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.

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.

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.

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.

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.

Cell division interference in newly fertilized ovules induces stenospermocarpy in cross-pollinated citrus fruit.

Seedlessness is a highly desirable characteristic in fresh fruits. However, post-fertilization seed abortion of cross-pollinated citrus fruit is uncommon. The factors regulating stenospermocarpy in citrus are unknown. In this research, we induced stenospermocarpy interfering in newly fertilized ovule cell division. The research also elucidates the most sensitive stage for ovule/seed abortion in citrus. Experiments were conducted with 'Afourer' mandarin that cross-pollinates with several cultivars and species. Cross-pollinated fruitlets were treated with maleic hydrazide (MH), a systemic growth regulator that specifically interferes in cell division. MH reduced ovule growth rate, the number of cell layers in nucella and inhibited embryo sac expansion; moreover, the treatment increased callose accumulation in nucella and surrounding the embryo sac. Fruits developed an early-aborted seed type with an immature, soft and edible seed coat. Seed number (-80%) and seed weight (-46%) were reduced in mature fruits. MH also hampered cell division in ovary walls, mesocarp and endocarp, thus reducing daily fruitlet growth and increasing fruit abscission. Stenospermocarpy could only be induced for a short period of time in the progamic phase of fertilization, specifically, when ovules are ready to be fertilized (7 days after anthesis) to early stages of embryo sac development (14 days after anthesis).

[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.

FISH in analysis of gamma ray-induced micronuclei formation in barley.

A micronucleus test in combination with fluorescent in situ hybridization (FISH) using telomere-, centromere-specific probes and 5S and 25S rDNA was used for a detailed analysis of the effects of gamma ray irradiation on the root tip meristem cells of barley, Hordeum vulgare (2n=14). FISH with four DNA probes was used to examine the involvement of specific chromosomes or chromosome fragments in gamma ray-induced micronuclei formation and then to explain their origin. Additionally, a comparison of the possible origin of the micronuclei induced by physical and chemical treatment: maleic hydrazide (MH) and N-nitroso-N-methylurea (MNU) was done. The micronuclei induced by gamma ray could originate from acentric fragments after chromosome breakage or from whole lagging chromosomes as a result of a dysfunction of the mitotic apparatus. No micronuclei containing only centromeric signals were found. An application of rDNA as probes allowed it to be stated that 5S rDNA-bearing chromosomes are involved in micronuclei formation more often than NOR chromosomes. This work allowed the origin of physically- and chemically-induced micronuclei in barley cells to be compared: the origin of micronuclei was most often from terminal fragments. FISH confirmed its usefulness in the characterization of micronuclei content, as well as in understanding and comparing the mechanisms of the actions of mutagens applied in plant genotoxicity.

[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.

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.

Cytotoxicity and mode of action of maleic hydrazide in root tips of Allium cepa L.

Maleic hydrazide (MH) is an herbicide and is a regulator of the growth of buds in vegetables during storage. It is used in agriculture-in despite its known effect as a mutagenic and clastogenic agent. In this research the effect of MH on the root tips of Allium cepa L. was determined; correlations between the effects of different concentrations and exposure times on the mitotic index (MI) and induction of chromosomal aberrations (ChA) were also examined. Experiments were carried out in triplicate, using aqueous solutions of MH to concentrations of 10(-6), 10(-5), 10(-4) and 10(-3)M, at intervals of 0, 4, 8, 12, 24, and 48 h, with a control for each combination (with the MH substituted by distilled water). The results revealed an inhibition of the MI linked to the concentration and time of treatment (F=845.51, P<0.01 and F=427.58, P<0.01, respectively). For all the concentrations used and exposure periods longer than 12 h, different types of ChA were present, with significantly increased frequencies with increases in the concentration and time of exposure (P<0.01). To determine the mechanism through which the herbicide exerts its toxicity, ultrastructural electron microscopy was conducted. The results reveal nucleolar alterations, suggesting an inhibitory effect of biosynthetic activity.

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.

Specific inhibition of type II inosine monophosphate dehydrogenase activity of Tmolt4 T cell human leukaemia cells by 3-methoxy and di-benzohydroxamic acids, maleic hydrazide and malonic acids.

Small-molecular-weight benzohydroxamic and malonic acids and maleic hydrazide proved to be potent inhibitors of the activity of human Tmolt4 leukaemia Type II IMP (inosine monophosphate) dehydrogenase (IMPDH) activity. They were competitive inhibitors with respect to IMPDH demonstrating Ki values in the range 2.57-41.3 microM, less than half the values of the IC50 (microM) for the inhibition of Type II IMPDH. The IC50 microM values positively correlated with the ability of each compound to inhibit crude IMPDH activity, de-novo purine and DNA syntheses and growth of the T leukaemia cell line. Compounds were not inhibitors of Type I IMPDH. Type I IMPDH predominates in normal resting cells compared with Type II which is found in rapidly proliferating cells. Discovery of agents which would selectivity target IMPDH found in proliferating cells should eliminate any antineoplastic therapeutic toxic effects in normal cells of the body.

[A study on inhibitory effect of plant growth retardants on earlier bolting of Chinese Angelica].

OBJECTIVE: Seeking chemical ways of controlling the earlier bolting of Chinese Angelica (CA). METHOD: PP333, B9 and MH in different concentrations and combinations were sprayed into the CA leaves in different periods. RESULTS: In leaf proliferating period the spray could inkibit the earlier bolting, shortening the length of the stem foot, increasing the stem diameter and enhancing the leaf chlorophy 11 contents. The quality of CA production was increased as a result of greater root weight and main root volume. In leaf luxuriatino period the spray showed no marked effect. The growth of the plant would be inhibited by any spray in excessive concentrations. CONCLUSION: Leaf spray could control the earlier bolting of CA.

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.

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.

Unscheduled DNA synthesis in growing roots and stored embryos of Vicia faba after the action of maleic hydrazide and methyl methanesulphonate.

Growing roots of Vicia faba were treated with MH for 5 h, washed for 2 h and exposed to 3H-thymidine (3H-TdR) for additional 2-h periods at 7 h, 24 h and 32 h after the onset of MH treatment, to label DNA. As the replicative DNA synthesis was suppressed by HU, an enhancement of 3H-TdR incorporation into nuclear DNA above the control, as determined by microautoradiography, was considered to be due to unscheduled DNA synthesis induced by the mutagen. A significantly higher incorporation of 3H-TdR into DNA of MH-treated roots occurred, when labelling was applied 7 h after the MH action, whereas at 24 h only slight and at 32 h no enhancement of DNA labelling above control was registered. A 3-14-day storage with 50% water content of V. faba seeds exposed to MH or MMS resulted in a recovery from mutagen-induced chromosomal damage and a significantly higher incorporation of 3H-TdR into nuclear DNA. This supports the hypothesis that recovery from MH- and MMS-induced chromosomal damage is mediated by excision repair during seed storage.