*2.6. Quantitative Analysis of Phosphorus Content*

The lpa1\* mutant and wild type control seeds were tested for total P, free P and phytic acid content. No significant alterations in total P amount were observed between the putative new lpa1 mutant and the control: in fact, even if the new *lpa1* mutation caused approximately an eight-fold increase in the amount of free phosphate, this was balanced by a reduction of phytic acid that was nearly halved in comparison to the wild type content (1.28mg/g and 3mg/g, respectively) (Figure 4). In the lpa1 mutant, total phosphorus is 50% free P and 50% phytic P.

**Figure 4.** (**a**) Chen assay performed on F4 families mutant seeds. Microtiter rows 1, 2 and 3 show the results of the assay performed on nine mutant seeds, st row correspond to the standard used for the semiquantitative classification. (**b**) lpa1-5525 mutant and wild type control mature dry kernels were analyzed for total P, free inorganic P and phytic acid P amount. Values are expressed as milligrams of P (atomic weight 31) in 1 g of flour. Confidence intervals at 95% are shown.

### *2.7. Structure Analysis of the ZmMRP4 Locus*

The lpa1\* mutant was isolated through a transposon-mediated mutagenesis experiment with the *Ac* element. To check for the presence of the 4.6 kb sequence of the *Ac* transposon into the *ZmMRP4* locus, the sequence of *lpa1* gene, spanning from the 5'UTR (nucleotide -313) to the 3'UTR (nucleotide 6460) (Figure 5a) was amplified in the lpa1\* mutant and in wild type. The gel electrophoresis of the amplicons obtained failed to reveal any difference in length between wild type and the lpa1\* mutant (also in the promoter region till about -1500bp, data not shown), thus suggesting the absence of the *Ac* transposon inside the *ZmMRP4* locus (Figure 5b).

**Figure 5.** (**a**) Structure of the *ZmMRP4* gene. The primers used to amplify the coding sequence of *ZmMRP4* gene are indicated by the arrows. (**b**) Agarose gel showing the amplicons obtained amplifying wt (B73) and mutant (lpa1-5525) genomic DNA with primers 5L/ZM2r and ZM2F/51R.
