The Dof transcription factor OBP3 modulates phytochrome and cryptochrome signaling in Arabidopsis

Abstract

Plants perceive subtle changes in light quality and quantity through a set of photoreceptors, including phytochromes and cryptochromes. Upon perception, these photoreceptors initiate signal transduction pathways leading to photomorphogenic changes in development. Using activation-tagging mutagenesis to identify novel light-signaling components, we have isolated a gain-of-function mutant, sob1-D (suppressor of phytochrome B-4 [phyB-4] dominant), which suppresses the long-hypocotyl phenotype of the phyB missense allele, phyB-4. The sob1-D mutant phenotype is caused by the overexpression of a Dof (DNA binding with one finger) transcription factor, OBF4 Binding Protein 3 (OBP3). A translational fusion between OBP3 and green fluorescent protein is nuclear localized in onion (Allium cepa) cells. Tissue-specific accumulation of an OBP3:OBP3-beta-glucuronidase translational fusion is regulated by light in Arabidopsis thaliana. Hypocotyls of transgenic lines with reduced OBP3 expression are less responsive to red light. This aberrant phenotype in red light requires functional phyB, suggesting that OBP3 is a positive regulator of phyB-mediated inhibition of hypocotyl elongation. Furthermore, these partial-loss-of-function lines have larger cotyledons. This light-dependent cotyledon phenotype is most dramatic in blue light and requires functional cryptochrome 1 (cry1), indicating that OBP3 is a negative regulator of cry1-mediated cotyledon expansion. These results suggest a model where OBP3 is a component in both phyB and cry1 signaling pathways, acting as a positive and negative regulator, respectively. An alternate, though not mutually exclusive, model places OBP3 as a general inhibitor of tissue expansion with phyB and cry1, differentially modulating OBP3's role in this response.

Figure 1.

Phenotypic Analysis of the sob1-D Mutant and Cloning of the SOB1 Gene. (A) Seedlings were grown in continuous white light (35 μM/m²/s) or in the dark for 5 d. (B) Three-week-old plants were grown in long-day (16 h light/8 h dark) growth conditions. (C) A diagram of the DNA that was cloned by plasmid rescue. The diagram shows the insertion site relative to the OBP3 coding region and the restriction sites used to clone the DNA. The red ovals represent the four enhancers from the CaMV 35S promoter present in the T-DNA, the blue and green bars represent pBluescript KS+ and nptII, respectively. (D) Total RNA was isolated from 5-d-old seedlings grown in continuous white light. PCR was performed on cDNA using OBP3-specific primers for 32 cycles. The UBQ10 cDNA, amplified for 24 cycles, was used to normalize the amount of cDNA in each of the samples. (E) RT-PCR was performed on total RNA isolated from 5-d-old seedlings grown in continuous white light or in the dark. PCR was performed as described in (D).

Figure 2.

Subcellular and Tissue-Specific Localization of OBP3. (A) to (C) Onion epidermal cells were cobombarded with constructs containing either UBQ:dsRed (A) or 35S:OBP3-GFP (B). The two images are merged to show the subcellular localization of OBP3 (C). (D) to (I) Wild-type Arabidopsis was transformed with a T-DNA containing genomic DNA encoding OBP3 translationally fused to the GUS gene. Expression of this fusion gene was controlled by ∼1.4 kb of DNA 5′ of the OBP3 gene. OBP3:OBP3-GUS localization was observed in 5-d-old seedlings grown either in the dark ([D] and [E]) or in continuous white light ([F] to [I]). GUS staining was observed in the apex and cotyledons of dark-grown seedlings ([D] and [E]). In light-grown seedlings, GUS staining was observed in the apex ([F] and [I]) and in the vascular tissue of the cotyledons ([F] and [G]), hypocotyl ([F] and [I]), and roots (H).

Figure 3.

Identification of the OBP3-RNAi Mutants. (A) Total RNA was isolated from 5-d-old seedlings grown in continuous white light. PCR was performed on cDNA by amplifying with OBP3-specific primers for 32 cycles. cDNA was normalized by amplifying with UBQ10 primers for 24 cycles. (B) The wild type and OBP3-RNAi homozygous, single locus insertion lines were grown in long-day growth conditions for 3 weeks. Bar = 10 mm.

Figure 4.

Fluence Rate Response Analysis for Hypocotyl Length of the OBP3-RNAi1-4-3 Mutant. Five-day-old seedlings were grown in the dark or varying fluence rates of continuous white (A), red (B), blue (C), and far-red (D) light. Bars = ±1 se. (*) equals P < 0.5, and (**) equals P < 0.001 from a Student's unpaired two-tail t test comparing the OBP3-RNAi mutant and the wild type from the same light treatment.

Figure 5.

Photoreceptor Double Mutant Analysis for Hypocotyl Length. Five-day-old seedlings were grown in the dark or continuous red light (29 μM/m²/s). The phyB-9 allele is a phyB-null mutant, and the phyB-4 allele is a phyB-missense mutant. The hypocotyls of the dark-grown double mutant were longer when compared with the wild type, so the data are normalized to the average dark-grown hypocotyl length for each line. Bars = ±1 se. (*) equals P < 0.5, and (**) equals P < 0.001 from a Student's unpaired two-tail t test comparing the mutant with its control.

Figure 6.

Cotyledon Cell Expansion in White Light. (A) Photographs of cotyledons from seedlings grown in continuous white light for 5 d. (B) Epidermal imprints of cotyledons from seedlings grown in continuous white light for 5 d. (C) Cotyledon area from 5-d-old seedlings grown in the dark or continuous white light (35 μM/m²/s). Error bars = ±1 se.

Figure 7.

Cotyledon Expansion in Response to Different Wavelengths of Light. (A) Cotyledon area from 5-d-old seedlings grown in continuous blue light (17 μM/m²/s). The cry1-102 allele is a cry1-missense mutant, and the cry1-103 allele is a cry1-null mutant. (B) Cotyledon area from 5-d-old seedlings grown in continuous red light (29 μM/m²/s). (C) Cotyledon area from 5-d-old seedlings grown in continuous far-red light (33 μM/m²/s). Error bars = ±1 se. (*) equals P < 0.5, and (**) equals P < 0.001 from a Student's unpaired two-tail t test comparing the mutant with its control.

Figure 8.

Possible Models Describing OBP3's Involvement in phyB and cry1 Signaling. (A) OBP3 acts as a negative regulator of cry1-mediated cotyledon expansion in blue light and a positive regulator of phyB-mediated inhibition of hypocotyl elongation in red light. (B) OBP3 is a negative regulator of expansion in the cotyledons and elongation in the hypocotyl. This regulation is modulated both negatively and positively by cry1 and phyB, respectively.