A possible origin of the middle phase of polyphasic chlorophyll fluorescence transient

The rise of the chlorophyll fluorescence of a whole leaf as induced by high-intensity actinic light comprises three distinct phases, and is termed the O-J-I-P polyphasic rise. The initial rise (the O-J phase) was found to be the most sensitive to light intensity, being slower and smaller with decreasing irradiation. The leaf was also found to be transparent for chlorophyll fluorescence to a considerable extent, so that the fluorescence originating from deep inside the sample could still be detected. In contrast, the actinic light used to induce fluorescence was strongly absorbed by chlorophylls, so that a steep light gradient was created along the light path. The fluorescence transient of a leaf, thus, was always a mixture of the fluorescence from the surface of the sample as well as that from the inside of the sample, whose O-J phase is slower as it is induced by a weaker actinic light. We have provided evidence suggesting that, in an intact leaf, the middle phase of the measured polyphasic fluorescence transient (the J-I phase) might actually reflect the initial rise of the transient coming from the abaxial layer of the leaf. Moreover, if the polyphasic fluorescence transient is used as an analytical tool for accessing information on the photosynthetic activities of leaves, the factors of concentration and thickness of the sample must be taken into account. To obtain the 'true' fluorescence transient of a sample, both the chlorophyll concentration and thickness of the sample must be kept as low as possible.