Ultraweak Photon Emission from the Seed Coat in Response to Temperature and Humidity—A Potential Mechanism for Environmental Signal Transduction in the Soil Seed Bank

Abstract Seeds beneath the soil sense the changing environment to time germination and seedling emergence with the optimum time of year for survival. Environmental signals first impact with the seed at the seed coat. To investigate whether seed coats have a role in environmental sensing we investigated their ultraweak photon emission (UPE) under the variable temperature, relative humidity and oxygen conditions they could experience in the soil seed bank. Using a custom‐built luminometer we measured UPE intensity and spectra (300–700 nm) from Phaseolus vulgaris seeds, seed coats and cotyledons. UPE was greatest from the internal surface of the seed coat. Seed coat UPE increased concomitantly with both increasing temperature and decreasing relative humidity. Emission was oxygen dependent and it was abolished by treatment with dinitrophenylhydrazine, demonstrating the key role of seed coat carbonyls in the phenomenon. We hypothesize that beneath the soil surface the attenuation of light (virtual darkness: low background noise) enables seeds to exploit UPE for transducing key environmental variables in the soil (temperature, humidity and oxygen) to inform them of seasonal and local temperature patterns. Overall, seed coats were found to have potential as effective transducers of key fluctuating environmental variables in the soil.

System overview (upper panel) and cross section of the chamber (lower panel). The luminometer consists of a two-part circular structure machined from black PVC. The lower part has a central plinth with a hole to engage the photomultiplier tube (PMT) holder. The PMT holder consists of a hollow PVC cylinder jutting 2 cm over the plinth and protruding 5 cm below the chamber bottom. The PMT is slid in from below and secured in place. That part of the PMT holder extending into the chamber is indented to hold the specimen 4 mm away from the PMT photocathode (for PMT characteristics see text).
The sample holder (A) consisted of a 12 ml capacity borosilicate glass (Pyrex) beaker.
Light transmission properties of the sample holder were checked with a Beckman DU-65 spectrophotometer and found to be >80% in the PMT sensitivity range.
The upper part (lid) of the luminometer, has an inner and outer wall bearing two hose adapters for water circulation (I). An air vent in the lid (G) enables the composition of the atmosphere to be modulated inside the luminometer. To prevent the entry of light after passing through the lid, the vent tube is coiled to produce a 2-turn spiral with the open end turned upwards, almost in contact with the internal lid surface. This air vent allows slow, gradual atmosphere changes in the luminometer chamber. In this study (G) was kept plugged and atmosphere changes were via a two-way injector system in the lid (F) allowing liquid/gas to be delivered into the sample chamber, near to the specimen, by hand-operated syringes (liquids) or controlled gaseous fluxes.
The atmosphere composition inside the luminometer was controlled by pump forced fluxes (1-3 L m-1) of ambient air, nitrogen or oxygen. Humid or dry atmosphere (about 90% and 10%RH, respectively) was obtained by either extensively bubbling air/ gases in distilled water or by passing them through coupled granular CaCl2 and silica gel columns, respectively. Ambient air at different %RH was obtained by mixing different percentages of humid and dry air. Nitrogen and oxygen were obtained from standard N2 and O2 cylinders, respectively. Humidity (%RH) in the proximity of the sample was monitored with a humidity probe  Different regions of the light spectrum were identified (red asterisks) and photon emission and TDOP in each region were derived by difference.
Normalized photon emission was then calculated by dividing the differential photon emission by the corresponding differential TDOP.

Example:
The normalized photon emission in the 415-460 region was calculated as follows:    Figure S4. Effect of seed coat damage on photon emission from intact seeds. Photon emission was measured at 35°C through a 60-20-60 %RH cycle in intact seeds (a), seeds with damaged coat (b), isolated coats and naked cotyledons (c). Sample weights were 1000-1200, 390 and 32.7 mg for whole seeds (2X for each trace), naked cotyledons (2 fragments from 1 seed) and isolated seed coats (2 fragments from 1 seed).

Figure S5
Considering the Arrhenius equation: where k = the rate constant of a chemical reaction T = temperature (K) E a = activation energy R = universal gas constant, value 8.314 * 10 -3 kJ mol -1 K -1 A = constant factor depending on the nature of the chemical reaction and assuming k = photon emission intensity (luminescence intensity is proportional to the rate constant of the chemical reaction producing excited, emitting molecules) Arrhenius plots were constructed by using coat UPE measured at two%RH (10-15% e >60%) For each plot, the best fit was calculated according to the equation Y = a + b*X and the slope value (b) was used to derive the activation energy (E a ) according to the formula: , indicating that the same mechanism is responsible for the emission. The procedure for Ea calculation is also showed.