Article Figures & Data
Figures
- Figure 1.
Evaluation of the Microarray Assay.
(A) Scatterplot of signal values from two replicates on microarray. Total RNA from 6-day-old white light– and dark-grown wild-type Arabidopsis seedlings was labeled with Cy5 and Cy3, respectively. The Log2 values of the Cy5-to-Cy3 ratios were plotted for the two replicates.
(B) Overlay image of two subarrays of a microarray hybridized with probes originating from 6-day-old white light–grown wild-type seedlings labeled with Cy5 and 6-day-old dark-grown wild-type seedlings labeled with Cy3.
(C) Overlay image of the same two subarrays as in (B) but hybridized with probes originating from 6-day-old white light–grown wild-type seedlings labeled with Cy3 and 6-day-old dark-grown wild-type seedlings labeled with Cy5.
(D) Distribution of average ratios of expression from white light– and dark-grown wild-type seedlings. Total RNA from 6-day-old white light– and dark-grown wild-type seedlings was labeled reciprocally with Cy3 and Cy5. The ratio for each clone is the average from four replicates.
- Figure 2.
Representative Seedlings Grown under Defined Light Conditions and Cluster Analysis of Their Light-Regulated Gene Expression.
(A) Wild-type Arabidopsis seedlings grown under different light conditions. WT, wild-type seedlings; W, white light; FR, far-red light; R, red light; B, blue light; 36D, white light–grown seedlings or leaves transferred to darkness for 36 hr; 36W, dark-grown seedlings transferred to white light for 36 hr. All light conditions were continuous illumination. All seedlings were 6 days old at the time of harvesting.
(B) Hierarchical cluster display of expression ratios from wild-type seedlings grown under different light qualities versus dark-grown siblings and seedlings or leaves before and after light/dark transitions. Only those ESTs that exhibited at least twofold differential change in at least one sample pair among the seven pairs tested were included for comparison. There are 4326 EST entries included in the cluster. WTL, leaves of 5-week-old white light–grown wild-type seedlings; vs, versus.
(C) and (D) Two subclusters of ESTs showed distinct patterns of regulation for selected light conditions. SOD, superoxide dismutase; ccr, cytokinin repressed; Cyt, cytochrome.
- Figure 3.
Developmental Characteristics of Far-Red-Light–Grown Seedlings and Cluster Analysis of Phytochrome A–Mediated Far-Red Light Regulation of Gene Expression in Arabidopsis Seedlings.
(A) Phenotypic characteristics of 6-day-old wild type (WT), phyA mutant, and phytochrome A overexpression line (PHYAOE) grown under far-red light (FR). A 6-day-old dark-grown seedling (WT/D) was included for comparison. Wild types of both Landsberg erecta and No-O ecotypes were used for direct comparison with the phyA mutant and PHYAOE seedlings in their respective ecotypes.
(B) Overview of the hierarchical cluster display. Lane 1, expression ratios of far-red-light–grown phytochrome A overexpression and wild-type seedlings (PHYAOE/FR versus WT/FR). Lane 2, expression ratios of far-red-light–grown wild-type and phyA mutant seedlings (WT/FR versus phyA/FR). Lane 3, expression ratios of far-red-light– and dark-grown wild-type seedlings (WT/FR versus WT/D). A total of 2062 ESTs that had at least twofold differential expression in one of the three sample pairs were included in the cluster.
(C) Two sample subclusters of ESTs displaying antagonistic regulation by phytochrome A overexpression and phytochrome A–mediated far-red light regulation in wild type.
(D) Interloping diagrams of the number of differentially expressed EST clones that exhibited twofold or greater upregulation (induction) or downregulation (repression) for each of the three sample pairs. The numbers in the overlapping areas indicate the shared number of EST clones that exhibited twofold or greater differential expression in either two or three sample pairs. vs, versus.
- Figure 4.
Developmental Characteristics of Red Light–Grown Seedlings and Cluster Analysis of Phytochrome B–Mediated Red Light Regulation of Gene Expression in Arabidopsis Seedlings.
(A) Phenotypic characteristics of 6-day-old wild type (WT), phyB mutant, and phytochrome B overexpression line (PHYBOE) grown under red light (R). A 6-day-old dark-grown seedling (WT/D) was included for comparison. Wild types of both Landsberg erecta and No-O ecotypes were used for direct comparison with the phyB mutant and PHYBOE seedlings in their respective ecotypes.
(B) Overview of the hierarchical cluster display. Lane 1, expression ratios of red light–grown phytochrome B overexpression and wild-type seedlings (PHYBOE/R versus WT/R). Lane 2, expression ratios of red light–grown wild-type and phyB mutant seedlings (WT/R versus phyB/R). Lane 3, expression ratios of red-light– and dark-grown wild-type seedlings (WT/R versus WT/D). A total of 2204 EST entries that had at least twofold differential expression in one of the three sample pairs were included in the cluster.
(C) Two sample subclusters of ESTs displaying antagonistic regulation by phytochrome B overexpression and phytochrome B–mediated red light regulation in wild type. ccr, cytokinin repressed.
(D) Interloping diagrams of the number of differentially expressed EST clones that exhibited twofold or greater upregulation (induction) or downregulation (repression) for each of the three sample pairs. The numbers in the overlapping areas indicate the shared number of EST clones that exhibited twofold or greater differential expression in either two or three sample pairs. vs, versus.
- Figure 5.
Developmental Characteristics of Blue Light–Grown Seedlings and Cluster Analysis of Cryptochrome-Mediated Blue Light Regulation of Gene Expression in Arabidopsis Seedlings.
(A) Phenotypic characteristics of 6-day-old wild type (WT), cry1 cry2 double mutant, and cryptochrome 1 overexpression line (CRY1OE) grown under blue light (B). A 6-day-old dark-grown seedling (WT/D) was included for comparison. Wild types of both Columbia and Wassilewskija ecotypes were used for direct comparison with the cry1 cry2 double mutant and CRY1OE seedlings in their respective ecotypes.
(B) Overview of the hierarchical cluster display. Lane 1, expression ratios of blue light–grown cryptochrome 1 overexpression and wild-type seedlings (CRY1OE/B versus WT/B). Lane 2, expression ratios of blue light–grown wild-type and cry1 cry2 double mutant seedlings (WT/B versus cry1 cry2/B). Lane 3, expression ratios of blue light– and dark-grown wild-type seedlings (WT/B versus WT/D). A total of 2444 EST entries that had at least twofold differential expression in one of the three sample pairs were included in the cluster.
(C) Two sample subclusters of ESTs displaying antagonistic regulation by cryptochrome 1 overexpression and cryptochrome-mediated blue light regulation in wild type.
(D) Interloping diagrams of the number of differentially expressed EST clones that exhibited twofold or greater upregulation (induction) or downregulation (repression) for each of the three sample pairs. The numbers in the overlapping areas indicate the shared number of EST clones that exhibited twofold or greater differential expression in either two or three sample pairs. vs, versus.
- Figure 6.
Cluster Analysis of ESTs Displaying Antagonistic Effects by Overexpression of Individual Photoreceptors.
(A) Overview of the hierarchical cluster display. Lane 1, expression ratios of far-red-light– and dark-grown wild-type seedlings (WT/FR versus WT/D). Lane 2, expression ratios of far-red-light–grown phytochrome A overexpression and wild-type seedlings (PHYAOE/FR versus WT/FR). Lane 3, expression ratios of red light– and dark-grown wild-type seedlings (WT/R versus WT/D). Lane 4, expression ratios of red light–grown phytochrome B overexpression and wild-type seedlings (PHYBOE/R versus WT/R). Lane 5, expression ratios of blue light– and dark-grown wild-type seedlings (WT/B versus WT/D). Lane 6, expression ratios of blue light-grown cryptochrome 1 overexpression and wild-type seedlings (CRY1OE/B versus WT/B). A total of 977 ESTs that had at least twofold expression changes in both light-regulated expression and antagonistic effect in a photoreceptor overexpression line were included in the cluster.
(B), (C), and (D) show three representative subclusters that contain ESTs displaying distinct patterns of antagonistic effects by different photoreceptor overexpression. The accession numbers and gene identities are listed. Subcluster B contains ESTs that displayed downregulation in all three light quality conditions and antagonist effects by overexpression of each of the three photoreceptors. Subcluster C shows ESTs that were upregulated in all three light quality conditions but were positively or antagonistically regulated by overexpression of phytochrome A and cryptochrome 1. The effects of phytochrome B on this group of ESTs were either nonantagonistic or antagonistic. Subcluster D shows ESTs that were downregulated by three light quality conditions and that were antagonistically affected by overexpression of both phytochrome A and phytochrome B but that were minimally affected by cryptochrome 1 overexpression. GST, glutathione S-transferase; ccr, cytokinin repressed; ACC, 1-aminocyclopropane-1-carboxylic acid; HPD, 4-hydroxyphenypyruvatev dioxygenase.
- Figure 7.
Diagrammatic Summary of Cellular Metabolic and Regulatory Pathways Controlled by Light in Arabidopsis.
The cellular metabolic and regulatory pathways that are upregulated by light are listed in red type, whereas the cellular metabolic and regulatory pathways that are downregulated by light are listed in green type. Note that some pathways involve multiple cellular compartments. TCA, trichloroacetic acid.
Tables
- Table 1.
Correlation Coefficient of Ratios of Data for Four Replicates from Wild-Type Seedlings Grown in White Light Versus Darka
Replicate W-Cy5 versus D-Cy3 (1) W-Cy5 versus D-Cy3 (2) W-Cy3 versus D-Cy5 (1) W-Cy3 versus D-Cy5 (2) W-Cy5 versus D-Cy3 (1)b —c 0.978 0.948 0.946 W-Cy5 versus D-Cy3 (2) 0.978 — 0.950 0.951 W-Cy3 versus D-Cy5 (1) 0.948 0.950 — 0.984 W-Cy3 versus D-Cy5 (2) 0.946 0.951 0.984 — a W, white light; D, darkness; Cy3, Cy3-deoxyUTP; Cy5, Cy5-deoxyUTP.
b The numbers 1 and 2 in the parentheses represent replicate 1 and replicate 2 of the same microarray slide.
c N/A, not applicable.
- Table 2.
Microarray Results from Some Well-Known Light- Regulated Genes Reported in the Literature
Fold Regulated by Light in the Microarraya Accession Number Gene Name A B C D E F G Reference N37414 D1 48.0 10.1 36.0 12.8 1.9 9.1 −1.4 Thompson et al. (1983) T76714 Lhb1B2 30.2 19.6 27.7 13.3 3.7 7.8 2.9 Tobin and Silverthorne (1985) N65655 Lhb1B1 30.2 18.2 32.1 18.3 5.0 7.9 2.4 Tobin and Silverthorne (1985) R89925 Lhca5 18.5 12.1 18.9 19.2 8.2 14.4 4.7 Tobin and Silverthorne (1985) N97182 Lhcb2 14.1 13.3 16.4 16.4 4.6 5.5 3.1 Tobin and Silverthorne (1985) N96851 Lhca2 6.5 3.0 4.3 3.5 1.9 3.8 2.0 Tobin and Silverthorne (1985) R90003 PC 14.6 3.6 7.7 9.0 5.9 10.2 4.1 Helliwell et al. (1997) AA067471 Rubisco activase b 14.5 6.5 11.5 11.5 4.2 9.0 3.8 Orozco and Ogren (1993) T20492 RBCS 3b 14.3 3.8 10.4 9.0 6.3 11.2 5.6 Tobin and Silverthorne (1985) T45355 RBCS 2B 14.2 2.9 8.4 7.1 5.4 8.6 4.8 Tobin and Silverthorne (1985) R30465 RBCS 1B 14.2 4.5 8.9 6.3 5.0 9.7 5.0 Tobin and Silverthorne (1985) N65785 RBCS 1A 10.9 5.1 4.2 8.4 4.8 7.2 4.6 Tobin and Silverthorne (1985) N37467 GAPDH 10.0 2.2 8.2 4.7 2.7 4.7 3.0 Cerff and Kloppstech (1982) H36324 CHS 10.9 2.7 6.0 3.9 2.3 2.6 1.8 Batschauer et al. (1991) T45207 PAL1 3.0 −1.3 1.5 1.2 1.3 1.2 3.1 Schroder et al. (1979) N65492 POR A −2.4 −2.1 −1.1 −2.5 −2.6 −1.2 −0.83 Apel (1981) T20944 PHYA −3.1 −1.4 −2.4 −1.9 −2.8 −2.1 −1.8 Bruce et al. (1989) N96756 AS −5.6 1.5 −2.9 −3.6 −3.4 −20 −16.7 Shi et al. (1997) H36556 AS −3.1 −1.3 −1.7 −1.6 −1.4 −1.6 −2.2 Shi et al. (1997) W43629 TUB1 −2.7 −1.9 −1.3 −1.8 −1.8 −2.2 −2.0 Leu et al. (1995) a The seven light treatment comparisons are as follows: A, 6-day-old wild-type seedlings grown in white light versus in dark (WT/W versus WT/D); B, 6-day-old wild-type seedlings grown in far-red light versus in dark (WT/FR versus WT/D); C, 6-day-old wild-type seedlings grown in red light versus in dark (WT/R versus WT/D); D, 6-day-old wild-type seedlings grown in blue light versus in dark (WT/B versus WT/D); E, white light–grown 6-day-old wild-type seedlings versus white light–grown 4.5-day-old wild-type seedlings transferred to dark for 36 hr (WT/W versus WT/36D); F, 4.5-day-old dark-grown wild-type seedlings transferred to white light for 36 hr versus 6-day-old dark-grown wild-type seedlings (WT/36W versus WT/D); G, white light–grown 5-week-old wild-type plant leaves versus white light–grown 5-week-old wild-type plant leaves transferred to dark for 36 hr (WTL/W versus WTL/36D).
b Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase.
- Table 3.
Summary of the Number of ESTs Induced or Repressed Twofold or More by Light Qualities, Light/Dark Transitions, and Individual Photoreceptors
Number of ESTs Induced by Light or Photoreceptor Number of ESTs Repressed by Light or Photoreceptor Experimentsa Fold >30 30 to 20 20 to 10 10 to 5 5 to 3 3 to 2 Total >30 30 to 20 20 to 10 10 to 5 5 to 3 3 to 2 Total WT/W versus WT/D 3 24 271 428 467 515 1708 1 3 26 153 373 712 1268 WT/FR versus WT/D 0 0 26 121 256 450 853 0 0 0 8 44 287 339 WT/R versus WT/D 3 11 85 277 320 506 1202 0 1 3 51 249 646 950 WT/B versus WT/D 0 1 71 305 247 472 1096 0 0 1 40 141 434 616 WT/W versus WT/36D 0 0 2 148 308 443 901 1 0 5 20 96 399 521 WT/36W versus WT/D 0 5 68 299 359 578 1309 2 5 5 80 306 653 1051 WTL/W versus WTL/36D 1 1 1 74 241 417 735 2 4 10 83 209 472 780 WT/FR versus phyA/FR 0 0 0 12 202 522 736 0 0 1 35 92 359 487 WT/R versus phyB/R 0 0 0 0 5 97 102 0 0 0 0 8 64 72 WT/B versus cry1 cry2/B 0 2 3 185 252 434 876 0 0 2 7 84 290 383 PhyAOE/FR versus WT/FR 9 6 19 24 50 276 384 0 0 1 6 68 189 264 PhyBOE/R versus WT/R 0 0 0 1 5 66 72 0 0 0 0 7 77 84 CRY1OE/B versus WT/B 1 0 3 6 49 281 340 0 0 9 30 127 357 523 a WT, wild type; W, white light; D, darkness; FR, far-red light; R, red light; B, blue light; 36, 36-hr transition; L, 5-week-old leaves; phyA, phyA mutant; phyB, phyB mutant; cry1 cry2, cry1 and cry2 double mutants; PHYAOE, PHYA overexpression; PHYBOE, PHYB overexpression; CRY1OE, CRY1 overexpression.
- Table 4.
Number of Clones Involved in Different Functional Groups Upregulated or Downregulated at Least Two-Fold by White Light
Pathways Gene Number Representative Gene Namesa Upregulated Photosynthetic light reactions 56 17 CAB genes, 15 PSI proteins, 14 PSII proteins, 10 proteins involved in electron transport and ATP synthesis Photosynthetic carbon metabolism 25 4 RBCS genes, 2 phosphoglyceate kinases, 2 GAPDHs, triose-phosphate isomerase, aldolase, fructose-1,6-phosphate phosphatase, 3 fructose-1,6-bisphosphate aldolases, transketolase, sedoheptulose 1,7-bisphosphate phosphatase, ribulose-5-phosphate epimerase, 2 ribose-5-phosphate isomerases, ribulose-5-phosphate kinase, ferredoxin, 2 thioredoxins, 2 ferredoxin-thioredoxin reductases, Rubisco activase Starch synthesis 3 Fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphate phosphatase, pyrophosphorylase Sucrose synthesis 5 Phosphate/triose phosphate translocator, cytosolic triose-phosphate isomerase, cytosolic fructose-1,6-bisphosphate aldolase, phosphofructokinase, sucrose synthase Photorespiration 8 4 RBCS genes, glycolate oxidase, catalase, hydroxypyruvate reductase, glycine decarboxylase Glycolysis 5 Fructokinase, triose phosphate isomerase, pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase Trichloroacetic acid cycle 5 Pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, phosphopyruvate hydratase, malate dehydrogenase Cell wall synthesis and cell wall protein 8 Cellulose synthase, glucosyltransferase, β-1,2-xylosyltransferase, UDP-glucose:glycoprotein glucosyltransferase, cell wall–plasma membrane linker protein, proline-rich protein, extensin, AGP Protein synthesis in chloroplast 56 11 50S ribosomal proteins, 4 30S ribosomal proteins, 7 other ribosomal proteins, 9 initiation and elongation factors, 9 aminoacyl-tRNA synthetases, 6 RNA helicases, 10 chaperonines Protein synthesis in cytoplasm 60 15 60S ribosomal proteins, 3 40S ribosomal proteins, 8 other ribosomal proteins, 9 initiation and elongation factors, 9 aminoacyl-tRNA synthetases, 6 RNA helicases, 10 chaperonins Phenylpropanoid biosynthesis 8 PAL1, CHS, caffeoyl-CoA O-methyltransferase, cinnamyl-alcohol dehydrogenase, O-methyltransferase, GST, glutathione-conjugate transporters, cytochrome P450 Amino acid synthesis pathways 15 Dehydrogenase, glutathione peroxidase, Δ-1-pyrroline-5-carboxylate synthetase, phosphoribosylglycinamide synthetase, histidinol dehydrogenase, acetolactate synthase, aminotransferase, adenine phosphoribosylanthranilate transferase, phosphoribosylanthranilate transferase, uridylyl transferase, glutamine synthetase, glutamate/ornithine acetyltransferase, 3-isopropylmalate dehydratase, glutathione reductase, tryptophan synthase Chlorophyll and heme synthesis 4 Ferrochelatase, glutamyl-tRNA reductase, protoporphyrinogen IX oxidase, glutamate-1-semialdehyde 2,1-aminomutase 1 Transcription factors 15 G-box binding factor, B-box zinc finger, 2 bHLH transcription factors, 3 MYB-related transcription factors, SCARECROW-like factor, zinc finger transcription factor–like protein, AP2 domain transcription factor-like protein, bZIP transcription factor, transcription factor HUA2, transcription factor II homolog, homeobox leucine zipper protein, putative transcription factor Uniquitin-proteasome pathway 23 4 polyuniquitin and ubiquitin genes, 9 26S proteasome subunits, 2 proteasome subunit–related proteins, E1, E2, and 6 E3 genes Downregulated Ethylene biosynthesis 3 ACC synthase, ACC oxidase, SAM synthase BR biosynthesis 4 24-sterol C-methyltransferase, C-8,7-sterol isomerase, 3-β-hydroxysteroid dehydrogenases, steroid sulfotransferase Cell wall degradation and cell wall proteins 26 Cellulase, XET, 2 expansins, xylosidase, xyloglucan endo-1,4-β-d-glucanase, endo-1,4-β-d-glucanase, 1,3-β-glucanase, xylose isomerase, pectinesterase, pectate lyase, 7 peroxidases, 2 extensin homologs, glycine-rich protein, AGP4, 2 proline-rich cell wall proteins, hydroxyproline-rich protein, pollen surface protein homolog Water transport across tonoplast 5 5 aquaporins in tonoplast Water transport across plasma membrane 5 5 water channel and membrane intrinsic proteins in plasma membrane Sulfur assimilation 9 2 GSTs, sulfate adenylyltransferase, sulfate transporter, phytochelatin synthase, cysteine synthase, cytochrome P450 monooxygenase, 2 ABC transporters Nitrogen assimilation 9 3 nitrate transporters, 2 AS proteins, glutamine synthetase, glutamate dehydrogenase, serine carboxypeptidase, cysteine synthase Fatty acid oxidation 5 Glycerol-3-phosphate dehydrogenase, inorganic pyrophosphatase, ketoacyl-CoA thiolase, malic enzyme, acyl-CoA oxidase Glyoxylate cycle 4 isocitrate lyase, citrate synthase, malate synthase, malate dehydrogenase Transcription factors 18 3 AP2 domain transcription factors, 2 MYB-related transcription factors, transcription factor L2, transcription factor tga1, transcription factor EREBP, MADS box transcription factor–like protein, TATA box binding protein, transcription factor–like protein, putative HLH DNA binding protein, homeobox leucine zipper proteins HAT4 and HAT5, homeodomain transcription factor–like protein, CONSTANS-like B box zinc finger protein–like protein, abscisic acid–responsive element binding factor, homeobox protein Uniquitin-proteasome pathway 16 6 polyuniquitin and ubiquitin genes, 2 26S proteasome subunits, 5 E2 and 3 E3 genes a PSI and PSII, photosystems I and II; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; GST, glutathione S-transferase; ACC, 1-aminocyclopropane-1-carboxylic acid; SAM, S-adenosyl methionine.
