Maize Enzyme Research Articles

The Effects of Tillage and the Combined Application of Organic and Inorganic Fertilizers on the Antioxidant Enzyme Activity and Yield of Maize Leaves

The aim of this study was to explore the characteristics of the combined application of organic fertilizer and inorganic fertilizer using different tillage methods to delay the senescence of maize leaves. The yield and activities of GDH, CAT, APX, GR, and GSH enzymes in maize leaves were measured at different growth stages by using two tillage methods, three organic and inorganic combined applications (P1, P2, and P3), and four control treatments. (1) During the growth period, the R + S and R treatments were P1 treatments, with the highest enzyme activities noted for GDH, CAT, APX, GR, and GSH, which were 36.79–103.22% higher than those of CK. (2) The average yield of all R + S treatments was higher than that of R treatments, and the average yield of P1 treatment was the highest under R + S, which was 13,663.79 kg hm−2, which was 6.39%, 7.90%, and 14.67% higher than that of P2, P3, and CK, respectively, which was lower than that of R. The yield of P1 treatment was 2.53% higher. (3) There was a significant positive correlation between APX activity, CAT activity, GR activity, GDH activity, GSH activity, grain number per ear, ear length, and 100-grain weight of maize leaves at the grain filling stage, and a significant negative correlation between bald tip length and yield. The treatment details had the strongest enzyme activity and the highest yield when using the rotary tillage + subsoiling (R + S) P1 method, which was the most suitable tillage method and the best fertilizer ratio combination, which could be demonstrated and popularized in a large area in the dry farming area of spring maize in Shanxi Province.

A maize enzyme from the 2-oxoglutarate-dependent oxygenase family with unique kinetic properties, mediates resistance against pathogens and regulates senescence.

In plants, salicylic acid (SA) hydroxylation regulates SA homoeostasis, playing an essential role during plant development and response to pathogens. This reaction is catalysed by SA hydroxylase enzymes, which hydroxylate SA producing 2,3-dihydroxybenzoic acid (2,3-DHBA) and/or 2,5-dihydroxybenzoic acid (2,5-DHBA). Several SA hydroxylases have recently been identified and characterised from different plant species, but no such activity has yet been reported in maize. In this work, we describe the identification and characterisation of a new SA hydroxylase in maize plants. This enzyme, with high sequence similarity to previously described SA hydroxylases from Arabidopsis and rice, converts SA into 2,5-DHBA; however, it has different kinetic properties to those of previously characterised enzymes, and it also catalysers the conversion of the flavonoid dihydroquercetin into quercetin in in vitro activity assays, suggesting that the maize enzyme may have different roles in vivo to those previously reported from other species. Despite this, ZmS5H can complement the pathogen resistance and the early senescence phenotypes of Arabidopsis s3h mutant plants. Finally, we characterised a maize mutant in the S5H gene (s5hMu) that has altered growth, senescence and increased resistance against Colletotrichum graminicola infection, showing not only alterations in SA and 2,5-DHBA but also in flavonol levels. Together, the results presented here provide evidence that SA hydroxylases in different plant species have evolved to show differences in catalytic properties that may be important to fine tune SA levels and other phenolic compounds such as flavonols, to regulate different aspects of plant development and pathogen defence.

Comparison of physio-biochemical and antioxidant enzymes in maize during early growth stage in response to salt stress

Comparison of physio-biochemical and antioxidant enzymes in maize during early growth stage in response to salt stress

Soluble and insoluble α-glucan synthesis in yeast by enzyme suites derived exclusively from maize endosperm.

Molecular mechanisms that distinguish the synthesis of semi-crystalline α-glucan polymers found in plant starch granules from the synthesis of water-soluble polymers by nonplant species are not well understood. To address this, starch biosynthetic enzymes from maize (Zea mays L.) endosperm were isolated in a reconstituted environment using yeast (Saccharomyces cerevisiae) as a test bed. Ninety strains were constructed containing unique combinations of 11 synthetic transcription units specifying maize starch synthase (SS), starch phosphorylase (PHO), starch branching enzyme (SBE), or isoamylase-type starch debranching enzyme (ISA). Soluble and insoluble branched α-glucans accumulated in varying proportions depending on the enzyme suite, with ISA function stimulating distribution into the insoluble form. Among the SS isoforms, SSIIa, SSIII, and SSIV individually supported the accumulation of glucan polymer. Neither SSI nor SSV alone produced polymers; however, synergistic effects demonstrated that both isoforms can stimulate α-glucan accumulation. PHO did not support α-glucan production by itself, but it had either positive or negative effects on polymer content depending on which SS or a combination thereof was present. The complete suite of maize enzymes generated insoluble particles resembling native starch granules in size, shape, and crystallinity. Ultrastructural analysis revealed a hierarchical assembly starting with subparticles of approximately 50 nm diameter that coalesce into discrete structures of approximately 200 nm diameter. These are assembled into semi-crystalline α-glucan superstructures up to 4 μm in length filling most of the yeast cytosol. ISA was not essential for the formation of such particles, but their abundance was increased dramatically by ISA presence.

Bioprotective potential of maize apoplastic fluid bacterium (Bacillus amyloliquefaciens) and arbuscular mycorrhizal fungi (Glomus intraradices) against Spodoptera frugiperda infestation in maize

Spodoptera frugiperda is an invasive polyphagous pest of cereal crops and responsible for less maize production in the past. Development of bioprotective agents to reduce the impact of this biotic stress is essential. The present investigation was done to evaluate whether co-inoculation of maize with two endophytes such as Bacillus amyloliquefaciens and Glomus intraradices could alter the level of antioxidants and defense enzymes in maize and affect the feeding behavior of S. frugiperda on maize. The results indicated higher activity of antioxidants (catalases - 106.26%, peroxidases - 62.71%, superoxide dismutase −17.39% and ascorbate oxidase - 160.00%) and defensive enzymes (polyphenol oxidases - 205.86%, phenylalanine ammonia lyases - 126.00% and lipoxygenases - 33.33%) in leaves of B.amyloliquefaciens and G.intraradices treated maize after fed with S. frugiperda (96 h) over control. The total phenolics and dry biomass content were also higher in B. amyloliquefaciens and G. intraradices inoculated plants after S. frugiperda infestation (96 h). Leaf feeding bioassay indicated reduction in relative consumptive rate (18.25%), relative growth rate (19.76%) and increased feeding deterrence index (15.50%) of S. frugiperda when fed on B. amyloliquefaciens and G. intraradices inoculated maize for 24 h Inoculation of maize with B. amyloliquefaciens and G. intraradices significantly altered the defense parameters of maize which are directly linked with reduced feeding and growth rate of S. frugiperda. Therefore, these endophytes can be used to reduce the usage of chemical insecticides or be a part in integrated pest management systems for sustainable agriculture production.

Influence of Arbuscular mycorrhizal fungi and Pseudomonas in the activity of Phosphatase enzyme in maize (Zea mays) in response to cadmium contamination

Influence of Arbuscular mycorrhizal fungi and Pseudomonas in the activity of Phosphatase enzyme in maize (Zea mays) in response to cadmium contamination

Metabolic link between auxin production and specialized metabolites in Sorghum bicolor.

Aldoximes are amino acid derivatives that serve as intermediates for numerous specialized metabolites including cyanogenic glycosides, glucosinolates, and auxins. Aldoxime formation is mainly catalyzed by cytochrome P450 monooxygenases of the 79 family (CYP79s) that can have broad or narrow substrate specificity. Except for SbCYP79A1, aldoxime biosynthetic enzymes in the cereal sorghum (Sorghum bicolor) have not been characterized. This study identified nine CYP79-encoding genes in the genome of sorghum. A phylogenetic analysis of CYP79 showed that SbCYP79A61 formed a subclade with maize ZmCYP79A61, previously characterized to be involved in aldoxime biosynthesis. Functional characterization of this sorghum enzyme using transient expression in Nicotiana benthamiana and stable overexpression in Arabidopsis thaliana revealed that SbCYP79A61 catalyzes the production of phenylacetaldoxime (PAOx) from phenylalanine but, unlike the maize enzyme, displays no detectable activity against tryptophan. Additionally, targeted metabolite analysis after stable isotope feeding assays revealed that PAOx can serve as a precursor of phenylacetic acid (PAA) in sorghum and identified benzyl cyanide as an intermediate of PAOx-derived PAA biosynthesis in both sorghum and maize. Taken together, our results demonstrate that SbCYP79A61 produces PAOx in sorghum and may serve in the biosynthesis of other nitrogen-containing phenylalanine-derived metabolites involved in mediating biotic and abiotic stresses.

Characterization of Cytochrome P450s with Key Roles in Determining Herbicide Selectivity in Maize.

Safeners such as metcamifen and benoxacor are widely used in maize to enhance the selectivity of herbicides through the induction of key detoxifying enzymes, notably cytochrome P450 monooxygenases (CYPs). Using a combination of transcriptomics, proteomics, and functional assays, the safener-inducible CYPs responsible for herbicide metabolism in this globally important crop have been identified. A total of 18 CYPs belonging to clans 71, 72, 74, and 86 were safener-induced, with the respective enzymes expressed in yeast and screened for activity toward thiadiazine (bentazon), sulfonylurea (nicosulfuron), and triketone (mesotrione and tembotrione) chemistries. Herbicide metabolism was largely restricted to family CYP81A members from clan 71, notably CYP81A9, CYP81A16, and CYP81A2. Quantitative transcriptomics and proteomics showed that CYP81A9/CYP81A16 were dominant enzymes in safener-treated field maize, whereas only CYP81A9 was determined in sweet corn. The relationship between CYP81A sequence and activities were investigated by splicing CYP81A2 and CP81A9 together as a series of recombinant chimeras. CYP81A9 showed wide ranging activities toward the three herbicide chemistries, while CYP81A2 uniquely hydroxylated bentazon in multiple positions. The plasticity in substrate specificity of CYP81A9 toward multiple herbicides resided in the second quartile of its N terminal half. Further phylogenetic analysis of CYP81A9 showed that the maize enzyme was related to other CYP81As linked to agrochemical metabolism in cereals and wild grasses, suggesting this clan 71 CYP has a unique function in determining herbicide selectivity in arable crops.

Use of pullulanase as a biocatalyst for starch hydrolysis: Part 1. Study of the effect of pullulanase on maize amylopectin starch

The use of debranching enzymes in starch hydrolysis is a topical direction for obtaining new types of starch products with controlled properties and a potential for the further use. The aim of the work was to study an effect of pullulanase (EC3.2.1.41) on maize amylopectin starch in the native and gelatinized state. The objects of the research were maize amylopectin starch and enzyme preparation Promozyme D6 (Novozymes, Denmark). High-performance liquid chromatography (HPLC) was used to determine the carbohydrate composition of hydrolysates. The mass fraction of reducing substances (RS) was determined by the Lane and Eynon method. A rotational viscometer was used to measure dynamic viscosity of the starch hydrolysis products. It was found that analyzed starch in the native state showed low enzymatic sensitivity to the action of pullulanase with insignificant changes in viscosity, solubility and iodine binding capacity of the samples. Pullulanase showed the highest effect on gelatinized starch during the first eight hours of incubation. After eight hours, the maximum degree of starch hydrolysis by pullulanase at a dose of 10 units/g dry matter (DM) was 4.7% on DM basis, iodine binding capacity of the hydrolysate was D600 = 0.343 (in the control experiment D600 = 0.154), and the viscosity of the hydrolysate decreased from 7887 mPa · s to 4.3 mPa · s. Hydrolysates cooled to 8 °C and held for 20 hours along with hydrolysates that were not cooled showed high susceptibility to attack by glucoamilase (97–98%) at 60 °C and 24 hours of saccharification, which suggested the absence of their resistance to the action of glucoamilase in the conditions of the experiment. The use of pullulanase in dextrinization of the analyzed starch, which was gelatinized and partly hydrolyzed by α-amylase (RS6.1%), enabled obtaining hydrolysates with the mass fraction of reducing substances in a range of 10–24% on DM basis with the process duration of 2 to 24 hours and the enzyme dose of 2–10 units, which contained mainly maltotriose, maltohexose and maltoheptose with their total amount of 45–60% on DM basis. The results indicate a need for further research of the biocatalytic action of pullulanase to develop new methods for enzymatic modification of starch.

Overexpression of the homeobox-leucine zipper protein ATHB-6 improves the drought tolerance of maize (Zea mays L.)

Homeo-Leucine Zipper (HD-Zip) proteins are a class of transcription factors unique to higher plants and are involved in plant stress responses and regulation of growth and development. However, the function of maize HD-Zip genes in enhancing drought tolerance is unknown. Here, Sub-Cellular Localization results showed that ATHB-6 fusion proteins were only localized in the nucleus. The malondialdehyde content was lower than the wild type under drought tolerance, proving that the introduction of the ATHB-6 gene can improve the drought tolerance of plants. Follow-up analysis showed that ATHB-6 could promote root growth and activities of a series of ROS-scavenging enzymes in maize. Moreover, overexpression of ATHB-6 in maize activated the expression of critical genes in the ROS signals pathway and ABA-dependent pathway under drought tolerance.Our results provides a significant advancement in undestanding the functions of HD-Zip transcription factors in maize.

Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme

The photosynthetic phosphoenolpyruvate carboxylase isozyme from C4 plants (PEPC-C4) has a complex allosteric regulation, involving positive cooperativity in binding the substrate phosphoenolpyruvate as well as positive and negative allosteric effectors. Besides the proposed R- and T-states, previous kinetic results suggested functionally relevant different R-states of the maize enzyme (ZmPEPC-C4) elicited by PEP or its two kinds of activators, glucose 6-phosphate or glycine. To detect these different R-state conformations, we used as conformational probes the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS), near-UV circular dichroism (CD) spectroscopy, and limited proteolysis by trypsin. Phosphoenolpyruvate and malate binding caused distinct concentration-dependent fluorescence changes of ZmPEPC-C4/ANS, suggesting that they elicited conformational states different from that of the free enzyme, while glucose 6-phosphate or glycine binding did not produce fluorescence changes. Differences were also observed in the near UV CD spectra of the enzyme, free or complexed with its substrate or allosteric effectors. Additionally, differences in the trypsin-digestion fragmentation patterns, as well as in the susceptibility of the free and complexed enzyme to digestion and digestion-provoked loss of activity, provided evidence of several ZmPEPC-C4 conformations in solution elicited by the substrate and the allosteric effectors. Using the already reported ZmPEPC-C4 crystal structures and bioinformatics methods, we predicted that the most probable trypsin-cleavage sites are located in superficial flexible regions, which seems relevant for the protein dynamics underlying the function and allosteric regulation of this enzyme. Together, our findings agree with previous kinetic results, shed light on this enzyme's complex allosteric regulation, and place ZmPEPC-C4 in the growing list of allosteric enzymes possessing an ensemble of closely related R-state conformations.

Indole-3-glycerolphosphate synthase, a branchpoint for the biosynthesis of tryptophan, indole, and benzoxazinoids in maize.

The maize (Zea mays) genome encodes three indole-3-glycerolphosphate synthase enzymes (IGPS1, 2, and 3) catalyzing the conversion of 1-(2-carboxyphenylamino)-l-deoxyribulose-5-phosphate to indole-3-glycerolphosphate. Three further maize enzymes (BX1, benzoxazinoneless 1; TSA, tryptophan synthase alpha subunit; and IGL, indole glycerolphosphate lyase) convert indole-3-glycerolphosphate to indole, which is released as a volatile defense signaling compound and also serves as a precursor for the biosynthesis of tryptophan and defense-related benzoxazinoids. Phylogenetic analyses showed that IGPS2 is similar to enzymes found in both monocots and dicots, whereas maize IGPS1 and IGPS3 are in monocot-specific clades. Fusions of yellow fluorescent protein with maize IGPS enzymes and indole-3-glycerolphosphate lyases were all localized in chloroplasts. In bimolecular fluorescence complementation assays, IGPS1 interacted strongly with BX1 and IGL, IGPS2 interacted primarily with TSA, and IGPS3 interacted equally with all three indole-3-glycerolphosphate lyases. Whereas IGPS1 and IGPS3 expression was induced by insect feeding, IGPS2 expression was not. Transposon insertions in IGPS1 and IGPS3 reduced the abundance of both benzoxazinoids and free indole. Spodoptera exigua (beet armyworm) larvae show improved growth on igps1 mutant maize plants. Together, these results suggest that IGPS1 and IGPS3 function mainly in the biosynthesis of defensive metabolites, whereas IGPS2 may be involved in the biosynthesis of tryptophan. This metabolic channeling is similar to, though less exclusive than, that proposed for the three maize indole-3-glycerolphosphate lyases.

Two forms of NAD-malic enzyme in maize leaves are regulated by light in opposite ways via promoter methylation

NAD-malic enzyme (EC 1.1.1.39) activity, expression and methylation of promoters of its two genes was studied in maize (Zea mays L.) leaves depending on light regime. The total activity was high in darkness and upon irradiation by far-red light and suppressed by white light and by red light. The changes in the levels of transcripts of the genes Me1 and Me2 encoding NAD-malic enzyme revealed their dependence on irradiation in opposite ways. White and red light decreased the quantity of mRNA of the gene Me1, while far-red light led to the increase of its transcripts. The opposite pattern was observed for the transcripts of Me2, the level of which was low in darkness and upon irradiation by far-red light, and was higher in white light and after irradiation by red light. The study of methylation of the promoters of the genes encoding NAD-ME showed a strong dependence between the levels of transcripts and the state of methylation of CG dinucleotides. The two isoforms of NAD-malic enzyme were partially purified from maize leaves and characterized. The first isoform had a pH optimum of 6.4 while the second had a pH optimum of 6.9; in the reverse reaction, the pH optimum was ∼0.5 units higher. It is concluded that the two genes encode different isoforms of NAD-malic enzyme with different kinetic properties. The role of both isoforms in the operation of the tricarboxylic acid cycle in the open mode is discussed.

ZmPBF and ZmGAMYB transcription factors independently transactivate the promoter of the maize (Zea mays) β-carotene hydroxylase 2 gene.

The maize (Zea mays) enzyme β-carotene hydroxylase 2 (ZmBCH2) controls key steps in the conversion of β-carotene to zeaxanthin in the endosperm. The ZmBCH2 gene has an endosperm-preferred and developmentally regulated expression profile, but the detailed regulatory mechanism is unknown. To gain insight into the regulation of ZmBCH2, we isolated 2036bp of the 5'-flanking region containing the 263bp 5'-untranslated region (5'-UTR) including the first intron. We linked this to the β-glucuronidase reporter gene gusA. We found that high-level expression of gusA in rice seeds requires the 5'-UTR for enhanced activation. Truncated variants of the ZmBCH2 promoter retained their seed-preferred expression profile as long as a prolamin box and AACA motif were present. We identified candidate genes encoding the corresponding transcription factors (ZmPBF and ZmGAMYB) and confirmed that their spatiotemporal expression profiles are similar to ZmBCH2. Both ZmPBF and ZmGAMYB can transactivate ZmBCH2 expression in maize endosperm. To eliminate potential confounding effects in maize, we characterized the regulation of the minimal promoter region of ZmBCH2 in transgenic rice. This revealed that ZmPBF and ZmGAMYB independently transactivate the ZmBCH2 promoter. The mechanism that underpins our data provides an exciting new strategy for the control of target gene expression in engineered plants.

Effect of exogenous nitric oxide on sulfur and nitrate assimilation pathway enzymes in maize (Zea mays L.) under drought stress

The present study aimed at investigating the effects of foliar applied nitric oxide (as SNP [sodium nitroprusside]) on sulfur (glutathione reductase, guaiacol peroxidase, and glutathione S-transferase) and nitrate assimilation (nitrite and nitrate reductase) pathway enzymes in maize (Zea mays L.) exposed to water deficit conditions. The seedlings of a drought tolerant (NK8711) and sensitive (P1574) maize hybrid were applied with various SNP doses (0, 50, 100, 150, and 200 µM) under normal and drought stress conditions. Foliar spray of 100 µM markedly improved water status and chlorophyll contents and alleviated drought-induced oxidative damages through increased antioxidant (catalase, ascorbate peroxidase, and superoxide dismutase) activities in both maize hybrids. Moreover, exogenous SNP supply increased nitrite and nitrate reductase activities and upregulated glutathione reductase, glutathione S-transferase, and guaiacol peroxidase compared to no SNP supply. Interestingly, the negative effects of excess NO generation at high SNP doses (150, 200 µM) were more pronounced in P1574 than NK8711 leading to lower biomass accumulation in drought-sensitive hybrid.

Induction of Herbicide Detoxifying Enzyme in Maize by Chiral 3-Dichloroacetyl Oxazolidine

Safeners are important tools used to ensure to safe useof herbicide. The aim of this paper is to evaluate the protective effect of four safeners (R-28725, 3-dichloroacetyl oxazolidine and its two optical isomers) and investigate the mechanism of herbicide detoxication by safener. Laboratory studies were conducted to evaluate the effectiveness of safeners for protecting maize from the residues of preemergent herbicide fomesafen in Northeast Agricultural University, China. Physiological and biochemical tests were herein conducted under laboratory conditions, by using seed treatment with safeners and soil treatment with fomesafen, respectively. R-28725 provoked high glutathione level, glutathione-Stransferase activity and affinity of glutathione-S-transferase than other safeners, but R-isomer treatment resulted in complete reversal of injury caused by fomesafen.

Allosteric Regulation of the Photosynthetic C4 Isoenzyme of Phosphoenolpyruvate Carboxylase: A Comparative Study Between Enzymes from Monocot and Dicot Plants

In the present study, we have investigated the complex allosteric regulation of the non-phosphorylated forms of the photosynthetic phosphoenolpyruvate carboxylase isoenzymes (PEPC-C4) from amaranth (AhPEPC-C4) and maize (ZmPEPC-C4) leaves. Previous studies showed that glycine (Gly) only activates PEPC-C4 from monocot plants, as maize, but not from dicot plants, as amaranth. Our initial velocity data confirm this, in spite that AhPEPC-C4 binds Gly with much higher affinity than ZmPEPC-C4. In AhPEPC-C4, the lack of Gly activation is overcome mainly by its higher affinity for the substrate phosphoenolpyruvate and its lower affinity for the inhibitor malate compared with ZmPEPC-C4. We have also explored the structural determinants of the differences in Gly activation by performing multiple alignments between the known monocot and dicot PEPC-C4 sequences and by modeling, in both the AhPEPC-C4 and ZmPEPC-C4 isoenzymes, the three-dimensional structure of the loop proposed as the Gly binding site, which was not observed in the crystal structure of the maize enzyme due to its high flexibility. The models suggest that conserved lysyl and aspartyl residues are important for binding to the activator molecule, and that a nearby non-conserved residue may be responsible for differences between the amaranth and maize enzymes in the loop conformation, which would account for the poorer affinity for Gly of the maize enzyme as well as for its higher degree of activation.

Purification of Maize Nucleotide Pyrophosphatase/Phosphodiesterase Casts Doubt on the Existence of Zeatin Cis-Trans Isomerase in Plants.

Almost 25 years ago, an enzyme named zeatin cis–trans isomerase from common bean has been described by Bassil et al. (1993). The partially purified enzyme required an external addition of FAD and dithiothreitol for the conversion of cis-zeatin to its trans- isomer that occurred only under light. Although an existence of this important enzyme involved in the metabolism of plant hormones cytokinins was generally accepted by plant biologists, the corresponding protein and encoding gene have not been identified to date. Based on the original paper, we purified and identified an enzyme from maize, which shows the described zeatin cis–trans isomerase activity. The enzyme belongs to nucleotide pyrophosphatase/phosphodiesterase family, which is well characterized in mammals, but less known in plants. Further experiments with the recombinant maize enzyme obtained from yeast expression system showed that rather than the catalytic activity of the enzyme itself, a non-enzymatic flavin induced photoisomerization is responsible for the observed zeatin cis–trans interconversion in vitro. An overexpression of the maize nucleotide pyrophosphatase/phosphodiesterase gene led to decreased FAD and increased FMN and riboflavin contents in transgenic Arabidopsis plants. However, neither contents nor the ratio of zeatin isomers was altered suggesting that the enzyme is unlikely to catalyze the interconversion of zeatin isomers in vivo. Using enhanced expression of a homologous gene, functional nucleotide pyrophosphatase/phosphodiesterase was also identified in rice.

Effect of Patulin from Penicillium vulpinum on the Activity of Glutathione-S-Transferase and Selected Antioxidative Enzymes in Maize.

The mycotoxin patulin (PAT) was purified from Penicillium vulpinum CM1 culture that has been isolated from a soil cultivated with maize. The effect of PAT and of a fungal culture filtrate on the activities of glutathione-S-transferase (GST) and some antioxidant enzymes viz. ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) was investigated in roots and shoots of 8-day-old maize seedlings. PAT and culture filtrate caused significant reduction effects in a dose-related manner on the total GST activity. Upon application of the high PAT concentration (25 μg·mL−1) and of the concentrated fungal filtrate (100%, v/v), the reduction in GST activity of roots was 73.8–76.0% and of shoots was 60–61.7%. Conversely, significant increases in the activities of antioxidant enzymes were induced. Application of 25 μg·PAT·mL−1 increased APX, GR, DHAR, and MDHAR activity of root by 2.40-, 2.00-, 1.24-, and 2.16-fold, respectively. In shoots, the enzymatic activity was increased by 1.57-, 1.45-, 1.45-, and 1.61-fold, respectively. Similar induction values of the enzymatic activity were obtained upon application of the concentrated fungal filtrate. This is the first report describing the response of GST and antioxidant enzyme activities of plant cells to PAT toxicity.

Physiological basis for isoxadifen-ethyl induction of nicosulfuron detoxification in maize hybrids.

Isoxadifen-ethyl can effectively alleviate nicosulfuron injury in the maize. However, the effects of safener isoxadifen-ethyl on detoxifying enzymes in maize is unknown. The individual and combined effects of the sulfonylurea herbicide nicosulfuron and the safener isoxadifen-ethyl on the growth and selected physiological processes of maize were evaluated. Bioassays showed that the EC50 values of nicosulfuron and nicosulfuron plus isoxadifen-ethyl for maize cultivar Zhengdan958 were 18.87 and 249.28 mg kg-1, respectively, and were 24.8 and 275.51 mg kg-1, respectively, for Zhenghuangnuo No. 2 cultivar. Evaluations of the target enzyme of acetolactate synthase showed that the I50 values of nicosulfuron and nicosulfuron plus isoxadifen-ethyl for the ALS of Zhengdan958 were 15.46 and 28.56 μmol L-1, respectively, and were 0.57 and 2.17 μmol L-1, respectively, for the acetolactate synthase of Zhenghuangnuo No. 2. The safener isoxadifen-ethyl significantly enhanced tolerance of maize to nicosulfuron. The enhanced tolerance of maize to nicosulfuron in the presence of the safener, coupled with the enhanced injury observed in the presence of piperonyl butoxide, 1-aminobenzotriazole, and malathion, suggested cytochrome P450 monooxygenases may be involved in metabolism of nicosulfuron. We proposed that isoxadifen-ethyl increases plant metabolism of nicosulfuron through non-P450-catalyzed routes or through P450 monooxygenases not inhibited by piperonyl butoxide, 1-aminobenzotriazole, and malathion. Isoxadifen-ethyl, at a rate of 33 mg kg-1, completely reversed the effects of all doses (37.5–300 mg kg-1) of nicosulfuron on both of the maize cultivars. When the two compounds were given simultaneously, isoxadifen-ethyl enhanced activity of glutathione S-transferases (GSTs) and acetolactate synthase activity in maize. The free acid 4,5-dihydro-5,5-diphenyl-1,2-oxazole-3-carboxylic was equally effective at inducing GSTs as the parent ester and appeared to be the active safener. GST induction in the maize Zhenghuangnuo No. 2 was faster than in Zhengdan 958.