| Generally, no chlorophyll has been detected in mature angiosperm seeds, but lotus (Nelumbo nucifera Gaertn.) is unusual in that it has a clear chlorophyllous embryo. In this study, we first investigated the composition of chlorophyll and photosystem in lotus embryo in detail, and then studied the biogenesis of chlorophyll and photosystem during lotus embryo maturation and dark-germination process. The main results were summarized as followings:1. The chlorophyll and photosystem compositions in lotus embryo were different from those of other angiosperms. The chla/b ratio in lotus embryo was about 0.8, which is far lower than that of other angiosperms (~3). No p-carotene could be detected in lotus embryo either, which is another unique characteristic of lotus embryo. The photosystem in lotus embryo had no electron transfer activity and its fluorescence induction kinetics results showed only higher FO and no Fy. In situ fluorescence spectrum at 77 k showed that lotus embryo had single fluorescence emission peak at 679nm and no normal PSII and PSI fluorescence peaks. Partial denaturing SDS-PAGE results demonstrated that lotus embryo had only monomeric and dimeric LHCII, and no other chlorophyll-protein complexes. Absence of PSI core and LHCI proteins inlotus embryo was further evidenced by Western Blots. LHCII protein in lotus embryo exhibited similar molecular weight to that of LHCII in lotus leaf, but spectrum analysis showed that the chla/b ratio and chlorophyll binding status were different between them, which were caused mainly by some chla molecules instead of chlb.2. By monitoring the chlorophyll and photosystem development in lotus embryo during its maturation, we proposed that this process could be divided into three phases: formation (0~20d), stabilization (20-30d), and degradation (30~40d). During the first two phases, the integuments covering lotus embryo might not be light-tight enough, so lotus embryo was able to sense light signal and both chlorophyll and photosystem biogenesis could take place. But later, when lotus seed capsule lignified gradually and became impossible for light to penetrate, the chlorophyll synthesis in lotus embryo stopped and the pre-formed photosystem began to degrade. All chlorophyll-protein complexes except LHCII were completely degraded before lotus embryo maturation. This might explain why lotus embryo has unusual pigment and photosystem compositions. Covering lotus pod with foil during its development process, we found lotus embryo turned into etiolated completely, which indicated that lotus embryo could not synthesize chlorophyll without light. To further test whether lotus embryohas the ability to form chlorophyll in the dark, we tried to amplify the homologous gene for DPOR in lotus genome, which catalyses the reduction from Pchlide to Chlide in the dark. The PCR results clearly excluded the possibility that lotus embryo can synthesize chlorophylls in a light-independent pathway.3, Dark-grown lotus seedling could keep its chlorophyll for a long time, especially its chla content was stable within 10 days. The in situ fluorescence spectrum at 77 k showed that both PSII and PSI fluorescence emission appeared during dark-grown process. The dark-grown 10 days lotus seedling had strongest PSII and PSI fluorescence emission, but compared to light-grown lotus seedling, it was still very weak. The changes of in situ fluorescence spectrum at 77k during the greening and freeze-thaw processes showed that the photosystem developed in dark-grown lotus seedling was unstable and uncompleted. The absorption spectrum indicated that the chloroplast in dark-grown lotus seedling is undeveloped compared to that of lotus leaf. The results from chlorophyll-protein complex separation, SDS-PAGE, Western Blots, and fluorescence induction kinetics all demonstrated the photosystem development, especially the PSI appearance in dark-grown lotus seedlings. The light-driven electron transfer activity measurements showed that both PSI and PSII in dark-grown lotus seedling is photochemicall...
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