Abstract
The small vegetative cells of a synchronous Chlorella pyrenoidosa culture subjected to a heat pre-treatment (46.5ºC for 1 h in the dark) and cultured again under continuous illumination thereafter started to disintegrate within hours. Chlorophylls were degraded via photooxidation, started after a 2-h-delay and completed within 8 h. However, no free radicals were released from the sites of photooxidation, but a FeSOD gene from Chlorella pyrenoidosa 211-8b was cloned. The full length of cDNA is 1063 base pairs, with an open reading frame (ORF) of 609 base pairs (203 amino acids), and compared with NCBI gene databank. It revealed 73.5% identity in nucleic sequence and 70.5% identity in amino acids sequence, respectively, with the FeSOD sequence of Chlamydomonas reinhardtii, an eukaryotic unicellular green alga. The nucleic sequence of FeSOD (609 bps ORF) along with the expression vector was transformed into E. coli expression host. An over-expressed protein of about 22.3 kDa was detected. The activity of the expressed protein was assayed by using a method of gel activity staining, and a clear band was observed at about 44 kDa, likely a dimer form of FeSOD. Besides, DNA in nucleus and chloroplast disappeared along with chlorophyll pigments, but mitochondrial DNA appeared to decay at a much slower rate. In addition, a novel nuclease activity was detected in heat-treated cells undergoing DNA degradation. The decomposition of DNA rendered the disintegration process irreversible. Contrary to the programmed cell death of higher plants, these heat-treated Chlorella cells failed to exhibit DNA laddering and massive protein degradation, but retained their cell membrane integrity. Thus, it might be in a way very different from the programmed cell death observed in many higher plants. However, study of chlorophyll fluorescence induction of higher plants, Ficus microcapa L. f. cv. Golden-leaves. The rise of the chlorophyll fluorescence of a whole leaf as induced by high intensity actinic light comprises three distinct phases, termed 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 evidences 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.