ABSTRACT
Pea seeds contain two major storage proteins, legumin and vicilin, in proportions that are genetically and environmentally determined. They are synthesized from at least 40 genes and at least 10 different genetic loci. Mutant alleles at loci involved in starch synthesis, which result in perturbations in starch accumulation, also affect the expression of legumin genes, thereby influencing the legumin: vicilin ratio within the total seed protein. Examples of such alleles include r (starch-branching enzyme) and rb (ADP-glucose pyrophosphorylase), both of which result in a reduction in legumin synthesis; double mutants (rrb) show a particularly severe reduction in the amount of legumin. The effects of such mutations are specific to legumins. The amounts of vicilin are unaffected by mutations at r or rb. One of the consequences of the production of legumin from many genes is structural heterogeneity that is believed to preclude the purification of homogeneous legumin for crystallization and 3D-structure determination. Expression of cloned legumin cDNA in E. coli can result in sequence homogeneity, but E. coli is unable to carry out the normal proteolytic processing of legumin precursors and consequently such material is different from that produced in pea seeds. This paper describes the high-level synthesis, processing and assembly of pea legumin in transgenic wheat seeds, leading to the spontaneous in vitro formation of paracrystalline arrays of legumin, which may be attributed to the fact that the legumin consists of a single type of subunit. Such material might be used as a source of single-sequence, processed and assembled pea legumin for structural investigation.
Subject(s)
Gene Expression Regulation, Plant , Pisum sativum/genetics , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Triticum/genetics , Mutation , Pisum sativum/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Plants, Genetically Modified/chemistry , Plants, Genetically Modified/metabolism , Seed Storage Proteins , Seeds/chemistry , Triticum/chemistry , Triticum/metabolism , LeguminsABSTRACT
We have characterized an almond (Prunus dulcis) lipoxygenase (LOX) that is expressed early in seed development. The presence of an active lipoxygenase was confirmed by western blot analysis and by measuring the enzymatic activity in microsomal and soluble protein samples purified from almond seeds at this stage of development. The almond lipoxygenase, which had a pH optimum around 6, was identified as a 9-LOX on the basis of the isomers of linoleic acid hydroperoxides produced in the enzymatic reaction. A genomic clone containing a complete lipoxygenase gene was isolated from an almond DNA library. The 6776-bp sequence reported includes an open reading frame of 4667 bp encoding a putative polypeptide of 862 amino acids with a calculated molecular mass of 98.0 kDa and a predicted pI of 5.61. Almond seed lipoxygenase shows 71% identity with an Arabidopsis LOX1 gene and is closely related to tomato fruit and potato tuber lipoxygenases. The sequence of the active site was consistent with the isolated gene encoding a 9-LOX.
Subject(s)
Gene Expression Regulation, Plant , Lipoxygenase/genetics , Lipoxygenase/metabolism , Rosales/enzymology , Seeds/enzymology , Amino Acid Sequence , Arabidopsis/enzymology , Base Sequence , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA Probes/genetics , DNA Probes/metabolism , Exons , Fatty Acids, Unsaturated/metabolism , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Hydrogen-Ion Concentration , Introns , Linoleic Acids/metabolism , Lipid Peroxides/metabolism , Lipoxygenase/chemistry , Microsomes/enzymology , Molecular Sequence Data , Molecular Weight , Phylogeny , Rosales/genetics , Rosales/growth & development , Seeds/genetics , Seeds/growth & development , SolubilityABSTRACT
We identified a lipoxygenase expressed early during almond seed development. Biochemical and molecular characterisation showed that the enzyme produces almost exclusively 9-hydroperoxides which have been demonstrated to be important factors for the production of characteristic aromas in several fruits. An almond LOX cDNA was identified by RT-PCR using RNA extracted from immature almond seeds. Sequence analysis revealed that the identified gene is closely related to tomato fruit and potato tuber lipoxygenases. The isolated cDNA was cloned into pET24a and the expression of recombinant protein was induced in E. coli. The presence of an active LOX was confirmed in cells containing the recombinant vector. HPLC analysis of the reaction products of recombinant almond LOX confirmed that the isolated cDNA encodes a 9-LOX.
