--S. Mapelli, S. Faranda and L. A. Manzocchi
Developing and germinating maize seeds have been extensively investigated for auxin content and metabolism, and represent a widely employed model in studies on auxin physiology and biochemical pathways (Reinecke, DM and Bandurski, RS, in "Plant Hormones and Their Role in Plant Growth and Development", P. J. Davies, ed., M. Nijhoff Pub., pp. 24-42, 1987); a new experimental approach would be represented by the use of mutations with altered auxin content or responses.
de*-B18 (from the Istituto Sperimentale per la Cerealicoltura, Stezzano, Bergamo) is a viable defective endosperm mutation of maize, monogenic, recessive, characterized by normal germination and auxin content in plant tissues, but extremely reduced levels of both free and bound indole-acetic acid (IAA) in the endosperm during the grain filling period (Torti et al., Maydica 29:335-343,1984; Theor. Appl. Genet. 72:602-605, 1986).
We have established cell suspension cultures derived from maize A69Y wildtype and de*-B18 endosperms; endosperm cell cultures represent a good tool for the study of biochemical events in developing seeds, as cells do not completely de-differentiate in culture and maintain tissue-specific synthesis such as starch, anthocyanins, and zeins (rev. in Manzocchi, LA et al., Plant Cell Rep. 7:639-643, 1989). Endosperm cultures have also been reported to express in vitro endosperm mutant phenotypes, such as waxy, amylose extender (Sarawitz, CH and Boyer, CD, Theor. Appl. Genet. 73:489-495, 1987) and opaque-2 (Manzocchi, LA, Plant Cell Rep. 9:555-558, 1991).
Calli were obtained from endosperms excised from ears harvested 10 days after pollination and cultured on agar medium without auxin addition (Manzocchi, LA et al., 1989); a good cell proliferation was obtained also for mutant endosperms, in spite of their low auxin content (Castelli, S, Manzocchi, LA, and Torti, G, in "Plant Cell Biotechnology", M. S. S. Pais, ed., NATO ASI Series, Vol. H18, Springer-Verlag, Berlin-Heidelberg, pp. 63-67, 1988). Liquid suspension cultures were obtained and subcultured every two weeks with hormone-free medium (Manzocchi, LA, Plant Cell Rep. 9:555-558, 1991), for 18 months; no substantial differences are evident between growth curves of wildtype and mutant cell cultures.
Cultured cells were analyzed for their content in auxins, extracted by acidified methanol. Methanol extract was partitioned against ethyl acetate for free indole compounds; indole ester-bound compounds were extracted in ethyl acetate after mild alkaline hydrolysis of water soluble residue. Analyses were carried out on reverse-phase C18 columns by a HPLC system equiped with fluorometric and electrochemical detectors.
Figure 1 reports auxin content of A69Y wt and de*-B18 cultured cells in logarithmic growth, and of developing endosperms (14 days after pollination), as a reference.
Cultured de*-B18 endosperm cells are characterized by a reduced content of both free and esterified IAA, with respect to wildtype cells, and they appear therefore to maintain in vitro the expression of the mutant phenotype.
Indole-pyruvic acid (IPyA), both free and bound, is found in growing cells of de*-B18, and is present in detectable amounts throughout all stages of cell growth. In wildtype cells, trace amounts of IPyA are detected only in the plateau phase. The presence of IPyA in mutant cells may give an indication of alterations in the IAA pathway; IPyA has been reported to be present in maize kernels (Stowe, BB and Thimann, KV, Arch. Biochem. Biophys. 51:499-516, 1954); in our experiments we did not detect it in developing endosperms, either normal or mutant, but we found it in mature seeds (not reported) when biochemical pathways are slowed down or stopped.
Analysis of free substances released by the cells to culture medium
confirms the different metabolism between wt and de*-B18. IAA is
released by wildtype cells, while only IPyA is detected in culture media
from mutant cells.
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