Development of Ribulose 1,5-Diphosphate Carboxylase in Nonphotosynthetic Endosperms of Germinating Castor Beans

Abstract

Previous reports have described the presence and properties of RuDP2 carboxylase in castor bean endosperms (4, 15). These are the only reports of the occurrence of this enzyme in nonphotosynthetic higher plant tissue. RuDP carboxylase has been localized in the proplastids of germinating castor beans (15) and the enzyme reaches a peak activity in the dark in 4 days (4). Dark-grown etiolated leaves contain low amounts of RuDP carboxylase activity which increases substantially only after exposure of the leaves to light (6, 10, 13). The purpose of this paper was to study the unique development of RuDP carboxylase and proplastids in the endosperms of germinating castor beans. were dusted with Arasan. The seeds were imbibed in H20 for 24 hr and germinated in moist vermiculite at 35 C in the dark. For developmental studies, the seedlings were harvested from 0 to 8 days. For antibiotic studies, the seedcoats were removed, and the seeds were imbibed in H20, 2 Ag/ml CHI, or 50 Atg/ml CAP. MATERIALS AND METHODSThe imbibed seeds were germinated at 35 C in the dark in Petri dishes in H20 or antibiotic solution. To study the effect of light on the development of RuDP carboxylase, the imbibed seeds, without seedcoats, were germinated in the light or dark at 35 C. The light source was General Electric cool-white 20-w fluorescent bulbs. The light intensity at the seed surface was 30 ,ueinsteins m-2 sec-1.Previously (4), it has been shown that RuDP carboxylase activity peaks in 4 day old germinating castor beans. We found that both the rate of growth of the castor bean seedlings and the rate of development of RuDP carboxylase in the seedlings are influenced by (a) removal of the hard seedcoat prior to germination and (b) the watering regime during the growth of the seedlings. Removing the seedcoats, prior to imbibing and germinating the seeds, increases the rate of seedling growth. In these seedlings growing in Petri dishes, RuDP carboxylase activity peaks 48 hr after germination. Depending on the watering regime, the peak carboxylase activity can be extended 1 to 2 days in seeds germinating in vermiculite. Because of these variables, in most experiments we have recorded the chronological age of the seedlings and the fresh weight of the endosperms. In most experiments, maximal RuDP carboxylase activity developed in seedlings with an endosperm weight between 0.60 and 0.84 g fresh weight/seed. Enzyme Isolation and Purification. RuDP carboxylase was isolated and partially purified from endosperms of 5-day-old seedlings. (0.75 g endosperm/seed). All steps were carried out at 4 C. The endosperms were separated from the testa, hypocotyl, epicotyl, and cotyledons and rinsed with deionized H20. The endosperms were homogenized for 60 sec in a prechilled Waring Blendor in 0.1 M tris buffer, pH 7.5, containing 0.1 mm GSH. The homogenizing medium contained 1 ml buffer/g fresh weight of endosperm. The homogenate was filtered through two layers of cheesecloth and centrifuged 15 min at 1OOOg in a RC-5 Sorvall refrigerated centrifuge. Th...

Development of Ribulose-1,5-Diphosphate Carboxylase in Castor Bean Cotyledons

“…Vol. 59,1977 sperm was substantially similar to that described by Benedict (3,8) and Osmond et al (16). However, as RuDP Case activity declined in the endosperm there was a rapid increase in activity in the cotyledons (Fig.…”

Photochrome-mediated regulation of messenger RNAs for the small subunit of ribulose 1, 5-bisphosphate carboxylase and the light-harvesting chlorophyll a/b-protein in Lemna gibba

“…It is also clear that in many species phytochrome action does not regulate the amount of this enzyme. As an extreme example, Gillen et al [15] found that the amount of RuBP carboxylase increased in the dark in the nonphotosynthetic endosperm of germinating castor beans.…”

Mapping the castor bean endosperm proteome revealed a metabolic interaction between plastid, mitochondria, and peroxisomes to optimize seedling growth

“…An alternative pathway to provide pyruvate for plastidial fatty acid biosynthesis could be via RubisCO. The activity of this enzyme has been demonstrated in the endosperm of germinating castor beans (Benedict, 1973;Osmond et al, 1975;Gillen et al, 1976) and the corresponding protein subunits were found in our plastid proteome (B9SYG1 and G1D766; Figure 9, No. 1).…”