Overexpression of a mutant basic helix-loop-helix protein HFR1, HFR1-deltaN105, activates a branch pathway of light signaling in Arabidopsis

Abstract

The HFR1, a basic helix-loop-helix protein, is required for a subset of phytochrome A-mediated photoresponses in Arabidopsis. Here, we show that overexpression of the HFR1-deltaN105 mutant, which lacks the N-terminal 105 amino acids, confers exaggerated photoresponses even in darkness. Physiological analysis implied that overexpression of HFR1-deltaN105 activated constitutively a branch pathway of light signaling that mediates a subset of photomorphogenic responses, including germination, de-etiolation, gravitropic hypocotyl growth, blocking of greening, and expression of some light-regulated genes such as CAB, DRT112, PSAE, PSBL, PORA, and XTR7, without affecting the light-responsiveness of anthocyanin accumulation and expression of other light-regulated genes such as CHS and PSBS. Although the end-of-day far-red light response and petiole elongation were suppressed in the HFR1-deltaN105-overexpressing plants, flowering time was not affected by HFR1-deltaN105. In addition, the HFR1-deltaN105-overexpressing plants showed hypersensitive photoresponses in the inhibition of hypocotyl elongation, dependently on phytochrome A, FHY1, and FHY3 under FR light or phyB under R light, respectively. Moreover, our double mutant analysis suggested that the hypersensitive photoresponse is due to functional cooperation between HFR1-deltaN105 and other light-signaling components including HY5, a basic leucine zipper protein. Taken together, our results of gain-of-function approach with HFR1-deltaN105 suggest the existence of a complex and important basic helix-loop-helix protein-mediated transcriptional network controlling a branch pathway of light signaling and provide a useful framework for further genetic dissection of light-signaling network in Arabidopsis.

Figure 1.

Constitutive photomorphogenic phenotypes of HFR1-ΔN105-overexpressing transgenic Arabidopsis. A, Fluence rate responses of inhibition of hypocotyl elongation under FR light. The data are expressed as average relative hypocotyl length from at least 20 seedlings, normalized to their respective hypocotyl length in darkness ± sd. The average length of dark-grown seedlings was 9.8, 8.9, and 10.2 mm for wild-type, hfr1HFR1 ox-39, and hfr1-201 seedlings, respectively. Error bars = sds. WT, Wild type. B, Hypocotyl elongation phenotypes in wild-type and transgenic plants. The seedlings were grown for 4 d under FR light (21 μW cm^-2) or in darkness. Each measurement was performed on at least 20 seedlings. The data are expressed as average hypocotyl length ± sd. C, Morphology of representative seedlings. The seedlings were grown for 5 d in darkness. Scale bar = 5 mm. D, RNA gel-blot analysis of wild-type and transgenic plants overexpressing full-length HFR1 or HFR1-ΔN105. Total RNA (10 μg) was loaded and subject to RNA gel-blot analysis. The blot was hybridized with a ³²P-labeled HFR1-ΔN105 probe. The 18S rRNA was used as a loading control. The signals were visualized with a phosphor imager (FLA2000, Fuji, Tokyo).

Figure 2.

Phytochrome-dependent germination response. A, Summary of seeds used in the germination experiment. WT, Wild type. B, Germination frequencies of wild-type or various transgenic plant lines overexpressing HFR1 or HFR1-ΔN105 seeds were measured. The seeds were treated with FR light (21 μW cm^-2) for 15 min just after imbibition and then transferred to darkness without or with exposure to R light (33 μW cm^-2) for 10 min. Seeds were then incubated in darkness for an additional 5 d. Each experiment was performed with at least 150 seeds. Similar results were obtained from three independent experiments. C, Representative plates from the germination experiments described in B. The plates were given a pulse of FR light and kept in darkness. D, Representative plates from the germination experiments described in B. The plates were given a pulse of R light after a pulse of FR light and then kept in further darkness.

Figure 3.

Gravitropic response of hypocotyl growth, greening response, and anthocyanin accumulation of transgenic plants overexpressing HFR1-ΔN105. A, Gravitropic response of hypocotyl growth. The seedlings were vertically grown in darkness or FR light (21 μW cm^-2) for 4 d. Upper, Representative seedlings. Lower, Bars = sds of the hypocotyl growth orientations from at least 70 seedlings. The higher scores indicate more reduced gravitropism of hypocotyl growth. Error bars = ses from three independent experiments. WT, Wild type. B, FR-preconditioned blocking of greening. The seedlings were grown on Murashige and Skoog medium for 5 d in FR light (21 μW cm^-2) and then irradiated with W light for indicated times. Data are expressed as average chlorophyll content (in milligrams) from 50 seedlings ± sd and were derived from three independent measurements. C, Accumulation of anthocyanin under different fluence rate of FR light. Anthocyanin measurement was performed on seedlings grown for 3 d under FR light at the indicated fluence rates. Bars = sds from three independent measurements.

Figure 4.

phyA-dependent gene expression of light-regulated genes in the transgenic plants overexpressing HFR1-ΔN105. A, RNA gel-blot analysis. Seedlings were grown on Murashige and Skoog-Suc medium (2% w/v) for 4 d in darkness and then transferred to FR light (14 μW cm^-2) or kept in darkness (D) for an additional 12 h before extraction of total RNA. Total RNA (10 μg) was loaded and subject to RNA gel-blot analysis. The 18S rRNA was used as a loading control. Similar results were obtained in two independent experiments. WT, Wild type. B, Quantitative measurement of individual transcripts shown in A from seedlings kept in darkness (black bars) or given by 12 h of FR light (hatched bars). The values denotes relative expressions and were calculated by first normalizing each signal against 18S rRNA and then against the lowest amount of expression for each particular gene. The signals were visualized and quantified with a phosphor imager (FLA2000, Fuji). A similar trend was repeated in another independent experiment.

Figure 5.

Photoresponses of transgenic plants overexpressing HFR1-ΔN105 under W. A, EOD FR responses of wild-type and transgenic plants. The average hypocotyl lengths ± sds are shown from at least 20 seedlings in each group. White bars, No EOD-FR treatments; black bars, EOD-FR treatments. WT, Wild type. B, Morphology in adult wild type and transgenic plants overexpressing HFR1-ΔN105. The plants were grown for 20 d under long-day (LD; 16 h of light/8 h of darkness) or for 24 d under SD (8 h of light/16 h of darkness) conditions. Scale bar = 5 mm. C, Flowering time responses of wild-type and transgenic plants overexpressing HFR1-ΔN105. The flowering time was defined as the number of days from seed sowing until opening of the first flower. LD consists of 16 h of fluorescent lighting and 8 h of darkness. SD consists of 8 h of fluorescent lighting and 16 h of darkness.

Figure 6.

Hypocotyl elongation responses of transgenic plants overexpressing HFR1-ΔN105 and double mutant analysis. A, Fluence rate responses of inhibition of hypocotyl elongation under R light. The data are expressed as average relative hypocotyl length from at least 20 seedlings, normalized to their respective hypocotyl length in darkness ± sd. The average length of dark-grown seedlings was 10.6 and 7.1 mm for the wild-type and HFR1-ΔN105 ox-9 seedlings, respectively. Error bars = sds. WT, Wild type. B, Fluence rate responses of inhibition of hypocotyl elongation under FR light. The data are expressed as average relative hypocotyl length from at least 20 seedlings, normalized to their respective hypocotyl length (Legend continues on facing page.) (Legend continued from facing page) in darkness ± sd. The average length of dark-grown seedlings was 9.9 and 7.1 mm for the wild-type and HFR1-ΔN105 ox-9 seedlings, respectively. C, Hypocotyl elongation phenotypes in wild-type and transgenic plants. The seedlings were grown for 4 d under R light (33 μW cm^-2), FR light (14 μW cm^-2), or in darkness (D). Each measurement was performed on at least 20 seedlings. The data are expressed as average hypocotyl length ± sd. D, Hypocotyl elongation phenotypes in wild-type and transgenic plants. The seedlings were grown for 4 d under FR light (14 μW cm^-2) or in darkness (D). Each measurement was performed on at least 20 seedlings. The data are expressed as average hypocotyl length ± sd. E, RNA gel-blot analysis. Seedlings were grown on Murashige and Skoog-Suc medium (2% w/v) for 4 d in darkness (D) or under FR light (14 μWcm^-2) before extraction of total RNA. Total RNA (10 μg) was loaded and subject to RNA gel-blot analysis using HFR1-ΔN105 probe.