The in vivo function of Chup

4.2.1 Confirmation of the T-DNA insertion in chup1 knockout plants

For a phenotypic characterization of Chup1 function, a mutant line harbouring a T-DNA insertion in the CHUP1 gene was obtained from the SALK institute and screened by PCR

genotyping. To verify the insertion of the T-DNA into the CHUP1 gene (see Figure 10A), a

gene-specific primers CHUP1 LP and CHUP1 RP should yield a WT band of 884 bp. In a heterozygous mutant plant with a T-DNA insertion in the CHUP1 gene on one chromosome

and no insertion on the other, two bands should be visible: the PCR product resulting from the combination of the T-DNA–specific left border primer (Lba1) and the gene-specific primer CHUP1 RP, which give a product of 590 bp. An additional band results from the gene- specific primers CHUP1 LP and CHUP1 RP (WT band). For a homozygous plant, only the PCR product from the Lba1 primer and the CHUP1 RP primer is amplified and results again in a product of 590 bp. The PCR products of the homozygous, heterozygous and wild type plants can be seen in Figure 10B, where the predicted bands of the correct size appear. The T- DNA insertion site in the third exon of the CHUP1 gene is depicted in Figure 10 A.

Figure 10 A Intron structure of the CHUP1 gene (At3g25690), not to scale. The white triangle marks the

insertion of the T-DNA. Back arrows mark the position of the gene specific left and right primer (LP, RP); grey arrow the position of Lba1 primer B PCR products from HZ (heterozygous, lane 1), WT (wild type, lane 2) and

HM (homozygous, lane 3) plants from the progeny of the SALK line 129128 are shown

The knock-out status of the ∆chup1 line was further confirmed by microarray analysis (see

chapter 4.1.1). A complete downregulation of the CHUP1 gene was observed (Figure 12 A).

4.2.2 White Band Assay

A chloroplast movement deficient phenotype was discovered in a screen by Kasahara et al. (2002) resulting from a mutation in the CHUP1 gene. To verify this phenotype the ∆chup

mutant was tested for loss of chloroplast movement. To assay the chloroplast movement deficient phenotype of ∆chup1, the white band assay was used (Kagawa et al. 2001). With

this screening method, defects in chloroplast movement can be made visible on a macroscopic level. At the illuminated area wild type plants show a paler green colour (Figure 11). This results from an increased transmittance of light through the leaf owing to a lower density of chloroplasts at the periclinial walls (walls perpendicular to the light) and a higher chloroplast density at the anticlinial walls (walls parallel to the light) (see Figure 2, Introduction).

Figure 11 White Band Assay. Leaves from WT (up) or ∆chup1 (down) were illuminated with strong white

light within a narrow area on the leaf.The pale band on the WT leaf denotes the chloroplast movement in this area. On the ∆chup1 leaf, no pale band is appearing upon illumination with strong light

As can be seen in Figure 11, the leaf of a ∆chup1 plant does not show any sign of chloroplast

movement, as no pale band is appearing after illumination. Thus, this is an independent confirmation of the chloroplast movement deficient phenotype of the chup1 knockout plant

which was described by Oikawa et al. (2003).

4.3

Light regulation

To obtain information about the changes in gene expression induced through high light conditions and to explore a mode of function of Chup1 in the network of adaptation to enhanced light intensities, an expression analysis was conducted. A comparison of global gene expression changes in ∆chup1 and wild type plants was obtained through microarray

analysis of mRNA from high light treated plants.

Figure 12 Expression analysis of ∆chup1 plants A The averaged signal intensity of gene expression (three

independent experiments) in wild type and ∆chup1 plants at day 30 with a detection P-value equal or smaller

than 0.005 in at least one plant type is shown. Lines indicate the border for an at least three fold signal difference and the number of significantly up- or downregulated genes are given (listed in Suppl. Table 1). The signal for CHUP1 is circled. B The averaged signal intensity (three independent experiments) at day 30 of the genes in

wild type plants before (wt) and after illumination (wtL) with a detection P-value equal to or smaller than 0.005 in at least one plant type is given. Lines indicate the border for an at least threefold signal difference. C

Comparison of the number of genes significantly up- (U) or down-regulated (D) in response to light in wild type (black) or mutant plants (grey). D The slope ratio values (slr) for signals found in wild type versus mutant and

mutant versus mutant after light treatment are shown for the genes significantly regulated in wild type in response to light (see panel B). Black dots indicate genes found to be downregulated and grey dots found to be upregulated in wild type in response to light. Positive values correspond to downregulation, negative values to upregulation.

A comparison of the expression signals of wild type and ∆chup1 (Figure 12 A) shows, that

only a small number of genes (3) are downregulated. The most drastically downregulated gene is CHUP1, which demonstrates the knock-out status of the mutant. The genes that were found to be upregulated in ∆chup1 are for the most part involved in stress response. This may

reflect adaptation to environmental conditions of the mutant and might give a link of the signal cascades of stress induced changes and light stimulus. As the behaviour of gene expression in response to high light in terms of the regulation of chloroplast movement was of major interest, plants treated with strong white light (400µmol) were analysed. The light treatment caused – not surprisingly - a significant change in the expression profile of the wild type compared to non treated wild type (Figure 12 B). With a significance criterion of a three fold enhanced expression change, 282 genes were found to be upregulated and 211 genes downregulated. For high light treated ∆chup1 plants, a similar observation was made (Figure

12 C) while not all of the regulated genes were entirely the same as in wild type.

The most interesting observation was made when the genes found to be regulated in wild type upon light treatment were analysed for their behaviour in the ∆chup1 mutant background

(Figure 12 D). Genes that were downregulated in the WT in response to light were not found to be regulated in the non-treated mutant. But this population of genes was slightly downregulated in the mutant in response to light (Figure 12 D black dots). A more drastic differential regulation was observed for genes that were upregulated in high light treated WT: Genes of this population that were found to be upregulated in the mutant compared to WT were found to be not regulated in the mutant after illumination. This is due to the fact that their expression before illumination was already at a comparable high level as reached in WT after illumination. By contrast, genes of that population again (upregulated in high light treated WT), that were downregulated in the mutant in comparison to wild type did strongly enhance their expression in the mutant in response to light to reach a similar expression level after light treatment as obtained in wild type after light treatment (Figure 12 D, grey dots). The exclusive differential regulation of genes in the mutant, which are usually upregulated in response to light in the wild type plants, demonstrates that the mutation causes a shift of the sensing light intensity.