All endosperm mutants genotypes were converted to the A69Y background through six backcrossing cycles, following by several rounds of self pollination, they are phenotypically uniform and appear genetically homogeneous ARQ197 NSCLC as expected, because after six backcross generations the mutant inbred lines should share, on average, 99% of the recurrent parent genome. The homozygous o2o7 double mutant was obtained by cross ing the above mentioned o2 and o7 A69Y lines, and selecting for the homozygous double mutant kernels. A minimum of 8 well filled ears for each genotype were sampled at 14 days after pollination, a stage where storage protein and starch syntheses commence, and frozen immediately in liquid nitrogen. Kernels were taken from the centre of each ear, the endosperm was dissected from the embryo and pericarp and stored at 80 C.

Mature kernels were harvested after physiological maturity and dried in a forced air oven. To minimize the effect of biological variation between ears on gene expression, equal numbers of dissected endosperms from 4 ears were pooled and treated as one sample, thus a minimum of three replicated samples was used for each experiment. Total Nitrogen, protein and amino acid analysis Protein analyses were performed with endosperm from mature kernels. Samples were freeze dried, ground in a mortar, and analyzed for total nitrogen content on an automated N analyzer follow ing the method of Dumas. Total endosperm proteins were extracted in duplicate, from 10 20 endosperms and fractionated as previously described by.

The percen tage of total protein was calculated by subtract ing the value of non protein N evaluated from the value obtained for total N content. Amino acids analysis was performed at the analytical facility of the University of Milan. Measurements were made with pooled samples of 15 kernels for each genotype, the data pre sented are the means of four independent assays. 2 D SDS PAGE Isoelectric focusing was performed with a Multi phor II System. 0. 5 mm thick IEF gels containing 3. 3% acrylamide bis, 0. 04% ammo nium persulfate, 0. 07% TEMED, Ampholine carrier ampholytes, pH 3. 5 10, pH 4 6, pH 5 7, pH 7 9, pH 8 10. 5, and 6 M urea, were cast onto a gel support med ium. Electrodes were placed at a distance of 13 cm. Wicks were soaked in 0. 5 M H3PO4 and 0. 5 M NaOH.

Sample wells were placed 1 cm from the anode and loaded with protein samples dissolved in IEF resus pension buffer and with 10 ul pI markers. IEF was performed at 8 W for 2 h. After IEF separation, one gel strip per well was cut out and equilibrated for 30 min. in 1. 12 M glycerol, 75mM Tris HCl pH 6. 8, 2. 4% SDS and 2. 5% 2 mercaptoethanol. For the second dimension, GSK-3 a 15% Laemmli gel with a 2 cm stacking gel was cast without slot former and the IEF strip was then mounted at the cathodic end. After SDS PAGE, gels were stained and dried.