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Graminicide insensitivity correlates with herbicide-binding co-operativity on acetyl-CoA carboxylase isoforms.

ABSTRACT: The sensitivity of grass species to important classes of graminicide herbicides inhibiting ACCase (acetyl-CoA carboxylase) is associated with a specific inhibition of the multifunctional ACCase located in the plastids of grasses. In contrast, the multisubunit form of ACCase found in the chloroplasts of dicotyledonous plants is insensitive and the minor cytosolic multifunctional isoforms of the enzyme in both types of plants are also less sensitive to inhibition. We have isolated, separated and characterized the multifunctional ACCase isoforms found in exceptional examples of grasses that are either inherently insensitive to these graminicides, or from biotypes showing acquired resistance to their use. Major and minor multifunctional enzymes were isolated from cell suspension cultures of Festuca rubra and the 'Notts A1'-resistant biotype of Alopecurus myosuroides, and their properties compared with those isolated from cells of wild-type sensitive A. myosuroides or from sensitive maize. Purifications of up to 300-fold were necessary to separate the two isoforms. The molecular masses (200-230 kDa) and K(m) values for all three substrates (ATP, bicarbonate and acetyl-CoA) were similar for the different ACCases, irrespective of their graminicide sensitivity. Moreover, we found no correlation between the ability of isoforms to carboxylate propionyl-CoA and their sensitivity to graminicides. However, insensitive purified forms of ACCase were characterized by herbicide-binding co-operativity, whereas, in contrast, sensitive forms of the enzymes were not. Our studies on isolated individual isoforms of ACCase from grasses support and extend previous indications that herbicide binding co-operativity is the only kinetic property that differentiates naturally or selected insensitive enzymes from the typical sensitive forms usually found in grasses.

SUBMITTER: Price LJ

PROVIDER: S-EPMC1223688 | biostudies-other | 2003 Oct

REPOSITORIES: biostudies-other

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Project description:BackgroundPolyadenylation is a crucial process that terminates mRNA molecules at their 3'-ends. It has been observed that alternative polyadenylation (APA) can generate multiple transcripts from a single gene locus, each with different polyadenylation sites (PASs). This leads to the formation of several 3' untranslated regions (UTRs) that vary in length and composition. APA has a significant impact on approximately 60-70% of eukaryotic genes and has far-reaching implications for cell proliferation, differentiation, and tumorigenesis.ResultsIn this study, we conducted long-read, single-molecule sequencing of mRNA from peanut seeds. Our findings revealed that over half of all peanut genes possess over two PASs, with older developing seeds containing more PASs. This suggesting that the PAS exhibits high tissue specificity and plays a crucial role in peanut seed maturation. For the peanut acetyl-CoA carboxylase A1 (AhACCA1) gene, we discovered four 3' UTRs referred to UTR1-4. RT-PCR analysis showed that UTR1-containing transcripts are predominantly expressed in roots, leaves, and early developing seeds. Transcripts containing UTR2/3 accumulated mainly in roots, flowers, and seeds, while those carrying UTR4 were constitutively expressed. In Nicotiana benthamiana leaves, we transiently expressed all four UTRs, revealing that each UTR impacted protein abundance but not subcellular location. For functional validation, we introduced each UTR into yeast cells and found UTR2 enhanced AhACCA1 expression compared to a yeast transcription terminator, whereas UTR3 did not. Furthermore, we determined ACC gene structures in seven plant species and identified 51 PASs for 15 ACC genes across four plant species, confirming that APA of the ACC gene family is universal phenomenon in plants.ConclusionOur data demonstrate that APA is widespread in peanut seeds and plays vital roles in peanut seed maturation. We have identified four 3' UTRs for AhACCA1 gene, each showing distinct tissue-specific expression patterns. Through subcellular location experiment and yeast transformation test, we have determined that UTR2 has a stronger impact on gene expression regulation compared to the other three UTRs.

Project description:The steady-state kinetics of two multifunctional isoforms of acetyl-CoA carboxylase (ACCase) from maize leaves (a major isoform, ACCase1 and a minor isoform, ACCase2) have been investigated with respect to reaction mechanism, inhibition by two graminicides of the aryloxyphenoxypropionate class (quizalofop and fluazifop) and some cellular metabolites. Substrate interaction and product inhibition patterns indicated that ADP and P(i) products from the first partial reaction were not released before acetyl-CoA bound to the enzymes. Product inhibition patterns did not match exactly those predicted for an ordered Ter Ter or a random Ter Ter mechanism, but were close to those postulated for an ordered mechanism. ACCase2 was about 1/2000 as sensitive as ACCase1 to quizalofop but only about 1/150 as sensitive to fluazifop. Fitting inhibition data to the Hill equation indicated that binding of quizalofop or fluazifop to ACCase1 was non-cooperative, as shown by the Hill constant (n(app)) values of 0.86 and 1.16 for quizalofop and fluazifop respectively. Apparent inhibition constant values (K' from the Hill equation) for ACCase1 were 0.054 microM for quizalofop and 21.8 microM for fluazifop. On the other hand, binding of quizalofop or fluazifop to ACCase2 exhibited positive co-operativity, as shown by the (napp) values of 1.85 and 1.59 for quizalofop and fluazifop respectively. K' values for ACCase2 were 1.7 mM for quizalofop and 140 mM for fluazifop. Kinetic parameters for the co-operative binding of quizalofop to maize ACCase2 were close to those of another multifunctional ACCase of limited sensitivity to graminicide, ACC220 from pea. Inhibition of ACCase1 by quizalofop was mixed-type with respect to acetyl-CoA or ATP, but the concentration of acetyl-CoA had the greater effect on the level of inhibition. Neither ACCase1 nor ACCase2 was appreciably sensitive to CoA esters of palmitic acid (16:0) or oleic acid (18:1). Approximate IC50 values were 10 microM (ACCase2) and 50 microM (ACCase1) for both CoA esters. Citrate concentrations up to 1 mM had no effect on ACCase1 activity. Above this concentration, citrate was inhibitory. ACCase2 activity was slightly stimulated by citrate over a broad concentration range (0.25-10 mM). The significance of possible effects of acyl-CoAs or citrate in vivo is discussed.

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Graminicide insensitivity correlates with herbicide-binding co-operativity on acetyl-CoA carboxylase isoforms.

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