Soybean Cell Suspension Research Articles

UV-B Radiation Exhibited Tissue-Specific Regulation of Isoflavone Biosynthesis in Soybean Cell Suspension Cultures.

Isoflavones, a class of substances with high biological activity, are abundant in soybeans. This study investigated isoflavone biosynthesis in soybean cell suspension cultures under UV-B radiation. UV-B radiation enhanced the transcription level and activity of key enzymes involved in isoflavone synthesis in cell suspension cultures. As a result, the isoflavone contents significantly increased by 19.80% and 91.21% in hypocotyl and cotyledon suspension cultures compared with the control, respectively. Meanwhile, a significant difference was observed in the composition of isoflavones between soybean hypocotyl and cotyledon suspension cultures. Genistin was only detected in hypocotyl suspension cultures, whereas glycitin, daidzein, and genistein accumulated in cotyledon suspension cultures. Therefore, UV-B radiation exhibited tissue-specific regulation of isoflavone biosynthesis in soybean cell suspension cultures. The combination of suspension cultures and abiotic stress provides a novel technological approach to isoflavone accumulation.

Development and validation of a novel and robust cell culture system in soybean (Glycine max (L.) Merr.) for promoter screening.

A novel soybean cell culture was developed, establishing a reliable and rapid promoter assay to enable high-throughput automated screening in soybean protoplasts relevant to shoot tissues in whole plants. Transient reporter gene assays can be valuable to rapidly estimate expression characteristics of heterologous promoters. The challenge for maximizing the value of such screens is to combine relevant cells or tissues with methods that can be scaled for high-throughput screening, especially for crop-rather than model species. We developed a robust and novel soybean cell suspension culture derived from leaf-derived callus for protoplast production: a platform for promoter screening. The protoplasts were transfected with promoter-reporter constructs, of which were chosen and validated against known promoter expression profiles from tissue-derived protoplasts (leaves, stems, and immature cotyledons) and gene expression data from plants. The cell culture reliably produced 2.82 ± 0.94 × 108 protoplasts/g fresh culture mass with a transfection efficiency of 31.06 ± 7.69% at 48h post-incubation. The promoter-reporter gene DNA expression levels of transfected cell culture-derived protoplasts were most similar to that of leaf- and stem-derived protoplasts (correlation coefficient of 0.99 and 0.96, respectively) harboring the same constructs. Cell culture expression was also significantly correlated to endogenous promoter-gene expression in leaf tissues as measured by qRT-PCR (correlation coefficient of 0.80). Using the manual protocols that produced these results, we performed early stage experiments to automate protoplast transformation on a robotic system. After optimizing the protocol, we achieved up to 29% transformation efficiency using our robotic system. We conclude that the soybean cell culture-to-protoplast transformation screen is amenable to automate promoter and gene screens in soybean that could be used to accelerate discoveries relevant for crop improvement. Key features of the system include low-cost, facile protoplast isolation, and transformation for soybean shoot tissue-relevant molecular screening.

Methyl Jasmonate Increases Isoflavone Production in Soybean Cell Cultures by Activating Structural Genes Involved in Isoflavonoid Biosynthesis

Isoflavonoids are a class of biologically active natural products that accumulate in soybean ( Glycine max L.) seeds during development, play vital roles in plant defense, and act as phytoestrogens with important human health benefits. Plant cell suspension cultures represent an excellent source of biologically important secondary metabolites. We found that methyl jasmonate (MJ) treatment increased isoflavone production in soybean suspension cell cultures. To investigate the underlying mechanism, we examined the expression of structural genes ( CHS6, CHS7, CHI1, IFS1, IFS2, IFMaT, and HID) in the isoflavonoid biosynthesis pathways in soybean suspension cells under various abiotic stress conditions. MJ treatment had the most significant effect on gene expression and increased the production of three glycosidic isoflavones (daidzin, malonyldaidzin, and malonylgenistin), with the maximum total isoflavone production (∼10-fold increase) obtained on day 9 after MJ application. MJ treatment significantly increased total phenolic contents and upregulated isoflavonoid biosynthesis genes, shedding light on the underlying mechanism.

Production of free and glycosylated isoflavones in in vitro soybean (Glycine max L.) hypocotyl cell suspensions and comparison with industrial seed extracts

Glycine max is one of the major sources of phytochemicals, in particular of isoflavones, a class of phytoestrogens with ascertained beneficial effects on human health. In the present study, in vitro callus production from soybean hypocotyl seedling explants and cell suspensions were optimized. Time-courses having 20, 40 and 60 g/L of initial cell inoculum were performed to determine the concentration most suitable for isoflavone production. The amount of total polyphenols and total flavonoids as well as the antioxidant capacity of both cell and culture media fractions were measured by means of spectrophotometric methods. The levels of aglycone and glycosylated isoflavones (didzein, genistein, glycitein, didzin, genistin, glycitin), as well as of ferulic acid, vanillic acid and vanillin, were determined by HPLC–DAD. On average, 93.5 % of the total (cells plus media) isoflavones in soybean cell suspensions were detected as aglycones. Concentrated cell cultures as well as industrial soybean seed extracts were enzymatically hydrolyzed to release the aglycones and their metabolic profiles were analysed by HPLC–DAD. In contrast to cell suspensions, in undigested seed extract the aglycon form represented only 16.8 % of the total isoflavones amount. After enzymatic treatment, the antioxidant capacity increased by 30 and 33 %, respectively, in concentrated cell and seed extracts, demonstrating the presence of a larger amount of bioactive metabolites after digestion. At the present extraction conditions, soybean concentrated cell suspensions yielded 5.8-fold more total isoflavones (mostly in the free form) than seed extracts, leading to hypothesise their possible use as ingredients for cosmetic and nutraceutical applications.

Isoflavone Augmentation in Soybean Cell Cultures Is Optimized Using Response Surface Methodology

Glycine max contains potential therapeutic isoflavones, and its productivity in plants is considerably influenced worldwide by several biotic and abiotic factors. Optimization of soybean cell suspension cultures (Indian variety, JS 335) to maximize the cell growth and isoflavone production in the present study was performed using response surface methodology (RSM) with three independent variables of plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthalene acetic acid (α-NAA), and kinetin (Kn). The maximum biomass achieved was 70.62 g/L dry weight (dw) using the optimized medium of 2.10 mg/L 2,4-D, 5.52 mg/L α-NAA, and 0.35 mg/L Kn supplemented in the Murashige and Skoog (MS) basal medium. The total isoflavone content of 38.59 mg/g of dw was obtained in the medium with optimized conditions of 1.33 mg/L 2,4-D, 1.76 mg/L α-NAA, and 0.15 mg/L Kn. In comparison to field-grown soybean seeds, the cell suspension cultures profoundly augmented isoflavone concentrations. The optimized conditions for both biomass and total isoflavone content were evaluated by superimposing the contour plots. The results suggested that the optimized medium of cell suspension cultures possibly be used for scale-up studies in bioreactors to offer a high content of bioactive isoflavones.

Identification of Alternaria alternata mycotoxins by LC-SPE-NMR and their cytotoxic effects to soybean (Glycine max) cell suspension culture.

This present work describes the application of liquid chromatograpy-solid phase extraction-nuclear magnetic resonance spectroscopy to analyse Alternaria alternata crude extracts. Altenusin (1), alternariol (2), 3'-hydroxyalternariol monomethyl ether (3), and alternariol monomethyl ether (4), were separated and identified. High-resolution mass spectrometry confirmed the proposed structures. The cytotoxic effects of these compounds towards plants were determined using soybean (Glycine max) cell cultures as a model. EC50 values which range from 0.11 (±0.02) to 4.69 (±0.47) μM showed the high cytotoxicity of these compounds.

Fast Transmission Electron Microscope (TEM) Technique for Observing Isolated Mitochondria of Soybean (Glycine max (L.) Merr.) in about Six Hours

In his PhD thesis presented different studies designed to define methods for the study of the basic, intracellular mode of action of herbicides at very low doses, and to test these on two commercial herbicides simazine and bentazone, in soybean cell suspensions, consisting primarily of single cells with a few clumps. To determine the progressive herbicidal damage of fine structure as dose increased a fast technique for TEM observations was developed. The technique is based on procedures already proved for human leukocytes and in animal tissue. This technique, presented here in detail, allowed to treat the single cells with the herbicides mentioned above for 40 minutes, fix, dehydrate, embed, and get the blocks ready for sectioning in about six hours. The same fast technique also allowed to observe the integrity of isolated soybean mitochondria previous to the citochrome c oxidase studies. The great similarity existing among plant, human and animal mitochondria has being observed long time ago. It seems reasonable to think that the fast technique, mentioned above, to observe the soybean mitochondria, could be useful, once adapted, to study human and animal mitochondria, due to the great similarity among the mentioned organelles.

GmPep914, an Eight-Amino Acid Peptide Isolated from Soybean Leaves, Activates Defense-Related Genes

Only a handful of endogenous peptide defense signals have been isolated from plants. Herein, we report a novel peptide from soybean (Glycine max) leaves that is capable of alkalinizing the media of soybean suspension cells, a response that is generally associated with defense peptides. The peptide, DHPRGGNY, was synthesized and found to be active at 0.25 nM and requiring only 5 to 10 min to obtain a maximal pH change. The peptide is located on the carboxy-terminal end of a 52-amino acid precursor protein (Glyma12g00990) deduced from the soybean genome project. A search of the soybean databank revealed a homolog (Glyma09g36370) that contained a similar peptide, DLPRGGNY, which was synthesized and shown to have identical activity. The peptides, designated GmPep914 (DHPRGGNY) and GmPep890 (DLPRGGNY), were capable of inducing the expression of both Glyma12g00990 (GmPROPEP914) and Glyma09g36370 (GmPROPEP890) in cultured soybean suspension cells within 1 h. Both peptides induced the expression of defense genes, including CYP93A1, a cytochrome P450 gene involved in phytoalexin synthesis, chitinaseb1-1, a chitinase involved in pathogen defense, and Glycine max chalcone synthase1 (Gmachs1), chalcone synthase, involved in phytoalexin production. Both GmPROPEP914 and GmPROPEP890 were highly expressed in the roots, relative to the aerial portions of the plant. However, treatment of the aerial portion of soybean plants with hormones involved in elicitation of defense responses revealed a significant increase in expression levels of GmPROPEP914 and GmPROPEP890. A search of gene databases revealed homologous sequences in other members of the Fabales and also in the closely related Cucurbitales but not in any other order of plants.

High external phosphate (Pi) increases sodium ion uptake and reduces salt tolerance of ‘Pi‐tolerant’ soybean

Previous studies on the interaction between environmental inorganic phosphate (Pi) and salinity stress using soybean cultivars sensitive to high external Pi had two limitations: (1) the phenotype was dominated by overaccumulation of phosphorus (P); and (2) no detailed analysis was performed for sodium ion uptake. In this study, we focused on the effects of high external Pi on the sodium ion uptake in 'Pi-tolerant' soybean cultivars. The P accumulation in Pi-tolerant soybean Union was much lower [9.0 mg g(-)(1) dry weight (DW); contrasting to 38-76 mg g(-)(1) DW in the 'Pi-sensitive' soybean cultivars]. At in planta level, high level of external Pi significantly (P < 0.001) increased net sodium ion uptake and aggravated salinity stress symptoms. The effects of high external Pi diminished when de-rooted plants were used, suggesting that root is the primary organ interacting with Pi in the growth medium. Two-cell models, including soybean suspension cells and the tobacco Bright Yellow-2 cell line, were also employed to study the effects of high external Pi at the cellular level. Consistent to in planta results, high external Pi uplifted cellular sodium ion uptake and reduced cell viability under salinity stress. Gene expression analyses further showed that HPi (2 mM Pi supplements; excessive level of Pi) could reduce the fold of induction of GmSOS1 and GmCNGC under salinity stress, suggesting that they may be possible molecular targets involved in the interaction between high external Pi and Na(+) uptake.

A New Branch of Endoplasmic Reticulum Stress Signaling and the Osmotic Signal Converge on Plant-specific Asparagine-rich Proteins to Promote Cell Death

NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene glycol treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activation of the UPR by ER stress inducers, but did not affect activation of NRPs. We also found that this integrated pathway transduces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senescence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmentation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytokinin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.

Analysis of the limitations of hepatitis B surface antigen expression in soybean cell suspension cultures

Soybean cell suspension cultures were transformed using Agrobacterium tumefaciens harboring pHBS/pHER constructs to express hepatitis B surface antigen (HBsAg). The transformed colonies were selected and analyzed for the expression of HBsAg by PCR, reverse transcription (RT) PCR, Western blot and ELISA analysis. The maximum expression of 700 ng/g F.W. was noted in pHER transformed cells. The highest expressing colonies were used to initiate the cell suspension cultures and the expression of HBsAg was estimated periodically. The expression levels were reduced drastically in cell suspension cultures compared to the colonies maintained on semi-solid medium. Various parameters were studied to maximize the cell growth and to retain the expression levels. The supplementation of culture medium with a protease inhibitor, leupeptin hemisulfate could restore up to 50% of HBsAg expression in cell suspension cultures. This is the first report to investigate the possible cause and solution to the loss of recombinant protein expression levels in plant cell suspension cultures.

Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride.

BackgroundCalcium is commonly involved as intracellular messenger in the transduction by plants of a wide range of biotic stimuli, including signals from pathogenic and symbiotic fungi. Trichoderma spp. are largely used in the biological control of plant diseases caused by fungal phytopathogens and are able to colonize plant roots. Early molecular events underlying their association with plants are relatively unknown.ResultsHere, we investigated the effects on plant cells of metabolite complexes secreted by Trichoderma atroviride wild type P1 and a deletion mutant of this strain on the level of cytosolic free Ca2+ and activation of defense responses. Trichoderma culture filtrates were obtained by growing the fungus alone or in direct antagonism with its fungal host, the necrotrophic pathogen Botrytis cinerea, and then separated in two fractions (>3 and <3 kDa). When applied to aequorin-expressing soybean (Glycine max L.) cell suspension cultures, Trichoderma and Botrytis metabolite mixtures were distinctively perceived and activated transient intracellular Ca2+ elevations with different kinetics, specific patterns of intracellular accumulation of reactive oxygen species and induction of cell death. Both Ca2+ signature and cellular effects were modified by the culture medium from the knock-out mutant of Trichoderma, defective for the production of the secreted 42 kDa endochitinase.ConclusionNew insights are provided into the mechanism of interaction between Trichoderma and plants, indicating that secreted fungal molecules are sensed by plant cells through intracellular Ca2+ changes. Plant cells are able to discriminate signals originating in the single or two-fungal partner interaction and modulate defense responses.

Effects of exogenous nitric oxide on the antioxidant capacity of cadmium-treated soybean cell suspension

Nitric oxide (NO) is a small, ubiquitous molecule, whose physiological function in plants has recently been widely investigated. It seems that one of its pivotal properties is the antioxidant capacity, enabling plants to alleviate the effects of the oxidized stress. In this work we investigated the role of NO in soybean (Glycine max L. Cv. Navico) cell suspension treated with cadmium. Sodium nitroprusside (SNP), nitric oxide donor, markedly decreased the negative influence of Cd2+ on cell growth. It was also found to stimulate superoxide dismutase (SOD, EC 1.15.1.1). Using specific fluorochromes — dihydroethidine (DHE) and 2′,7′- dichlorofluorescein (DCFH-DA) it was shown that NO was very effective in reducing the level of superoxide anion (O2·−) and hydrogen peroxide, respectively. Furthermore, as evaluated by means of NO specific fluorochrome 4,5-diaminofluorescein diacetate (DAF-2DA), increased production of NO was found in Cd-treated cells. In cadmium-stressed cells SNP lowered the level of oxidized proteins.

Identification and subcellular localization of the soybean copper P 1B-ATPase GmHMA8 transporter

We have identified a copper P 1B-ATPase transporter in soybean ( Glycine max), named as GmHMA8, homologue to cyanobacterial PacS and Arabidopsis thaliana AtHMA8 (PAA2) transporters. A novel specific polyclonal anti- GmHMA8 antibody raised against a synthetic peptide reacted with a protein of an apparent mass of around 180–200 kDa in chloroplast and thylakoid membrane preparations isolated from soybean cell suspensions. Immunoblot analysis with this antibody also showed a band with similar apparent molecular mass in chloroplasts from Lotus corniculatus. Immunofluorescence labelling with the anti- GmHMA8 antibody and double immunofluorescence labelling with anti- GmHMA8 and anti-RuBisCo antibodies revealed the localization of the GmHMA8 transporter within the chloroplast organelle. Furthermore, the precise ultrastructural distribution of GmHMA8 within the chloroplast subcompartments was demonstrated by using electron microscopy immunogold labelling. The GmHMA8 copper transporter from soybean was localized in the thylakoid membranes showing a heterogeneous distribution in small clusters.

A light-sensitive mechanism differently regulates transcription and transcript stability of ω3 fatty-acid desaturases ( FAD3, FAD7 and FAD8) in soybean photosynthetic cell suspensions

The ω3 fatty-acid desaturases: FAD7 and FAD8 (plastid) and FAD3 (reticular) are responsible for trienoic fatty-acid (TA) production in plants. The expression of these enzymes seemed to be regulated differently in response to light. Darkness leads to a decrease in total TA level. Under such conditions, FAD3 and FAD8 transcript levels were undetectable but increased after re-illumination concomitant with TA levels, indicating a transcriptional control. On the contrary, FAD7 transcript levels were similar to illuminated control cells, suggesting the presence of a post-transcriptional control mechanism. Furthermore, FAD7 mRNA stability increased dramatically in darkness. Analysis of FAD7 protein accumulation using specific antibodies revealed that FAD7 was very stable whatever the light or darkness conditions. These results indicate that FAD7 enzyme availability is not limiting for 18:3 production in darkness. Our data point to an additional post-translational regulatory mechanism that controls the activity of FAD7 in response to light.

Isolation of a novel ABC-transporter gene from soybean induced by salicylic acid

This paper reports on the identification and characterization of a new ATP-binding cassette (ABC) transporter which was identified as a salicylic acid-induced gene from soybean (Glycine max cv. Williams 82) in a subtractive suppression hybridization approach. A fragment of an ABC-transporter gene was used to isolate a full-length cDNA clone for this gene with a length of 4750 bp. The encoded protein has a length of 1447 amino acids and is composed of two similar repeat units typical of full-size ABC transporters. The sequence displays a close relationship to plant pleiotropic drug resistance (PDR)-type transporters and, on a homology basis, clusters together with the Arabidopsis thaliana PDR12 gene, suggesting GmPDR12 as a name for the gene isolated from soybean. GmPDR12 is rapidly responsive to salicylic acid and methyl jasmonate. The mRNA starts to accumulate 30 min after the addition of the signalling compounds. Salicylic acid is required for the execution of the hypersensitive reaction in soybean cell suspension cultures inoculated with Pseudomonas syringae pv. glycinea. It has been demonstrated previously that salicylic acid can be substituted by a variety of functional analogues of salicylic acid. All of these compounds lead to a strong and rapid transcriptional activation of GmPDR12, suggesting a shared signalling pathway.

Excess copper effect on growth, chloroplast ultrastructure, oxygen‐evolution activity and chlorophyll fluorescence in Glycine max cell suspensions

The influence of excess copper on soybean photosynthetic cell suspensions was investigated. The cell suspensions grew well in the presence of 5–20 µM CuSO4 and developed tolerance to even higher levels of CuSO4 (i.e. up to 50 µM), indicating that copper was not toxic to the cells at that high concentrations. Cu‐adapted cell suspensions grew faster than the control in limiting light conditions and had higher content of chlorophyll per dry weight of cells. Copper was accumulated within the cells, and this event was accompanied by (1) increased oxygen evolution activity; (2) increased number of chloroplasts per cell, smaller chloroplasts, increased thylakoid stacking and grana size; (3) higher fluorescence emission of photosystem II antenna complexes and (4) stimulation of plastocyanin protein synthesis compared with untreated cells. Microanalysis of cross‐sections revealed an increase of copper content in chloroplasts as well as vacuole, cytoplasm and cell wall in Cu‐adapted cells. No antagonist interaction between copper and iron uptake took place in these cell suspensions. On the other hand, copper at subtoxic concentrations stimulated oxygen evolution activity in thylakoids from control cells, but this event did not take place in those from Cu‐adapted ones. Furthermore, the loss of activity by copper inhibitory action at toxic concentrations was two‐fold slower in thylakoids from Cu‐adapted cells compared with the control ones. The data strongly indicate that copper plays a specific positive role on photosynthesis and stimulates the growth and the oxygen evolution activity in soybean cell suspensions.

Effects of Feeding Spodoptera littoralis on Lima Bean Leaves. III. Membrane Depolarization and Involvement of Hydrogen Peroxide

In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H(2)O(2)) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H(2)O(2) was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H(2)O(2) was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H(2)O(2) in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H(2)O(2) prompted a transient transmembrane potential (V(m)) depolarization, with a V(m) depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca(2+)-sensing aequorin system, increasing amounts of added H(2)O(2) correlated with a higher cytosolic calcium ([Ca(2+)](cyt)) concentration. In MD and HW leaves, H(2)O(2) also triggered the increase of [Ca(2+)](cyt), but MD-elicited [Ca(2+)](cyt) increase was more pronounced when compared to HW leaves after addition of exogenous H(2)O(2). The results clearly indicate that V(m) depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.

Apoplastic redox metabolism: Synergistic phenolic oxidation and a novel oxidative burst

The plant apoplast is an important mediator of communication between the cell cytoplasm and its surroundings. Plant cell suspensions offer a convenient model system to gain insight into apoplastic physiology. Here, we describe a novel phenomenon that took place when two naturally occurring phenolics were added together to either soybean or tobacco cell suspensions. Acetosyringone (AS) and/or hydroxyacetophenone (HAP), phenolics found in the extracellular/apoplast of tobacco cells, were added to soybean or tobacco cell suspensions undergoing an oxidative burst. Individually, AS appeared to be utilized as a typical peroxidase substrate to scavenge hydrogen peroxide, while HAP was utilized at a much lower rate. However, when added together the rate of utilization of both phenolics increased and surprisingly resulted in the production of hydrogen peroxide. We have further characterized this novel phenomenon in suspension cells. This study demonstrates that certain phenolics in plants can cause co-oxidation which, as in animals, could alter the structure and bioactivity of surrounding phenolics.

Proteins induced by cadmium in soybean cells

The cadmium (Cd)-induced changes in protein pattern and identification of metal-stimulated polypeptides were analyzed in soybean cell suspension culture. The cell cultures were treated with various concentrations of Cd(2+) (3-10microM) for 24, 48 and 72h. The synthesis of [(35)S]-labeled proteins and their accumulation were analyzed by SDS-PAGE, whereas the identification of selected protein bands was performed by mass spectrometry. It is shown that Cd induced the appearance of the following proteins in soybean cells: superoxide dismutase, histone H2B, chalcone synthase and glutathione transferase.