C.N.R. - Istituto Biosintesi Vegetali

Nitrogen requirements of wildtype and opaque-2 endosperm cultures

--L. A. Manzocchi and S. Faranda

Cells from immature endosperms differentiate in culture into storage cells, corresponding in most physiological features to the intact tissue (reviewed by Felker and Goodwin, Plant Physiol. 88:1235, 1988). After the first reports on the presence of zein and protein bodies in cultures (Shimamoto et al., Plant Physiol. 73:915, 1983; Felker, Am. J. Bot. 74:1912, 1987), recent results on endosperm suspension cultures have evidenced that active zein accumulation is associated with the negative logarithmic phase of cell growth cycle, and therefore coincides with the termination of cell divisions (Lyznik and Tsai, Plant Sci. 63:105, 1989; Manzocchi, Plant Cell Rep. 1991, in press). This further analogy to the in vivo condition makes suspension endosperm cell cultures a promising in vitro system for the study of the regulation of reserve protein synthesis and nitrogen metabolism in maize endosperms at the cellular level. With respect to kernel cultures (Cully et al., Plant Physiol. 74:389, 1984) and endosperm tissue in vitro maturation (Balconi et al., Plant Sci. 1990, in press), endosperm cell suspension cultures offer, together with an easily controlled environment, the possibility to obtain protoplasts able to express foreign DNA in transient expression experiments (Manzocchi, Plant Cell Rep. 1991, in press).

Although nutritional requirements for endosperm cell growth have been described (Shannon, in Sheridan (ed.), Maize for Biological Research, p. 397, 1982), we began experiments on nitrogen requirements for cell division and zein accumulation in A69Y wt and o2 cultures. A medium containing 30 g/l sucrose, 0.4 mg/l thiamine, 200 mg/l inositol and Murashige and Skoog salts (MS-N) was used to grow cells with inorganic nitrogen supply (40 mM NO3-, 20 mM NH4+). Amino acid supplemented media were prepared from MS-N medium by addition of 15 mM asparagine (MS-A) or glutamine (MS-G), proline (MS-P) or asparagine and glutamine (7.5+7.5 mM: MS-AG). Control growth curves were obtained with medium MS-O, with the components of MS-N with the exception of nitrogen salts.

MS-N medium supported for both wt and o2 cells a growth rate approximately one half of growth obtained with MS-A (the medium normally used for endosperm cell culture); nitrogen salts, and not amino acids eventually derived from dead cells in the cultures, represent the nitrogen source, as can be stated from the complete absence of growth in MS-O medium (Fig. 1). Cells have been cultured in MS-N medium for over 5 months without modifications of growth rate; their ability to accumulate zein (expressed as protein/mg cells) was maintained through subcultures, although slightly reduced with respect to cells grown in MS-A.

MS-A, MS-G, MS-AG stimulated cell growth rate at the same extent; MS-P was less effective. Zein accumulation was similar in MS-A, MS-G and MS-P; a considerable increase was observed in MS-AG. Asparagine and glutamine, the amino acids supplied to the developing kernels by maize plants (Lyznik et al., Phytochemistry 24:425, 1985) represent a good nitrogen source for wt and o2 cultured cells, as reported for A636 cultures (Lyznik and Tsai, Plant Sci. 63:105, 1989). The ability of cells to grow and accumulate zein in the absence of an organic nitrogen source suggests the capacity of endosperm cells for a consistent amino acid synthesis from inorganic nitrogen; experiments are in progress to elucidate the levels in cultured cells of nitrogen metabolism enzymes, whose levels during endosperm development have been reported with conflicting results (reviewed by Muhitch, Physiol. Plant. 74:176, 1988).

Figure 1A and 1B. Growth curves of wildtype (wt) and opaque-2 (o2) A69Y endosperm cell cultures in MS-A (square), MS-N (triangle right-side up) and MS-O (triangle upside down) medium.

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