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First published online 5 October 2005
doi: 10.1242/dev.02055

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The WEREWOLF MYB protein directly regulates CAPRICE transcription during cell fate specification in the Arabidopsis root epidermis

¹ Department of Biology, Yonsei University, Sinchon 134, Seoul 120-749, Korea
² Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea
³ Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA

View larger version (51K): [in a new window]   Fig. 1. Subcellular localization of the WER-GFP. (A) Schematic diagrams of PWER:GFP and PWER:WER-GFP gene constructs. The stop codon of the WER was deleted for the translational fusion with the GFP. (B) Complementation of the wer mutation with PWER:WER-GFP. The wer-1 mutant was transformed with PWER:WER-GFP. wt, wild type. (C) WER-GFP accumulation in the Arabidopsis root epidermal cells. The wer-1 mutant transformed with PWER:WER-GFP and the wild-type plant transformed with PWER:GFP were grown on agarose-solidified media. Fluorescence was observed in the 4-day-old seedlings using confocal microscopy.

View larger version (40K): [in a new window]   Fig. 2. Effect of P35S:WER-VP16 on cell fate specification in the root epidermis. (A) Schematic diagram of P35S:WER-VP16. The stop codon of the WER was deleted. (B) Comparison of the transcriptional activation activity of the WER-VP16 recombinant protein with the activity of the native WER protein. Error bars indicate s.d. (C) Expression pattern of PGL2:GUS in the epidermis of 4-day-old seedlings. Seedlings were stained for 4 hours. wt, wild type.

View larger version (59K): [in a new window]   Fig. 3. Direct regulation of CPC transcription by the WER. (A) Schematic diagram of P35S:WER-GR gene construct. The stop codon of the WER was deleted for the translational fusion with the hormone-binding domain of the glucocorticoid receptor (GR HBD; from the 519th amino acid to the end). (B) Expression of the PCPC:GUS reporter gene in the epidermis of 4-day-old seedlings. Seedlings were stained for 8 hours. (C) Expression of the PGL2:GUS reporter gene in the epidermis of 4-day-old seedlings. (Top) The seedlings were stained for 6 hours without any treatment. wt, wild type. (Bottom) The seedlings harboring P35S:WER-GR (P35S:WER-GR wer-1) were untreated (–DEX), treated with 10 µM of cycloheximide (CHX) alone, treated with 1 µM of dexamethasone (+DEX) alone, or treated with both (+DEX, +CHX). Seedlings were pre-treated with 10 µM of CHX for 20 minutes before treatment with CHX and DEX. Seedlings were stained for 6 hours. (D) Accumulation of CPC transcripts in transgenic plants harboring P35S:WER-GR. P35S:WER-GR wer-1 seedlings (4-day-old) were treated as described in C. Northern blot analysis was carried out with total RNA extracted from the root tips using radiolabeled CPC DNA fragment as a probe (Lee and Schiefelbein, 2002). Resulting signal was detected using BAS-2500 (Fujifilm).

View larger version (55K): [in a new window]   Fig. 4. Binding of the WER to CPC promoter. (A) EMSA using the purified WER protein and two 20 bp long DNA fragments (WBSI and WBSII) from the CPC promoter. Lane 1 of each gel contains the free DNA probe without the WER protein, and lane 2, 3 and 4 of each gel contain increasing amount of the WER protein (1x, 3x and 6x). (B) Sequences of mutated WBSI and WBSII used in this experiment. Each double-stranded probe contains a single base substitution as indicated. Lines indicate no change. (C) EMSA using the purified WER protein and the mutated probes as shown in B. (D) Competitive EMSA. Competitions were performed using increasing amounts of wild-type DNA fragments or some mutated derivatives as shown in B. Lane 1 of each gel contains the free DNA probe without the WER protein and lane 2 contains the WER protein and the radiolabeled wild-type DNA fragment (WBSI or WBSII) as a probe without a competitor. Increasing amounts (1x, 10x and 50x) of the unlabeled wild type DNA fragments (lanes 3, 4 and 5), and the unlabeled mutated versions of WBSI or WBSII (lanes 6, 7, 8 and lanes 9, 10, 11) were added as a competitor. (E) Binding competition between WBSI and WBSII. Increasing amounts of the wild-type or mutated derivative (m10) of WBSII, and increasing amounts of the wild-type or mutated derivative (m6) of WBSI were used as an unlabeled competitor for the binding of the WER protein to radiolabeled WBSI and to radiolabeled WBSII, respectively. Lane 1 contains only free probe (radiolabeled WBSI or WBSII) and lane 2 contains the WER protein and the radiolabeled wild-type DNA fragment (WBSI or WBSII) as a probe without a competitor. Lanes 3, 4 and 5 of each gel contain increasing amounts of the unlabeled wild-type DNA fragments (unlabeled WBSI for radiolabeled WBSII, and unlabeled WBSI for radiolabeled WBSII) as a competitor (1x, 10x and 50x). Lanes 6 and 7 contain the same components as lane 3, 4 and 5, except that they contain increasing amounts of unlabeled mutated versions of WBSII (m10) or WBSI (m6) instead of the unlabeled wild-type DNA fragments as a competitor.

View larger version (22K): [in a new window]   Fig. 5. Binding of the WER protein to WBSI in yeast. (A) Schematic diagrams of the reporter genes and the effectors used in this yeast one-hybrid assay. Three tandem repeats of each DNA fragment were inserted upstream of a reporter gene (lacZ) in the vector pLacZi and then yeast reporter strains were made by genomic integration of these reporters. (B) Yeast one-hybrid assay with WBSI as a cis-acting element. AD-WER was expressed in the yeast reporter strains harboring a reporter gene that has the wild-type or one of the mutant derivatives of WBSI in its upstream as shown in A. A ß-galactosidase assay is performed to verify the DNA-protein interaction using CPRG as a substrate.

View larger version (29K): [in a new window]   Fig. 6. Effect of the WER protein on CPC expression in Arabidopsis. (A,C) Schematic diagrams of the reporter genes and the effectors used in this transient expression assay (A) and stable transgenic study (C). For reporter constructs, several versions of CPC promoters that are 700 bp long (from the translational start site) were inserted upstream of the luciferase gene (A) or ß-glucuronidase gene (C). Each mutant reporter contains mutations at WBSI or WBSII, or both. For the effector construct, the genomic DNA fragment of WER-coding region was inserted downstream of the 35S promoter (A). (B) Importance of WBSI and WBSII (refer to A) in CPC expression in the Arabidopsis protoplast transient expression assay. Protoplasts were transfected with UBQ10-GUS as an internal control, each of the reporters (wild type, M1, M2 or M3) and an effector (WER). (D) Importance of WBSI and WBSII (refer to C) in the proper expression of CPC in the Arabidopsis root epidermis. The seedlings were stained for 24 hours.