Cell Suspension Cultures Of Wheat Research Articles

Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures.

Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium-mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8'-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective means of Sanger sequencing and bioinformatic analysis. We discuss advantages and potential future developments of this method in contrast to other in vitro approaches.

Zearalenone‐malonyl‐glucosides as phase II metabolites in plant cell suspension cultures

AbstractBackground and objectivesConjugation of mycotoxins in the phase II metabolism of plants results in modified mycotoxins such as glucosides and malonyl‐glucosides. However, malonyl‐glucosides have not yet been completely elucidated for zearalenone (ZEN). Thus, the aim of this study was to produce and isolate malonyl‐glucosides of ZEN for an unambiguous identification by NMR spectroscopy.FindingsZearalenone was incubated in plant cell suspension cultures of wheat, soy, and tobacco, and phase II metabolites were analyzed by using LC‐DAD‐MS, ‐HRMS, and NMR spectroscopy. Four main metabolites of ZEN were detected in the cell extracts and identified as two glucosides (attached in positions 14 and 16) and their 6´‐malonyl derivatives.ConclusionsZearalenone‐malonyl‐glucosides should be incorporated in future analyses of modified mycotoxins because of their potential relevance for food and feed safety.Significance and noveltyFor the first time, the structures of the two malonyl‐glucosides of ZEN were unambiguously identified by NMR spectroscopy after preparative isolation as 14‐O‐(6’‐O‐malonyl‐β‐D‐glucopyranosyl)ZEN and 16‐O‐(6’‐O‐malonyl‐β‐D‐glucopyranosyl)ZEN.

Metabolism of 14C-labeled polychlorinated biphenyl congeners by wheat cell suspension cultures

The metabolism of [UL-14C]-2,2′,5,5′-tetrachlorobiphenyl (14C-PCB-52), [UL-14C]-2,2′,4,4′,5,5′-hexachlorobiphenyl (14C-PCB-153, and a congeneric mixture of [UL-14C]-labeled polychlorinated biphenyls (14C-PCB-Mix) was studied in cell suspension cultures of wheat (Triticum aestivum L. cv. ‘Heines Koga II’). About 50% of applied 14C-PCB-52 (20 μg/assay) was transformed during 96 h of incubation. While 7.6% on non-extractable residues emerged, turnover of 14C-PCB-52 was mainly due to soluble polar metabolites. These were subjected to chemical glycoside cleavage. In the resulting hydrolysate, four aglycons were identified by GC-EIMS, namely four tetrachloro-hydroxy-biphenyl isomers (C6H6Cl4O, M+· at m/z = 306, 308, 310 and 312), and one trichloro-hydroxy-biphenyl (C6H7Cl3O, M+· at m/z = 272, 274 and 276). Number and character of hydroxylated products pointed to cytochromes P450 as enzymatic catalysts of hydroxylation. 14C-PCB-153 was metabolized by wheat to minor degree if at all. Due to GC-EIMS analysis, of 14C-PCB-Mix consisted of biphenyl, one mono-, four di-, seven tri-, eleven tetra-, and four pentachlorobiphenyls besides traces of further mono- and hexachlorobiphenyls. Among these were PCB-28, PCB-52, PCB 101, and PCB-118 (identified by seven key congeners standard). The mixture resembled industrial products Clophen A30 or Aroclor 1016. Metabolic turnover of applied 14C-PCB-Mix (15 μg/assay) was 30% after 96 h; 8.4% of non-extractable residues emerged. Using DDE (p,p’-dichlorodiphenyl-dichloroethylene) as internal standard it was demonstrated that biphenyl, one monochloro-, two dichloro-, and one trichlorobiphenyl were completely metabolized to polar products. Partial metabolization occurred with one di-, five tri-, and four tetrachlorobiphenyls. Two tri-, four tetra-, and all pentachlorbiphenyls proved to be stable. Due to strong interference by matrix, evaluation of three congeners was not possible. In addition to wheat, results of similar experiments with cell cultures of other species are briefly mentioned.

The xylanase inhibitor TAXI-III counteracts the necrotic activity of a Fusarium graminearum xylanase in vitro and in durum wheat transgenic plants.

The xylanase inhibitor TAXI-III has been proven to delay Fusarium head blight (FHB) symptoms caused by Fusarium graminearum in transgenic durum wheat plants. To elucidate the molecular mechanism underlying the capacity of the TAXI-III transgenic plants to limit FHB symptoms, we treated wheat tissues with the xylanase FGSG_03624, hitherto shown to induce cell death and hydrogen peroxide accumulation. Experiments performed on lemmas of flowering wheat spikes and wheat cell suspension cultures demonstrated that pre-incubation of xylanase FGSG_03624 with TAXI-III significantly decreased cell death. Most interestingly, a reduced cell death relative to control non-transgenic plants was also obtained by treating, with the same xylanase, lemmas of TAXI-III transgenic plants. Molecular modelling studies predicted an interaction between the TAXI-III residue H395 and residues E122 and E214 belonging to the active site of xylanase FGSG_03624. These results provide, for the first time, clear indications in vitro and in planta that a xylanase inhibitor can prevent the necrotic activity of a xylanase, and suggest that the reduced FHB symptoms on transgenic TAXI-III plants may be a result not only of the direct inhibition of xylanase activity secreted by the pathogen, but also of the capacity of TAXI-III to avoid host cell death.

Untargeted profiling of tracer-derived metabolites using stable isotopic labeling and fast polarity-switching LC-ESI-HRMS.

An untargeted metabolomics workflow for the detection of metabolites derived from endogenous or exogenous tracer substances is presented. To this end, a recently developed stable isotope-assisted LC–HRMS-based metabolomics workflow for the global annotation of biological samples has been further developed and extended. For untargeted detection of metabolites arising from labeled tracer substances, isotope pattern recognition has been adjusted to account for nonlabeled moieties conjugated to the native and labeled tracer molecules. Furthermore, the workflow has been extended by (i) an optional ion intensity ratio check, (ii) the automated combination of positive and negative ionization mode mass spectra derived from fast polarity switching, and (iii) metabolic feature annotation. These extensions enable the automated, unbiased, and global detection of tracer-derived metabolites in complex biological samples. The workflow is demonstrated with the metabolism of 13C9-phenylalanine in wheat cell suspension cultures in the presence of the mycotoxin deoxynivalenol (DON). In total, 341 metabolic features (150 in positive and 191 in negative ionization mode) corresponding to 139 metabolites were detected. The benefit of fast polarity switching was evident, with 32 and 58 of these metabolites having exclusively been detected in the positive and negative modes, respectively. Moreover, for 19 of the remaining 49 phenylalanine-derived metabolites, the assignment of ion species and, thus, molecular weight was possible only by the use of complementary features of the two ion polarity modes. Statistical evaluation showed that treatment with DON increased or decreased the abundances of many detected metabolites.

Metabolism of Strobilurins by Wheat Cell Suspension Cultures

Strobilurin fungicides are a leading class of antifungal chemicals used today in agricultural applications. Although degradation of some strobilurin fungicides has been assessed in plant residues, little information has appeared in the literature concerning the rates of metabolism of these fungicides in plants. In this study, we explored plant metabolism of three strobilurin fungicides, azoxystrobin, kresoxim-methyl, and trifloxystrobin, using wheat cell suspension cultures. Trifloxystrobin and kresoxim-methyl were completely metabolized within 24 h, whereas the metabolism of azoxystrobin was relatively slow with half-lives up to 48 h depending on specific experimental conditions. Metabolic rates of these fungicides were affected by the amounts of compound and cells added to the media. Structural analysis of metabolites of trifloxystrobin and kresoxim-methyl by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR) indicated that trifloxystrobin was first demethylated followed by subsequent hydroxylation, whereas kresoxim-methyl was largely demethylated. In contrast, a number of minor metabolites of azoxystrobin were present suggesting a differential metabolism of strobilurins by wheat cells.

Rapid production of wheat cell suspension cultures directly from immature embryos

The aim of this study was to produce suspension cultures of winter wheat directly from immature embryos bypassing the callus stage, and to determine their capacity for growth and regeneration in comparison to suspension cultures produced from callus. The study was carried out using Polish winter wheat varieties: ‘Grana’ and ‘Rosa’. Immature embryos were isolated, homogenized and transferred directly to liquid medium supplemented with 2,4-D. Actively dividing cell cultures were obtained within 2 months after the cultures were started. Suspension cultures from callus of immature embryos was also produced. With both cultivars, faster growth was observed in the suspension cultures produced directly from embryos than in the suspensions produced from callus. Metabolic activity was higher in the suspension culture produced directly from embryos than in the suspension derived from callus only in ‘Grana’. The production of 1-amiocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, was lower in the suspension cultures produced directly from embryos than in the suspensions produced from callus. Morphogenic capacity was significantly higher in aggregates derived directly from embryos than in aggregates derived from callus. With ‘Rosa’, about one third of the aggregates derived directly from embryos regenerated shoots. Production of ACC was lower in ‘Rosa’ cell culture that regenerated then in other cell cultures that did not. Photosystem II reactions were more efficient in dark green aggregates than in light green or pale green aggregates which were unable to regenerate. With the method presented, wheat cell suspension cultures with a regeneration potential can be produced in 2 or 3 months less time than with traditional methods.

Transformation of the mycotoxin ochratoxin A in plants: 1. Isolation and identification of metabolites formed in cell suspension cultures of wheat and maize.

The metabolism of the mycotoxin ochratoxin A in plant cells was investigated using cell suspension cultures of wheat and maize. A number of metabolites were detected by HPLC-chromatography with fluorescence detection. The main metabolites were ochratoxin alpha, ochratoxin A methyl ester, two isomers of hydroxyochratoxin A, and the glucosides and methyl esters of both hydroxyochratoxin A isomers. The compounds were isolated by TLC and preparative HPLC and identified by mass spectrometry and specific enzymic reactions.

Characterization of the bound residues of the fungicide cyprodinil formed in plant cell suspension cultures of wheat

The non-extractable residues of the fungicide cyprodinil formed in heterotrophic cell suspension cultures of wheat were studied by application of [2-pyrimidyl-14C] or [2-pyrimidyl-13C]cyprodinil. The main objective was to examine whether solid-state and liquid 13C NMR spectroscopy can be used to examine plant bound residues of pesticides. For 14C experiments, wheat suspensions grown on glucose as carbon source were treated with 10 mg litre(-1) of 14C-cyprodinil. After incubation for 12 days, 20% of applied 14C was detected as non-extractable residues. The cell debris were treated with 0.1 M HCl (reflux), 1.0 M HCl (reflux), buffer, or 2 M NaOH (50 degrees C); Björkman lignin and acidolysis lignin fractions were also prepared from the debris. Radioactivity liberated and solubilized by these procedures was examined by thin-layer chromatography and high-performance liquid chromatography. The results showed that cyprodinil and primary metabolites contributed to the fungicide's bound residues. Most of the residues (12% of applied 14C) remained associated with polar or polymeric/oligomeric endogenous cell materials in a stable manner. For the study with 13C-cyprodinil, wheat suspensions were cultivated on 13C-depleted glucose for four growth cycles, resulting in maximum 13C depletion of the natural cell components to about 0.10%. During the fourth cycle, 13C-labelled cyprodinil was applied, and cells were incubated (12 days). Cell debris was prepared and examined by solid-state 13C NMR spectroscopy. Debris was then treated as described above in the 14C experiment. Solubilized fractions were analyzed by liquid 13C NMR spectroscopy. However, none of the 13C NMR spectra recorded gave utilizable or unambiguous results, and all exhibited large inconsistencies, especially concerning the data from the conventional 14C experiment.

Agrobacterium tumefaciens-mediated transformation of wheat using suspension cells as a model system and green fluorescent protein as a visual marker

Conditions for Agrobacterium-mediated transformation of wheat (Triticum aestivum L.) were defined using wheat suspension cells as a model system and green fluorescent protein (GFP) as a visual marker. Different strains of Agrobacterium tumefaciens were compared using established wheat cell suspension cultures, where the frequency of cell clusters showing transient activity of GFP ranged from 2 to 52%. High levels of transient GFP activity and stable transformed callus lines were obtained with plasmid pTO134 containing a gfp gene with an enhanced CaMV 35S promoter and a bar gene with a 35S promoter in combination with Agrobacterium strain AGL0. These results suggest that the important variables in Agrobacterium-mediated transformation of wheat cells include media composition, Agrobacterium strain, plasmid vector and the addition of virulence-inducing agents such as acetosyringone. The conditions deemed optimal for transformation of wheat suspension cell lines were applied to scutella isolated from immature embryos and scutella-derived calli. Transient GFP expression in these tissues ranged from 10 to 75% and, while quite variable among and within cultivars, stably transformed scutellum-derived callus was obtained. Further studies with scutellum-derived calli suggested that variables such as duration of pre-inoculation culture and co-cultivation, as well as co-cultivation temperature, were also important. Optimisation of these variables resulted in the recovery of transformed wheat plants at a transformation frequency of 1.8%, which is comparable with other reports.

Research Articles

The metabolism of phenanthrene was studied both in cell suspension cultures of wheat (Triticum aestivum) and soybean (Glycine max), and in intact plants of the water mossFontinalis antipyretica. Metabolism in cell suspension cultures strongly differed between the monocotyle and the dicotyle plant. Only small amounts oftrans-phenanthrene-9,10-dihydrodiole and phenanthrene-9,10-dione were detectable in the wheat culture. Soybean cultures, in contrast demonstrated a strong turnover resulting in a 75% reduction of the initial phenanthrene concentration. Metabolites were phenanthrene-9,10-dione, not further characterized polar metabolites and bound residues. Intact plants ofFontinalis antipyretica metabolized only small amounts of phenanthrene. Data obtained from cell cultures did not provide information for the metabolic potential in intact plants. Therefore standardized tests with model systems like suspension cultures lead to inadequate assessment of the ecological risk of certain xenobiotics.

3,4-Dichloroaniline N-glucosyl- and N-malonyltransferase activities in cell cultures and plants of soybean and wheat

The activities of UDP-glucose: 3,4-dichloroaniline N-glucosyltransferase ( N-GT; EC 2.4.1.71) and malonyl-CoA: 3,4-dichloroaniline, N-malonyltransferase ( N-MT) were determined in soybean and wheat cell suspension cultures, and in individual organs of the respective plants. In the soybean suspensions, N-GT and N-MT activities were quite constant over the subculture interval, and ranged from 0.48 to 1.65, and 34.3 to 80.1 pkat·mg −1 protein, respectively. No N-MT activity was detected in wheat cultures, while the specific N-GT activities were between 1.3 and 4.5 pkat·mg −1 protein. In soybean plants, N-MT was most active in the roots (9.6 pkat·mg −1 protein) and N-GT activity was maximal in the cotyledons (1.7 pkat·mg −1 protein). No N-MT activity was detected in the wheat plants, whereas N-GT activities of the wheat roots and cotyledons amounted to 1.4 and 1.0 pkat·mg −1 protein, respectively.

Detoxification of Formaldehyde by the Spider Plant (Chlorophytum comosum L.) and by Soybean (Glycine max L.) Cell-Suspension Cultures.

The phytotoxicity of formaldehyde for spider plants (Chlorophytum comosum L.), tobacco plants (Nicotiana tabacum L. cv Bel B and Bel W3), and soybean (Glycine max L.) cell-suspension cultures was found to be low enough to allow metabolic studies. Spider plant shoots were exposed to 7.1 [mu]L L-1 (8.5 mg m-3) gaseous [14C]-formaldehyde over 24 h. Approximately 88% of the recovered radioactivity was plant associated and was found to be incorporated into organic acids, amino acids, free sugars, and lipids as well as cell-wall components. Similar results were obtained upon feeding [14C]formaldehyde from aqueous solution to aseptic soybean cell-suspension cultures. Serine and phosphatidylcholine were identified as major metabolic products. Spider plant enzyme extracts contained two NAS+-dependent formaldehyde dehydrogenase activities with molecular mass values of about 129 and 79 kD. Only the latter enzyme activity required glutathione as an obligatory second cofactor. It had an apparent Km value of 30 [mu]M for formaldehyde and an isoelectric point at pH 5.4. Total cell-free dehydrogenase activity corresponded to 13 [mu]g formaldehyde oxidized h-1 g-1 leaf fresh weight. Glutathione-dependent formaldehyde dehydrogenases were also isolated from shoots and leaves of Equisetum telmateia and from cell-suspension cultures of wheat (Triticum aestivum L.) and maize (Zea mays L.). The results obtained are consistent with the concept of indoor air decontamination with common room plants such as the spider plant. Formaldehyde appears to be efficiently detoxified by oxidation and subsequent C1 metabolism.

A Similar Metabolism of Chlorotoluron in Cell Suspension Cultures from Near-Isogenic Susceptible and Tolerant Lines of Wheat

The metabolism of the herbicide chlorotoluron was followed in cell suspension cultures of wheat. The cultures originated from plants of the susceptible variety Corin, the tolerant variety Clément, and six near-isogenic lines, 9S, 10S, 16S, 17T, 18T, and 24T [susceptible (S) and tolerant (T), respectively]. The six lines had the genetic background of the susceptible variety Chinese Spring, but the three lines 17T, 18T, and 24T contained a gene for tolerance (Sul) transferred from the variety Cappelle-Desprez. The cultures from Corin and Clément produced identical patterns of metabolites which were also identical to those found in plants. The rate of metabolism of the herbicide was slightly higher in the cell culture of Clément (T) than in Corin (S), as reported for the corresponding plants. Patterns of metabolites were similar in the cell cultures from the six isogenic lines. The rate of metabolism was the same in 9S, 10S, 16S, 18T, and 24T, and lower in 17T, so that a differential rate of metabolism was not found between T and S cell cultures. The occurrence of a differential rate of metabolism as the primary mechanism of varietal selectivity of wheat to chlorotoluron is discussed.

Plant metabolism of herbicides with CP bonds: Glyphosate

Plants have been reported not to metabolize glyphosate. In order to reexamine these literature findings, cell suspension cultures of wheat ( Triticum aestivum L.), soybean ( Glycine max L.), and maize ( Zea mays L.) as well as whole-plant samples of spruce ( Picea abies L. Karst), tobacco ( Nicotiana tabacum sp.), wheat, soybean, and maize have now been incubated with [3- 14C]glyphosate. Appreciable uptake of glyphosate was observed with seedlings and leaves and to a lesser extent with cultured cells. The cell extracts contained most of the radioactivity in the form of nonmetabolized glyphosate with aminomethylphosphonic acid (AMPA) as the only detectable metabolite. The ratio of both compounds varied between the different plant species. The highest percentages of AMPA (49.7% of applied radioactivity) were present in soybean cell cultures. In soybean cell cultures 14CO 2-evolution of about 1% of the applied radioactivity occurred within 4 days. A low incorporation into the polar group region of soybean phospholipids also occurred. Incorporation into the insoluble residue fraction was more significant (up to 27% in soybean leaves). These bound residues consisted largely of cell wall material and were sequentially solubilized. The incorporated radioactivity was associated with all solubilized residue fractions. In the case of soybean cell cultures it could be shown that the bound radioactivity in the amylase, pronase, and pectinase solubilized fractions was to ≥90% due to AMPA.

Stably Transformed Callus Lines from Microprojectile Bombardment of Cell Suspension Cultures of Wheat

We have obtained stably transformed callus lines by direct delivery of DNA into plated suspension culture cells of wheat (Triticum aestivum L.) using high velocity microprojectile bombardment. Three different reporter or selectable marker genes, jointly present or on separate plasmids, were introduced: neomycin phospho-transferase (NPTII), β-glucuronidase (GUS) and 5-enolpyruvylshikimate phosphate (EPSP) synthase. Kanamycin was used for the selection of resistant calli, which were screened for GUS expression by a histochemical stain. Southern analysis confirmed that the NPTII, GUS and EPSP synthase genes had been stably integrated in all of the kanamycin resistant and GUS positive lines, and NPTII and EPSP synthase activities were demonstrated in the transformed calli.

Interactions of various agrochemicals with cytochrome P-450-dependent monooxygenases of wheat cells

Levels of cytochrome P-450s and rates of monooxygenase activities were studied in microsomes prepared from wheat cell suspension cultures. Cytochrome contents and enzymatic activities, namely, the enzymatic systems responsible for chlorotoluron ring-methyl hydroxylation and N-demethylation, lauric acid hydroxylase, and cinnamic acid 4-hydroxylase, were enhanced after pretreatment of cells with 2,4-D, procloraz, mecoprop, chlorotoluron, and oxime ether safeners. Effects of various agrochemicals were also determined on chlorotoluron ring-methyl hydroxylase and N-demethylase, following their direct addition to microsomal preparations. Plant growth regulators and fungicides, as well as piperonyl butoxide decreased both activities, tetcyclacis, and procloraz being markedly inhibitory. Naphthalic anhydride, oxime ether safeners, dichlormid, and tridiphane had only weak effects. The substrate specificities of chlorotoluron ring-methyl hydroxylase and N-demethylase were also investigated using structural analogues of the herbicide. Diuron was the strongest inhibitor among the tested phenylureas. Other herbicides that can be metabolized by wheat affected both activities to different extents. However, diclofop enhanced only chlorotoluron N-demethylase.

Metabolism of pentachlorophenol in cell suspension cultures of wheat (Triticum aestivum L.). Tetrachlorocatechol as a primary metabolite

Metabolism of pentachlorophenol in cell suspension cultures of wheat (Triticum aestivum L.). Tetrachlorocatechol as a primary metabolite

Metabolism of the persistent plasticizer chemical bis(2-ethylhexyl) phthalate in cell suspension cultures of wheat (Triticum aestivum L.). Discrepancy from the intact plant

Metabolism of the persistent plasticizer chemical bis(2-ethylhexyl) phthalate in cell suspension cultures of wheat (Triticum aestivum L.). Discrepancy from the intact plant

Metabolism of DDT and related compounds in cell suspension cultures of soybean ( Glycine max L.) and wheat ( Triticum aestivum L.)

Cell suspension cultures of wheat and soybean were incubated with [ 14C]-1,1,1-trichloro-2,2-bis-(4-chlorophenyl)ethane (DDT), [ 14C]-1,1-dichloro-2,2-bis-(4-chlorophenyl)ethene (DDE), and [ 14C]-2,2-bis-(4-chlorophenyl)acetic acid (DDA) under standardized conditions. Polar metabolites were formed in yields of 1–2.5% in the cases of DDT and DDE, and of 56% in the case of DDA. A nonpolar metabolite was only observed in the case of DDT in soybean. This metabolite was identified as DDE on the basis of cochromatography and mass spectroscopy. By the same methods DDA was identified as a major polar DDT metabolite of both soybean and wheat. The further conversion of DDA to hexose esters was demonstrated by chromatographic and mass spectroscopic comparison with synthetic DDA-β- d-glucopyranosyl tetraacetate. These studies suggest the metabolic sequence, DDT → DDA → DDA-hexose ester.