ABSTRACT
Plant metallothioneins (MTs) constitute a family of small Cys-rich proteins capable of coordinating metal ions, significantly differing from microbial and animal MTs. They are divided into four subfamilies depending on the Cys pattern in their sequence. In this work, the MT system of the sunflower plant (Helianthus annuus) has been defined, with ten genes coding for MTs (HaMT) belonging to the four plant MT subfamilies; three HaMT1, four HaMT2, one HaMT3 and two HaMT4 isoforms. The gene expression pattern and capacity to confer metal resistance to yeast cells have been analysed for at least one member of each subfamily. The divalent metal ion-binding abilities of HaMT1-2 and HaMT2-1 (the isoforms encoded by the most abundantly expressed HaMT1 and HaMT2 isogenes) have been characterised, as HaMT3 and HaMT4 were previously studied. Those isoforms constitute an optimum material to study the effect of Cys number variability on their coordination abilities, as they exhibit additional Cys residues regarding the canonical Cys pattern of each subfamily. Our results show that the variation in the number of Cys does not drastically modify their M(II)-binding abilities, but instead modulates the degree of heterogeneity of the corresponding recombinant syntheses. Significantly, the Zn(II)-HaMT1 complexes were highly susceptible to proteolytic cleavage. The recombinant Cd-MT preparations of both isoforms exhibit significant acid-labile sulphide content-Cd6S8 or Cd7S7 species. Overall results suggest that HaMT2-1 is probably associated with Cd(II) detoxification, in contrast to HaMT1-2, which may be more related to physiological functions, such as metal ion transport and delivery.
Subject(s)
Cadmium/metabolism , Helianthus/metabolism , Metallothionein/metabolism , Plant Proteins/metabolism , Zinc/metabolism , Amino Acid Sequence , Cadmium/chemistry , Cadmium/pharmacology , Circular Dichroism , Drug Resistance/genetics , Gene Expression Regulation, Plant , Genetic Complementation Test , Helianthus/genetics , Metallothionein/chemistry , Metallothionein/genetics , Molecular Sequence Data , Mutation , Plant Proteins/chemistry , Plant Proteins/genetics , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Sequence Homology, Amino Acid , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, Ultraviolet , Zinc/chemistry , Zinc/pharmacologyABSTRACT
Cadmium is a highly toxic heavy metal for both plants and animals. The presence of Cd in agricultural soils is of major concern regarding its entry into the food chain, since Cd compounds are readily taken up by plants, and accumulated in edible parts due to their high solubility. In this study, we first demonstrate the high capacity for Cd concentration of soybean grains. Consequently, we considered the study and characterization of the molecular determinants of Cd accumulation -such as metallothioneins (MT)- to be of major practical importance. We report here the first characterization of the soybean MT system, with the identification of nine genes (one of which is a truncated pseudogene), belonging to the four plant MT types. The most highly expressed of each type was chosen for further function analysis. All of them are expressed at high levels in soybean tissues: GmMT1, GmMT2 and GmMT3 in roots, shoots and seeds, and GmMT4 only in seeds. The corresponding recombinant soybean MTs, synthesized in Escherichia coli cells cultured in metal supplemented media, exhibit greater cadmium than zinc binding capacity. These results suggest a definite role of GmMTs in Cd(II) accumulation as one of the main responses of soybean to an overload of this metal.
Subject(s)
Cadmium/toxicity , Glycine max/metabolism , Metallothionein/metabolism , Plant Proteins/metabolism , Cadmium/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Metallothionein/antagonists & inhibitors , Plant Proteins/antagonists & inhibitors , Plant Roots/metabolism , Polymerase Chain Reaction , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
The physicochemical properties of the protein and starch fractions of flour obtained from buckwheat grains that were previously dehulled or puffed after dehulling were investigated. Dehulling removed most of the nonprotein, nonstarch components of the grain, without affecting the chemical and structural features of the protein and starch components, as made evident by microstructural and spectroscopic measurements. Puffing resulted in extensive modifications of the interprotein network as well as in most of the properties of the buckwheat starch. Flours obtained from dehulled or puffed after dehulling grains were blended with 60-80% wheat flour and tested for their dough-making ability. Blends containing dehulled and puffed buckwheat flours gave dough of much lower quality than dehulled, but had water-holding properties that may be of interest for the shelf life of baked products.
