Assaying maize pollen phytic acid

--Victor Raboy, Glenn M. Magyar, Paolo Gerbasi

As part of a survey of pollen phytic acid (myo-inositol hexaphosphate) levels in 3 gymnosperm and 25 angiosperm species (the only survey of its kind in the literature), Jackson et al. (Phytochem. 21:1255, 1982) reported that the maize pollen sampled contained 2.6 mg phytic acid/gm dry weight. Since phytic acid is typically thought of (by plant biologists at least) primarily as a phosphorus (P) storage compound, these authors also assayed pollen total P, and determined the percent of total P represented by phytic acid P. Their maize pollen sample contained 5.3 mg total P/gm dry weight and phytic acid P (0.73 mg/gm) represented 14% of total P. They did not identify the maize stock tested in their survey.

To assay pollen phytic acid, these authors first ground pollen in liquid N2, extracted in 0.02 M Na4EDTA, heated the extracts in a boiling water bath, and following centrifugation, subjected aliquots of supernatant to high-volt-

age paper electrophoresis (0.1 M oxalic acid pH 1.5; 13 V/cm; 2 hr; Whatman 3mm paper). P-containing compounds were visualized using an acid molybdate stain. Spots co-migrating with standard phytic acid were visually quantified by comparison with a set of standards. They report that their lower limit of sensitivity was a spot containing approximately 0.66 �g phytic acid (1.0 n mol).

Our laboratory has been using an essentially identical method of paper electrophoresis with a similar lower limit of sensitivity to screen for EMS-induced mutations which perturb kernel phytic acid synthesis (Raboy et al., Maydica 35:383, 1990). We would like to use similar methods to screen for mutations at the level of pollen as well as seed. So, as a preliminary experiment, we used the methods of Jackson et al. (faithfully) to test pollen sampled from F2 and F3 progeny of an A632 x Mol7 cross, and from an "Early ACR composite," both kindly provided by M. G. Neuffer and used in our work. Despite repeated efforts, we could not detect phytic acid in these pollen samples using the methods of Jackson et al.

We therefore decided to use a more reliable approach to quantifying phytic acid, the "ferric-precipitation" method (described in Raboy< V et al., Maydica 35:385, 1990). Using this method, we found that the A632xMo17 progenies' pollen contained from 0.49 to 0.56 mg phytic acid/gm dry weight, and the "Early ACR composite" lines contained from 0.49 to 0.82 mg phytic acid/gm dry weight. These samples therefore contained from one-fifth to one-third the level of pollen phytic acid reported for maize by Jackson et al. However, we found similar levels of pollen total P, ranging from 5.2 to 6.2 mg total P/gm dry weight. Thus the lower levels of pollen phytic acid we observed relative to the report of Jackson et al. were not due to dramatically reduced supply of P to the developing pollen grain. In our hands, maize pollen phytic acid P represents from 3% to 4% of pollen total P.

Since we still may not have been efficiently extracting and assaying maize pollen phytic acid, we continued to test a variety of combinations of methods (grinding in liquid N2, different extraction media, freeze-thawing, heating, ferric precipitation, barium precipitation, paper electrophoresis, etc.). All tests gave values in the 0.5 to 1.0 mg phytic acid/gm pollen range, confirming our initial "ferric-precipitation" results.

Another possibility is that the discrepancy between the value reported by Jackson et al. and our results reflects a genotypic difference. We plan to conduct an informal survey this summer to test this hypothesis, and to continue efforts to develop rapid and reliable methods to assay pollen phytic acid.

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