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Simultaneous photocalorimetric and oxygen polarographic measurements on Dunaliella maritima cells reveal a thermal discrepancy that could be due to nonphotochemical quenching (NPQ)
Photocalorimetry has rarely been employed to investigate the processes in photosynthesis. Yet, especially when complemented by simultaneous oxygen polarographic measurements, it can probe: (i) the light-excited decay of chlorophyll molecules to the ground state by the photochemical process in photosynthesis; (ii) light emission as fluorescence; and (iii) thermal dissipation. The possibility of detecting the latter two was examined by these methods. The halotolerant chlorophytic microalga, Dunaliella maritima, was grown routinely at 25 mol photon m−2 s−1 incident light and in the experiments it was exposed to higher light intensity and salinity in order to observe the energetic consequences of stress in terms of the net heat and oxygen fluxes.
The results showed that at 50 and 90 mol photon m−2 s−1 the net heat flow varied from rapidly negative (endothermic) to sharply positive (exothermic). This was different to the simultaneous data from the oxygen polarographic sensor that always showed oxygen evolution in the light. Energy balances to correct for the slight imbalance in the response to incident radiant light and to convert the oxygen evolution to energy values by the oxycaloric equivalent for glucose (−470 kJ mol−1 O2) revealed an extra source of heat at 15.5±3.3 (S.E.) and at 9.4±3.2 pW per cell for the control and treated cells, respectively, at 90 mol photon m−2 s−1. This was thought to be due mainly to nonphotochemical quenching (NPQ), characteristically with a small contribution from chlorophyll fluorescence.
Fig. 1. The unicellular green chlorophyte alga, D. maritima, from the collection of the Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia ,was grown in T-75 vented flasks suspended in sterile Bold’s Basal Medium (BM) in 1 and 3% saline buffered to pH 6.8 in a constant temperature room at 25 ◦C. The only source of carbonwas atmospheric CO2. The illumination measured as PFD with a Skye Quantum Sensor (Skye Instruments Ltd., Llandrindod Wells, LD1 6DF Wales, UK) for photosynthetically active radiation (PAR, 400–700 nm) was 25 mol m−2 s−1 and the photoperiod was 12 h.
Fig. 2. The rates of respirometric oxygen uptake in the dark and photosynthetic oxygen evolution in the light were measured in parallel to the heat flows using an Oroboros Oxygraph (Oroboros Instruments, A-6020 Innsbruck, Austria) two-chamber oxygen polarographic sensor at 25 ◦C. A 2.3 cm3 aliquot of the same cell suspension used in the calorimetry was added to the glass vessel of each chamber and magnetically stirred at 100 rev/min. The rate of oxygen flow was measured under the same light conditions as for the calorimetry. It was corrected for the salinity of the medium and the cell count at the end of the experiment to express the results as the scalar flux.
Fig. 3. The photocalorimeter is a customised module of the commercial TAM heat conduction batch microcalorimeter (Thermometric AB, SE-17561 J¨arf¨alla, Sweden). Itwas modified from a design used for estimating the enthalpy changes of photochemical solutions and the rate of photosynthesis in spinach leaves but differs from the latter in several respects. Using the twin differential principle, 5 cm3 of the cells suspended in Bold’s BM containing 1 or 3% saline was placed in a 20-cm3 stainless steel titration vessel (test) and stirred at 60 rev/min with a Thermometric KelF turbine while the reference vessel contained Bold’s BMin 1% saline. Measurements were undertaken at 25 ◦C and another cell count was taken at the end of the experiment to obtain the heat flux. The instrument was chemically calibrated by the heat flow of the slow hydrolysis of triacetin. Light was introduced into each vessel through a 5-mm quartz rod (Suprasil 1, Hereaus Quartzglas, GmbH, D-63450 Hanau, Germany). As in earlier experiments, the optical part consisted of an Oriel 150 W Xe lamp in the housing (LOT-Oriel Ltd., Leatherhead KT22 7BA, UK). The light was focussed with a condenser and this also acted to attenuate the PPFD before it passed through a continuously cooled water filter and a glass filter to remove IR and UV radiations, respectively. Light was transferred from the housing exit slit through an Oriel bifurcated silica fibre bundle connected to the quartz rods which, unlike in the Johansson–Wads¨o setup that employed two calorimeters, are in the test and reference vessels of one calorimeter.
Fig. 4. Principles of differential photocalorimetry of microalgal culture. In the dark, the output signal is always positive due to cell respiration and heat production (left image). In the light (right), photon influx to both the vessels is equivalent, and light heats them. However, a part of light energy in the test vessel is converted to chemical forms as a result of photosynthesis. Thus, the test vessel is heated less than the reference one. After switching light on, the heat flow curve goes down, sometimes below zero (at photosynthesis rate > respiration rate).
Fig. 5. Results of two experiments in which dark/light heat and oxygen fluxes were measured at the same time for control (a) and treated (b) D. maritima in Bold’s medium containing 1 and 3% NaCl, respectively. The oxygen flux measurements were made in two replicates. The arrows indicate the time points of switching on the light at 90 mol photon m−2 s−1 (1) and 50 mol photon m−2 s−1 (2) and switching off the light.
Fig. 6. In the light, plants and other autophototrophs generally display net oxygen evolution because, in order for them to grow, there must be sufficient carbon compounds (photosynthate) for biomass production (p) as well as for combustion (r), i.e. p/r > 1. Applying this logic to direct photocalorimetry means that photosynthetic material should "absorb" energy as heat to reflect the endothermic reaction. However, the NPQ part of photoprotection is accompanied by thermal dissipation which manifests itself in the calorimeter as heat production. In some cases, this diminishes or even abolishes the photosynthetic heat absorption to give net heat production.