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doi: 10.1242/10.1242/dev.00281

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Analysis of the mutational effects of the COP/DET/FUS loci on genome expression profiles reveals their overlapping yet not identical roles in regulating Arabidopsis seedling development

¹ Peking-Yale Joint Center of Plant Molecular Genetics and Agrobiotechnology, College of Life Sciences, Peking University, Beijing 100871, Peoples Republic of China
² Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT06520-8104, USA
³ Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut 06520, USA

View larger version (71K): [in a new window]   Fig. 1. A summary of the seedling phenotypes of the photomorphogenic mutants and their genome expression profile relatedness. (A) Morphological comparison of dark-grown Arabidopsis wild-type (WT), pleiotropic cop/det/fus and partial photomorphogenic mutant seedlings. All seedlings were 6-day-old. Scale bars: (in cop1-1 panel for top row) 1 mm. (B) Morphological comparison of continuous white light-grown wild-type (WT) and pleiotropic cop/det/fus mutant seedlings. All seedlings were 6-day-old and photographed at the same magnification. Some of the mutants shown in A and B were used in studies reported in C and D, while some were used in studies reported in subsequent Figures. (C) Hierarchical clustering analysis of overall relatedness for expression ratios from wild-type seedlings grown under normal white light (WL) versus dark-grown (D) siblings, and dark-grown pleiotropic cop/det/fus and partial photomorphogenic mutants versus dark-grown wild-type seedlings of the same ecotype. An expression profile from dark-grown tir1-1 versus dark-grown wild-type seedlings is also included for comparison. Only those genes that exhibited twofold or more differential expression in at least one sample pair of the 13 tested were included. There are 3057 genes included in the cluster analysis (see supplementary data at http://dev.biologists.org/supplemental/ or http://plantgenomics.biology.yale.edu/ for more information). Asterisks denote the ecotype of wild-type Arabidopsis: *for Col-0 and **for WS. (D). Hierarchical clustering analysis of overall relatedness for expression ratios from wild-type seedlings grown under normal white light (WL) versus dark-grown siblings, and high intensity light-grown (HWL) pleiotropic cop/det/fus mutants versus normal white light-grown wild-type seedlings of the same ecotype. Only those genes that exhibited twofold or more differential expression at least in one sample pair of the nine tested were included for comparison. There are 2608 genes included in the cluster (see supplementary data at http://dev.biologists.org/supplemental/ or http://plantgenomics.biology.yale.edu/ for more information). Asterisks denote the ecotype of wild-type Arabidopsis as in A.

View larger version (44K): [in a new window]   Fig. 2. A comparison of the expression profiles of 15 representative genes encoding photosynthetic light reaction proteins. (A) Hierarchical clustering display of expression ratios from dark-grown pleiotropic cop/det/fus mutants seedlings versus dark-grown wild-type seedlings, in comparison with normal and high-light regulation and CRY1 overexpression affected by blue-light regulation. Lane 1, normal white light and dark-grown wild-type seedlings; lane 2, dark-grown cop1-6 and wild-type seedlings; lane 3, dark-grown cop1-1 and wild-type seedlings; lane 4, dark-grown cop1-8 and wild-type seedlings; lane 5, dark-grown det1-1 and wild-type seedlings; lane 6, of dark-grown det1-6 and wild-type seedlings; lane 7, blue light-grown CRY1 OE and wild-type seedlings; lane 8, high intensity (2500 µmol/m2/second) and normal intensity (150 µmol/m2/second) light-grown wild-type seedlings. (B) Hierarchical clustering display of expression ratios of normal white light-grown pleiotropic cop/det/fus mutants seedlings versus white light-grown wild-type seedlings, in comparison with normal white light regulation. Lane 1, white light and dark-grown wild-type seedlings; lane 2, light-grown cop1-6 and wild-type seedlings; lane 3, light-grown cop1-1 and wild-type seedlings; lane 4, light-grown cop1-8 and wild-type seedlings; lane 5, light-grown det1-1 and wild-type seedlings; lane 6, light-grown det1-6 and wild-type seedlings; lane 7, high intensity (2500 µmol/m2/second) and normal intensity (150 µmol/m2/second) light-grown wild-type seedlings. The color scale for A and B is shown at the bottom of B. Positive numbers represent fold of induction and negative numbers represent fold of repression.

View larger version (31K): [in a new window]   Fig. 3. A comparison of overall genome expression patterns among pleiotropic cop/det/fus mutations and high light intensity. (A) The number of differentially expressed genes (2-fold regulation) in representative cop/det/fus mutations or high-intensity light. Six-day-old dark-grown (hatched bars) and white light-grown (white bars) mutant seedlings were examined. Lane 1, cop1-8 vs. WT; lane 2, det1-6 vs. WT; lane 3, fus6-1 vs. WT; lane 4, cop10-1 vs. WT; lane 5, high light vs. dark (hatched bar) or normal light (white bar). (B) The number of oppositely regulated and newly regulated genes by the pleiotropic cop/det/fus mutations in darkness and high light intensity. Lane 1, cop1-8/D vs. WT/D; lane 2, det1-6/D vs. WT/D; lane 3, fus6-1/D vs. WT/D; lane 4, cop10-1/D vs. WT/D; lane 5, WT/HWL vs. WT/D. Only those genes that exhibited twofold or more differential expression in each sample pair and showed oppositely regulation by a cop/det/fus mutation and normal light were selected as oppositely regulated genes. Those genes that exhibited twofold or more differential expression in cop/det/fus mutants in darkness but did not change (ratio between 0.8 and 1.2) in a normal light sample pair were selected as newly regulated genes. (C) The number of oppositely regulated genes and newly regulated genes by the cop/det/fus mutations in normal and high light intensity. Lane 1, cop1-8/WL vs. WT/WL; lane 2, det1-6/WL vs. WT/WL; lane 3, fus6-1/WL vs. WT/WL; lane 4, cop10-1/WL vs. WT/WL; lane 5, WT/HWL vs. WT/WL. Only those genes that exhibited twofold or more differential expression in each sample pair and showed opposite regulation between light-grown cop/det/fus mutants and light-grown wild type were selected as oppositely regulated genes. Those genes that exhibited twofold or more differential expression in light-grown cop/det/fus mutants as compared to wild type but did not change (ratio between 0.8 and 1.2) in normal light versus dark-grown seedlings were selected as newly regulated genes.

View larger version (47K): [in a new window]   Fig. 4. Comparison of the morphogenic patterns and genome expression profiles of Arabidopsis seedlings grown under different light intensity conditions. (A) Phenotype of different intensity white light-grown wild-type seedlings. The seedlings were grown under 1.5 (1.5), 15 (15) or 150 (150) µmol/m2/second continuous white light or in the dark (D) for 6 days, or under 150 µmol/m2/second continuous white light for 5.5 days, then transferred to 2500 µmol/m2/second continuous white light for another 12 hours (2500). Seedlings were photographed at the same magnification. (B) An overview of the hierarchical cluster display of the genome expression profiles for all these different intensity light- and dark-grown wild-type sample pairs is shown in A. The dark-grown det1-6 or cop1-8 and wild-type sample pairs were used for comparison. Lane 1, 1.5 µmol/m2second white light- and dark-grown wild-type seedlings; lane 2, 15 µmol/m2second white light- and dark-grown wild-type seedlings; lane 3, 150 µmol/m2/second white light- and dark-grown wild-type seedlings; lane 4, 2500 µmol/m2/second white light- and dark-grown wild-type seedlings; lane 5, dark-grown det1-6 and wild-type seedlings; lane 6, dark-grown cop1-8 and wild-type seedlings. Only those genes that exhibited twofold or more differential expression in at least one sample pair of the six pairs examined were included for comparison. 2614 genes were included in the cluster analysis (see supplementary data at http://dev.biologists.org/supplemental/ or http://plantgenomics.biology.yale.edu/ for detail). (C) The number of differentially regulated genes by distinct light intensities and different alleles of COP1, in comparison with oppositely regulated genes in each case. Lane 1, 1.5 µmol/m2/second white light- and dark-grown wild-type seedlings; lane 2, 15 µmol/m2/second white light- and dark-grown wild-type seedlings; lane 3, normal 150 µmol/m2/second white light- and dark-grown wild-type seedlings; lane 4, 2500 µmol/m2/second white light- and dark-grown wild-type seedlings; lane 5, dark-grown cop1-6 and wild-type seedlings; lane 6, dark-grown cop1-1 and wild-type seedlings; lane 7, dark-grown cop1-8 and wild-type seedlings. Only those genes that exhibited twofold or more differential expression in each sample pair and showed opposite regulation toward normal light regulation were selected as oppositely regulated genes.

View larger version (33K): [in a new window]   Fig. 5. The 12 distinct response patterns of gene expression to light intensity changes. The profiles of 12 representative genes that showed a different gene expression pattern from wild-type seedlings grown under different intensities of white light. The total number of genes (n) that fit each of the 12 patterns is indicated. The representative genes are: A, At4g10340: chlorophyll a/b binding protein; B, At3g14415: glycolate oxidase; C, At1g11260: glucose transporter; D, At3g47960: peptide transporter PTR1; E, At3g47340: glutamine-dependent asparagine synthetase; F, At2g45210: auxin-regulated protein; G, At2g03340: WRKY DNA-binding protein; H, At4g14130: XET-related protein XTR-7; I, At5g46180: ornithine aminotransferase; J, At5g13760: unknown protein; K, At1g49760: polyA binding protein; L, At5g15410: cyclic nucleotide-gated cation channel. The bars in each graph are: 1, WT/1.5 µmol/m2/second WL vs. WT/D; 2, WT/15 µmol/m2/second WL vs. WT/D; 3, WT/150 µmol/m2/second WL vs. WT/D; 4 WT/2500 µmol/m2/second WL vs. WT/D.

View larger version (73K): [in a new window]   Fig. 6. Clustering analysis of light-regulated transcription factor genes. (A) An overview of the hierarchical cluster display for those transcription factor genes in our microarray regulated by light and COP1. Only those genes that exhibited twofold or more differential change in at least one sample pair of the four light intensity pairs tested were included for comparison. There are 53 genes included in the cluster (see supplementary data at http://dev.biologists.org/supplemental/ or http://plantgenomics.biology.yale.edu/ for detail). The gray color indicates that high quality data was not available. The lanes are as follows: 1, WT/1.5 µmol/m2/second WL vs. WT/D; 2, WT/15 µmol/m2/second WL vs. WT/D; 3, WT/150 µmol/m2/second WL vs. WT/D; 4 WT/2500 µmol/m2/second WL vs. WT/D. (B) The expression profiles of 5 representative genes from wild-type seedlings grown at four different light intensities and dark-grown cop1 alleles. The four lanes in the left panels of B are the same as in A, for the four light intensities used. The four lanes in the right panels of B are different cop1 alleles with increasing severity. Lane 1, dark-grown N282 and wild-type seedlings; lane 2, dark-grown cop1-6 and wild-type seedlings; lane 3, dark-grown cop1-1 and wild-type seedlings; lane 4, dark-grown cop1-5 and wild-type seedlings.

View larger version (49K): [in a new window]   Fig. 7. Hierarchical clustering display of expression profiles of cytosolic ribosomal protein genes from normal light grown and pleiotropic cop/det/fus mutations in darkness. Lane 1, expression ratios of white light and dark-grown wild-type seedlings; lane 2, dark-grown det1-6 and wild-type seedlings; lane 3, dark-grown cop1-8 and wild-type seedlings; lane 4, dark-grown cop9-1 and wild-type seedlings; lane 5, dark-grown cop10-1 and wild-type seedlings. RPL, 60S ribosomal protein genes; RPS, 40S ribosomal protein genes.

View larger version (62K): [in a new window]   Fig. 8. Comparison of genome expression profiles of COP4 and DET2 with the pleiotropic cop/det/fus mutants and light regulation. (A) Hierarchical clustering display of expression profiles for cop4, wild type and four representative pleiotropic cop/det/fus mutants. The six lanes in the cluster analysis are: 1, cop4/D vs. WT/D D; 2, det1-1/D vs. WT/D; 3, WT/WL vs. WT/D; 4, cop1-1/D vs. WT/D; 5, cop9-1/D vs. WT/D; 6, cop10-1/D vs. WT/D. Those genes that exhibited twofold or more differential expression in cop4 mutant and corresponding genes in other pairs were included for comparison. A total of 496 genes were included in the cluster analysis. (B) Hierarchical clustering display of expression profiles for det2, wild type and four representative pleiotropic cop/det/fus mutants. The six lanes are: 1, det2/D vs. WT/D; 2, WT/WL vs. WT/D; 3, cop9-1/D vs. WT/D; 4, det1-1/D vs. WT/D; 5, cop1-1/D vs. WT/D; 6, cop10-1/D vs. WT/D. Only those genes that exhibited twofold or more differential expression in the det2 mutant were included for comparison. A total of 253 genes were included in the cluster. (C,D). The percentage of up- (C) or down-regulated (D) genes (exhibiting twofold or more differential expression) in the cop4 mutant that are similarly or oppositely regulated by light or each of the 4 representative pleiotropic cop/det/fus mutations. The six sample pairs selected (bars 1-6) are the same as in the lanes in A. (E,F) The percentage of up- (E) or down-regulated (F) genes (exhibiting a twofold or more differential expression) in the det2 mutant that were similarly or oppositely regulated by light or each of the 4 representative pleiotropic cop/det/fus mutations. The six bars in the diagram correspond to the lanes in B.