THE MAIZE VP*404 MUTANT IS IMPAIRED IN MOCO-S BIOSYNTHESIS.

part 1: DESICCATION TOLERANCE In maize, desiccation tolerance is acquired by the embryo at a precise developmental stage, between 20 and 25 DAP (days after pollination) and is probably related to the maturation process characterized by the accumulation of storage products and LEA (late embryogenesis abundant) proteins, that have a protective role. In viviparous (vp) mutants, that are deficient in ABA synthesis or lack an active vp1 factor, embryos do not express the normal set of maturation phase proteins; it is also conceivable that these embryos do not acquire the desiccation tolerance. To verify this, we applied premature desiccation to developing vp embryos about 25 DAP, and compared their germination capacity to embryos not subjected to such treatment. When cultured immediately after their excision, immature embryos of all mutants tested germinated with a high frequency (95-100%) similarly to their wild type counterparts. if they were cultured following a premature dehydration treatment, only vp1 and vp10 maintained a partial desiccation tolerance while the other mutants lost it. These results seem to suggest that acquisition of desiccation tolerance requires the completion of the steps between carotenoid production and the later stages of ABA biosynthesis, with a possible protective role of carotenoid against oxidative phenomena that take place even during desiccation, because the metabolism is still active. part 2: THE MAIZE vp*404 MUTANT is IMPAIRED in Moco BIOSYNTHESIS In maize the vp10 and vp15 genes have been isolated; they encode the orthologs of the Arabidopsis Cnx1, and Cnx7 respectively, which catalyse the last steps of Moco-O biosynthesis. Moco-O is required for the activity of both Nitrate Reductase and Sulfite Oxidase. Maize mutants defective in Moco-S are instead unknown, but Moco-S mutant plants have been described for Arabidopsis (ABA3), tomato (flacca) and tobacco (ABA1), which lack AO and XDH activities, but show normal NR enzymatic activity. These mutants show a wilty phenotype and are impaired in stress responses. vp*404 is a viviparous mutant with light green seedlings, reduced content of chlorophyll, carotenoids and ABA.Complementation tests with all viviparous mutants with green seedlings reported in literature showed that vp*404 defines a new vp gene. Moco enzyme activities analysis showed normal value for SO and an extremely reduced activity for both AO and XDH, suggesting that vp*404 is likely a Moco-Sulfurase defective mutant. In Arabidopsis the ABA3 gene encodes the Moco-Sulfurase. Blast analysis performed with the ABA3 sequence , gave two sequences with high Identity on chromosome 6. The two sequences, which are 30kb distal, encode the Moco-S N-terminal and the Moco-S C terminal domain respectively. However,since in all Eukaryotes analysed the two protein domains are encoded by the same gene, we refer to the gene in Sorghum as a model, and we have reconstructed in silico the structure of a maize unique theoretical gene, in which the C terminal domain was correctly oriented and 30,000bp between the two domains were arranged. Specific primers were designed on the basis of the theoretical gene and the corresponding cDNA sequence was analysed by means of a RT-PCR approach. Preliminary data seem to confirm the proposed gene model, however gene isolation and sequence analysis will be necessary to confirm our hypothesis.