Arabidopsis thaliana life without phytochromes

Abstract

Plants use light as a source of energy for photosynthesis and as a source of environmental information perceived by photoreceptors. Testing whether plants can complete their cycle if light provides energy but no information about the environment requires a plant devoid of phytochromes because all photosynthetically active wavelengths activate phytochromes. Producing such a quintuple mutant of Arabidopsis thaliana has been challenging, but we were able to obtain it in the flowering locus T (ft) mutant background. The quintuple phytochrome mutant does not germinate in the FT background, but it germinates to some extent in the ft background. If germination problems are bypassed by the addition of gibberellins, the seedlings of the quintuple phytochrome mutant exposed to red light produce chlorophyll, indicating that phytochromes are not the sole red-light photoreceptors, but they become developmentally arrested shortly after the cotyledon stage. Blue light bypasses this blockage, rejecting the long-standing idea that the blue-light receptors cryptochromes cannot operate without phytochromes. After growth under white light, returning the quintuple phytochrome mutant to red light resulted in rapid senescence of already expanded leaves and severely impaired expansion of new leaves. We conclude that Arabidopsis development is stalled at several points in the presence of light suitable for photosynthesis but providing no photomorphogenic signal.

Fig. 1.

Germination of the quintuple phytochrome mutant is not light responsive and requires exogenous GA. (A) Seeds were plated on Murashige and Skoog (MS) salts agar, stratified for 3 days at 4 °C, and incubated for 5 days under different light regimes at 23 °C before counting germinated seeds (radicle emergence). Light conditions: white light, 80 μmol m⁻² s⁻¹; red light, 10 μmol m⁻² s⁻¹; far-red light, 60 μmol m⁻² s⁻¹; blue light, 10 μmol m⁻² s⁻¹; and darkness. Data are averages ± SE of six or three (far-red) independent experiments with 50 seeds each. None of the light treatments promoted germination of the quintuple phytochrome mutant (one-way ANOVA, P = 0.43). (B) Seeds of the quintuple phytochrome mutant were sown on moistened filter paper containing 100 μM GA₃, GA₄ (Sigma), or no hormone and stratified for 3 days at 4 °C before the induction of germination at 23 °C under red light. Data scored 5 days later are averages ± SE of five independently collected seed pools (50 seeds each). One-way ANOVA followed by Bonferroni tests indicated significant differences between the control and +GA₄ (P < 0.01). (C) Seeds of the WT, ft, and phyA phyB phyC phyD phyE ft mutants were sown on agar containing MS salts and stratified at 4 °C for the times indicated on the abscissa. Germination was induced and scored as in (B). Data are averages ± SE of five independently collected seed pools for the quintuple phytochrome mutant and two for the WT and ft controls. A t test indicates that the quintuple phytochrome mutant responded to stratification (P < 0.001).

Fig. 2.

The quintuple phytochrome mutant does not develop under red light and requires blue light for developmental progression. (A) Seedlings were grown on MS agar plates under blue- or red-light photoperiods (10 μmol m⁻² s⁻¹; 16 h light/8 h dark) for 4 days at 23 °C. Hypocotyl length was measured at the end of the treatment. Data are averages ± SE of at least 15 seedlings in three independent experiments. One-way anova followed by Bonferroni posttests indicated that the effect of blue light was significant in the quadruple and quintuple phytochrome mutants (P < 0.01), and the effect of red light was significant in the quadruple mutant (P < 0.05) but not in the quintuple phytochrome mutant (P > 0.05). (B) phyA phyB phyC phyD phyE ft quintuple phytochrome mutant grown for 4 days under blue light (Upper Left), 9 days under red light (Upper Right) or 9 days in the dark (Lower Right). (B Lower Left) phyA phyC phyD phyE ft quadruple phytochrome mutant grown for 9 days under red light. Light and growth conditions were as in (A). (C) Seedlings were grown under continuous fluorescent white light (30 μmol m⁻² s⁻¹) for 20 h to allow germination and then moved to continuous white light (30 μmol m⁻² s⁻¹) with or without the addition of far-red light (24 μmol m⁻² s⁻¹; λ max 735 nm) for 7 days. Data are averages ± SE of four independent replicate dishes with 10–15 seedlings each. One-way ANOVA followed by Bonferroni posttests indicate significant differences (P < 0.001) between high and low red/far-red ratios for the quadruple phytochrome mutants but not for the quintuple phytochrome mutant. (D) Detail of the apical portion of the phyA phyB phyC phyD phyE ft sextuple mutant grown on MS salts agar plus 2% sucrose under 50 μmol m⁻² s⁻¹ continuous red light for 36 days (three Left), or 60 days (Right). (E Left) A flowering phyA phyB phyC phyD phyE ft sextuple mutant grown on MS salts agar plus 2% sucrose, under 50 μmol m⁻² s⁻¹ continuous blue light for 40 days. (E Center and Right) Flowering phyB phyC phyD phyE ft mutant and phyA phyB double mutant grown under 50 μmol m⁻² s⁻¹ continuous red light. Arrows indicate flower buds. (F) Flowering plants of the ft (Upper Right), phyA phyB phyC phyD phyE ft mutant (Upper Left), and phyA phyB phyC phyD phyE quintuple mutant in the FT background (Lower) grown in long days (100 μmol m⁻² s⁻¹ cool white light). The stem-length of the quintuple mutant in the FT background is 1.5 cm; note the yellow micropipette tip for reference. (G) Chlorophyll accumulation in the absence of phytochrome. Quintuple phytochrome mutants were grown for 8 days at 23 °C in the dark or under red light (50 μmol m⁻² s⁻¹). See Fig. S5 for controls and full details. (H) Chlorophyll synthesis in the absence of phytochrome. Seedlings were grown on MS agar plates in the dark for 4 days at 23 °C and either treated with 15 min of red light (50 μmol m⁻² s⁻¹; Right) or kept in darkness (Left) before harvest. Protochlorophyllide was extracted as described in ref. , and emission spectra were recorded every 0.5 nm with excitation with light of 433 nm. The data are averages of three independent plates with 20 seedlings each. For clarity only the SE of the peaks are presented. (I) Sextuple mutants phyA phyB phyC phyD phyE ft and ft controls were grown in white-light photoperiods on MS plus 2% sucrose for 14 days and then moved to either 50 μmol m⁻² s⁻¹ blue light or 50 μmol m⁻² s⁻¹ red light for the other 13 days. These plants are representative of six plants. The arrow points to an older leaf that turned yellow during the treatment despite the fact that it was green before moving the plants to red-light conditions. (Scale bars: B and D, 1 mm; E, F, and I, 1 cm.)

Fig. 3.

The circadian clock still functions in the absence of phytochromes. Seeds of the ft and phyA phyB phyC phyD phyE ft mutants were sown on soil and entrained in 12-h white light/12-h dark photoperiods at 23 °C for 10 days. After entrainment, lightning was set to continuous mode (LL) and the angle between primary leaves was measured every 2 h for 3 days. The data represent the average ± SE of four independent experiments with three seedlings each.