Light-Induced Transformation of Amyloplasts into Chloroplasts in Potato Tubers'
Received for publication November 9, 1983 and in revised form January 19, 1984
YU S. ZHU, DENISE L. MERKLE-LEHMAN, SHAIN D. KUNG*
Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, Maryland 21228
The transformation of amyloplast into chloroplasts in potato (Solawum tuberosum L.) tuber tissue can be induced by light. Excised potato tuber discs illuminated with white light of 3000 lux bepgn to synthesize chlorophyll after a lag period of 1 day, and continued to synthesize chlorophyll for 3 weeks. In this paper we present evidence, based on ultracentrifugal sedimentation and immunoprecipitation, that the lightmediated synthesis of Ribulose-1,5-bisphosphate carboxylase began 1 day after illumination with white light. When illuminated the chloroplasts isolated from light-grown potato tuber tissue incorporated I3SImethionine into polypeptides, one of which has been idenified as the large subunit of Ribulose-1,5-bisphosphate carboxylase. These chloroplasts are functional as determined by 02 evolution in the Hill reaction.
A great deal of data is now available on the biochemistry of the development of chloroplasts from etioplasts (6). Much less is known about the development of chloroplasts from amyloplasts, a starch storing organeile, which exists in storage tissue, roots, and some callus. It is well known that potatoes turn green on exposure to light for several days. Not only does the study of the organelle transformation and light-regulated gene expression stimulate theoretical interest, but the greening of potato also has some practical importance, since the greening of the potato is accompanied by the formation of the poisonous alkaloid, solanine (5). The morphological and ultrastructural changes in the development of chloroplasts from amyloplasts were described in several electron microscopic studies (3, 11). In this communication we present some biochemical evidence to show the function of chloroplasts from potato tuber based on the biosynthesis of Chl, RuBPCase,2 and Hill activity.
plates in sterile Petri dishes (9 cm), 40 discs per dish. The discs were illuminated at room temperature by fluorescent tubes with intensity of 3000 lux.
Chlorophyll and RuBPCase Determination. Chl was determined according to Arnon (2). RuBPCase was detected with a Model E analytical ultracentrifuge, as previously described (9). Schlieren pictures were taken at 44,770 rpm, 10 min after
attaining this speed. An Ouchterlony double diffusion test was employed to determine the RuBPCase specifically. The antiserum was prepared against RuBPCase from tobacco (15). Light-Driven Protein Synthesis in Choroplasts. Ten g of green potato tuber discs which had been exposed to white light for 7 d were homogenized in a Waring Blendor with cold isolation buffer (sucrose 0.35 M, Hepes-NaCl 25 mm, EDTA 2 mM, isoascorbateNa 2 mm, pH 7.6) and filtered through 2 layers of Miracloth. The resultant filtrate was centrifuged at 30g for 1 min to remove starch granules, and the supernatant was then centrifuged at 2500g for 1 min. The pellet was resuspended in 1 ml of KCI
suspension (KCI 0.2 M, Tricine-KOH 66 mm, MgCl2 6.6 mM).
The chloroplast suspension was transferred to a Petri dish (3 cm in diameter) and incubated with [135S]methionine (10 ,uCi) at room temperature under white light (4000 lux). During a 2 h
incubation, the incorporation of [35S]methionine into proteins was measured. After the 2 h incubation the radioactive polypeptides were separated on an SDS-polyacrylamide gradient gel (815%), followed by fluorography as described by Blair and Ellis
02 Evolution Assay. The ability of chloroplasts from potato
tuber discs to evolve 02 was measured in the Hill reaction
according to the procedure of Marsho et al. (12). The incubation system...