UV-B signaling pathways with different fluence-rate response profiles are distinguished in mature Arabidopsis leaf tissue by requirement for UVR8, HY5, and HYH

Abstract

UV-B signaling is an important but poorly understood aspect of light responsiveness in plants. Arabidopsis (Arabidopsis thaliana) UV RESISTANCE LOCUS8 (UVR8) is a recently identified UV-B-specific signaling component that regulates UV-protective responses. Using the uvr8 mutant, we defined genetically distinct UVR8-dependent and UVR8-independent pathways that stimulate different sets of genes in mature Arabidopsis leaf tissue. Both pathways operate at 1 micromol m(-2) s(-1) UV-B and above, but the UVR8-dependent pathway is able to stimulate UV-protective genes even in response to 0.1 micromol m(-2) s(-1) UV-B. Both pathways function in mutants lacking phytochromes, cryptochromes, or phototropins. Genes encoding the ELONGATED HYPOCOTYL5 (HY5) and HY5 HOMOLOG (HYH) transcription factors are induced at low UV-B fluence rates (0.1 micromol m(-2) s(-1)). Experiments with hy5 and hyh mutants reveal that both these factors mediate responses of the UVR8-dependent pathway, acting with partial or complete redundancy to stimulate expression of particular genes. Furthermore, evidence is presented that all UVR8 pathway genes are likely to be regulated by HY5/HYH and that these transcription factors do not mediate UV-B responses independent of UVR8. Finally, we highlight the functions of HY5 and HYH in UV protection and show that HY5 plays the more critical role. This research provides evidence that, in UV-B signaling, UVR8, HY5, and HYH act together in a photoregulatory pathway and demonstrates a new role for HYH in UV-B responses.

Figure 1.

Transcript accumulation in wild-type and uvr8 mutant under different UV-B fluence rates. A, Wild-type Ler and uvr8-2 mutant plants grown for 3 weeks in a low fluence rate (20 μmol m⁻² s⁻¹) of white light were transferred to various fluence rates of UV-B light for 4 h. Leaf tissue was harvested, RNA isolated, and cDNA synthesized. Transcript levels for the genes indicated were measured by RT-PCR. ACTIN2 transcript levels are shown as a loading control. The data shown are representative of up to four independent experiments. B, Levels of selected transcripts were quantified from RT-PCR products obtained as in A using imaging software. The images show combined data from three independent experiments. Each point shows the transcript level relative to ACTIN2 in the same experiment. In each image, the data from the three experiments are normalized relative to the transcript level in wild type at 3 μmol m⁻² s⁻¹ UV-B, set at 1.0. Black circles, Wild type; white squares, uvr8-2.

Figure 2.

Transcript accumulation in response to UV-B in photoreceptor mutants. Wild-type, phyA phyB, cry1 cry2, hy1-100, and phot1 phot2 mutant plants grown for 3 weeks in a low fluence rate (25 μmol m⁻² s⁻¹) of white light were transferred to 3 μmol m⁻² s⁻¹ UV-B light for 4 h. Leaf tissue was harvested and transcript levels measured as in Figure 1.

Figure 3.

Transcript accumulation in hy5, hyh, and hy5 hyh mutants under different UV-B fluence rates. Wild-type, hy5, hyh, and hy5 hyh mutant plants grown for 3 weeks in a low fluence rate (25 μmol m⁻² s⁻¹) of white light were transferred to various fluence rates of UV-B light for 4 h. Leaf tissue was harvested and transcript levels measured as in Figure 1.

Figure 4.

Redundancy of HY5 and HYH in the regulation of gene expression by UV-B. Wild-type, hy5, hyh, and hy5 hyh mutant plants grown for 3 weeks in a low fluence rate (25 μmol m⁻² s⁻¹) of white light were transferred to various fluence rates of UV-B light for 4 h. Leaf tissue was harvested and transcript levels measured as in Figure 1.

Figure 5.

Failure to identify genes regulated by HY5/HYH, but not UVR8. A, Wild-type (Ler), uvr8-2, and hy5 mutant plants grown for 3 weeks in a low fluence rate (20 μmol m⁻² s⁻¹) of white light were transferred to various fluence rates of UV-B light for 4 h. B, Wild-type (Ws) and hy5 hyh mutant plants grown for 3 weeks in a low fluence rate (25 μmol m⁻² s⁻¹) of white light were transferred to various fluence rates of UV-B light for 4 h. Leaf tissue was harvested and transcript levels measured as in Figure 1.

Figure 6.

UV-B sensitivity assay. Wild-type and mutant plants were grown in 120 μmol m⁻² s⁻¹ white light for 12 d and then exposed to above-ambient (5 μmol m⁻² s⁻¹) UV-B with supplementary 120 μmol m⁻² s⁻¹ white light for the durations shown. Untreated control plants (0 h, - UV-B) were left in 120 μmol m⁻² s⁻¹ white light throughout. Plants were photographed after return to white light for approximately 5 d.

Figure 7.

Model showing the UVR8-dependent and UVR8-independent UV-B signaling pathways and representative genes they regulate in mature Arabidopsis leaf tissue. The broken arrow leading from HYH to the CHS, ELIP1, and CRYD genes indicates that HYH cannot completely compensate for the absence of HY5 in this pathway. The large arrow leading from UV-B to UVR8 overlaps the low fluence and high fluence range, indicating that both high and low UV-B fluence rates can activate UVR8.