Results

Chlorophyll Synthesis in Light-Illuminated Potato Tuber.Our experiments showed that the greening of potato tuber depended on varieties，storage temperature，light intensity，and wavelength of light.Out of more than ten varieties of potatoes tested，two varieties，Centennial and Katahdin，which turn green more quickly under light，were selected in this study.Storage of potato tuber below 4℃retarded or inhibited the transformation of amyloplasts into chloroplasts.Blue light was most effective in inducing greening of potato tubers.No red light stimulation was observed.

Potato tuber discs illuminated with white light began to green with a lag period of about 1 d.The greening continued for 3 weeks after culture（see Figure 1）.After illumination for 3 weeks，the potato tuber tissue contained 10 μg Chl/g of fresh tissue，about one hundredth of the Chl content in normal leaves.This is primarily due to fewer chloroplasts per cell.The chloroplasts were not distributed uniformly in discs and were probably linked to some specific ultrastructure in the cortex.Microscopic observation revealed that the amyloplasts were comprised of two types：large（55×80 μm）and small（5—20 μm）.Most of them were stainable with I2-KI.Upon exposure of the discs to light the small amyloplasts only turned greenish.

Figure 1　Time course of the synthesis of chlorophyll in potato tuber discs during continuous illumination with white light

RuBPCase Biosynthesis in Potato Tuber during Greening.Since RuBPCase is the most abundant soluble protein in the chloroplasts［10］，it can be used as an important biochemical marker of chloroplasts.The de novo synthesis of RuBPCase was clearly demonstrated by ultracentrifugal sedimentation（data not shown）and specific immunoprecipitation（see Figure 2）.The Schlieren pattern of extracts from normal leaves consists of four peaks representing 80S cytoplasmic ribosomes，70S chloroplast ribosomes，18S Fraction I protein（RuBPCase），and 4 to 6S Fraction Ⅱ proteins［8，9］.The extract from potato tuber stored in the dark lacked the peak of RuBPCase，whereas a small peak was observed in discs exposed to 3 d of light，indicating the light initiated the synthesis of RuBPCase（data not shown）.

Figure 2　Biosynthesis of RuBPCase in potato tuber tissue as assayed by Ouchterlony double diffusion 0 h（1），6 h（2），12 h（3），24 h（4），48 h（5），and 72 h（6）after illumination.The central well contained antibody to LS.One g of potato tuber discs was homogenized with 0.1 ml of Tris 80 mmol/L，MgCl2 20 mmol/L，KCl 40 mmol/L，pH 8.5.After centrifugation at 12，000 g for 10 min，20 μl of the supernatant were used for assay of RuBPCase

The biosynthesis of RuBPCase was also demonstrated by an Ouchterlony double diffusion assay（see Figure 2）.The light-mediated synthesis of RuBPCase started at 1 d after illumination and increased during greening.The RuBPCase content was estimated to be～6 μg/g fresh potato tuber tissue，whereas the RuBPCase content of a typical green leaf is 5 to 10 mg/g fresh tissue［10］.

Further evidence for the de novo synthesis of RuBPCase in light-treated potato tuber was obtained from the experiment on light-driven protein synthesis in chloroplasts.The chloroplasts，isolated from potato tuber discs after illumination for 7 d，exhibited a higher activity of protein synthesis，as demonstrated by the incorporation of［35 S］methionine into proteins（see Figure 3）.In contrast to light-driven protein synthesis in chloroplasts，the chloroplasts in the dark and especially amyloplasts，either in the light or dark，exhibited a very low protein synthesis activity.A number of radioactive polypeptides synthesized in the chloroplasts in the light were recognized on SDS-polyacrylamide gels followed by fluorography（see Figure 4）.It was observed that some chloroplast polypeptides（mol wt 39，000，50，000，52，000，55，000，94，000，96，000）were synthesized more readily in the light than in the dark.One of these polypeptides co-migrated with purified unlabeled LS of RuBPCase（mol wt 52，000），and was identified as the LS，based on this and results presented in Figure 2.There is also one strongly light-initiated polypeptide（mol wt 64，000）which does not appear in the dark.

O2 Evolution of Chloroplasts from Light-Induced Potato Tubers.Upon illumination with red light，the chloroplasts，isolated from light-treated potato tuber discs after 3 weeks of light treatment，evolved O2 using ferricyanide as an electron acceptor.This Hill reaction activity is comparably high（177 μmol O2/mg Chl·h），indicating that an active electron transport reaction took place in this chloroplast preparation.

Figure 3　Incorporation of［35 S］methionine into proteins in the light-driven chloorplast protein synthesis system.5-μl samples were added to 20%TCA containing 10 μg/ml unlabeled methionine.Proteins were precipitated with 5%TCA containing 10 μg/ml unlabeled methionine，and counted with a Mark I scintillation counter.ct，chloroplasts；At，amyloplasts（control）；L，light-incubated；D，dark-incubated.