Isolation and characterization of the two structural genes coding for phosphofructokinase in yeast

Summary Yeast phosphofructokinase is an octamer composed of two different kinds of subunit. The genes coding for these subunits have been isolated by means of functional complementation in a pfk1 pfk2 double mutant. As a source of DNA the genomic library of Nasmyth and Tatchell (1980) constructed in the yeast multicopy vector YEp13 was used. Plasmids containing the information of one or the other gene were identified by back-transformation into pfk single mutants (pfk1 PFK2, PFK1 pfk2). Restriction maps of the respective insertions are provided. The genomic organization was confirmed by Southern analysis. Northern analysis showed hybridization to mRNAs of about 3.6 kb for both genes, corresponding to the molecular weight of the protein subunits. Transformation with one of the plasmids did not lead to an increase in phosphofructokinase activity. Subcloning of both genes in one multicopy vector (YEp13) and reintroduction into the yeast cell resulted in a 3.5-fold higher specific activity compared to the wild type. Overproduction of the protein subunits in this transformant was confirmed by SDS-polyacrylamide electrophoresis of crude extracts stained with Coomassie-blue. This was not accompanied by an increased ethanol production. The sequences encoding the two subunits were shown to share homology.     

Selectable tolerance to herbicides by mutated acetolactate synthase genes integrated into the chloroplast genome of tobacco

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicide-resistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3'-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.     

Isolation and purification of acetolactate synthase and acetolactate decarboxylase from the culture ofLactococcus lactis

Enzymes catalyzing the synthesis and subsequent transformation of α-acetolactate (AcL)—acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)—were isolated and partially purified from the cells of lactic acid bacteriaLactococcus lactis ssp.lactis biovar.diacetylactis, strain 4. The preparation of AcLS, purified 560-fold, had a specific activity of 358 300 U/mg protein (9% yield). The preparation of AcLDC., purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield). The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer). The values of apparentK _m, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM. AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration. In the case of AcLDC, this dependence was hyperbolic and the affinity of the enzyme for its substrate was high (K _m = 20 mM). Leucine, valine, and isoleucine were shown to be activators of AcDLC.     

Isolation and purification of acetolactate synthase and acetolactate decarboxylase from a Lactococcus lactis culture

Enzymes catalyzing the synthesis and subsequent transformation of alpha-acetolactate (AcL)--acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)--were isolated and partially purified from the cells of lactic acid bacteria Lactococcus lactis ssp. lactis biovar. diacetylactis strain 4. The preparation of AcLS, purified 560-fold, had a specific activity of 358,300 U/mg protein (9% yield). The preparation of AcLDC, purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield). The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer). The values of apparent Km, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM. AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration. In the case of AcLDC, this dependence was hyperbolic, and the affinity of the enzyme for its substrate was high (Km = 20 mM). Leucine, valine, and isoleucine were shown to be activators of AcDLC.     

Isolation, purification and properties of acetolactate synthase from cultured Lactococcus lactis

Acetolactate synthase catalyzing the synthesis of alpha-acetolactate was isolated from lactic acid bacteria Lactococcus lactis subsp. lactis biovar. diacetylactis 4 and purified. Acetolactate synthase was shown to be an allosteric enzyme with low affinity for the substrate: the Km for pyruvate was 70 mM. The curve relating the dependence of enzyme activity on pyruvate concentration had a sigmoid shape. The enzyme activity persisted for 24 h in the presence of stabilizers, pyruvate, and thiamine pyrophosphate. Acetolactate synthase had the pH optimums of 5.8 and 6.5-7.0 in acetate and phosphate buffers, respectively. The temperature optimum for this enzyme was 38-40 degrees C at pH 6.5. The molecular weight of acetolactate synthase was 150 kDa. In Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consisted of three identical subunits with a molecular weight of 55 kDa.     

Isolation and characterization of the two distinct genes for human glutamate dehydrogenase

Two genomic clones containing a part of the glutamate dehydrogenase gene were isolated from a human genomic library. The restriction map of both clones were distinctly different from one another, although the nucleotide sequences of the three exons that they contained were virtually the same in each clone. Southern blotting analysis of the genomic DNAs from several unrelated human individuals revealed that in every case the probe hybridized with at least two DNA fragments of different sizes, each characteristic to one of the two clones. These results strongly suggest that the two clones presently obtained do not result from polymorphism but are generated from two different gene loci for glutamate dehydrogenase on the human chromosome.     

Inheritance and mechanism of resistance to herbicides inhibiting acetolactate synthase in Sonchus oleraceus L.

A biotype of Sonchus oleraceus L. (Compositae) has developed resistance to herbicides inhibiting acetolactate synthase (ALS) following field selection with chlorsulfuron for 8 consecutive years. The aim of this study was to determine the inheritance and mechanism of resistance in this biotype. Determination of ALS activity and inhibition kinetics revealed that K_m and V_max did not vary greatly between the resistant and susceptible biotypes. ALS extracted from the resistant biotype was resistant to five ALS-inhibiting herbicides in an in vitro assay. ALS activity from the resistant biotype was 14 19, 2, 3 and 3 times more resistant to inhibition by chlorsulfuron, sulfometuron, imazethapyr, imazapyr and flumetsulam, respectively, than the susceptible biotype. Hybrids between the resistant and a susceptible biotype were produced, and inheritance was followed through the F₁, F₂ and F₃ generations. F₁ hybrids displayed a uniform intermediate level of resistance between resistant and susceptible parents. Three distinct phenotypes, resistant, intermediate and susceptible, were identified in the F₂ generation following chlorsulfuron application. A segregation ratio of 121 was observed, indicative of the action of a single, nuclear, incompletely dominant gene. F₃ families, derived from intermediate F₂ individuals, segregated in a similar manner. Resistance to herbicides inhibiting ALS in this biotype of S. oleraceus is due to the effect of a single gene coding for a resistant form of the target enzyme, ALS.     

Isolation and characterization of two wound-regulated tomato extensin genes

Extensins comprise a family of structural cell wall hydroxyproline-rich glycoproteins in plants. Two tomato genomic clones, Tom J-10 and Tom L-4, were isolated from a tomato genomic DNA library byin situ plaque hybridization with extensin DNA probes. Tom J-10 encoded an extensin with 388 amino acid residues and a predicted molecular mass of 43 kDa. The Tom J-10 encoded extensin lacked a typical signal peptide sequence, but contained two distinct protein domains consisting of 19 tandem repeats of Ser-Pro₄-Ser-Pro-Lys-Tyr-Val-Tyr-Lys at the amino terminus which were directly followed by 8 tandem repeats of the consensus sequence Ser-Pro₄-Tyr₃-Lys-Ser-Pro₄-Ser-Pro at the carboxy terminus. RNA blot hybridization analysis with the Tom J-10 extensin probe demonstrated the presence of a 4.0 kb tomato stem mRNA which accumulated markedly in response to wounding. Tom L-4 encoded an extensin with 322 amino acid residues and a predicted molecular mass of 35 kDa. The Tom L-4 encoded extensin contained a typical signal peptide sequence at the amino terminus and was followed by at least 3 distinct domains. These domains consisted of an amino terminal domain containing several Lys-Pro and Ser-Pro₄ repeat units, a central domain with repeats of the consensus sequence Ser-Pro_2–5-Thr-Pro-Ser-Tyr-Glu-His-Pro-Lys-Thr-Pro, and a carboxy terminal domain containing repeats of the consensus sequence Ser-Ser-Pro₄-Ser-Pro-Ser-Pro₄-Thr-Tyr_1–3. RNA blot hybridization analysis with the Tom L-4 extensin probe demonstrated the presence of a 2.6 kb tomato stem mRNA which accumulated in response to wounding.     

Localization within chloroplasts of protoporphyrinogen oxidase, the target enzyme for diphenylether-like herbicides.

Plant protoporphyrinogen oxidase is of particular interest since it is the last enzyme of the common branch for chlorophyll and heme biosynthetic pathways. In addition, it is the target enzyme for diphenyl ether-type herbicides, such as acifluorfen. Two distinct methods were used to investigate the localization of this enzyme within Percoll-purified spinach chloroplasts. We first assayed the enzymatic activity by spectrofluorimetry and we analyzed the specific binding of the herbicide acifluorfen, using highly purified chloroplast fractions. The results obtained give clear evidence that chloroplast protoporphyrinogen oxidase activity is membrane-bound and is associated with both chloroplast membranes, i.e. envelope and thylakoids. Protoporphyrinogen oxidase specific activity was 7-8 times higher in envelope membranes than in thylakoids, in good agreement with the number of [3H]acifluorfen binding sites in each membrane system: 21 and 3 pmol/mg protein, respectively, in envelope membranes and thylakoids. On a total activity basis, 25% of protoporphyrinogen oxidase activity were associated with envelope membranes. The presence of protoporphyrinogen oxidase in chloroplast envelope membranes provides further evidence for a role of this membrane system in chlorophyll biosynthesis. In contrast, the physiological significance of the enzyme associated with thylakoids is still unknown, but it is possible that thylakoid protoporphyrinogen oxidase could be involved in heme biosynthesis.     

Oxidative stress is not related to the mode of action of herbicides that inhibit acetolactate synthase

Imazethapyr (IM) is an imidazolinone herbicide which inhibits the biosynthesis of branched chain amino acids, by blocking acetolactate synthase (ALS; EC 4.1.3.18), the first common enzyme of the pathway. To study new aspects of the mode of action of ALS-inhibiting herbicides, pea plants grown in hydroponic cultures were supplied with IM and were analysed with reference to the antioxidant system and oxidative markers. A slight lipid peroxidation was detected in leaves after IM treatment, but no changes were noted in electrolyte leakage or carbonyl content. The ascorbate pool of leaves was oxidized under IM treatment. The analysis of the antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT) and guaiacol peroxidase (GPX), showed that IM treatment only caused an enhancement of GPX activity in leaves. In roots, the herbicide caused a decrease in lipid peroxidation. The enhancement of the reduced glutathione content detected in IM-treated roots can be related to the detected increase of GR activity. The lack of more noticeable effects on antioxidant enzymatic activities could be explained by the inability of IM-treated plants to respond to oxidative stress with modifications in their protein synthesis. Our results suggest that oxidative stress is not related to the mode of action of ALS-inhibitors. The slight changes detected in the antioxidative status of treated plants are too secondary in time and intensity to be related to the lethality caused by ALS-inhibitors     

Isolation,functional characterization and stress responses of raffinose synthase genes in sugar beet

Raffinose (sucrosylgalactoside oligosaccharide) is a water soluble carbohydrate and accumulates in response to abiotic stresses in plants. Plant raffinose synthases are poorly characterized, and the genes involved in raffinose biosynthesis are unknown in sugar beet. Here, we report the isolation of two genes encoding raffinose synthase (BvRS1 and BvRS2) as well as a gene encoding galactinol synthase (BvGolS1) from sugar beet. BvRS1 and BvRS2 show high homologies to Arabidopsis raffinose synthase AtRS5. BvRS1 and BvGolS1 were expressed in Escherichia coli. Crude extracts showed the activities of raffinose synthase and galactinol synthase. The K _m values of BvRS1 for galactinol and sucrose and the K _m values of BvGolS1 for UDP-galactose and myo-inositol were determined. The expression levels of BvRS1 were significantly higher than that of BvRS2. The mRNA for BvRS1 was rapidly induced by cold stress whereas the mRNA for BvRS2 was slowly induced by cold and salt stresses. These data suggest that BvRS1 and BvRS2 encode raffinose synthase genes responsible to cold and salt stress, respectively.     

New aspects on inhibition of plant acetolactate synthase by chlorsulfuron and imazaquin

The sulfonylurea herbicide chlorsulfuron and the imidazolinone herbicide imazaquin were shown to be noncompetitive and uncompetitive inhibitors, respectively, of purified acetolactate synthase from barley (Hordeum vulgare L.) with respect to pyruvate. From double-reciprocal plots of the time-dependent biphasic inhibition by chlorsulfuron, an initial apparent inhibition constant of 68 nanomolar was calculated (a 0 to 4 minute assay was used for the initial inhibition), and a final steady-state dissociation constant of 3 nanomolar was estimated. The corresponding constants for imazaquin were 10 and 0.55 micromolar. Specific binding of [¹⁴C]chlorsulfuron and [¹⁴C]imazaquin to purified acetolactate synthase from barley and partially purified enzyme from corn (Zea mays L.) could be demonstrated by gel filtration and equilibrium dialysis. Evidence is presented that the binding of the inhibitors to the enzyme follows the previously described mechanism of slow reversibility once excess inhibitor has been removed. However, after formation of the slowly reversible complex and subsequent dissociation, both chlorsulfuron and imazaquin seem to permanently inactivate acetolactate synthase. These results add a new feature to the mode of action of these herbicides with respect to their high herbicidal potency.     

Isolation and characterization of maize genes coding for zein proteins of the 21000 dalton size class

Cloning in lambda gt WES phage of EcoRI fragments from maize seedlings DNA led to the isolation of four fragments containing genes coding for 21000 dalton zein proteins. The zein genes, identified by electron microscopic analysis, do not contain intervening sequences detectable by this method. The flanking sequences were analyzed by restriction sites mapping and hybridization and showed areas of homology between each other and with sequences surrounding a previously isolated gene of the 19000 dalton zein class.     

Comparison of increased expression of wild-type and herbicide-resistant acetolactate synthase genes in transgenic plants, and indication of posttranscriptional limitation on enzyme activity

Genes encoding wild type acetolactate synthase (ALS) and a sulfonylurea herbicide-resistant form of the enzyme, isolated from Arabidopsis thaliana, were expressed in transgenic Nicotiana tabacum plants under the control of their native promoters or of the highly active cauliflower mosaic virus 35S promoter. Expression of the wild type coding region from the 35S promoter resulted in a small, threefold increase in sulfonylurea tolerance above the levels measured in tissue expressing the native wild type gene. A much larger, 300-fold increase in herbicide tolerance was conferred by the mutant gene encoding a herbicide-resistant ALS. An additional 10-fold increase in tolerance was attained by expressing this coding region from the 35S promoter. The increase in both wild type and mutant gene expression directed by the 35S promoter resulted in over 25-fold higher levels of ALS messenger RNA in some transformants as compared with those expressing the native genes. However, ALS specific activity increased at most twofold, indicating that the amount of functional enzyme and messenger RNA are not correlated.     

Isolation and characterization of two knotted-like homeobox genes from tomato

Homeobox genes are known to play a role in developmental regulation. The knotted-like homeobox (knox) genes fall into two classes. The class I knox genes like kn1, stm1, and knat1 are involved in maintaining meristem identity in cells. The function of class II knox genes is at yet undetermined. We have characterized two knox genes from tomato. LeT6 and LeT12 map to distinct chromosome locations that are different from the location for a recently cloned knox gene from tomato, tkn1, confirming that plant homeobox genes are not clustered on chromosomes. These genes have a distinct expression pattern. Unlike other class I kn1-like genes, LeT6 is expressed in developing lateral organs and developing ovaries in flowers. LeT12 is more ubiquitously expressed in the mature plant. RNA in situ localization data suggest that both these genes may have a role to play in formative events in ovule and embryo morphogenesis.