Lyase Synthesis Research Articles

Optimizing pectin lyase production using the one-factor-at-a-time method and response surface methodology.

Pectinases are commonly industrially synthesized by molds. This study aimed to optimize pectin lyase synthesis by a bacterium, Pseudomonas fluorescens, using both the one-factor-at-a-time (OFAT) method and response surface methodology. First, on optimization of pectin lyase fermentation by the OFAT method, the effects of pectin, peptone, yeast extract, (NH4)2SO4, pH, and salts were investigated. The highest pectin lyase activity was determined to be 28.63U/mL at pH 8, 30°C, with 1% (w/v) pectin and 0.14% (w/v) (NH4)2SO4 concentration at the 90th hour. The effect of substrate inhibition on the microbial growth was also investigated, and the results showed that the process can be described by noncompetitive inhibition model. The values of kinetic constants were determined as µm=0.175h-1, KS=6.931g/L, and, KI=6.932g/L by nonlinear regression analysis. It was reported that pectin lyase enzymes exhibited peak activity at 50°C and pH 8. Finally, response surface methodology (RSM) was utilized to optimize pH, concentrations of ammonium sulfate, and pectin, which were chosen as independent variables. The interactions between these variables were also examined. According to RSM, the optimum values of the parameters to achieve a maximum pectin lyase activity of 35.62U/mL were determined to be pH 7.97, 1.25% (w/v) pectin concentration, and 0.25% (w/v) (NH4)2SO4 concentration.

Boosting NAD blunts T H17 inflammation via arginine biosynthesis and redox regulatory control in healthy control and psoriasis human subjects

Abstract Although NAD +levels modulate metabolism, how this regulates immunometabolism/inflammation remains unclear. Employing Nicotinamide Riboside (NR) to boost NAD, we explored T H17-linked inflammatory in Psoriasis. Primary Psoriasis CD4 +T cells exposed to NR reduced IL-17 secretion and RNA-seq/pathway analysis implicated NR in modulating sequestosome 1 (SQSTM1/p62)-coupled oxidative stress. In parallel, NR reduced CD4 +T cell reactive oxygen species (ROS) levels and activated the NRF2 transcription factor. Depletion of NRF2 or SQSTM1 diminished NR effects on ROS and T H17 activation. Metabolomic analysis implicated NR in arginine biosynthesis and L-arginine replicated NR CD4 +T cell effects. Genetic disruption of the argininosuccinate lyase synthesis enzyme ameliorated NR antiinflammatory effects, supporting that NR, via arginine biosynthesis orchestrates NRF2 activation, CD4 +T cell antioxidant defenses and blunted T H17 responsiveness. A pilot in-vivo human study similarly showed that NR increased arginine, antioxidant enzyme gene expression and blunted T H17 regulation, implicating NAD metabolism in CD4 +T cell-linked inflammation. NHLBI Division of Intramural Research (MNS – ZIA-HL005102), NIH Bench to Bedside award (MNS and RT, HL-129510-04S1) and the NIH Office of Dietary Supplementation (JT) and the UK MRC (JLG – MR/P011705/2; UKDRI-5002; MAP UK).

Induction of the glycolysis product methylglyoxal on trimethylamine lyase synthesis in the intestinal microbiota from mice fed with choline and dietary fiber.

The present study investigated the induction of the glycolysis product methylglyoxal by trimethylamine (TMA) lyase synthesis in the intestinal microbiota and investigated the intervention mechanism of the effects of dietary fiber on methylglyoxal formation. Intestinal digesta samples, collected from the ceca of mice fed with choline-rich and fiber-supplemented diets, were incubated in an anaerobic environment at 37 °C and pH 7.0 with choline, glycine, and methylglyoxal as inductive factors. The differences between the gut microbiota and its metagenomic and metabonomics profiles were determined using 16S rRNA gene sequencing analysis. The results elucidated that the different dietary interventions could induce differences in the composition of the microbiota, gene expression profiles associated with glycine metabolism, and glycolysis. As compared to the gut microbiota of choline-diet fed mice, fiber supplementation effectively altered the composition of the microbiota and inhibited the genes involved in choline metabolism, glycine and methylglyoxal accumulation, and TMA lyase expression, and improved the methylglyoxal utilization by regulating the pathway related to pyruvate production. However, the intervention of exogenous methylglyoxal significantly decreased these effects. These findings successfully revealed the correlations between the TMA lyase expression and glycine level, as well as the inhibitory effects of dietary fiber on the glycine level, thereby highlighting the role of common glycolytic metabolites as a potential target for TMA production.

Hyperinduction of pectate lyase in Dickeya chrysanthemi EC16 by plant-derived sugars

ABSTRACTPectate lyase (Pel) synthesis in Dickeya chrysanthemi has been reported to be hyperinduced in planta and also in the medium containing plant extract in addition to polygalacturonate. In this study, the major components of Pel-hyperinducing fractions were found to be glucose, fructose, and sucrose by TLC and NMR. From the analysis of the sugars and their derivatives, it was found that acyclic d-hexoses with the trans relationship between C-3 and C-5 hydroxyl groups were found to be basic structure required for hyperinducing the expression of a major isozyme in infected plants (i.e. pelE). From the fact that some non-metabolizable sugars such as 2-deoxy-d-glucose and d-fucose could lead to hyperinduction and that the hyperinduction was observed only in the medium containing low concentration (<0.25%) but not higher of the sugars was added, these sugars may be considered to participate in hyperinduction as the signal rather than through their metabolism.

Utilization of Aspergillus oryzae to produce pectin lyase from various agro-industrial residues

The present study was aimed to investigate the culture influence on pectin lyase production potential of fungal strain Aspergillus oryzae. The enzyme profile of A. oryzae showed highest activity of pectin lyase after 3rd day of incubation on lemon peel waste under solid state fermentation conditions. To induce the pectin lyase synthesis capability of A. oryzae at optimal level various culture variables including physical and nutritional parameters were optimized by adopting classical optimization technique. Therefore, through fermentation process optimization the production of pectin lyase was substantially induced up to the level of 875 U/mL, when fermentation medium of lemon peel waste inoculated with 5 mL spore suspension of A. oryzae. The optimal fermentation conditions for maximum pectin lyase yield were as: optimum pH 5, 70% moisture level and incubated at 40 °C in addition with 1% sterile glucose solution as readily available carbon source and 0.2% yeast extract as an inexpensive nitrogen supplement (1%). The results obtained in current investigation so far demonstrated that culture conditions have great influence on the pectin lyase production potential of A. oryzae.

Potato signal molecules that activate pectate lyase synthesis in Pectobacterium atrosepticum SCRI1043

A new type of plant-derived signal molecules that activate extracellular pectate lyase activity in phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043 was revealed. These compounds were characterized and partially purified by means of several approaches including RT-PCR analysis, luminescence bioassay and HPLC fractionation. They were smaller than 1 kDa, thermoresistant, nonproteinaceous, hydrophilic, and slightly negatively charged molecules. Using gene expression analysis and bacterial biosensor assay the mode of activity of revealed compounds was studied. The possibility of their action through quorum sensing- and KdgR-mediated pathways was analyzed.

PecS and PecT coregulate the synthesis of HrpN and pectate lyases, two virulence determinants in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi strain 3937 is a necrotrophic bacterial plant pathogen. Pectinolytic enzymes and, in particular, pectate lyases play a key role in soft rot symptoms; however, the efficient colonization of plants by E. chrysanthemi requires additional factors. These factors include HrpN (harpin), a heat-stable, glycine-rich hydrophilic protein, which is secreted by the type III secretion system. We investigated the expression of hrpN in E. chrysanthemi 3937 in various environmental conditions and different regulatory backgrounds. Using lacZ fusions, hrpN expression was markedly influenced by the carbon source, osmolarity, growth phase, and growth substrate. hrpN was repressed when pectinolysis started and negatively regulated by the repressors of pectate lyase synthesis, PecS and PecT. Primer extension data and in vitro DNA-protein interaction experiments support a model whereby PecS represses hrpN expression by binding to the hrpN regulatory region and inhibiting transcript elongation. The results suggest coordinated regulation of HrpN and pectate lyases by PecS and PecT. A putative model of the synthesis of these two virulence factors in E. chrysanthemi during pathogenesis is presented.

Virulence, accumulation of acetyl-coenzyme A, and pectate lyase synthesis are controlled by PhoP-PhoQ two-component regulatory system responding to organic acids of Erwinia chrysanthemi 3937

The PhoP-PhoQ two-components regulatory system is involved in the pathogenesis of animal, plant, and insect pathogenic bacteria in response to various environmental factors. To elucidate how this system contributes to the plant pathogenesis of Erwinia chrysanthemi 3937 (Ech 3937), marker-exchanged mutants of phoP and phoQ were constructed. Their role in the regulation of a major virulent factor, pectate lyase (Pel), in response to various organic acids was then tested. These mutants synthesized more Pel than did the wild type in the medium containing acetate or citrate as the sole source of carbon, but they synthesized less Pel than did the wild type in pyruvate or malate as the sole source of carbon. Synthesis of Pel did not differ in succinate, fumarate, or glycerol from the wild type. The phoP and phoQ mutants grown and resuspended in acetate or citrate also caused more maceration, and the wild type pretreated in pyruvate or malate caused more maceration than did the mutants. The level of intracellular acetyl-coenzyme A (acetyl-CoA) almost paralleled the synthesis of Pel in the wild type and in the mutants of the phoP and phoQ. These results suggested that acetyl-CoA may be involved in regulation of Pel synthesis through two-independent regulatory cascades via the PhoP-PhoQ system (in an opposite manner) in response to acetate/citrate and pyruvate/malate. However, ackA and pta genes, involved in the synthesis of acetyl-CoA in Escherichia coli, were not expressed as predicted on the basis of the level of acetyl-CoA. Thus there may be an additional regulation or pathway for the synthesis of acetyl-CoA in Ech 3937.

Role of L-phenylalanine ammonia Lyase in the induced resistance and susceptibility of sotato plants

Biogenic elicitors (chitosan and its complex with salicylic acid) and an immunosuppressor (laminarin) were shown to increase the activity of L-phenylalanine ammonia lyase (EC 4.3.1.5) and protein synthesis in potato tubers. Laminarin did not decrease L-phenylalanine ammonia lyase activity. It is unlikely that the activity of this enzyme may serve as a criterion for the induced resistance.

Role of the nucleoid-associated protein H-NS in the synthesis of virulence factors in the phytopathogenic bacterium Erwinia chrysanthemi.

The ability of the enterobacterium Erwinia chrysanthemi to induce pathogenesis in plant tissue is strongly related to the massive production of plant-cell-wall-degrading enzymes (pectinases, cellulases, and proteases). Additional factors, including flagellar proteins and exopolysaccharides (EPS), also are required for the efficient colonization of plants. Production of these virulence factors, particularly pectate lyases, the main virulence determinant, is tightly regulated by environmental conditions. The possible involvement of the protein H-NS in this process was investigated. The E. chrysanthemi hns gene was cloned by complementation of an Escherichia coli hns mutation. Its nucleotide sequence contains a 405-bp open reading frame that codes for a protein with 85% identity to the E. coli H-NS protein. An E. chrysanthemi hns mutant was constructed by reverse genetics. This mutant displays a reduced growth rate and motility but an increased EPS synthesis and sensitivity toward high osmolarity. Furthermore, pectate lyase production is dramatically reduced in this mutant. The hns mutation acts on at least two conditions affecting pectate lyase synthesis: induction of pectate lyase synthesis at low temperatures (25 degrees C) is no longer observed in the hns mutant and induction of pectate lyase production occurs in the late stationary growth phase in the hns background, instead of in the late exponential growth phase as it does in the parental strain. Moreover, the E. chrysanthemi hns mutant displays reduced virulence on plants. Taken together, these data suggest that H-NS plays a crucial role in the expression of the virulence genes and in the pathogenicity of E. chrysanthemi.

The TRAP-like SplA protein is a trans-acting negative regulator of spore photoproduct lyase synthesis during Bacillus subtilis sporulation.

UV resistance of bacterial endospores derives from a unique DNA photochemistry in which the major UV photoproduct is the thymine dimer 5-thyminyl-5,6-dihydrothymine (spore photoproduct [SP]) instead of cyclobutane pyrimidine dimers. Repair of SP during spore germination is due in large part to the activity of the enzyme SP lyase encoded by splB, the second cistron of the splAB operon. Expression of the splAB operon in Bacillus subtilis is transcriptionally activated by the Esigma(G) form of RNA polymerase during morphological stage III in the developing forespore compartment, and SP lyase is packaged into the dormant spore. In addition to temporal and compartmental control of splAB expression, a second regulatory circuit which modulates the level of expression of splB-lacZ fusions without altering their developmental timing or compartmentalization is reported here. This second regulatory circuit involves the negative action of the splA gene product, a 79-amino-acid protein with approximately 50% similarity and 17% identity to TRAP, the tryptophan RNA-binding attenuation protein from B. subtilis and Bacillus pumilus.

The PecT Repressor Coregulates Synthesis of Exopolysaccharides and Virulence Factors in Erwinia chrysanthemi

Erwinia chrysanthemi 3937 synthesizes an exopolysaccharide (EPS) composed of rhamnose, galactose, and galacturonic acid. Fourteen transcriptional fusions in genes required for EPS synthesis, named eps, were obtained by Tn5-B21 mutagenesis. Eleven of them are clustered on the chromosome and are repressed by PecT, a regulator of pectate lyase synthesis. In addition, expression of these fusions is repressed by the catabolite regulatory protein, CRP, and induced in low osmolarity medium. The three other mutations are located in genes that are not regulated by pecT. A 13-kb DNA fragment containing pecT-regulated eps genes has been cloned. All the genes identified on this fragment are transcribed in the same orientation and could form a large operon. The promoter region of this operon has been sequenced. It contains a JUMP-start sequence, a sequence required for the expression of polysaccharide-associated operons. E. chrysanthemi 3937 produces a systemic soft rot on its host Saintpaulia ionantha. An eps mutant was less efficient than the wild-type strain in initiating a maceration symptom, suggesting that production of EPS is required for the full expression of the E. chrysanthemi virulence.

Characterization of the pecT control region from Erwinia chrysanthemi 3937.

Erwinia chrysanthemi synthesizes and secretes pectate lyases that attack components of the plant cell wall and, therefore, play a major role in the pathogenesis of soft rot disease. We isolated a new mutant (designated pec-1), by Tn5 mutagenesis, that displays weak pectate lyase production and decreased motility and mucoidicity. Maceration and pathogenicity tests done on different plant organs showed that the pec-1 strain displays a reduced virulence compared to that of the parental strain. The Tn5 insertion was localized between the pelL and the out loci and defines a new regulatory region. Sequencing of the pec-1::Tn5 insertion revealed that pec-1 is tightly linked to the pecT regulatory gene that also controls pectate lyase synthesis. Moreover, the pecT mutation is dominant over the pec-1 mutation, suggesting that these two loci are involved in the same regulatory network. We demonstrated, by Northern blot analysis, that the pec-1::Tn5 insertion provokes derepression of pecT transcription and defines a cis-acting element. Introduction of the pecT gene in trans of a pecT::uidA fusion induced a decrease of pecT::uidA transcription, indicating a negative autoregulation. Band shift experiments confirmed that the PecT repressor specifically interacts with the pecT regulatory region. We also demonstrated that the PecT protein interacts with the regulatory region of the pelD gene encoding a pectate lyase. Therefore, the abolition of the pecT autoregulation in the pec-1 mutant provokes an overproduction of the PecT repressor that is responsible for the decrease of pectate lyase synthesis. Mutagenesis of the pecT regulatory region revealed the presence of two sites in which insertions reproduced the pec-1 phenotype. This result suggests that pecT autoregulation requires the presence of two functional operator sites. From this study, we propose that the PecT repressor binds to these two sites, generating a loop that blocks pecT transcription.

The Erwinia chrysanthemi pecT gene regulates pectinase gene expression.

A new type of Erwinia chrysanthemi mutant displaying a derepressed synthesis of pectate lyase was isolated. The gene mutated in these strains, pecT, encodes a 316-amino-acid protein with a size of 34,761 Da that belongs to the LysR family of transcriptional activators and presents 61% identity with the E. coli protein LrhA. PecT represses the expression of pectate lyase genes pelC, pelD, pelE, pelL, and kdgC, activates pelB, and has no effect on the expression of pelA or the pectin methylesterase genes pemA and pemB. PecT activiates its own expression. The mechanism by which PecT regulates pectate lyase synthesis is independent of that of the two characterized regulators of pectate lyase genes, KdgR and PecS. In contrast to most of the members of the LysR family, pecT is not transcribed in a direction opposite that of a gene that it regulates. pecT mutants are mucoid when grown on minimal medium plates and flocculate when grown in liquid minimal medium, unless leucine or alanine is added to the medium. Thus, pecT may regulate other functions in the bacterium.

Quantification of citrate lyase by enzyme-linked immunosorbent assay for determining the population of Lactococcus lactis subsp. Lactis biovar diacetylactis in pure and mixed cultures

Citrate lyase production by Lactococcus lactis subsp. lactis biovar diacetylactis DRC2 was quantified by an enzyme-linked immunosorbent assay (ELISA). The citrate lyase reached a concentration equivalent to 41 ± 4 μg/ml purified citrate lyase in pure culture. When the strain DRC2, grown in mixed culture with L. lactis subsp. cremoris AM2, represented around 70% (DC culture) or 30% (CD culture) of the total initial population, the level of citrate lyase decreased to 21 ± 7 μg/ml and 4.5 ± 1.5 μg/ml respectively. The maximum bacterial concentration of strain DRC2 in pure culture reached 2.6 × 109 cfu/ml and decreased to 1.5 (± 0.2) × 109 cfu/ml and 0.5 (± 0.3) × 109 cfu/ml in DC and CD mixed cultures respectively. In mixed cultures, the proportion of the strain DRC2 was 8.5 ± 5.0% lower at the end of the fermentation than immediately after inoculation, thus showing that this strain was clearly inhibited. However, the maximum rate of citrate consumption was the same during pure DRC2 culture and CD mixed culture (2.5 ± 0.3 mmol/h) and slightly highre in DC culture (3.07 mmol/h). The maximum rate of acidification was 0.37 ± 0.04 pH unit/h regardless of the culture. A good correlation was obtained between the population of the strain DRC2 and the citrate lyase concentration determined by ELISA but no relationship was found between citrate consumption and citrate lyase synthesis. Therefore an ELISA test of this kind can be used to monitor the growth of L. lactis subsp. lactis biovar diacetylactis in mixed cultures.

Transcriptional regulation of the isocitrate lyase encoding gene in Saccharomyces cerevisiae.

In this work, we studied the transcriptional regulation of isocitrate lyase synthesis. In Northern blot analyses we first showed that the steady-state ICL1 mRNA levels depend on the carbon source used for growth. In addition, we determined the kinetics of transcriptional repression upon a shift of ethanol-grown cells to glucose and of the induction when cells were transferred from glucose to ethanol. By deletion analyses as well as by studying the influence on expression of different fragments cloned into the heterologous CYC1 promoter lacking its own UAS sequences, we defined UAS and URS elements in the ICL1 promoter. A region mediating the control by CAT3, a gene also involved in the control of expression of other genes subject to carbon catabolite repression, was found to overlap with one of these UAS elements.

Regulation of the expression of a pelA::uidA fusion in Erwinia chrysanthemi and demonstration of the synergistic action of plant extract with polygalacturonate on pectate lyase synthesis.

The phytopathogenicity of Erwinia chrysanthemi is chiefly supported by the production of pectate lyase isoenzymes, encoded by the pel genes. One of these enzymes, PelA, encoded by the pelA gene, seems to represent only a small part of the total pectate lyase activity, but is required for full bacterial pathogenicity. To study the regulation of pelA gene expression, a pelA::uidA gene fusion was constructed. Expression of this fusion was analysed under various growth conditions and in various genetic backgrounds. Whatever the culture conditions, the pelA gene was weakly expressed. Moreover, pelA expression seems not to be regulated by the pecS gene product, but essentially by the kdgR gene product. In many plant-associated bacteria, genes involved in pathogenicity are induced by certain plant compounds. In this work, we demonstrate that E. chrysanthemi pel genes are induced in the presence of plant extracts, but only in synergy with known pectate lyase inducers (KDG: 2-keto-3-deoxygluconate; DKII: 2,5-diketo-3-deoxygluconate). However, different pel genes did not exhibit the same sensitivity to plant signal molecules. Partial purification of inducing plant compounds suggested that plant extracts contain at least one molecule involved in pectate lyase induction. This compound is thermoresistant, and has a low molecular mass and a very hydrophilic behaviour.

Selection of glucose-assimilating variants ofAcinetobacter calcoaceticus LMD 79.41 in chemostat culture

Glucose metabolism has been studied in two strains of Acinetobacter calcoaceticus. Strain LMD 82.3, was able to grow on glucose and possessed glucose dehydrogenase (EC 1.1.99.17). Glucose oxidation by whole cells was stimulated by PQQ, the prosthetic group of glucose dehydrogenase. PQQ not only increased the rate of glucose oxidation and gluconic acid production but also shortened the lag phase for growth on glucose. Strain LMD 79.41 also possessed glucose dehydrogenase but was unable to grow on glucose. Batch cultures and carbon-limited chemostat cultures growing on acetate in the presence of glucose oxidized the sugar to gluconic acid, which was not further metabolized. However, after prolonged cultivation on mixtures of acetate and glucose, carbon-limited chemostat cultures suddenly acquired the capacity to utilize gluconate. This phenomenon was accompanied by the appearance of gluconate kinase and a repression of isocitrate lyase synthesis. In contrast to the starter culture, cells from chemostats which had been fully adapted to gluconate utilization, were able to utilize glucose as a sole carbon and energy source in liquid and solid media.

Influence of nar (nitrate reductase) genes on nitrate inhibition of formate-hydrogen lyase and fumarate reductase gene expression in Escherichia coli K-12.

In Escherichia coli, aerobiosis inhibits the synthesis of enzymes for anaerobic respiration (e.g., nitrate reductase and fumarate reductase) and for fermentation (e.g., formate-hydrogen lyase). Anaerobically, nitrate induces nitrate reductase synthesis and inhibits the formation of both fumarate reductase and formate-hydrogen lyase. Previous work has shown that narL+ is required for the effects of nitrate on synthesis of both nitrate reductase and fumarate reductase. Another gene, narK (whose function is unknown), has no observable effect on formation of these enzymes. We report here our studies on the role of nar genes in fumarate reductase and formate-hydrogen lyase gene expression. We observed that insertions in narX (also of unknown function) significantly relieved nitrate inhibition of fumarate reductase gene expression. This phenotype was distinct from that of narL insertions, which abolished this nitrate effect under certain growth conditions. In contrast, insertion mutations in narK and narGHJI (the structural genes for the nitrate reductase enzyme complex) significantly relieved nitrate inhibition of formate-hydrogen lyase gene expression. Insertions in narL had a lesser effect, and insertions in narX had no effect. We conclude that nitrate affects formate-hydrogen lyase synthesis by a pathway distinct from that for nitrate reductase and fumarate reductase.

Consequences of metolachlor induced inhibition of gibberellin biosynthesis in sorghum seedlings

Forage sorghum [ Sorghum bicolor (L.) Moench] cultivars were field evaluated for response to seed protectants CGA 92194, CGA 133205, and flurazole for ability to reverse the activity of metolachlor (2.8 kg a.i./ha) and alachlor (3.36 kg a.i./ha). Funk “G102F” was “tolerant” to metolachlor and alachlor (∼17% decrease in forage production), R. C. Young “Red Top Kandy” was “intermediate” (37–50% inhibition), and AgraTech “GK 83S” was “susceptible” (60–65% inhibition). Average plant heights were decreased 0, 26, and 32% for “G102F,” “Red Top Kandy,” and “GK 83S,” respectively, while numbers of plants/4.57-m row were decreased 19, 70, and 90%, respectively. Resultant average individual plant fresh weights (g/plant) were 104, 200, and 410% of the untreated plants for G102F, Red Top Kandy, and GK 83S, respectively. Thus, metolachlor and alachlor lethal responses were exhibited at a very early stage of sorghum seedling development (<1 true leaf), and any plant which survived that stage of growth ultimately developed to the maximum attainable under extant competitive growing conditions. The seed applied protectants essentially reversed the growth inhibition by metolachlor and alachlor at this stage of development and forage production was not inhibited by metolachlor and alachlor when CGA 92194, CGA 133205, or flurazole were utilized as seed protectants. Greenhouse evaluation of the critical seedling stage of growth hypothesis demonstrated a massive shoot growth inhibition by metolachlor and alachlor for all three cultivars during the first 5 days after inhibition; the growth inhibition was reversed by all three seed protectants. Metolachlor inhibited the growth of sorghum root (96-hr bioassay) and acetate reversed that inhibition in Funk G-522 DR roots. Grass seedling (<1 true leaf) production of acetate is by the utilization of seed stored products via α-amylase and isocitric lyase, and the production of both of these enzyme systems was inhibited by metolachlor. The inhibition of α-amylase production by metolachlor was reversed by CGA 92194 and exogenous gibberellic acid (GA 3). These data support the concept that alachlor and metolachlor inhibit GA biosynthesis in seedling tissue which results in greatly decreased utilization of seed food reserves and failure of the juvenile seedling to grow sufficiently to become an independent plant. Alternatively, metolachlor inhibition of acetyl-coenzyme A synthesis results in decreased GA biosynthesis which induces a decreased α-amylase and isocitric lyase synthesis. Additionally, sorghum cv G-522 DR seeds were germinated (64 hr) in GA 3 (0, 2, 4, 6, 8, or 10 ppmw) in the dark. Coleoptiles were immersed in [ 14C]metolachlor (5 μ M metolachlor), GA 3 (≥4 ppmw) induced a decrease (DPM/g fresh wt) in total 14C absorption by G-522 DR sorghum coleoptiles. Conversion of [ 14C]metolachlor → [ 14C]metolachlorglutathione conjugate was increased as the GA concentration in the germination medium increased. GA induces the synthesis of enzymes requisite to starch degradation, β-oxidation of triglycerides, as well as influencing metolachlor uptake and GS conjugation.