A NEW TYPE OF LUMINESCENCE IN GREEN PLANTS

Until recently, only one type of luminescence in plants was known—fluorescence.

The fluorescence of green plants was discovered a hundred years ago by Stokes. At present, a considerable number of works have been devoted to the study of this phenomenon.

It has been established that the chlorophyll of leaves fluoresces. The most intense band of leaf fluorescence is due to chlorophyll a and has a maximum at approximately 682 mμ; in addition, there is a weaker band with a maximum at 656 mμ, due to chlorophyll b, and two weak bands of chlorophyll a with maxima at 740 mμ and 812 mμ.

The intensity of the fluorescent radiation of living leaves amounts to approximately 0.1% of the quantity of light absorbed; in chlorophyll solutions the fluorescence intensity is tens of times greater.

The main stimulus for the study of fluorescence was the belief that the data obtained could provide direct information about the mechanism by which plants use light energy in photosynthesis. However, the connection between fluorescence and the efficiency of light-energy utilization in photosynthesis has proved to be more complex than had previously been assumed.

At present there are grounds for believing that, irrespective of the wavelength of the absorbed light, the excited chlorophyll molecule undergoes a practically instantaneous transition to a lower energy level corresponding to the energy of the “red” quantum. From this state, various paths of transformation of the excitation energy are possible. First, a direct transition to the ground level is possible; this case corresponds to the emission of red fluorescence light. Much more probable, however, is a transition as a result of an internal-conversion process to a metastable level lying somewhat below the “red” level. It is apparently from this metastable level that the transfer of the excitation energy of chlorophyll to the photosynthetic acceptor takes place. Obviously, the probability of such transfer depends little on the probability of emission of a fluorescence quantum.

Theoretically, one might have expected that, as a result of fluctuations of thermal energy, some of the chlorophyll molecules in the long-lived metastable state would pass back to the “red” level, with subsequent emission of a light quantum. However, such delayed fluorescence was not observed experimentally. The direct transition from the metastable state to the ground state is also conceivable; this case would correspond to weak phosphorescent radiation lying in the near infrared region. Experiments specially designed to detect this radiation likewise gave no positive result.

Instead of these theoretically expected, purely “physical” types of luminescence, a new type of afterglow, possessing a number of interesting properties, was recently discovered by chance¹. It was found that if a sufficiently sensitive light detector is used, then after preliminary irradiation of plant leaves with light absorbed by chlorophyll (i.e., light lying practically entirely in the visible region of the spectrum), a very weak and prolonged afterglow of the leaves can be detected.

As a light detector, a photomultiplier was used in combination with an amplifier. Cooling the photomultiplier to the temperature of liquid nitrogen made it possible to observe the afterglow for several minutes. At 0.1 sec after cessation of irradiation, the luminescence intensity proved to be

in intensity than ordinary fluorescence. The low intensity of the glow should apparently explain the belated discovery of the new phenomenon.

As a result of detailed investigation, it proved possible to establish the basic properties of the new type of luminescence.

The following characteristics bring it closer to ordinary fluorescence:

1) In spectral composition, both types of radiation are similar and possibly identical; the measurements made, however, are not sufficiently accurate to prove the identity of the spectra.

2) The excitation spectra coincide with one another and are similar to the action spectrum of photosynthesis and, consequently, to the absorption spectrum of chlorophyll.

3) The dependence of the intensity of the afterglow on the concentration of carbon dioxide is the same for both types of luminescence; namely, an increase in the concentration of $\mathrm{CO_2}$ diminishes the intensity of the luminescence.

In other respects, the afterglow discovered differs substantially from ordinary fluorescence and at the same time is most directly connected with photosynthesis:

1) The rates of decay differ by $10^8$—$10^9$ times.

2) In incomplete solutions of chlorophyll, the afterglow (as well as photosynthesis) is not observed, while the intensity of fluorescence, as already noted, increases tens of times.

3) Ultraviolet rays of sufficient intensity destroy the ability of the leaf to emit prolonged luminescent radiation and at the same time completely stop photosynthesis; fluorescence under the same conditions is practically unchanged.

4) Low temperature (4—6° C) lowers the intensity of luminescence and simultaneously inhibits photosynthesis; the intensity of fluorescence, on the contrary, increases.

5) Light saturation of luminescence and photosynthesis occurs at approximately one and the same value of the intensity of the exciting light; the intensity of fluorescence, however, continues to increase at much larger values of the intensity of the exciting light.

6) Chemical agents that usually inhibit photosynthesis also reduce the intensity of luminescence and enhance fluorescence.

Such properties as temperature dependence, light saturation, and the influence of chemical agents indicate that the observed prolonged afterglow is coupled at least with one enzymatic chemical reaction and, consequently, cannot be explained purely by physical phenomena—delayed fluorescence or phosphorescence.

To explain the results obtained, the authors assume that in the general chain of photosynthetic reactions some of the early reactions are reversible up to the emission of the absorbed light quantum.

Additional data in favor of such a chemiluminescent mechanism were obtained in the study of prolonged afterglow in isolated chloroplasts². Chloroplasts, i.e., bodies containing chlorophyll, are ordinarily found in the cells of leaves and can be separated from the cells by grinding the leaves in a suitable liquid followed by filtration and centrifugation. An extremely interesting property of such “isolated” chloroplasts is their ability to release oxygen under the action of light in the presence of sufficiently active oxidizers (the Hill reaction). Let us note that carbon dioxide, which is the oxidizer in photosynthesis, is not a sufficiently active oxidizer for the Hill reaction.

The new afterglow was also detected in isolated chloroplasts. Such features as temperature dependence, the effect of chemical agents, the rate of decay, and light saturation proved to be similar to the properties observed in leaves. As might have been expected, carbon dioxide does not affect the intensity of the luminescence of isolated chloroplasts.

The most significant result was the quantitative demonstration that the intensity of luminescence and the rate of oxygen evolution depend in the same way on the intensity of the exciting light. In particular, saturation of both processes was observed at one and the same value of the intensity of the incident light. This fact is regarded by the author as decisive evidence in favor of the chemiluminescent nature of the prolonged afterglow.

At present it is difficult to assess the significance of the new phenomenon for the study of the mechanism of photosynthesis. However, the very fact of the existence of such a biological mechanism of chemiluminescence is undoubtedly of great fundamental interest.

L. B.

References

1. B. L. Strehler, W. T. Arnold, Gen. Phys. 34, 809 (1951).
2. B. L. Strehler, Arch. Biochem. Biophys. 34, 239 (1951).