Properties of mutant acetolactate synthases resistant to triazolopyrimidine sulfonanilide

Triazolopyrimidine sulfanilides are a class of highly active herbicides whose primary target is acetolactate synthase. Spontaneous mutants of tobacco (Nicotiana tabacum) (KS-43) and cotton (Gossypium hirsutum) (PS-3 and DO-2) resistant to triazolopyrimidine sulfonanilide were selected in tissue culture. Acetolactate synthase partially purified from the three mutants were 80- to 1000-fold less sensitive to inhibition by the compound compared with the corresponding wild-type enzyme. The mutants also varied in the cross-resistance pattern to other acetolactate synthase inhibiting herbicides in the sulfonylurea, imidazolinone, and pyrimidyl-oxy-benzoate chemical families. Thus, acetolactate synthase from KS-43, PS-3, and DO-2 cultures have different mutations. The affinities for pyruvate, thiamine pyrophosphate, as well as the activity of the mutant enzymes were found to be comparable to the corresponding wild-type enzymes. However, the enzyme from PS-3 was highly resistant to feedback inhibition by valine and leucine. In contrast, acetolactate synthase from KS-43 and DO-2 were inhibited by valine and leucine to nearly the same extent as the wild-type enzymes. Also, PS-3 cultures accumulated much higher levels of the branched chain amino acids compared to the wild-type cotton culture. The mutation in the PS-3 enzyme has therefore rendered it insensitive to feedback regulation by valine and leucine.     

Glutamine synthetase activity of the endosperm, embryo and pedicel-placento-chalazal regions of developing maize (Zea mays) kernels

Glutamine synthetase (GS, EC 6.3.1.2) activity in homogenates of the maize ( Zea mays L. hybrid A619 X W64A) kernel pedicel-placento-chalazal (PPCh), endosperm regions was characterized in order to optimize assay (hydroxylamine-dependent γ-glutamyl hydroxymate formation) conditions for quantitating maize kernel GS in crude extracts. The GS activities of all three tissue extracts exhibited optima at pH 7.0 with ATP:Mg²⁺ of 1:1.6. Assays of kernel tissue GS activity required relatively high concentrations of substrates to achieve saturation compared to GS from other plant tissue sources, a point which has not been considered in previous reports of maize kernel GS activity. When measured under optimal assay conditions. PPCh-GS increased to a peak of 51 nmol γ-glutamyl hydroxymate kernel⁻¹ min⁻¹ at 25 days after pollination and then declined throughout the remainder of kernel development. Embryo GS activity increased steadily throughout development to a maximum of 24 nmol γ-glutamyl hydroxymate embryo⁻¹ min⁻¹ by 50 days after pollination. In contrast, endosperm GS activity, which was 25 nmol γ-glutamyl hydroxymate endosperm⁻¹ min⁻¹ at 25 days after pollination, exhibited no discernable pattern of change during kernel development. These findings are discussed with respect to the possible roles PPCh, endosperm and embryo GS play in kernel development.     

Regulation of the Pool Size of Valine in Escherichia coli K-12

Three mutations (ilvH611, ilvH612, and ilvH613) are described which make Escherichia coli K-12 resistant to valine inhibition and are located near leu. The expression of the ilv genes appears to be normal in these mutants since the isoleucine-valine biosynthetic enzymes are not derepressed relative to the wild type. The intracellular concentration of valine is, however, higher in the mutants than in the isogenic ilvH(+) strain. These mutants also excrete valine, probably because of the high intracellular concentration of this amino acid. The pool size of valine is regulated independently from that of isoleucine and leucine. The increased intracellular concentration of valine is due to a decreased feedback inhibition that valine exerts on its own biosynthetic pathway. In fact, acetolactate synthase activity assayed in extracts of ilvH612 and ilvH613 mutants is more resistant to valine inhibition than the activity assayed in the ilvH(+) isogenic strain. Two forms of acetolactate synthase activity can be separated from these extracts by adsorption and elution on hydroxylapatite. One of them is as sensitive to valine inhibition as that of the wild type, the other is more resistant to valine inhibition.     

Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis.

The alpha-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp. lactis. It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter. The product of aldB is responsible for leucine sensibility under valine starvation. In the presence of more than 10 microM leucine, the alpha-acetolactate produced by the biosynthetic acetohydroxy acid synthase IlvBN is transformed to acetoin by AldB and, consequently, is not available for valine synthesis. AldB is also involved in acetoin formation in the 2,3-butanediol pathway, initiated by the catabolic acetolactate synthase, AlsS. The differences in the genetic organization, the expression, and the kinetics parameters of these enzymes between L. lactis and Klebsiella terrigena, Bacillus subtilis, or Leuconostoc oenos suggest that this pathway plays a different role in the metabolism in these bacteria. Thus, the alpha-acetolactate decarboxylase from L. lactis plays a dual role in the cell: (i) as key regulator of valine and leucine biosynthesis, by controlling the acetolactate flux by a shift to catabolism; and (ii) as an enzyme catalyzing the second step of the 2,3-butanediol pathway.     

Activity and distribution of nitrogen-metabolism enzymes in the developing maize kernel

The activities of glutamine synthetase (EC 6.3.1.2), glutamate dehydrogenase (EC 1.4.1.2), aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2) and soluble protein content in the developing endosperm and embryo of normal (Oh-43) and mutant (Oh-430₂) maize were investigated. Maize inbred lines were grown under field conditions and all plants were self-pollinated. Ears for experiments were harvested over the period of 15 lo 45 days after pollination. After pollination kernel capacity for soluble protein synthesis is located mainly in the endosperm. This progressively decreases and about 40 days after pollination soluble protein synthesis is taken over by the embryo. Comparative data on the activity of the investigated enzymes in the embryo and endosperm indicate that the capacity for synthesis of glutamine and glutamate predominates in the embryo tissue, whereas transamination processes at the initial stages of the embryo development are less intensive than their counterparts in the endosperm. The roles of embryo and endosperm subsequently interchange. Biosynthetic processes of soluble precursors for protein synthesis in the embryo and endosperm of the developing kernel are mutually coordinated.     

Amino acid composition of vascular sap of maize ear peduncle

The amino acid composition of the vascular sap of a high lysine maize mutant was determined during kernel development. With the exception of proline and cystine, all amino acids that occur in the endosperm were found in the vascular sap of the ear peduncle. Glutamine is the major amino acid transported to the endosperm varying from 30.6 to 20.6 μmol at 7 and 42 days after pollination, respectively. Aspartic acid, the second most important nitrogen form translocated to the seeds, was ca 10 μmol% during kernel filling. Glutamine and arginine content decreased with maturity, while valine, methionine, isoleucine, leucine, tyrosine and phenylalanine increased with kernel development. The remaining N forms were constant during endosperm growth.     

Structural Genes for a Newly Recognized Acetolactate Synthase in Escherichia coli K-12

Evidence is reported that shows the presence in Escherichia coli K-12 of a newly found acetolactate synthase. This enzyme is the product of two genes, ilvH and ilvI, both located very close to leu. Amber mutations have been found in both genes and therefore their products are polypeptides. Mutations in the ilvH gene cause the appearance of an acetolactate synthase activity which is relatively resistant to valine inhibition and can be separated by adsorption on hydroxylapatite from another activity present in the extract and more sensitive to valine inhibition than the former. A mutant altered in the ilvI gene was isolated among the revertants sensitive to valine inhibition of an ilvH mutant. Such a mutant lacks the resistant acetolactate synthase. A temperature-sensitive revertant of the ilvI mutant contained a temperature-sensitive acetolactate synthase. Thus ilvI is the structural gene for a specific acetolactate synthase. The activity of the ilvH gene product has been measured by adding an extract containing it to a purified ilvI acetolactate synthase, which, upon incubation, became more sensitive to valine inhibition. Conversely, a valine-sensitive acetolactate synthase (the product of the ilvH and the ilvI genes) became more resistant to valine inhibition upon incubation with an extract of a strain containing a missense ilvH gene product.     

The existence of three types of acetohydroxy acid synthetase in an isoleucine-requiring mutant of Aerobacter aerogenes.

The synthesis of the three types of acetolactate synthase (EC 4.1.3.18) which are responsible for the biosynthesis os isoleucine and valine, was observed in Aerobacter aerogenes I-12, an isoleucine-requiring mutant, when grown on the four kinds of media. When the cells were grown on isoleucine-rich medium, acetolactate synthase sensitive to feedback inhibition and having an optimum pH at 8.0 was formed. By increasing the amount of potassium phosphate in the medium, the catabolite repression of the enzyme having an optimum pH at 6.0 and which is insensitive to feedback inhibition, was released. In contrast, acetolactate synthase having an optimum pH at 8.0 and insensitive to feedback inhibition was formd when isoleucine was limited, irrespective of phosphate concentrations. Two insensitive enzymes were not regulated by isoleucine, leucine and valine, although sensitive pH 8.0 enzyme was repressed by them. Thus, it may be assumed that the synthesis of insensitive pH 8.0 enzyme were repressed by limiting the amount of isoleucine is still open.     

A valine-resistant mutant ofArabidopsis thaliana displays an acetolactate synthase with altered feedback control

A valine-resistant mutant line, VAL-2, ofArabidopsis thaliana (L.) Heynh. was identified by screening M 2 populations of ethylmethane-sulfonate-mutagenized seeds. The resistance was found to be due to a single, dominant, nuclear gene mutation. Assay of acetolactate synthase (ALS) indicated that the valine resistance in this mutant is caused by decreased sensitivity of ALS to the branched-chain amino acids, valine, leucine andisoleucine. A two fold decrease in apparentK _m value for pyruvate of the mutant ALS enzyme was detected compared with that of the wild type. The sensitivity of the ALS enzyme to sulfonylurea, imidazolinone and triazolopyrimidine herbicides was not altered in the mutant. At the plant growth level the mutant was also resistant to valine plus leucine, but was sensitive to leucine orisoleucine alone. The mutant gene,var1, maps, or is very closely linked, toCSR1, the gene encoding acetolactate synthase inArabidopsis.Abbreviations ALS acetolactate synthase - BCAA branched-chain amino acid - CS chlorsulfuron - IM imidazolinone - SU sulfonylurea - TP triazolopyrimidine We thank Dr. George W. Haughn for providing Arabidopsis lines MSU12, MSU15, MSU21, MSU22 and MSU23. This work was supported by a Research Grant from the Natural Sciences and Engineering Research Council of Canada to J.K., K.W. is grateful for a University of Saskatchewan Graduate Scholarship.     

Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.Abbreviations ALS acetolactate synthase; ile, isoleucine - leu leucine - NTG N-methyl-N-nitro-N-nitrosoguanidine - SM sulfometuron methyl - SM^r sulfometuron methyl resistant - val valine     

Movement of C-Labeled Assimilates into Kernels of Zea mays L: I. Pattern and Rate of Sugar Movement

Carbon-14, photosynthetically fixed in leaves of Zea mays L. and translocated to developing kernels, passed through specialized basal endosperm cells prior to movement into the starchy endosperm and embryo. Radioactivity migrated in the endosperm at a maximum rate of 2.7 millimeters per hour, and there was no difference in the rate of movement in kernels treated 14 to 30 days after pollination.     

The acetolactate synthase isoenzymes of Escherichia coli K-12.

Summary Strains of Escherichia coli K-12 possessing only one of the three genes coding for acetolactate synthetase activity present either in the wild type or in its ilv0603 derivative were prepared and analyzed. Extracts prepared from these strains show different values of acetolactate synthase specific activity and different sensitivity to valine inhibition. These strains show a unique pattern of growth inhibition by different substances.Temperature sensitive (ts) mutations in the ilvB and ilvG genes, have been isolated and characterized. Extracts of these strains were found to have an acetolactate synthase activity more heat labile than that of a strain containing the corresponding wild type allele. We conclude that ilvB and ilvG are the structural genes for two different forms of acetolactate synthase activity, most likely two isoenzymes. Moreover, since the strains containing a ts mutation show a temperature sensitive auxotrophy for isoleucine and valine, these two acetolactate synthases participate in isoleucine and valine biosynthesis. Similar evidence for a third acetolactate synthase, the product of the ilvHI genes, has been reported previously.We propose the following names for the acetolactate synthase isoenzymes: acetolactate synthase I (AHAS I), the product of the ilvB gene; acetolactate synthase II (AHAS II), the product of ilvG gene; and acetolactate synthase III (AHAS III), the product of the ilvHI genes.     

Redirection of the Reaction Specificity of a Thermophilic Acetolactate Synthase toward Acetaldehyde Formation

Acetolactate synthase and pyruvate decarboxylase are thiamine pyrophosphate-dependent enzymes that convert pyruvate into acetolactate and acetaldehyde, respectively. Although the former are encoded in the genomes of many thermophiles and hyperthermophiles, the latter has been found only in mesophilic organisms. In this study, the reaction specificity of acetolactate synthase from Thermus thermophilus was redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. Error-prone PCR and mutant library screening led to the identification of a quadruple mutant with 3.1-fold higher acetaldehyde-forming activity than the wild-type. Site-directed mutagenesis experiments revealed that the increased activity of the mutant was due to H474R amino acid substitution, which likely generated two new hydrogen bonds near the thiamine pyrophosphate-binding site. These hydrogen bonds might result in the better accessibility of H⁺ to the substrate-cofactor-enzyme intermediate and a shift in the reaction specificity of the enzyme.     

The Fertilization and Early Development of Embryo and Endosperm in Stevia rebaudiana Bertani

The mature embryo sac is surrounded by endothelium tapetum. It is composed or an egg apparatus, one central cell with secondary nucleus, and 1–6 antipodal cells. About the 6th hour after pollination, female and male nuclei fuse with each other. The syngamy occurred almost simultaneously with the fusion of an other sperm nucleus and the secondary nucleus, but the velocity of the latter is faster than that of the syngamy. The fertilization of Stevia rebaudiana Bertani belongs to the premitotic type. About the 8th hour after pollination, primary endosperm nucleus is in mitosis, its dividing orientation may parallel or at right angle to the long axis of the embryo sac, and gives rise to two initial endosperm cells. The first five divisions of the endosperm cells are of synchronism. At the stage of heart-shaped embryo, the endosperm cells show the signs of digestion and absorbed. The endosperm development is of the cellular type. About the 10th hour after pollination, zygote divides for the first time. The division of the zygote is always transverse. The embryo development conforms to the Asterad type.     

Characteristics and development of leucine transport activity in the scutellum of germinating barley grain

Scutella separated from grains of Himalaya barley after germination for 3 days rapidly took up l-leucine from aerated incubation media; with 1 millimolar leucine the rate varied between 4 and 14 micromoles per gram per hour and the pH optimum was at 3.5 to 5, both depending on buffer composition and prewashing time. The rate of the uptake increased with increasing concentration of leucine in a complex manner, which could be interpreted as multiphasic kinetics with apparent K(m) values of 3.4 and 15.5 millimolar below and above 3 millimolar leucine, respectively. The uptake took place against a concentration difference (highest estimated ratio 270: 1) and was strongly inhibited by dinitrophenol. Uptake was apparently due to active transport requiring metabolic energy.The development of the uptake activity during germination was studied using Pirkka barley. A low activity was present in the scutella of ungerminated grains. It began to increase after 6 hours imbibition, and the increase was biphasic, the major changes occurring during days 0 to 3 and 4 to 6. The total increase was about 20-fold.The regulation of the development was studied by allowing separated embryos to germinate on agar gel. The increase of uptake activity was strongly inhibited by inhibitors of RNA or protein synthesis. Increase did not require the presence of the embryo proper, and was not affected by gibberellic or abscisic acid. Removal of the endosperm greatly accelerated the increase of uptake activity, and the presence of 5 or 20 millimolar glutamine counteracted the removal of the endosperm. The results suggest that the availability of glutamine or amino acids in general in the endosperm may regulate the development or the activity of the transport system.     

Localization of sucrose synthase activity in developing maize kernels by in situ enzyme histochemistry

Sucrose synthase is usually localized by immunocytochemistry, but this method does not show the actual activity of the localized enzyme. A histochemical assay is presented here showing the activity of sucrose synthase by tetrazolium salt precipitation on sections of developing maize kernels. The advantages of the assay are a high sensitivity for low amounts of active sucrose synthase and the independence of specific antibodies.In this study the activity of endosperm sucrose synthase is shown to move gradually from the apical part of the endosperm to the basal endosperm during kernel development. This shift in sucrose synthase activity correlates well with the localization of starch synthesis during kernel development. The assay also shows the early loss of activity in the aleurone layer bordering the embryo, and a loss of activity in the apical aleurone during the final stage of kernel development while the enzyme was still found by immunocytochemistry. This is in contrast to a high sucrose synthase activity in the epithelium of the scutellum, where hardly any labelling was found with antibodies against maize sucrose synthase. Low sucrose synthase activities were found in the pericarp and pedicel parenchyma.Possible functions of the high and low activity patterns in the developing maize kernels and differences between the enzyme assay and immunocytochemistry are discussed.     

微胚乳玉米籽粒发育期间非胚部位（胚乳）中的腺苷二磷酸-葡萄糖焦磷酸化酶活性变化

检测微胚乳玉米非胚部位（胚乳）中腺苷二磷酸-葡萄糖焦磷酸化酶（ADPG—PPase）的活性结果表明：微胚乳玉米的非胚部位（胚乳）中ADPG．PPase活性在授粉后21-28d达到峰值,而对照的玉米品种‘高油115’的ADPG—PPase活性在授粉后14-21d达到峰值,滞后约7d。‘高油115’非胚部位（胚乳）中ADPG—PPase活性最大值极显著高于微胚乳玉米,其单粒ADPG—PPase活性最大值为微胚乳玉米的2．2-3．6倍,其每克干重ADPG—PPase活性最大值则为微胚乳玉米的2．4—3．8倍。     

An invertase inhibitor from maize localizes to the embryo surrounding region during early kernel development

Invertase activity is thought to play a regulatory role during early kernel development by converting sucrose originating from source leaves into hexoses to support cell division in the endosperm and embryo. Invertases are regulated at the posttranslational level by small protein inhibitors, INVINHs. We found that in maize (Zea mays), an invertase inhibitor homolog (ZM-INVINH1) is expressed early in kernel development, between 4 and 7 d after pollination. Invertase activity is reduced in vitro in the presence of recombinant ZM-INVINH1, and inhibition is attenuated by pre-incubation with sucrose. The presence of a putative signal peptide, fractionation experiments, and ZM-INVINH1::green fluorescent protein fusion experiments indicate that the protein is exported to the apoplast. Moreover, association of ZM-INVINH1 with the glycoprotein fraction by concanavalin A chromatogaphy suggests that ZM-INVINH1 interacts with an apoplastic invertase during early kernel development. ZM-INVINH1 was localized to the embryo surrounding region by in situ analysis, suggesting that this region forms a boundary, compartmentalizing apoplast invertase activity to allow different embryo and endosperm developmental rates.     

Two Additional Phosphorylases in Developing Maize Seeds

Two additional phosphorylases (III and IV) have been detected in developing seeds of maize. Phosphorylase IV is found only in the embryo (with scutellum). It is also present in the embryo of the germinating seed where its activity is 90-fold greater than the activity in the developing embryo 22 days after pollination. Phosphorylase IV is eluted from a DEAE-cellulose column in the same fraction as phosphorylase I of the endosperm, and the 2 enzymes are similar in many respects. Phosphorylase IV is distinguished from phosphorylase I by electrophoretic mobility, by pH optimum, and because its properties are not affected by the shrunken-4 mutation.Phosphorylase III is found both in the endosperms and embryos of developing seeds. Activity for this enzyme is not detected in crude homogenates nor eluates from a DEAE-cellulose column apparently because it complexes with a non-dialyzable, heat-labile inhibitor. High activity is found after protamine sulfate fractionation. Phosphorylase III is bound to protamine sulfate and is then removed by washing with 0.3 m phosphate buffer. Phosphorylase III activity in the endosperm is not detectable 8 days after pollination but is present 12 days after pollination. Phosphorylase III differs from phosphorylases I, II, and IV in several respects-pH optimum, pH-independent ATP inhibition, time of appearance in the endosperm, and because purine and pyrimidine nucleotides are equally inhibitory. In common with phosphorylase II, phosphorylase III apparently does not require a primer to initiate the synthesis of an amylose-like polymer.     

Role of small subunit (IlvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity of the enzyme to valine inhibition.

Most of the coding sequence for the IlvN polypeptide subunit of acetohydroxyacid synthase I was deleted from the ilvB+ ilvN+ plasmid pTCN12 by in vitro methods. Several ilvB+ delta ilvN derivatives of pTCN12 were identified among transformants of a strain otherwise lacking any acetohydroxyacid synthase. Deletion derivatives produced an enzymatically active IlvB polypeptide, as shown by the Ilv+ phenotype of transformed cells and by immunologic and enzymatic assays. However, whereas the growth of pTCN12 transformants was sensitive to valine inhibition, growth of the ilvB+ delta ilvN transformants was relatively resistant. Moreover, in vitro analyses confirmed that both acetolactate and acetohydroxybutyrate synthesis in extracts of the ilvB+ delta ilvN transformants was resistant to valine inhibition, in comparison with that in extracts of pTCN12 transformants or with that catalyzed by purified acetohydroxyacid synthase I. The IlvN polypeptide had a minimal effect, if any, on IlvB polypeptide accumulation as measured by immunoprecipitation, but its absence resulted in a greater than 10-fold reduction in enzyme specific activity.