Expression of Zein in Long Term Endosperm Cultures of Maize

Abstract

Continuous cultures, established 10 days after pollination from endosperms of inbred A636 Zea mays (L.) were extracted 21 months later with aqueous ethanol. The solubilized proteins were analyzed by polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Two protein bands co-migrated with zein, the major storage protein of maize. Immunoblotting of the gel followed by incubation of the immobilized proteins with anti-zein IgG provided evidence that the polypeptides were in fact zein. Electron microscopic studies showed that the cultures contained cells with protein bodies as found in developing endosperms. The protein bodies could be isolated from the cultures and were shown to contain zein. We conclude that the long term cultures described here synthesize zein and deposit it in the form of protein bodies of the type found in developing endosperms. Thus, certain endosperm characteristics and the production of tissue-specific proteins are retained in prolonged culture.Tissue cultures which express tissue-specific proteins are highly desirable for studying the regulatory mechanisms leading to the differential expression of these proteins. In higher plants such culture systems have, to our knowledge, not been described, mainly because few plant proteins have been studied for their tissue specificity. Among the better known ones are storage proteins of seeds accumulating in developing endosperms of cereal grains (14) or in cotyledons of legumes (17). Attempts to establish long term cultures of cereal endosperms have been described (9). The first report of a successful, continuous culture of maize endosperm appeared in 1949 (16). Several more reports on endosperm cultures followed some ofwhich have been shown to produce anthocyanin (7, 25) and starch (2). Production of storage proteins in such cultures has not been investigated. Zein, which constitutes more than 50% of the seed storage proteins in maize, is synthesized on membrane-bound polysomes and deposited in the form ofprotein bodies (1, 1 1). Immunohistochemical studies have shown that zein is specifically localized in the endosperm (5). Because of its tissue specifity and the rapidly expanding knowledge of its biochemistry and molecular biology (8,13,14,20) Initiation and Maintenance of Endosperm Cultures. After carefully removing all husks and silks, ears were surface-sterilized in 0.01% mercuric chloride for 20 min and rinsed three times with sterile distilled H20. The tops of the 10-d-old kernels were cut off with a scalpel, and the endosperms were scooped out with forceps carefully avoiding the embryo. Isolated endosperms were immediately placed on 6-cm-diameter plastic Petri dishes containing 10 ml agar medium and the dishes were sealed with Parafilm. Strauss medium (26) modified according to Shannon and Batey (23) to contain 2 g/l L-asparagine and 2 g/l yeast extracts (Merck, Munich, F.R.G.) was used. All the cultures were grown in the dark at 25°C and subcultured every 3 to 4 weeks.Electron Microscopy. Two mm3 sections of each culture were pr...

Sugar Uptake by Maize Endosperm Suspension Cultures

“…The lack of susceptibility of the sucrose analog 1 '-deoxy-l '-fluorosucrose (FS') to invertase hydrolysis (12) was exploited to distinguish between uptake of intact sucrose and that of hexoses liberated by cell wall invertase. (2,27) in that the tissue contained starch and zein (data not shown). Cultures increased from 3 to 16 g fresh weight during the culture period.…”

“…The differentiation of the suspension culture cells into storage cells corresponding to the intact endosperm is evidenced by their ability to synthesize and accumulate starch (2), zein (27), and anthocyanins (19). There are also ultrastructural similarities between the cultures and the intact parent tissue (8).…”

Surface Position, Not Signaling from Surrounding Maternal Tissues, Specifies Aleurone Epidermal Cell Fate in Maize

“…Since the first establishment of maize endosperm in vitro cultures, studies of starch, storage protein, and anthocyanin biosynthesis have been reported (Chu and Shannon, 1975;Shimamoto et al, 1983;Racchi and Manzocchi, 1988). However, these studies have provided limited insight into mechanisms in endosperm development.…”

“…Although procedures have varied over time, usually, 10 d after pollination (DAP), isolated endosperm has been grown on medium with 3% Suc. Whereas these cultures retained some characteristics of in planta endosperm, including some degree of starch, storage protein, and anthocyanin accumulation (Chu and Shannon, 1975;Shimamoto et al, 1983;Racchi and Manzocchi, 1988), their lack of histological organization has limited their value for developmental biology studies (Felker, 1987).…”

Agrobacterium tumefaciens-Mediated Transformation of Maize Endosperm as a Tool to Study Endosperm Cell Biology

“…Although several approaches for culturing maize (Zea mays) endosperm in vitro have been reported in the past years (Shimamoto et al, 1983), only recently a novel method developed by Odd-Arne Olsen and colleagues (Gruis et al, 2006) has proven to be successful in retaining endosperm tissue and cell type identity in in vitro conditions. Cultures derived from transgenic maize lines in which endosperm cell types are identified by the activity of specific promoters have shown that aleurone and starchy endosperm cell identity continues to be established in vitro (Gruis et al, 2006).…”