Time-lapse images of Arabidopsis thaliana photoreceptor mutants under darkness and blue-light conditions

Rapid cell expansion pushes the Arabidopsis hypocotyl (juvenile stem) through the soil until blue light acting first through phototropin 1 (phot1) and then through cryptochrome 1 (cry1) suppresses elongation to produce the short hypocotyl characteristic of established, photosynthetically capable seedlings.

To determine where these two different blue light receptors act to suppress hypocotyl elongation, we measured relative elemental growth rate specifically along the hypocotyl midline at 5-minute intervals before and during blue light using a machine learning-based image analysis pipeline designed specifically for this kinematic analysis of growth.

In darkness, hypocotyl material expanded most rapidly (approximately 4% h⁻¹) in a zone from 0.4 to 1.5 mm below the apical terminus of the hypocotyl (cotyledonary node). Blue light acting through phot1 rapidly inhibited expansion in this zone, while simultaneously stimulating unexpanded cells in a more apical region only 0.1–0.3 mm below the cotyledonary node. Nuclear cry1, and not its cytoplasmic pool, counteracted the phot1-initiated expansion of the small cells in this apical region, preventing them from entering the more basal elongation zone. In a cry1 mutant, activation of these apical cells proceeded unchecked, reaching rates as high as 6% h⁻¹ to produce the iconic cry1 long-hypocotyl phenotype.

In addition to showing where future cell and molecular studies of cry1 and phot1 signaling mechanisms should focus, the new spatial information indicates that a seedling may use an apical reservoir of elongation potential to reenter a lit environment should a natural darkening event such as soil disturbance deactivate cry1. 

Methods
A clear plastic Petri plate with seedlings growing vertically on the surface of agar was mounted perpendicular to the optical axis of a macro video zoom lens (18–108 mm f/2.5, Edmund Optics) fitted to a charge-coupled device camera (Marlin F-146B; Allied Vision) that was controlled by a computer. A close-up +4 lens (Tiffen) attached to the zoom lens increased magnification.

An infrared-pass filter (R72, Hoya Filter USA) permitted 948 nm infrared radiation emitted by a BL020201 backlight (Advanced Illumination) placed behind the seedlings to reach the camera, which was made sensitive to infrared by removal of the internal infrared-blocking filter. This platform produced images of growing seedlings at a resolution of 184 px mm⁻¹, even in the complete absence of visible light. This arrangement ensured that blue light produced by an LED source described in Wu and Spalding treated the seedlings with a fluence rate of approximately 80 μmol m⁻² s⁻¹ without affecting the image the camera collected.

All experiments were performed in a photobiology darkroom. Some manipulations required brief use of a dim green safelight. 

The camera collected images every 5 minutes. The indicated light treatment began at frame 24, 2 hours after the start of recording in complete darkness. Image collection continued every 5 minutes for 8 hours. The 96 images per trial were stored in Tagged Image File Format (TIFF). These time series of images were used to develop the analysis pipeline, and they were the raw experimental data used to characterize the kinematics of light responses in the wild type and the indicated mutant genotypes.