*4.2. Density Assay*

Previous analyses [35] indicated that the lpa1 seeds were characterized by a lower density in comparison to the corresponding wild type controls. Starting from these data, we developed a non-disrupting, fast, cheap and rapid test to screen the mutagenized populations looking for the putative mutants containing the *Ac* transposon, *lpa1-1*/*lpa1-Ac*.

The test was performed by putting the F1 seeds of the mutagenized populations in a concentrated sucrose solution, density 1.28 g/cm<sup>3</sup> or 1,122g/cm<sup>3</sup> at the temperature of 23 ◦C. This density assay allowed the isolation of the lpa1 putative mutants, which, because of their low density, were able to float while the wild type seeds stayed on the bottom of the beaker.

This assay is non-disrupting, i.e., the selected seeds can be recovered from the beaker, rinsed, dried and used for further analysis, stored or sown.

### *4.3. Assay for Free Phosphate Content in the Seeds*

The Chen assay [28] was performed with some little modifications. The seeds were ground in a mortar with a steel pestle, and 100 mg of the flour obtained was extracted for 1 h at room temperature with 1 mL of 0.4 M HCl solution. After overnight incubation in a shaker at room temperature, 100 μL of extract was used for the free phosphate assay, adding 900 μL of Chen's reagen<sup>t</sup> (6 N H2SO4, 2.5% ammonium molybdate, 10% ascorbic acid, H2O [1:1:1:2, *v*/*v*/*v*/*v*]) in microtiter plates. After incubation of 1 h at room temperature the blue-colored phosphomolybdate complex was observed: the intensity of the blue color is directly proportional to the free phosphate content. The free phosphate content was quantified by using a spectrophotometer (λ 650 nm) and adopting a series of calibration standards obtained from a stock solution of KH2PO4.
