Brassinosteroid mutants uncover fine tuning of phytochrome signaling

Abstract

Phytochromes (phy) A and B provide higher plants the ability to perceive divergent light signals. phyB mediates red/far-red light reversible, low fluence responses (LFR). phyA mediates both very-low-fluence responses (VLFR), which saturate with single or infrequent light pulses of very low fluence, and high irradiance responses (HIR), which require sustained activation with far-red light. We investigated whether VLFR, LFR, and HIR are genetically coregulated. The Arabidopsis enhanced very-low-fluence response1 mutant, obtained in a novel screening under hourly far-red light pulses, showed enhanced VLFR of hypocotyl growth inhibition, cotyledon unfolding, blocking of greening, and anthocyanin synthesis. However, eve1 showed reduced LFR and HIR. eve1 was found allelic to the brassinosteroid biosynthesis mutant dim/dwarf1. The analysis of both the brassinosteroid mutant det2 in the Columbia background (where VLFR are repressed) and the phyA eve1 double mutant indicates that the negative effect of brassinosteroid mutations on LFR requires phyA signaling in the VLFR mode but not the expression of the VLFR. Under sunlight, hypocotyl growth of eve1 showed little difference with the wild type but failed to respond to canopy shadelight. We propose that the opposite regulation of VLFR versus LFR and HIR could be part of a context-dependent mechanism of adjustment of sensitivity to light signals.

Figure 1

Phenotype of eve1 seedlings (A) and adult plants (B) and of the F₁ generation between eve1 and dim (C). In C, inset, a PCR marker for the dim (dwf1-2) allele was used in seedlings homozygous for eve1 (lane 1) or dim (lane 2). The presence of the dim allele in lane 3 demonstrates that the seedling in the photograph is product of a successful cross between eve1 (mother plant) and dim.

Figure 2

dwf1-101 (previously designated eve1) shows enhanced VLFR and reduced LFR of hypocotyl growth (A and B) and cotyledon unfolding (C). The seedlings were exposed to hourly R/FR pulses predicted to establish the calculated Pfr/P displayed in abscissas. In B, the difference between hypocotyl length in darkness and a given light condition is expressed relative to the length in darkness. Data are means ± se of 18 replicate boxes.

Figure 3

Reduced slope of the hypocotyl growth inhibition response to continuous FR in dwf1-101. Hypocotyl length in dark controls: WT = 11.4 ± 0.3; dwf1-101 = 4.6 ± 0.3. Data are means ± se of nine replicate boxes.

Figure 4

dwf1-101 shows enhanced VLFR and reduced HIR of hypocotyl growth inhibition (A), cotyledon unfolding (B), blocking of greening (C), and anthocyanin synthesis (D). The seedlings were grown in darkness, hourly pulses of FR or continuous FR (at equal total fluence) before measurements or transfer to white light (chlorophyll experiments). Hypocotyl length in dark controls: WT = 8.4 ± 0.2; dwf1-101 = 4.3 ± 0.2. Data are means ± se of 12 (A and B), nine (C), or six (D) replicate boxes.

Figure 5

Reduced HIR and LFR in the brassinosteroid mutant det2. In A, the seedlings were grown in darkness or under hourly FR, hourly R, or continuous FR (4 or 100 μmol m⁻² s⁻¹) In B, the seedlings were daily exposed to a R versus a FR pulse given in factorial combination with 3 h of FR, 3 h of blue light, or darkness. The LFR (angle between the cotyledons for seedlings receiving a R pulse minus angle between the cotyledons for the seedlings receiving a FR pulse) is indicated for the 3 h of FR, 3 h of blue light, or no previous light conditions. Data are means ± se of 21 replicate boxes.

Figure 6

Seedling phenotype of the phyA dwf1-101 double mutant (A) and adult phenotype of the phyA dwf1-101 and phyB dwf1-101 double mutants (B). In A, data are means ± se of six replicates. Hypocotyl length in dark controls: WT = 9.7 ± 0.6; dwf1-101 = 4.8 ± 0.1; phyA = 11.8 ± 0.5; phyA dwf1-101 = 5.0 ± 0.3.

Figure 7

Normal levels of inmunochemically detectable phyA in dwf1-101. The seedlings were grown in darkness for 4 d after the R pulse given for the induction of seed germination.

Figure 8

The dwf1-101 mutant fails to respond to the presence of a shading canopy. The seedlings were grown in pots under sunlight or canopy shade light. The length of the hypocotyl (mm) in dark controls (grown near the other seedlings inside dark boxes) was: WT = 11.4 ± 0.7; dwf1-101 = 8.1 ± 0.4. Data are means ± se of 12 replicate plants.

Figure 9

Model based on genetic and physiological data showing the proposed role of DWF1 in the phytochrome signaling network.