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The PsMTA gene from pea (Pisum sativum) shares similarity with metallothionein (MT) genes and related sequences have also been isolated from a number of other higher-plant species. The proteins encoded by these genes have not yet been purified from plants and their functions remain unclear although, by analogy to MT, roles in the metabolism and detoxification of metal ions have been proposed. By contrast, correlation between transcript abundance and Fe availability has led to an alternative proposal that these genes are involved in mechanisms of Fe efficiency. Phenotypic effects of constitutive PsMTA expression were examined in Escherichia coli and Arabidopsis thaliana. Copper accumulation by E. coli cells expressing recombinant PsMTA protein was approximately 8-fold greater than in control cells. No significant effects on the accumulation of Zn or Cd were detected. In segregating A. thaliana progeny, derived from a transgenic F1 parent containing the PsMTA gene under the control of a CaMV 35S promoter, 75% of individuals accumulated more Cu (several-fold in some plants) than untransformed, control plants. These data suggest that PsMTA protein binds Cu in planta and that uncoupled (constitutive) expression of the PsMTA gene causes enhanced Cu accumulation. Roots of P. sativum plants grown under conditions of low Fe availability showed elevated activity of root surface Fe(III) reductase and accumulated more Cu than roots of plants grown in an Fe-supplemented solution. Changes in the expression of MT-like genes, coincident with changes in Fe availability, are consistent with a role in Cu homoeostasis.
Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme specific to lysine biosynthesis in plants, was In plants and bacteria, DHPS2 catalyzes the first step specific to lysine synthesis in the general pathway for biosynthesis of aspartate-derived amino acids including threonine, isoleucine, and methionine (2). DHPS isolated from plants is feedback inhibited by relatively low concentrations of lysine, indicating that subcellular end product concentrations contribute to regulation of DHPS activity and metabolite flux through the lysine-specific branch. This potential regulatory role in lysine synthesis has attracted interest to DHPS as a target for selection of feedback-resistant mutants (17) This study was initiated to obtain purified maize DHPS for physical characterization and for kinetic and inhibitor studies. We obtained highly purified DHPS from fully expanded maize leaf blades and cell suspension cultures with 23 to 25% recovery. The native enzyme is composed of four subunits with mol wt of approximately 38,000 and is subject to substrate inhibition by high ASA concentrations (>2 mM) and to feedback inhibition by low lysine concentrations (<100 ltM).A partial N-terminal amino acid sequence of the purified DHPS was also used to corroborate the nucleotide sequence of a cDNA clone for maize DHPS (8). MATERIALS AND METHODS Plant MaterialBlack Mexican Sweet maize (Zea mays L.) cell suspension cultures were maintained on a modified Murashige and Skoog medium by a 1:20 dilution into fresh liquid medium every 7 d (5). Cells were harvested during mid-log phase 5 d after subculture. Plants of inbred line A619 and two homozygous mutant lines, Ask-LTJ9 (12) and Ask2-LT20 (4), were grown in the field nursery at St. Paul, MN, in 1989. The Ask-LTJ9 and Ask2-LT20 alleles are altered forms of the aspartate kinase structural genes (Ask and Ask2) which result in reduced feedback inhibition of aspartate kinase by lysine (7). The two or three youngest, fully expanded leaves were harvested from plants at the five-leaf stage.
We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity. A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44 degrees C on ampicillin was isolated. Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-glycerol-3-phosphate acyltransferase activity to the cells. Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells. Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in acyltransferase assays. The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42,543. The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids. Homologies between the SLC1 protein, the 1-acyl-sn-glycerol-3-phosphate acyltransferase of E. coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable.
Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants. Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism. To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA- auxotroph. The maize DHPS activity expressed in the complemented E. coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme. The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence. The molecular weight of 35,854, predicted from the deduced amino acid sequence, was similar to the 38,000 Mr determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize. DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues. Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome.
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