Li 190sa

We measured the light intensity at the top (5^th leaf), middle (3^rd leaf), and bottom (1^st leaf) of the canopy (Fig 2) with a quantum sensor (LI-190SA; Li-Cor). The sensor was angled the same as nearby leaves. The LED inter-lighting remained in use. Solar irradiance alone was measured as a control.

Within each of the two populations, we recorded the air temperature inside and outside of the OTCs using a thermal data logger with two external sensors (ThermaD-ta^® logger TB2F, Electronic Temperature Instruments Ltd., Worthing, UK). One of the sensors was placed inside, whereas the other was placed outside of the OTC. A total of three devices were employed per population. Temperature measurements were taken at 30 min intervals during the whole experimental period.
To assess the micro-climate conditions within individual clones, we recorded the following environmental variables inside and outside of each OTC, just before leaf sampling: air temperature (15 cm above ground), soil moisture and soil temperature (at 5 cm in depth), light intensity (photosynthetically active radiation, PAR), and Red/Far-Red ratio (RFR). Air and leaf lamina temperature were recorded with an infrared laser non-contact thermometer (Crop TRAK, Spectrum Technologies, Inc., Plainfield, IL, USA); soil moisture with a soil moisture sensor (ML3 ThetaProbe, Delta-T Devices Ltd., Cambridge, UK), soil temperature with a contact stab digital thermometer (SuperFast Thermapen, Electronic Temperature Instruments Ltd., Worthing, UK), PAR with a point quantum sensor (LI-190SA, LI-COR, Inc., Lincoln, NE, USA), and RFR with R: FR sensor (Skye SKR-110, Skye Instruments Ltd., Powys, UK).

Photosynthetic active radiation (PAR) sensor (Li-190SA, Li-COR, USA) was installed above the plants in the field to collect light intensity, and was connected to a data logger (Li-1400, Li-COR, USA). This enabled us to measure the PAR value, its maximum, and to calculate the total input and to obtain average values of PAR for each treatment during canopy development. The total PAR input of any leaf was calculated as a sum of incident PAR (in mols of photons per unit area per second) between the appearance of the leaf and the time of performing photosynthesis and fluorescence measurements and the HL treatment. The middle part of mature leaves of barley (which was measured) was almost in a horizontal position; hence, the measured values of PAR almost fully corresponded to light intensities incident on leaves.

UVB exposure was provided with UVB-fluorescent tubes (FL20SE; Toshiba or GL20SE; Sankyo Denki). Strong light exposure was provided by a xenon light source (MAX-303; Asahi Spectra) equipped with a mirror module (MAX-VIS; Asahi Spectra) to extract visible light (wavelength 385–740 nm) and a rod lens (RLQL80-1; Asahi Spectra) to emit light with uniform intensity in a chamber. The intensity of UVB or visible light was measured with a data logger (LI-1400; Li-Cor) equipped with a UVB sensor (SD204B; Li-Cor) or a photosynthetic photon flux density sensor (LI-190SA; Li-Cor), respectively. After the treatment, plants were cultivated in the indicated growth conditions until the analysis.
For ConA treatment to suppress lytic activity in the vacuole, MES-NaOH (pH 5.5) containing 1 �M ConA was infiltrated immediately after UVB treatment into leaves with a 1-ml syringe and the leaves incubated under the indicated growth conditions. After 1 or 2 d, leaf mesophyll cells were observed under the confocal microscopy.

Photosynthetically active radiation (PAR) was measured with a data logger (LI-1000; LI-COR, Inc., Lincoln, NE, USA) and a quantum sensor (LI-190SA; LI-COR, Inc., Lincoln, NE, USA). The radiation spectra were measured on the day of sampling in April and June using a portable spectrometer (Jaz Modular Optical Sensing Suite; Ocean Optics, Inc., Dunedin, FL, USA). A white reference panel (Spectralon; Labsphere, North Sutton, NH, USA) was used for calibration of the spectrometer to 100% reflectance prior to measurement. Ten measurements of PAR and ten measurements of solar radiation spectra from 200 to 1100 nm were performed at the plant level for each of the three habitats.

Haematococcus pluvialis UTEX 2505 was obtained from the Culture Collection of Algae at the University of Texas at Austin. The alga was cultivated on 1.5% v/v agar plates containing MES-volvox medium with a pH of 6.7. Cultures were incubated at 22°C. The cultures were continuously illuminated with cool-white fluorescent light at 30 µmol photons m⁻²·s⁻¹. Light was measured with a light meter (LI-250A, LI-COR, Inc.) and a photometric sensor (LI-190SA, LI-COR, Inc.) in the 400–700 nm region of the electromagnetic spectrum. H. pluvialis cells were induced to accumulate astaxanthin by exposure to low light conditions of 5 µmol photons m⁻²·s⁻¹ of cool-white fluorescent light.
H. pluvialis cells were lifted from the agar and quickly (30 s) immobilized in an 8% polyacrylamide gel [34] . The cells were immediately imaged with white light microscopy to check for uniform distributions of cells and then the cells were imaged with a nonlinear optical microscope in a temperature-controlled environment at 20°C.

The wild-type F. diplosiphon UTEX 481, hereafter UTEX 481; shortened-filament, wild-type pigmentation strain SF33 (Cobley et al., 1993 (link)), hereafter denoted SF33 WT; and RcaE-deficient mutant strain, i.e., ΔrcaE (Kehoe and Grossman, 1996 (link)), were used in this study. Strains were grown in BG-11 medium (Allen, 1968 (link)) containing 20 mM HEPES at pH 8.0 (hereafter BG-11/HEPES) in a GL growth chamber at an intensity of ∼15 μmol m^-2 s^-1 with continuous shaking at 175 rpm at 28°C. Exponentially growing cultures, which were diluted to an initial optical density of ∼0.2 at 750 nm (OD₇₅₀), were then transferred to either GL or RL at an intensity of ∼15 μmol m^-2 s^-1 at 28°C with continuous shaking at 175 rpm. The sources of monochromatic GL and RL were those previously described (Bordowitz and Montgomery, 2008 (link)). The intensity of the light was measured with a LI-250A light meter (LI-COR, Lincoln, NE) equipped with a quantum sensor (model LI-190SA).