Unraveling the DNA Methylation in the rDNA Foci in Mutagen-Induced Brachypodium distachyon Micronuclei.

Many years have passed since micronuclei were first observed then accepted as an indicator of the effect of mutagens. However, the possible mechanisms of their formation and elimination from the cell are still not fully understood. Various stresses, including mutagens, can alter gene expression through changes in DNA methylation in plants. In this study we demonstrate for the first time DNA methylation in the foci of 5S and 35S rDNA sequences in individual Brachypodium distachyon micronuclei that are induced by mutagenic treatment with maleic acid hydrazide (MH). The impact of MH on global epigenetic modifications in nuclei and micronuclei has been studied in plants before; however, no in situ analyses of DNA methylation in specific DNA sequence sites are known. To address this problem, we used sequential immunodetection of 5-methylcytosine and fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes on the non-dividing cells of B. distachyon. Such investigations into the presence or absence of DNA methylation within specific DNA sequences are extremely important in plant mutagenesis in the light of altering gene expression.

Maleic hydrazide elicits global transcriptomic changes in chemically topped tobacco to influence shoot bud development.

MAIN CONCLUSION: Transcriptomic analysis revealed maleic hydrazide suppresses apical and axillary bud development by altering the expression of genes related to meristem development, cell division, DNA replication, DNA damage and recombination, and phytohormone signaling. Topping (removal of apical buds) is a common agricultural practice for some crop plants including cotton, cannabis, and tobacco. Maleic hydrazide (MH) is a systemic suckercide, a chemical that inhibits shoot bud growth, used to control the growth of apical (ApB) and axillary buds (AxB) following topping. However, the influence of MH on gene expression and the underlying molecular mechanism of controlling meristem development are not well studied. Our RNA sequencing analysis showed that MH significantly influences the transcriptomic landscape in ApB and AxB of chemically topped tobacco. Gene ontology (GO) enrichment analysis revealed that upregulated genes in ApB were enriched for phosphorelay signal transduction, and the regulation of transition timing from vegetative to reproductive phase, whereas downregulated genes were largely associated with meristem maintenance, cytokinin metabolism, cell wall synthesis, photosynthesis, and DNA metabolism. In MH-treated AxB, GO terms related to defense response and oxylipin metabolism were overrepresented in upregulated genes. GO terms associated with cell cycle, DNA metabolism, and cytokinin metabolism were enriched in downregulated genes. Expression of KNOX and MADS transcription factor (TF) family genes, known to be involved in meristem development, were affected in ApB and AxB by MH treatment. The promoters of MH-responsive genes are enriched for several known cis-acting elements, suggesting the involvement of a subset of TF families. Our findings suggest that MH affects shoot bud development in chemically topped tobacco by altering the expression of genes related to meristem development, DNA repair and recombination, cell division, and phytohormone signaling.

Al-Tolerant Barley Mutant hvatr.g Shows the ATR-Regulated DNA Damage Response to Maleic Acid Hydrazide.

ATR, a DNA damage signaling kinase, is required for cell cycle checkpoint regulation and detecting DNA damage caused by genotoxic factors including Al3+ ions. We analyzed the function of the HvATR gene in response to chemical clastogen-maleic acid hydrazide (MH). For this purpose, the Al-tolerant barley TILLING mutant hvatr.g was used. We described the effects of MH on the nuclear genome of hvatr.g mutant and its WT parent cv. "Sebastian", showing that the genotoxic effect measured by TUNEL test and frequency of cells with micronuclei was much stronger in hvatr.g than in WT. MH caused a significant decrease in the mitotic activity of root cells in both genotypes, however this effect was significantly stronger in "Sebastian". The impact of MH on the roots cell cycle, analyzed using flow cytometry, showed no differences between the mutant and WT.

[Effect of different plant growth regulators on yield and quality of Angelica dahurica var. formosana development].

OBJECTIVE: To investigate the effect of plant growth regulators on the growth and quality of Angelica dahurica var. formosana. METHOD: Five plant growth regulators: chlormequat chloride (CCC), Mepiquat chloride (PIX), Gibberellic acid (GA3), Paclobutrazol (PP333) and Maleic Hydrazide (MH) were sprayed in rosette stage, the effects of these plant growth regulators (PGRs) on the growth, yield and quality of A. dahurica var. formosanaw were observed. The biological traits were first measured and then imperatorin and isoimperatorin contents in roots were determined by HPLC. RESULT: Low concentration GA3 increased the yield while not influenced the premature bolting rate and the coumarin content. CONCLUSION: Spraying of GA3 (30 mg x L(-1)) could guarantee the growth and development of A. dahurica var. formosana to have a higher yield and maintain the active ingredients content in the root as well.

[Determination of maleic hydrazide residues and its influence on the quality of Atractylodes macrocephala].

OBJECTIVE: To determine maleic hydrazide (MH) residues and discuss its influence on the quality of Atractylodes macrocephala. METHODS: At the bud stage, A. macrocephala different concentration of MH. Then MH residues,the contents of sugar and lactone were determined by HPLC and UV. The quality of A. macrocephala was comprehensively evaluated by independent sample t test and principal component analysis. RESULTS: The range of MH residues was 0.3-2.2 mg/kg. The results of independent sample t test revealed that the trend of the contents of lactone was low-high-low with the increase of MH, and the effect of MH on the content of sugar was barely obvious. Meanwhile, principal component analysis showed that comprehensive evaluation on the quality of A. macrocephala was the best when MH with 75 or 100 times water was applied. CONCLUSION: Proper concentration MH is applied to ensure low concentration MH residues and improve yield and quality of A. macrocephala.

Maleic hydrazide: sprout suppression of potatoes in the field.

In 2005, the active substance maleic hydrazide was released on the Belgian market. Maleic hydrazide is authorized in potatoes as foliar treatment for instore sprout suppression and control of volunteers. The mode of action is based on blocking cell division whilst cell elongation is not affected. The product must be applied at once during the growing season, only after at least 80% of the tubers have reached 25 mm diameter and not later than 3 weeks before haulm killing. The first 24 h after application, no meaningful precipitation should occur to insure sufficiently uptake of the product by the crop. Field trials were set up for 4 years (2005-2008) and 4 locations per year with application of maleic hydrazide in four different cultivars (Bintje, Fontane, Asterix and Cilena). After application, the cultivar Asterix showed almost every year a temporarily phytotoxicity (bronze discoloration). On the first place yield was determined. When maleic hydrazide was applied too early (80% tubers % 25mm diameter) yield was negatively affected (3 years on 4) except for the cultivar Cilena (fresh market). Internal quality (dry matter and fry quality) was not influenced by the application of maleic hydrazide. Only Fontane had a slightly lower dry matter content. Maleic hydrazide also influenced appearance of secondary growth. However, the results were very variable depending on cultivar, location and time of application. After harvest, the tubers were kept in storage and assessed monthly on germination. Potatoes treated late in the growing season, showed a shorter dormancy period. A part of the tubers was replanted the following spring to verify volunteer control. Additional trials were set up by the Flemish government for two years (2010-2011). The results of previous trials were confirmed. Additional, the influence of maleic hydrazide on internal germination during storage was examined on the cultivar Innovator. The tests clearly showed a positive effect for this parameter.

Brachypodium distachyon - A Useful Model in the Qualification of Mutagen-Induced Micronuclei Using Multicolor FISH.

Brachypodium distachyon (Brachypodium) is now intensively utilized as a model grass species in various biological studies. Its favorable cytological features create a unique foundation for a convenient system in mutagenesis, thereby potentially enabling the 'hot spots' and 'cold spots' of DNA damage in its genome to be analyzed. The aim of this study was to analyze the involvement of 5S rDNA, 25S rDNA, the Arabidopsis-type (TTTAGGG)n telomeric sequence and the Brachypodium-originated centromeric BAC clone CB33J12 in the micronuclei formation in Brachypodium root tip cells that were subjected to the chemical clastogenic agent maleic hydrazide (MH). To the best of our knowledge, this is the first use of a multicolor fluorescence in situ hybridization (mFISH) with four different DNA probes being used simultaneously to study plant mutagenesis. A quantitative analysis allowed ten types of micronuclei, which were characterized by the presence or absence of specific FISH signal(s), to be distinguished, thus enabling some specific rules governing the composition of the MH-induced micronuclei with the majority of them originating from the terminal regions of chromosomes, to be identified. The application of rDNA sequences as probes showed that 5S rDNA-bearing chromosomes are involved in micronuclei formation more frequently than the 25S rDNA-bearing chromosomes. These findings demonstrate the promising potential of Brachypodium to be a useful model organism to analyze the effects of various genotoxic agents on the plant nuclear genome stability, especially when the complex FISH-based and chromosome-specific approaches such as chromosome barcoding and chromosome painting will be applied in future studies.

Reduced clastogenic activity of maleic hydrazide in Vicia faba seedlings grown in a situation of overcrowding stress.

A pre-treatment stress situation of overcrowding of Vicia faba seedlings in the phase of germination and growth influenced their subsequent sensibility to treatment with the mutagenic herbicide maleic hydrazide. The seedlings showed a significant reduction in the frequency of micronucleated cells when they grew in a strongly crowded manner compared with scattered and uniformly distributed seedlings (3.83% versus 11.46%). The findings do not provide evidence for the involvement of phytochelatins in response to stress conditions in this process: pre-treatment with buthionine sulfoximine, a specific inhibitor of phytochelatin synthesis, did not modify the response of the seedlings to maleic hydrazide under conditions of overcrowding or under normal conditions. Scanning electron microscopy (SEM) was used to observe the root tip of V. faba grown in conditions of normal growth or overcrowding. SEM micrographs revealed differences between the tips with regards to root hair density and root surface morphology. Finally, we found a positive correlation between the frequency of micronucleated cells and the length of the primary root, for every time of growth considered (1, 3, 4 and 5 days).

Salicylic acid triggers genotoxic adaptation to methyl mercuric chloride and ethyl methane sulfonate, but not to maleic hydrazide in root meristem cells of Allium cepa L.

Salicylic acid (SA), 0.01 mM, a signalling phytohormone, was tested for induction of adaptive response against genotoxicity of methyl mercuric chloride (MMCl), 0.013 mM; ethylmethane sulfonate (EMS), 2.5 mM, or maleic hydrazide (MH), 5 mM, in root meristem cells of Allium cepa. Induction of adaptive response to EMS by hydrogen peroxide (H2O2), 1 mM, and yet another secondary signal molecule was tested for comparison. Assessed by the incidence of mitoses with spindle and/or chromosome aberration and micronucleus, the findings provided evidence that SA-conditioning triggered adaptive response against the genotoxic-challenges of MMCl and EMS, but failed to do so against MH. H2O2, which is known to induce adaptive response to MMCl and MH, failed to induce the same against EMS in the present study. The findings pointed to the possible role of signal transduction in the SA-induced adaptive response to genotoxic stress that perhaps ruled out an involvement of H2O2.

[Inhibitory effects of exogenous abscisic acid and maleic hydrazide on panicle sprouting in hybrid rice F1].

"On-panicle seed sprouting" is a serious obstacle in hybrid rice seed production. It was reported that exogenous ABA inhibited seed sprouting of hybrid rice F(1) seeds. It was shown that by ABA 1000 mg/L or MH 4000 mg/L application at 21 d after full heading (filling stage)(Fig.1), not only seed sprouting was inhibited, but also the seed energy was affected (Fig.2). The retardation of germination and lowing of germination rate were resulted from ABA treatment and MH treatment respectively (Fig.3). When the seeds were treated with ABA, GA(1) level was decreased (Fig.4). The expression of amylase was delayed and their levels become lowed by ABA treatment (Fig.5). However, those seeds maintained a certain level of GA(1) and amylase and still have germination ability. During germination, the GA(1) content and amylase activity did not decrease significantly by MH treatment, but the GA(1) content and amylase activity were significantly decreased in non-germination MH treated seeds (Fig.4, 5). Therefore, the sprouting retarding effects of ABA was considered as "post-harvest effects" and sprouting inhibiting effect of MH was considered as "inhibition effects" by the authors.

The Use of Maleic Hydrazide for Effective Hybridization of Setaria viridis.

An efficient method for crossing green foxtail (Setaria viridis) is currently lacking. S. viridis is considered to be the new model plant for the study of C4 system in monocots and so an effective crossing protocol is urgently needed. S. viridis is a small grass with C4-NADP (ME) type of photosynthesis and has the advantage of having small genome of about 515 Mb, small plant stature, short life cycle, multiple tillers, and profuse seed set, and hence is an ideal model species for research. The objectives of this project were to develop efficient methods of emasculation and pollination, and to speed up generation advancement. We assessed the response of S. viridis flowers to hot water treatment (48°C) and to different concentrations of gibberellic acid, abscisic acid, maleic hydrazide (MH), and kinetin. We found that 500 µM of MH was effective in the emasculation of S. viridis, whilst still retaining the receptivity of the stigma to pollination. We also report effective ways to accelerate the breeding cycle of S. viridis for research through the germination of mature as well as immature seeds in optimized culture media. We believe these findings will be of great interest to researchers using Setaria.

Genotoxicity of humic acid in cultured human lymphocytes and its interaction with the herbicides alachlor and maleic hydrazide.

The genotoxicity of humic acid and its possible interaction with the herbicides alachlor and maleic hydrazide have been evaluated in cultured human lymphocytes from two donors. Humic acid and the two herbicides have been tested (alone and combined) for sister-chromatid exchange (SCE) induction. In addition, the effect of two different preincubation times, 2 and 24 hr, was analyzed. The results indicate that humic acid and the herbicides alachlor and maleic hydrazide appear to significantly enhance the frequency of SCE, the effect of the herbicides being more pronounced. With reference to the possible interaction of humic acid with the herbicides, the results do not show a common pattern, although mainly an additive effect was obtained. Nevertheless, there is some evidence suggesting that antagonism may occur, especially in the combined treatment of humic acid and maleic hydrazide.

Anticlastogenic effect of Spirulina maxima extract on the micronuclei induced by maleic hydrazide in Tradescantia.

The aim of this investigation was to determine if extracts of Spirulina maxima reduce the genotoxic damage induced by maleic hydrazide (MH) using the Tradescantia biosssay. Two types of extracts from the alga were prepared: an aqueous extract with two different concentrations, 100 and 500 mg/ml, and a second one, the extract of a 1% solution of dimethyl sulfoxide (DMSO) which corresponded to 100 mg/ml of the alga. The capacity of MH to induce micronuclei (MN) was initially established by administering 0.005, 0.01, and 0.015 mg/ml of the chemical to the Tradescantia inflorescences, and observing its effect after 24 h.The results of this experiment showed a significant MN increase with the two high concentrations tested, although no dose-response effect was observed. For the anticlastogenic assay, the extracts of Spirulina were applied to the inflorescences alone or immediately before the application of MH (0.01 mg/ml) and the induced MN were observed 24 h later. We found that none of the extracts increased the MN level with respect to the untreated plants; also, that MH more or less doubled the basal micronuclei frequency, and finally, that all tested extracts reduced the genotoxic damage caused by MH. The inhibitory indices obtained for the aqueous extracts (100 and 500 mg/ml) and for the DMSO extract were respectively 59, 85, and 56.3%. These data indicate that Spirulina is an anticlastogenic agent and suggest that it is advisable to extend studies on this matter using other biological models.

Factors affecting the production of SCEs by maleic hydrazide in root-tip chromosomes of Allium cepa.

We have investigated the influence of pH on the induction of chromatid-type aberrations and sister-chromatid exchanges (SCEs) by maleic hydrazide (MH) in root-tip cells of Allium cepa. For both cytogenetic endpoints, the lower the pH of the treatment solution, the higher were the frequencies of chromosome alterations detected at metaphase. We have further studied the persistence of lesions giving rise to SCEs during successive cell cycles, as well as the influence of BrdU concentration in the post-treatment medium on the yield of MH-induced SCEs. Our results suggest that the cytogenetic action of MH in many respects resembles that of bifunctional alkylating agents.

Effects of novobiocin on heat shock protection against chromatid aberration induction by triethylenemelamine (TEM) and maleic hydrazide (MH) in Vicia faba.

Heat shock (10 min 40 degrees C) prior to challenge treatment with triethylenemelamine (TEM) or maleic hydrazide (MH) significantly reduced the frequency of induced chromatid aberrations in Vicia faba main root meristems. Novobiocin treatment before heat shock did not prevent heat shock protection against both clastogens; novobiocin application after heat shock prevented protective effects. These results and those obtained earlier for heat shock protection against X-ray challenge are used to discuss possible causes underlying the protective effects triggered by heat shock.

Are SCE frequencies indicative of adaptive response of plant cells?

Low-dose pretreatments with maleic hydrazide, mitomycin C, and N-methyl-N-nitrosourea or sublethal heat shock were tested with regard to their effect on sister-chromatid exchange (SCE) induction by high doses of the same mutagens administered 2 h later to root-tip meristems of Vicia faba. Consecutive treatments resulted in either additive or, in a minority of experiments, in below-additive SCE frequencies. A model is proposed to explain the conflicting data reported on adaptation to SCE and aberration induction.

Heat shock protection against induction of chromatid aberrations is dependent on the time span between heat shock and clastogen treatment of Vicia faba root tip meristem cells.

Variation of the time span between heat shock (hs) and clastogen treatment of V. faba root tip meristems showed that hs protection is a very quick response (effective after less than 10 min) and lasting for up to 240 min in the case of induction of chromatid aberrations by maleic hydrazide (MH). Analogous protective responses are significantly slower and shorter when TEM is used for aberration induction. This, together with absence of 'clastogenic cross-adaptation' to these agents and differential effects of benzamide (BA, an inhibitor of poly-ADP-ribosylation) pretreatment before hs on hs protection, suggests that hs before clastogen treatment triggers at least 2 clastogen-specific, protective functions which eventually result in protection against these 2 clastogens.

On the expressivity of aberration hot spots after treatment with mutagens showing delayed or non-delayed effects.

The intrachromosomal distribution patterns of chromatid aberrations induced by agents with delayed effects (as exemplified by ethanol and maleic hydrazide) were compared with those produced by agents with non-delayed effects (as exemplified by fast neutrons, X-rays and bleomycin). Despite nonrandomness of aberration distribution in all cases, the mutagens with nondelayed effects generally showed up with much less pronounced aberration hot spots than the mutagens with delayed effects. From the results obtained it is concluded that hot-spot expressivity is a characteristic "group-specific" feature of the two classes of mutagen and that aberration production during DNA replication (S-phase) by agents with delayed effects strongly favours a very pronounced aberration clustering, which is partly mutagen-specific. Possible causes of these differences with respect to hot-spot expressivity after treatment with mutagens showing non-delayed and delayed effects, respectively, are discussed.

Premature onset of mitosis and potentiation of chromosome damage induced by poly-D-lysine in plant cells: evidence for G2 repair.

Poly-D-lysine has been reported to induce a triggering of mitosis in plant cells due to a selective stimulatory effect on cells arrested in G2. Root-tip cells of Allium cepa L. were first exposed to maleic hydrazide (MH) early in the cell cycle and posttreated with different concentrations of the polycationic agent while in G2. The result was a dose-dependent potentiation of chromosome damage observed at metaphase without any apparent effect induced by poly-D-lysine itself. The enhancement of the yield of chromosomal aberrations was concomitant with an increase in the frequency of mitosis. In order to test further the stimulatory effect of poly-D-lysine on mitosis, as well as the consequences of a shortening of the time available for repair, cells synchronized by protracted treatment with 5-aminouracil (5-AU), which also induces chromosome damage, were allowed to recover in the presence of the polycationic compound. Our data show that a premature arrival at mitosis resulted in an increase in the frequency of damaged cells observed.

Effects of benzamide pretreatment on clastogenic adaptation of Vicia faba root-tip meristem cells to triethylenemelamine (TEM) and maleic hydrazide (MH).

Clastogenic adaptation to TEM or MH no longer occurred when benzamide, an inhibitor of nuclear ADP-ribosyltransferase (ADPRT), was applied prior to the low dose (conditioning) treatment which triggers this phenomenon. This may be indicative that inducible processes connected with ribosylation reactions are involved in the protective effects exerted by clastogenic adaptation. No increase by benzamide pretreatment was observed in the yield of metaphases with TEM- or MH-induced chromatid aberrations after conditioning and challenge treatment, respectively. High benzamide concentrations (1 h, 5 X 10(-3) M) exerted protective effects against TEM challenging but not against MH.