A comparative study of femtosecond pulsed and continuous wave lasers on physiological responses through activation of phytochromes in seeds

Abstract

Red light activates phytochrome photoreceptors, which mediate such key developmental steps as germination and seedling photomorphogenesis in Arabidopsis thaliana. To examine the details of these responses, we developed a novel experimental system and demonstrated that brief, high-intensity light pulses can elicit sustained physiological responses. We observed that the seeds responded to the femtosecond laser light pulses, but with lower sensitivity compared with continuous light sources having the same average fluence. We concluded that (i) phytochrome B photoreceptors within imbibed seeds efficiently absorb red and far-red photons from pulsed femtosecond laser pulses, with absorption occurring during approximately 10 orders of magnitude shorter amount of time than with conventional light sources; (ii) these treatments did not induce adverse effects during later plant development; and (iii) the effect of ultrashort light pulses in planta coincides with phytochrome photoconversion characteristics described during in vitro studies. Our findings demonstrate that seed germination and photomorphogenic development can be effectively triggered by light, regardless of whether it is delivered continuously or within extremely brief pulses. This research expands the potential applications of femtosecond laser technology and demonstrates the feasibility of investigating the effects of ultrafast physical phenomena on biological processes in vivo using diverse biological readouts.

Supplementary Information: The online version contains supplementary material available at 10.1038/s41598-025-11183-8.

Keywords: Femtosecond pulsed laser; Germination; Photomorphogenesis; Phytochrome B; Plant phenotyping; Ultrafast photoswitch; Ultrashort light pulse.

Fig. 1

Emission spectra of the applied light sources. (A) Continuous wave light sources: white light halogen lamp in the incubator (FWL, 425–750 nm), the safe green laser (SG, 532 nm), red diode laser (DLR, 660 nm), red LED (LEDR, 600–700 nm) and far-red LED (LEDFR, 670–780 nm). (B) Femtosecond pulsed laser sources as the fsWL (450–750 nm with FES0750 short pass filter), fsR (620–680 nm with FB650-40 band pass and FES0750 short pass filter) and fsFR (690–750 nm with FEL700 long pass and FES0750 short pass filter) were generated using the Titanium: Sapphire (Ti: Sa) laser.

Fig. 2

Seed germination protocol indicating the different light and temperature treatments. Schematic illustration of the light irradiation and incubation of seeds at different temperatures during experimentation. Seeds were imbibed in water at 4 °C then irradiated and germinated at 22 °C. The irradiation protocols were as follows: (A) The following short (< 1000 s) irradiation with cw LEDR or LEDFR (incoherent 660 nm R or 725 nm FR light, respectively); femtosecond red (fsR, 630–670 nm) or femtosecond far red (fsFR, 700–750 nm) pulsed coherent laser light at the Ti: Sa or HR1 laser. Subsequently, the samples were processed under safe green light and incubated in the dark for 72 h at 22 °C. (B) Full white light (FWL) irradiation (425–750 nm) for 72 h after imbibition. (C) Dark control with no irradiation; all sample handling was done under diffuse safe green light.

Fig. 3

The schematic drawing of the irradiation setup. (A) LED irradiation setup with LEDR or LEDFR panels collimating a single LED light with a plano-convex lens to adjust the beam diameter and a black metal lens tube for proper shadowing from other LEDs in the panel and magnetic stirring; (B) DLR irradiation setup adjusting the beam diameter with two lenses and directing the beam vertically downward into the sample vial with a periscope mirror. (C) fsWL (450–750 nm, with FES0750 short pass filter), fsR (620–680 nm, with FB650-40 band pass and FES0750 short pass filters) and fsFR (690–750 nm, with FEL700 long pass and FES0750 short pass filters) irradiation setup at the Titan: Sapphire (Ti: Sa, 782 nm, 1 kHz, ~ 100 fs) or HR1 (1030 nm, 100 kHz, ~ 30 fs) laser generated on a sapphire plate focusing the NIR beam on the crystal and collimated to the suitable beam diameter.

Fig. 4

Saturation of germination with red diode laser irradiation. Wild-type Arabidopsis seeds were irradiated with a cw red diode laser (DLR, 660 nm, 6000 µmol m^{– 2}s^{– 1}) for longer (A, 10–1000 s) or shorter (B, 0.1–10 s) irradiation time periods and germinated for 72 h in the dark at 22 °C or kept under constant white light (FWL). The percentage of germination was determined in three replicates with the total number of seeds per treatment group N > 300 and N > 65 per replicates in a subgroup (mean ± SE; n = 3). The line bisecting each box represents the median value; the lower and upper edge of the box indicate the minimum and the maximum values, respectively. The empty square within each box indicate the mean value. The letters a–d indicate significant differences in the means (p < 0.05 by ANOVA followed by a Tukey Test).

Fig. 5

Fluence dependent germination induced by different red-light sources. Arabidopsis wild-type seeds were irradiated with R light at 8.5 µmol m^{– 2} s^{– 1} fluence rate (100 µW average power), for 0, 10, 30, 100, 300 and 1000 s resulting in 85–8500 µmol m^{– 2} total fluence during the treatments using the following light sources: (A) Red LED irradiation (LEDR, 600–700 nm). (B) Red diode laser irradiation (DLR, 660 nm). (C) Femtosecond red pulsed light irradiation generated with the Ti: Sa laser (fsR, 620–680 nm) (D) LEDR, (E) DLR and (F) fsR Ti: Sa, based on the average values from panels A, B and C, respectively. Sigmoid curves were calculated using the dose response function of the Origin software based on the normalized data, setting the minimum values (dark) 0, and the maximum (Full White Light) 1.0. Each measurement was performed in three replicates (blue, red, green symbols) with the total number of seeds per treatment group being N > 200 and N > 50 per replicates in a subgroup.

Fig. 6

Germination is affected by ultrashort femtosecond R and FR pulses. Imbibed wild-type Arabidopsis seeds were pulse-irradiated with different R or FR light sources, or with R irradiation followed by FR irradiation (R + FR). The germination was determined after 72 h dark incubation. (A) The seeds were irradiated with R light at a fluence rate of 8.5 µmol m^{– 2} s^{– 1} (total fluence: 8.5 mmol m^{– 2}), or with FR light at a fluence rate of 95.3 µmol m^{– 2} s^{– 1} (total fluence: 11.4 mmol m^{– 2}) or with R and subsequently with FR light, using the following irradiation parameters: fsR Ti: Sa (100–120 µW, 1000 s); fsFR (1000–1100 µW, 120 s); LEDR (100 µW, 1000 s); LEDFR (1000 µW, 120 s). Non-irradiated control (Dark) was also included. The measurement was performed in two replicates with the total number of seeds per treatment group being N > 160 and N > 70 per replicates in a subgroup (mean ± SE; n = 2). The letters indicate significant differences of the means (p < 0.05 by ANOVA followed by a Tukey Test). (B) The seeds were irradiated with fsR Ti: Sa (80 µW, 1000 s) at a fluence rate of 6.8 µmol m^{– 2} s^{– 1} (total fluence of 6.8 mmol m^{– 2} ) or fsFR Ti: Sa (750 µW, 120 s) at a fluence rate 71.5 µmol m^{– 2} s^{– 1} (total fluence of 8.6 mmol m^{– 2}). Non-irradiated control (Dark) was also included. The measurement was performed in three replicates with the total number of seeds per treatment group being N > 170 and N > 50 per replicates in a subgroup (mean ± SE; n = 3). The line bisecting each box represents the median value; the lower and upper edge of the box indicate the minimum and the maximum values, respectively. The empty square within each box indicate the mean value. The different letters indicate significant differences in the means (p < 0.05 by ANOVA followed by a Tukey Test).

Fig. 7

PhyB induced germination after FWL, R and fsR pulses. Imbibed Arabidopsis seeds were irradiated as indicated and kept at 22 °C for 72 h before germination rates were determined. (A) The WT, aBcde and abcde mutant seeds were irradiated with white light (FWL) or were kept in the dark for 72 h. (B) The aBcde mutant seeds were irradiated with fsR Ti: Sa, fsR + fsFR Ti: Sa, LEDR, LEDR + LEDFR and LEDFR irradiations or were kept in the dark for 72 h. The average powers and irradiation times were: fsR: 80 µW, 1000 s; fsFR: 1000 µW, 120 s; LEDR: 100 µW, 1000 s; LEDFR: 1000 µW, 120 s; resulting in the following fluences 6.8 mmol m^− 2 (fsR), 11.4 mmol m^− 2 (fsFR); 8.5 mmol m^− 2 (LEDR) and 11.4 mmol m^− 2 (LEDFR). Both measurements (panel A and B) were performed in three replicates with the total number of seeds per treatment group being N > 85 and N > 35 per replicates of a sub-group (WT, aBcde, abcde). The line bisecting each box represents the median value; the lower and upper edge of the box indicate the minimum and the maximum values, respectively. The empty square within each box indicate the mean value. Different letters above the boxes indicate significant differences of the means (p < 0.05 by ANOVA followed by a Tukey Test).

Fig. 8

Elongation of hypocotyl is inhibited by fsR pulses. Wild type seeds were germinated in the dark with or without the indicated post-imbibition light treatments, except the FWL seeds that were kept under white light irradiation for 72 h at 22 °C. (A) Hypocotyl length of dark, fsFR, fsR, fsR + fsFR and FWL irradiated seedlings grown in 96-well plates. The applied fluence was 8.54 mmol m^− 2 for fsR or 11.4 mmol m^− 2 photon fluence for fsFR irradiation. (B) Hypocotyl length of fsR irradiated (8.5 mmol m^− 2 photon fluence) and non-irradiated seedlings that were grown in full darkness in Petri dishes for 72 h. For both panels asterisks indicate significant differences of the means (**** P < 0.0001, ns P > 0.05 by ANOVA followed by a Tukey Test).