Effect of circularly polarized light on germination, hypocotyl elongation and biomass production of arabidopsis and lettuce: Involvement of phytochrome B

Abstract

Circular dichroism (CD), defined as the differential absorption of left- and right-handed circularly polarized light (CPL), is a useful spectroscopic technique for structural studies of biological systems composed of chiral molecules. The present study evaluated the effects of CPL on germination, hypocotyl elongation and biomass production of Arabidopsis and lettuce. Higher germination rates were observed when Arabidopsis and lettuce seedlings were irradiated with red right-handed CPL (R-CPL) than with red left-handed CPL (L-CPL). Hypocotyl elongation was effectively inhibited when Arabidopsis and lettuce seedlings were irradiated with red R-CPL than with red L-CPL. This difference was not observed when a phytochrome B (phyB) deficient mutant of Arabidopsis was irradiated, suggesting that inhibition of elongation by red R-CPL was mediated by phyB. White R-CPL induced greater biomass production by adult Arabidopsis plants, as determined by their fresh shoot weight, than white L-CPL. To determine the molecular basis of these CPL effects, CD spectra and the effect of CPL on the photoreaction of a sensory module of Arabidopsis phyB were measured. The red light-absorbing form of phyB showed a negative CD in the red light-absorbing region, consistent with the results of germination, inhibition of hypocotyl elongation and biomass production. L-CPL and R-CPL, however, did not differ in their ability to induce the interconversion of the red light-absorbing and far-red light-absorbing forms of phyB. These findings suggest that these CPL effects involve phyB, along with other photoreceptors and the photosynthetic process.

Keywords: circularly polarized light; germination; hypocotyl elongation; photoreaction; phytochrome.

Figure 1. Effect of red CPL on germination of Arabidopsis (A) and lettuce (B) seeds. Cold-treated seeds were irradiated with red L-CPL (L) or R-CPL (R) for 10 min at 1.02 µmol/m²/s (A) and 0.36 µmol/m²/s (B) at 22°C. After incubation for 3 days in the dark at 22°C, the germinated seeds were counted and germination rates were calculated. * p<0.05 by t-tests, error bar=S.D., N=10.

Figure 2. Effect of red CPL on hypocotyl elongation. (A, B) Germinated seeds of Arabidopsis (A) and lettuce (B) were cultured in the growth chamber under continuous red L-CPL (white columns) or R-CPL (grey columns) for 7 or 10 days (A) and 7 days (B) at 22°C. The seedlings were cut and hypocotyl lengths were measured. * p<0.05 by t-tests, error bar=S.D., N=5. (C) Germinated seeds of Arabidopsis wild type (WT) and phyB deficient mutant (phyB) were cultured in the growth chamber under continuous red L-CPL (white columns) or R-CPL (grey columns) for 7 days at 22°C. The seedlings were cut and hypocotyl lengths were measured. * p<0.05 by t-tests, error bar=S.D., N=4.

Figure 3. Effect of white CPL (A) and red, green and blue CPL (B) on biomass production by Arabidopsis. (A) Seedlings planted in soil were cultured under unpolarized white light for 2 weeks with a 16 h light/8 h dark cycle (22 µmol/m²/s) at 22°C. The seedlings were subsequently cultured under white L- (L) or R- (R) CPL with a 16 h light/8 h dark cycle at 10.8 µmol/m²/s for 3 weeks at 22°C. Shoots of the adult plants were cut and their fresh weights were measured. p<0.07 by t-test, error bar=S.D., N=3. (B) Arabidopsis plants were grown under unpolarized white light for 2 weeks, as described in the legend to (A). The plants were subsequently grown under red (29.7 µmol/m²/s for 2 weeks), green (5.0 µmol/m²/s for 3 weeks) and blue (2.3 µmol/m²/s for 3 weeks) L- (L) and R- (R) CPLs, at total fluences of 35.6, 8.1 and 2.8 mol photons, respectively. Shoots of the adult plants were cut and their fresh weights were measured. p=0.128, 0.941 and 0.332 for red, green and blue light, respectively, by t-tests, error bar=S.D., N=3.

Figure 4. UV-Vis absorption spectra and CD spectra of AtphyB-N651. (A) UV-Vis absorption spectra of PCB-bound AtphyB-N651 in Pr (blue line) and a red light-induced photostationary state (orange line). Spectra of Pr and the red light-induced photostationary state were measured after saturating far-red LPL and under saturating red LPL, respectively, at 25°C. (B, C) UV-Vis absorption (B) and CD (C) spectra of in Pr (blue line) and 100% Pfr (orange line). The absorption spectrum of 100% Pfr was calculated from the absorption spectra of Pr and the red light-induced photostationary state shown in (A). The CD spectra of 100% Pr and 100% Pfr were calculated from the CD spectra of Pr and a red light-induced photostationary state measured at 25°C (Supplementary Figure S2) by correcting for the actinic effects of the light used to measure CD.

Figure 5. Changes in the UV-Vis absorption spectra of PCB-bound AtphyB-N651 during photoreactive conversion from Pr to a red light-induced photostationary state at 25°C. (A–C) Changes in the spectra of Pr (black thick lines) were monitored by repeat scanning every 1 min (thin lines) for 15 min (thick lines) after the onset of red light illumination of LPL (black lines in A), L- CPL (blue lines in B) and R-CPL (red lines in C). (D, E) Kinetics of photoreactions monitored at Pr (650 nm; D) and Pfr (715 nm; E) peaks. (●), (▲) and (▼) indicate absorbance changes induced by LPL, L-CPL and R-CPL, as determined by the changes in spectra in (A), (B) and (C), respectively. Black, blue and red lines are simulation curves fitted with a single exponential for the first order reaction, as described in the Results.