The seed coat is a maternal organ which surrounds the embryo and is involved in the control of its nutrition. This study with pea (Pisum sativum L.) was conducted to understand more fully the sucrose/starch interconversions occurring in the seed coat. The concentrations of soluble sugars, the starch content, and the activities of the sucrose-metabolizing enzymes, sucrose synthase (Sus; EC 2.4.1.13), alkaline and soluble acid invertase (EC 3.2.1.26) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) were compared at four developmental stages during seed filling. Among the four enzymes, only Sus activity was very high and strongly correlated with the starch concentration in the seed coat. Sucrose synthase catalyses the cleavage of sucrose in the presence of UDP into UDP-glucose and fructose. Sucrose synthase was purified from pea seed coats in a three-step protocol, consisting of diethylaminoethyl-Sephacel chromatography, gel filtration and affinity chromatography. The enzyme was characterized at the biochemical and molecular levels. Sucrose synthase exhibits biochemical properties which allow it to function in the direction of both sucrose cleavage and synthesis. The mass-action ratio of its four substrate was close to the theoretical equilibrium constant at the four developmental stages we studied. A labelling experiment on seed coats has shown that Sus activity is reversible in vivo and can produce 37% of neo-synthesized sucrose in the seed coat cells (minimum value). It is concluded that Sus could play a central role in the control of sucrose concentration in the seed coat cells in response to the demand for sucrose in the embryo during the development of the seed.

During germination, maize seedlings express a phytase able to hydrolyse the large amount of phytin stored in the dry seed. Previous studies allowed purification and characterization of this enzyme as a homodimer of 38 kDa subunits [Laboure, Gagnon and Lescure, Biochem. J. (1993) 295, 413-419]. In the present work, an antibody against the purified maize phytase has been used to screen a maize seedling cDNA expression library. Several positive clones containing an insert of about 1400 bp were isolated. The nucleotide sequence of the insert of one of these clones has been established. This cDNA, called phy S11, was 1335 bp long and contained an open reading frame of 387 amino acids. The sequence of N-terminal residues (23 amino acids) of the purified phytase has been established. These residues are found at positions 19-41 of the amino acid sequence encoded by phy S11. This confirms that this cDNA codes for the maize phytase. The deduced amino acid sequence appears to be very different from those of published Aspergillus niger phytases; however, an homologous region of 33 amino acids was detected. This region of the fungal sequence contains the RHGxRxP consensus motif found in various high molecular mass acid phosphatases and believed to be the acceptor site for phosphate. Expression of the phy S11 cDNA in Escherichia coli allowed the production of the phytase subunit and its assembly to give a protein of the same size as the native phytase. The time course of phy S11 mRNA accumulation during germination showed that no transcript was present in dry seeds. The mRNA accumulated during the first day of germination, to reach a maximum after 2 days (radicle protrusion), and then decreased in young seedlings. Genomic Southern blot analyses suggest the existence of at least two genes and genetic mapping reveals two loci separated by 1 cM on chromosome 3 of maize. The cloning of this first cDNA coding for a plant phytase, will allow the isolation of the corresponding genes and the study of their regulation during germination.

Up to 80% of Zea mays L. grain phosphorus is stored in the form of phytin in the embryo. Our objective is to determine the control of phytin mobilization during germination and seedling growth. A maize phytase cDNA, phy S11, has been previously characterized (Maugenest et al., Biochem J 322: 511-517, 1997). In the present work, phy S11 was used to screen a maize genomic library and two distinct genes, PHYT I and PHYT II, were isolated and sequenced. The transcribed sequences of these two genes presented a strong homology whereas the untranscribed upstream and downstream sequences appeared very different. Northern blot analysis and in situ hybridization showed a high accumulation of phytase mRNA at the early steps of germination in the coleorhiza, radicle cortex and coleoptile parenchyma. Phytase expression was also detected at a lower extent in the scutellum. In adult plants, northern blot analyses revealed low but significant levels of phytase mRNA in the roots. In situ hybridizations on root cross-sections localized phytase mRNA in rhizodermis, endodermis and pericycle layers. Immunolocalization analysis showed phytase accumulation at the same sites as its mRNA. A RT-PCR approach was used in an attempt to discriminate between the transcripts from each gene in the different situations. These experiments indicate that both genes are expressed during germination, whereas only PHYT I is expressed in adult roots. This suggests that signals responsible for phytase gene expression in roots are different from those responsible for gene expression during germination.