1 x pip5k2 PIP5K2 T500A-EYFP and pip5k1 x pip5k2 PIP5K2 T500D-EYFP were phenotypically analyzed (Fig. 3.22). Figure 3.22 A and C show wild type and pip5k1 pip5k2 double mutant plants, respectively. The expression of PIP5K2 T500A-EYFP in the pip5k1 pip5k2 double mutant background is able to rescue the phenotype (Fig. 3.22 B). pip5k1 x pip5k2 PIP5K2 T500A-EYFP mutants grew like wild type plants and also developed normal inflorescence. In contrast to that, pip5k1 x pip5k2 PIP5K2 T500D-EYFP mutant plants depicted the same phenotype as described for pip5k1 pip5k2 double mutants (Fig. 3.22 D).

69 Fig. 3.22: Expression of PIP5K2-EYFP T500A can rescue the phenotype of the Arabidopsis pip5k1 pip5k2 double mutant. PIP5K2::PIP5K2T500A:EYFP and PIP5K2::PIP5K2T500D:EYFP cDNA was expressed in the pip5k1 PIP5K1 pip5k2 pip5k2 mutant background. Transformed double mutant plants identified by PCR-based genotyping and analyzed for morphology . A, Wild type control. B, Double mutant expressing PIP5K2T500A-EYFP. C, Double mutant control displaying dwarfism. D, Double mutant expressing PIP5K2T500D-EYFP. All plants shown (A-D) have been grown side-by-side for the same period under identical conditions. E, PCR based genotyping. Black arrowheads indicate 1000 bp. F, genomic PIP5K2 sequence with exons (black) and introns. The white arrowhead indicates the site if the T-DNA insertion.

The PIP5K2 variants of T500 were selected for the mutant complementation tests because of the clear difference in catalytic activities between T500A and T500D (Fig. 3.15). In future experiments, it will be interesting to also test the effects of expressing PIP5K2 T430A and PIP5K2 T430D in the Arabidopsis pip5k1 pip5k2 mutant background.