An Ala205Val Substitution in Acetohydroxyacid Synthase of Eastern Black Nightshade (Solanum ptychanthum) Reduces Sensitivity to Herbicides and Feedback Inhibition

Twelve populations of eastern black nightshade from different locations in Ontario are resistant to imazethapyr. This study aimed at determining the molecular basis of resistance in these populations and the activity of the resistant acetohydroxyacid synthase (AHAS) enzyme compared to that of the sensitive AHAS in response to different herbicides and branched-chain amino acid concentration. The results of partialAHASsequencing indicated that all resistant populations had a cytosine331to thymine substitution coding for an alanine205to valine substitution.In vitroAHAS enzyme assays of one resistant population showed that the specific activity of the resistant enzyme was 56% less than that of the susceptible enzyme. AHAS from the resistant population was 72-, 70-, and 64-fold less sensitive than that of the susceptible population to imazethapyr, imazamox, and primisulfuron, respectively. Furthermore, the resistant enzyme was less sensitive to feedback inhibition from branched-chain amino acids compared to the susceptible enzyme. Results confirmed that resistance in resistant populations of eastern black nightshade was conferred by target-site modification and that the Ala205Val substitution alters the kinetics and regulation of branched-chain amino acid biosynthesis.

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The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.52 ◽

1995 ◽

Vol 15 (1) ◽

pp. 52-57 ◽

Cited By ~ 33

Author(s):

Y Hu ◽

T G Cooper ◽

G B Kohlhaw

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Glutamate Dehydrogenase ◽

Specific Activity ◽

Yeast Cells ◽

Homologous Sequence ◽

Biosynthetic Pathways ◽

Lacz Expression ◽

Branched Chain Amino Acid ◽

Branched Chain

The Leu3 protein of Saccharomyces cerevisiae has been shown to be a transcriptional regulator of genes encoding enzymes of the branched-chain amino acid biosynthetic pathways. Leu3 binds to upstream activating sequences (UASLEU) found in the promoters of LEU1, LEU2, LEU4, ILV2, and ILV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of alpha-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when alpha-isopropylmalate is absent. Following identification of a UASLEU-homologous sequence in the promoter of GDH1, the gene encoding NADP(+)-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UASLEU element. We then show that Leu3 is required for full activation of the GDH1 gene. First, the expression of a GDH1-lacZ fusion gene is three- to sixfold lower in a strain lacking the LEU3 gene than in an isogenic LEU3+ strain. Expression is restored to near-normal levels when the leu3 deletion cells are transformed with a LEU3-bearing plasmid. Second, a significant decrease in GDH1-lacZ expression is also seen when the UASLEU of the GDH1-lacZ construct is made nonfunctional by mutation. Third, the steady-state level of GDH1 mRNA decreases about threefold in leu3 null cells. The decrease in GDH1 expression in leu3 null cells is reflected in a diminished specific activity of NADP(+)-dependent glutamate dehydrogenase. We also demonstrate that the level of GDH1-lacZ expression correlates with the cells' ability to generate alpha-isopropylmalate and is lowest in cells unable to produce alpha-isopropylmalate. We conclude that GDH1, which plays an important role in the assimilation of ammonia in yeast cells, is, in part, activated by a Leu3-alpha-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.

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Heterologous Expression and Functional Characterization of Catalytic Subunit of Rice Acetohydroxyacid Synthase

Protein and Peptide Letters ◽

10.2174/0929866525666181114153727 ◽

2019 ◽

Vol 26 (3) ◽

pp. 176-183

Author(s):

Ghazaleh Arabzadeh ◽

Azar Shahpiri

Keyword(s):

Amino Acids ◽

Specific Activity ◽

Feedback Inhibition ◽

Functional Characterization ◽

Branched Chain Amino Acids ◽

Biochemical Properties ◽

Catalytic Subunit ◽

Acetohydroxyacid Synthase ◽

Branched Chain

Background: Acetohydroxyacid Synthase (AHAS) is the first enzyme in the biosynthesis pathway of the branched chain amino acids. AHAS is the common target site of five herbicide chemical groups: sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl-thiobenzoates, and sulfonyl-aminocarbonyl-triazolinone. </P><P> Objective: The purification of protein enabled us to study the physical and biochemical properties of the enzyme. In addition in vitro activity of this enzyme was tested in the presence of four different sulfonylureaherbicides and the feedback regulation of enzyme was analyzed in the presence of branched amino acids. Methods: The gene encoding catalytic subunit of rice AHAS (cOsAHAS) without part of the chloroplast transit sequence was cloned into the bacterial expression vector pET41a and heterologously expressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST).The soluble protein was purified using affinity chromatography. The measurement of GSTOsAHAS activity was performed under optimized conditions at present of branched-chain amino acids and sulfonylurea herbicides independently. Results: The optimum pH and temperature for GST-cOsAHAS activity was 8.0 and 37 °C, respectively. The specific activity and Km value of this enzyme toward pyruvate were 0.08 U/mg and 30 mM, respectively.GST-cOsAHAS was inhibited by herbicides tribenuron, sulfosulfuron, nicosulfuron and bensulfuron while the enzyme was insensitivieto end products. Conclusion: These results suggest that the recombinant form of GST-cOsAHAS is functionally active and carries the binding site for sulfynylurea herbicides. Furthermore, GST-cOsAHAS was insensitive to feedback inhibition by endproducts which indicates the existence of a regulator subunit in rice AHAS as previously has been described in other plant AHASs.

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