Coordinate repression of regulators of embryonic identity by PICKLE during germination in Arabidopsis

Abstract

In angiosperms, germination represents an important developmental transition during which embryonic identity is repressed and vegetative identity emerges. PICKLE (PKL) encodes a CHD3-chromatin-remodeling factor necessary for the repression of expression of LEAFY COTYLEDON1 (LEC1), a central regulator of embryogenesis. A candidate gene approach and microarray analysis identified nine additional genes that exhibit PKL-dependent repression of expression during germination. Transcripts for all three LEAFY COTYLEDON genes, LEC1, LEC2, and FUS3, exhibit PKL-dependent repression, and all three transcripts are elevated more than 100-fold in pkl primary roots that inappropriately express embryonic traits (pickle roots). Three other genes that exhibit PKL-dependent regulation have expression patterns correlated with zygotic or somatic embryogenesis, and one gene encodes a putative Lin-11, Isl-1, MEC-3 (LIM) domain transcriptional regulator that is preferentially expressed in siliques. Genes that exhibit PKL-dependent repression during germination are not necessarily regulated by PKL at other points in development. Our data suggest that PKL selectively regulates a suite of genes during germination to repress embryonic identity. In particular, we propose that PKL acts as a master regulator of the LEAFY COTYLEDON genes, and that joint derepression of these genes is likely to contribute substantially to expression of embryonic identity in pkl seedlings.

Figure 1

LEC1 and LEC2 transcripts are elevated in germinating pkl seeds. Quantitative RT-PCR was used to determine the relative transcript levels of the genes of interest in germinating wild-type and pkl seeds in the absence or presence of uniconazole-P. The transcripts examined represent genes known or suspected to be involved in embryogenesis (see text). 18s rRNA was used as a standardization control, and expression levels are normalized to wild-type seeds imbibed in the absence of uniconazole-P. Error bars represent the standard deviation of the mean.

Figure 2

Identification of genes that exhibit PKL-dependent transcript levels. The Venn diagram (top) indicates the number of loci for which the corresponding transcript is expressed at significantly different (p < 0.05) levels in wild-type (PKL) versus pkl seeds in the absence (- Uni-P) or presence (+ Uni-P) of uniconazole-P. The intersection of both data sets represents the 293 genes that showed significantly different expression (p < 0.05, uncorrected for multiple comparisons) in the pkl mutant regardless of the presence of uniconazole-P. The 293 genes constituted about 3.5% of the total number of loci interrogated with the array, and 54% of the genes identified have a known or putative function. Functional categories assigned to the 54% with known or putative functions are represented in the chart at the bottom. The smaller number in parentheses indicates the percentage of all of the genes represented on the microarray that were assigned to that functional category.

Figure 3

Transcript level of several genes is strongly PKL-dependent during germination. Quantitative RT-PCR was used to determine the relative transcript levels of the genes of interest in germinating wild-type and pkl seeds in the absence or presence of uniconazole-P. The transcripts examined represent twelve genes that exhibit robust PKL-dependent expression based on microarray analysis (see text). (a) Microarray data for the twelve candidate genes that were analyzed. Both the AGI code and the corresponding Affymetrix identification code are given. The mean values derived from the microarray analysis for these genes under the four treatments are shown. The last two columns indicate the name of the gene as referred to in the text of this paper along with the predicted function of the gene based on published data and/or sequence similarity. (b) Relative transcript levels of the twelve genes as indicated by microarray analysis. Expression levels are normalized to wild-type seeds imbibed in the absence of uniconazole-P. (c) Relative transcript levels of the twelve genes as indicated by quantitative RT-PCR. 18s rRNA was used as a standardization control, and expression levels are normalized to wild-type seeds imbibed in the absence of uniconazole-P. Genes are ordered by relative expression in pkl seeds from highest to lowest from left to right. Error bars represent the standard deviation of the mean.

Figure 4

The LIM-encoding gene is preferentially expressed in siliques. Quantitative RT-PCR was used to determine the relative transcript levels of the genes of interest in wild-type roots, rosette leaves, and siliques. The transcripts examined represent genes that exhibit PKL-dependent expression during germination. Genes are ordered by relative expression in siliques from highest to lowest from left to right. 18s rRNA was used as a standardization control, and expression levels are normalized to roots. Error bars represent the standard deviation of the mean.

Figure 5

The LEC genes are highly expressed in pickle roots. Quantitative RT-PCR was used to determine the relative transcript levels of the genes of interest in wild-type roots and pickle roots. The transcripts examined represent genes that exhibit PKL-dependent expression during germination. Genes are ordered by relative expression in pickle roots from highest to lowest from left to right. 18s rRNA was used as a standardization control, and expression levels are normalized to wild-type roots. Error bars represent the standard deviation of the mean.

Figure 6

The FUS3 transcript is elevated in pkl leaves. Quantitative RT-PCR was used to determine the relative transcript level of the genes of interest in wild-type leaves and pkl leaves. The transcripts examined represent genes that exhibit PKL-dependent expression during germination. Genes are ordered by relative expression in pkl leaf tissue from highest to lowest from left to right. The LEC1 transcript was not detected in either tissue and is therefore not included on the graph. 18s rRNA was used as a standardization control, and expression levels are normalized to wild-type leaves. Error bars represent the standard deviation of the mean.

Figure 7

Genes exhibit coordinate regulation by PKL during germination. Quantitative RT-PCR was used to determine the relative transcript level of the genes of interest in wild-type seeds and pkl seeds that were desiccated or imbibed for 12, 24, and 36 hours. The transcripts examined represent genes that exhibit PKL-dependent expression during germination. Genes are ordered by relative expression in pkl seeds at 36 hours after imbibition from highest to lowest from left to right. 18s rRNA was used as a standardization control, and expression levels are normalized to wild-type seeds at each time point. Error bars represent the standard deviation of the mean.